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Academic year 2002-2003

COMPARATIVE STUDY OF THE QUALITY OF MICROPROPAGATED C3, C4 AND CAM PLANTS DURING

THE ACCLIMATIZATION STAGES

VERGELIJKENDE STUDIE VAN DE KWALITEIT VAN GEMICROPROPAGEERDE C3-, C4- EN CAM-PLANTEN

GEDURENDE DE ACCLIMATISATIESTADIA

door

MSc. Pham Thi Sen

Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences:

cell and gene biotechnology

Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wetenschappen:

Cel- en genbiotechnologie

Op gezag van Rector Prof. dr. A. DE LEENHEER

Decaan Promotoren

Prof. dr. ir. H. VAN LANGENHOVE Prof. dr. ir. P. DEBERGH Prof. dr. HO Huu Nhi

Faculteit Landbouwkundige en

Toegepaste Biologische Wetenschapppen

The author and the promoters give authorization to consult and copy parts of this book for

personal use only. Any other use is limited by laws of Copyright. Permission to reproduce any

material contained in this work should be obtained from the author.

De auteur en de promotor geven de toelating dit doctoraatswerk voor consultatie beschikbaar

te stellen, en delen ervan te kopieren voor persoonlijk gebruik. Elk ander gebruik valt onder

de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting

uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten uit dit werk.

Prof. Dr. ir. Pierre DEBERGH Pham Thi Sen Prof. Dr. HO Huu Nhi Author Promotors

ACKNOWLEDGEMENTS

I am deeply grateful to my promoter, Prof. Pierre Debergh, for his kindness, generosity, supervision and assistance throughout the course of my PhD study. The immense efforts of Prof. Dirk de Waelle in developing cooperation between VLIR and VASI, and facilitating my studies are really appreciated. My sincere gratitude to Prof. Alan Cassells for his invaluable encouraging e-words, moral supports and talks throughout many years since. To Prof. Ho Huu Nhi, many thanks for his sincere assistance and sympathy.

The valuable comments and suggestions of the members of the reading and examination committee (Prof. dr. ir. Pierre Debergh, Prof. dr. Ho Huu Nhi, Prof. dr. ir. J. Viaene, Prof. dr. ir. D. Reheul, Prof. dr. R. Lemeur, Prof. dr. ir. M. Höfte, Dr. ir. J. Van Huylenbroeck, Dr. ir. S. De Schepper and Dr. ir. S. Roeland) are greatly appreciated.

I would like to thank Nicole for her patience with all sorts of the paper work related to my studies. My deepest appreciation to Thea, Els, Paul2, Pol, Adrien, Sandra, Hans and Sophie for their kind care of my cultures, plants, experiments and all kinds of troubleshooting during the preparation of this thesis. The assistance of Katrien with the difficult FID and TCD, Helga with the expensive PAM, and Diep with the LCA is unforgettable.

I wish also to say “sincere thank you ” to Koen, Patricia, to all the staff of the Department of Plant Production, UGent and the Department of Biotechnology, VASI, for their valuable friendship, assistance and encouragement. Lots of thanks to all my friends, too many to be named, for their continuing support, sympathy and generosity so that this thesis could be completed with easy and happy moments.

Last, but not least, I am greatly indebted to my husband, Viet Hung and our two children, Hai Van and Van Hai, who are always with me wherever I am, whatever I do.

Gent, May 2003.

TABLE OF CONTENTS

SUMMARY i SAMENVATTING iii LIST OF ABBREVIATIONS v CHAPTER 1 GENERAL INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 4

2.1. LIGHT, AND THE PLANT GROWTH AND DEVELOPMENT 5

2.1.1. Light and photosynthesis of plants 5 2.1.1.1. Photosynthetic systems and light reactions 5 2.1.1.2. Calvin-Benson cycle and C3 photosynthesis 8 2.1.1.3. Low efficiency of Rubisco and other photosynthesis pathways 9

2.1.2. Light and photomorphogenesis of plants 15

2.1.3. Photoinhibition and photoprotection of plants 17 2.1.3.1. Photodamages caused by the excess of light energy absorbed 17 2.1.3.2. Photosynthetic photoprotection and acclimatization of plants 20

2.2. EFFECTS OF SPECIAL IN VITRO CULTURE CONDITIONS ON THE IN VITRO CULTURED PLANT GROWTH AND DEVELOPMENT 25

2.2.1. Photosynthetic behaviour of plantlets in vitro 26 2.2.1.1. Effects of CO2 and O2 levels inside the vessel 27 2.2.1.2. Effects of light conditions 28 2.2.1.3. Effects of sugar in the culture medium 28 2.2.1.4. Relative humidity and temperature inside the vessel 30

2.2.2. Photoinhibition and photorespiration of plantlets in vitro 31

2.2.3. Photomorphogenesis of in vitro propagated plants 32

2.3. ADAPTATION OF TISSUE CULTURE DERIVED PLANTS TO THE EX VITRO CONDITIONS 34

2.3.1. Factors affecting ex vitro acclimatisation 34

2.3.2. The plantlet adaptation capacity and methods for its quantification 36 2.3.2.1. Water retention capacity and leaf structure 36 2.3.2.2. Net CO2 uptake and carboxylating enzymes 37 2.3.2.3. Chlorophyll fluorescence 38 2.3.2.4. Protective enzymes 41

CHAPTER 3 GENERAL EXPERIMENTAL MATERIALS AND METHODS 43

3.1. PLANT MATERIALS 44

3.1.1. Subject plants 44 3.1.2. Stock plant source 44

3.2. EXPERIMENTAL AND GROWTH CONDITIONS 45

3.2.1. In vitro conditions 45 3.2.1.1. General in vitro culture conditions 45 3.2.1.2. Production of microplants of different physiological quality 45

3.2.2. Greenhouse conditions 47

3.3. OBSERVATION METHODS 47

3.3.1. Plant growth parameters 47 3.3.2. Stomatal density and size 48 3.3.3. Study of leaf anatomy 48 3.3.4. Observation of gas evolution in the culture vessel headspace 49 3.3.5. Determination of photosynthetic pigment contents 50 3.3.6. Measurement of net photosynthetic rate 50 3.3.7. Determination of PEP-case activity 51 3.3.8. Determination of SOD- activity 52 3.3.9. Determination of catalase activity 53 3.3.10. Determination of total soluble proteins 53 3.3.11. Chlorophyll fluorescence study 54

3.4. EXPERIMENTAL DESIGN AND DATA ANALYSIS 54

CHAPTER 4 EXPERIMENTAL RESULTS AND DISCUSSION 55

4.1. GROWTH AND PHOTOMORPHOGENESIS OF MICROPROPAGATED PLANTS DURING THE ACCLIMATIZATION PERIOD; DIFFERENCES BETWEEN PLANTLETS PRODUCED UNDER DIFFERENT IN VITRO LIGHT QUALITY AND CULTURE MEDIUM CONDITIONS, AND BETWEEN C3, C4 AND CAM PLANT 56

4.1.1. Specific introduction and objectives 56

4.1.2. Specific materials and methods 59

4.1.3. Results 60 4.1.3.1. Rooting of plantletss in vitro 60 4.1.3.2. Leaves 62 4.1.3. 3. Plant weight and dry matter content 64 4.1.3.4. Stomata density and size 66 4.1.3.5. Leaf mesophyll structure 69 4.1.3.6. Survival of plantlets 70

4.1.4. Discussion 71 4.1.4.1. Effects of AC and Gelrite 71 4.1.4.2. Effects of light quality 72 4.1.4.3. Leaf mesophyll structure 74 4.1.4.4. Plant survival and overall performance 75

4.2. PHOTOSYNTHESIS OF MICROPROPAGATED PLANTS; DIFFERENCES BETWEEN PLANTLETS PRODUCED UNDER DIFFERENT IN VITRO LIGHT QUALITY AND CULTURE MEDIUM CONDITIONS, AND BETWEEN C3, C4 AND CAM PLANTS 76



4.2.3. Results and discussions 78 4.2.3.1. Evolution of gasses in the culture container headspace 78 4.2.3.2. Net photosynthetic rates of transplants 87 4.2.3.3. Maximal quantum yield of transplants 88 4.2.3.4. Overall photoquantum yield 90 4.2.3.5. Photochemical quenching 91 4.2.3.6. Specific activity of PEP-carboxylase 92

4.2.4. Conclusions 97

4.3. P H O T O I N H I B I T I O N A N D P H O T O P R O T E C T I O N O F

MICROPROPAGTED PLANTS DURING THE ACCLIMATIZATION PERIOD; DIFFERENCES BETWEEN PLANTLETS OF DIFFERENT PHYSIOLOGICAL QUALITY, AND BETWEEN C3, C4 AND CAM PLANTS 98



4.3.3. Results and discussion 100 4.3.3.1. Minimal chl a fluorescence (Fo) 100 4.3.3.2. Fm and Fm' of transplants 101 4.3.3.3. Non-photochemical quenching 102 4.3.3.4. Catalase activity 103 4.3.3.5. Total SOD-activity 105 4.3.3.6. Pigment concentration 107

4.3.4. Conclusions 111

CHAPTER 5 GENERAL DISCUSSION AND CONCLUSIONS 113

5.1. IN VITRO 114

5.2. EX VITRO 117

REFERENCES 123

i

SUMMARY

Next to quantity, the target of plant micropropagation is low losses and good

growth of plantlets during ex vitro reestablishment, and hence to improve the quality

of micropropagated plants has been the continuous concern of the people involved. For

this end, physiological competence of tissue culture derived plants should be

describable, and their quality improved through adjustment of the in vitro culture

conditions. Obviously no common standards can ever be set up for different plant

species, but methods and markers for the still problematic task of evaluating the

physiological quality of micropropagated plants can be developed. Once more this is

confirmed by data obtained in our present study on the behaviour of micropropagated

plants of different physiological quality from a C3 vs. a C4 and a CAM plant species

during the in vitro rooting stage and over the ex vitro acclimatization period.

The three model plants used in the present work included Prunus avium (a C3),

sugarcane (a C4), and pineapple (an obligate CAM) species. Micropropagated plants

of different physiological quality from each species were produced using different

culture media and variable light qualities during the in vitro rooting stage. Of each

species, plantlets from 8 treatments (combinations of agar vs. Gelrite; with or without

activated charcoal; high blue light, high red light or high far-red light) were used. The

plantlets’ growth and morphological characteristics, their net photosynthesis, activity

of phosphoenol pyruvate carboxylase (PEP-case), superoxide dismutase (SOD) and

catalase enzyme systems, leaf pigment contents, and chlorophyll fluorescence

transition were followed at the end of the in vitro culture stage and over the ex vitro

acclimatization period.

Results obtained demonstrate that the quality of micropropagated plants and

consequently their performance upon transplanting depended largely on the in vitro

environment, and that plants from different species reacted differently to variable in

vitro growth conditions. Depending also on the plant species, the effects of in vitro

treatments persisted longer or shorter upon transplanting, implying different abilities

ii

of microplants to “correct” abnormalities arising while in vitro and to adapt to ex vitro

growing conditions. The quality of micropropagated plants from each species could be

categorised by comparing measurements of various parameters related to growth,

photomorphology, photosynthesis and photoprotection, and especially their changing

patterns over the ex vitro acclimatization, within and between species.

In comparison with plants growing outdoors, at the end of the in vitro rooting

stage micropropagated plants of C3, C4 and CAM species were similar in leaf

mesophyll structure, stomata density and size, and Fv/Fm ratio. Abnormalities were

detected at different levels, depending on both the plant species and culture conditions,

in low chl a/b ratio, low photosynthetic efficiency and low photoprotection capacity.

Considering photosynthesis and chlorophyll fluorescence parameters (Pn, Φp, qP,

PEP-case, Fo, Fm), anti-oxidative stress enzymes’ activities (superoxide dismutase,

catalase), and pigment contents, addition of activated charcoal (1 g/l) to the in vitro

rooting medium significantly improved the quality of sugarcane and pineapple

microplants, and use of agar as the gelling agent of the in vitro rooting medium

resulted in better Prunus plantlets.

Upon transfer to soil, regardless of their species and quality, plantlets suffered

from stress, but could also develop different photoprotecting mechanisms, e.g.

scavenging of active oxygen species, non-photochemical quenching of chlorophyll

fluorescence and adjusting their photosystems’ size.

For all the three model species, plantlets with better quality, as categorised

according to the above mentioned parameters at the end of the in vitro culture period,

suffered less from stress and exhibited a faster recovery and better growth compared to

those categorised as poor quality. This confirms the usefulness of chlorophyll

fluorescence study, measurement of PEP-case, superoxide dismutase and catalase

activity and pigment levels in describing the physiological quality of tissue culture

derived plants.

iii

SAMENVATTING

Naast de productie van kwantiteit beoogt micropropagatie van planten het

minimaliseren van de verliezen evenals een goede hergroei tijdens de acclimatisatie.

Met dit voor ogen is het noodzakelijk de fysiologische status van de planten te

beschrijven en hun kwaliteit te verbeteren tijdens de cultuuromstandigheden in vitro.

Het is uiteraard onmogelijk algemeen geldende standaarden voorop te stellen voor de

verschillende plantensoorten, men kan wel methodes en merkers ontwikkelen voor de

evaluatie van de fysiologische gezondheid van de geproduceerde microplanten. In

onze studie werd vergelijkend onderzoek uitgevoerd met planten die een verschillende

fysiologische achtergrond hebben, C3, C4 en CAM-planten, tijdens de stadia van in

vitro-beworteling en ex vitro-acclimatisatie.

We selecteerden de volgende gewassen: Prunus (C3), Ananas comosus (obligaat

CAM) en Saccharum officinalis (C4). We produceerden planten met een verschillende

fysiologische status door gebruik te maken van verschillende cultuurmedia en

lichtkwaliteiten tijdens het stadium van de in vitro-beworteling. Enerzijds gebruikten

we media die gesolidifieerd werden met agar of met Gelrite, met of zonder

geactiveerde kool en we maakten tevens gebruik van hoog blauw, hoog verroood en

hoog rood licht; de combinatie van voornoemde factoren werd gewijzigd i.f.v. het

gewas. Tijdens de wortelontwikkeling in vitro en de acclimatisatie ex vitro werden

volgende parameters gevolgd: groei, morfologische kenmerken, fotosynthese, de

activiteit van phosphoenol pyruvaat carboxylase (PEP-case), superoxide dismutase

(SOD) en catalase, de inhoud aan bladpigmenten en de transitie van chlorofyl

fluorescentie.

Onze resultaten tonen aan dat de kwaliteit van de gemicropropageerde planten en

als gevolg daarvan hun performantie tijdens de uitplant, in grote mate afhankelijk

waren van de cultuuromstandigheden in vitro, en dat de verschillende plantensoorten

uiteenlopend reageerden op wijzigende in vitro-omstandigheden. Naargelang van de

plantensoort was het effect van de in vitro-behandelingen min of meer persistent na de

iv

uitplant, hetgeen wijst op een verschillende capaciteit van de planten om

abnormaliteiten te corrigeren en te adapteren aan de ex vitro-omstandigheden. Door

vergelijking van verschillende parameters die gerelateerd zijn aan groei,

fotomorfogenese, fotosynthese en fotoprotectie en hoofdzakelijk hun veranderingen

tijdens de ex vitro-acclimatisatie, was het mogelijk de planten van de verschillende

soorten onder te verdelen in verschillende categorieën.

Op het einde van de in vitro wortelfase waren de planten vergelijkbaar met

planten die buiten groeiden wat betreft mesofylstructuur, stomatadensiteit en –

afmetingen alsook voor de Fv/Fm-verhouding. Op verschillende niveau’s werden

afwijkingen geregistreerd, afhankelijk van de plantensoort en de

cultuuromstandigheden, o.a. lage chl a/b-verhouding, lage fotosynthetische efficiëntie

en een laag niveau van fotoprotectie. Bij de evaluatie van Pn, Φp, qP, PEP-case, Fo,

Fm, SOD, catalase en hoeveelheid pigmenten, blijkt dat het toevoegen van actieve

kool (1 g/l) aan het wortelinducerend medium in vitro op significante wijze de

kwaliteit van suikerriet en ananas verbetert; voor Prunus kon de performantie

verbeterd worden door het gebruik van agar als gelerend agent.

Bij transfer naar normale teeltomstandigheden, ongeacht de soort en de kwaliteit,

leden de microplanten onder stress, maar ze waren in staat verschillende fotoprotectie

mechanismen te ontwikkelen, b.v. het neutraliseren van actieve zuurstofsoorten, niet-

fotochemische quenching van chl-fluorescentie en het aanpassen van de omvang van

het fotosysteem.

Voor de drie soorten vertoonden microplanten van een betere kwaliteit, zoals

gecategoriseerd op het einde van de in vitro-periode op basis van de hierboven

vernoemde parameters, minder stressreacties en hernamen ze vlotter hun groei na

uitplanten, dan de microplanten van een lagere kwaliteitscategorie. Dit illustreert dat

chl-fluorescentie, het meten van PEP-case, SOD- en catalase-activiteit en de inhoud

aan pigmenten goed bruikbare parameters zijn om de fysiologische status van planten

uit weefselteelt te beschrijven.

v

LIST OF ABBREVIATIONS AND SYMBOLS ζ the photon ratio of red light to far-red light λ wavelength (nm) ΦF total yield of chlorophyll fluorescence Φp total quantum yield of primary photochemistry 3PG 3-phosphoglycerate A1 phylloquinone AB assay buffer AC activated charcoal ADP adenine di-phosphate Ao monomeric chl a molecule, the primary acceptor of electron from

photosystem 1 AOS active oxygen species ATP adenine tri-phosphate B/R ratio of blue to red photon flux [Σλ(400−500) / (Σλ(600−700)] BA in vitro treatment using high blue light, and agar-solidified medium BAP N6-benzylaminopurine BD in vitro treatment using high blue light, and AC-containing medium BG in vitro treatment using high blue light, and Gelrite-solidified medium BS bundle sheath BT in vitro treatment using high blue light, and AC-free medium CA in vitro treatment using control light, and agar-solidified medium CAM Crassulacean Acid Metabolism CD in vitro treatment using control light, and AC-containing medium CG in vitro treatment using control light, and Gelrite-solidified medium Chl chlorophyll Chl* chlorophyll in excited state CP43, CP47, PsaA, PseB, & PsbS are polypeptides in the thylakoid membrane

associated with antenna pigment molecules of photosystems CT in vitro treatment using control light, and AC-free medium cyt. cytochrome d. day DDT dithiothreitol DE dissipated energy flux, including heat dissipation, fluorescence, and

energy used in other than photosynthesis processes DM dry matter DW dry weight e- electron EDTA ethylene-diamino-tetra-acetic acid FAA fixative solution (containing 5 ml formalin 35%, 5ml glacial acetic

acid, and 90 ml 70 % ethyl alcohol) FAD flavin adenine dinucleotide Fdx ferredoxin Fm maximal chlorophyll fluorescence with maximal closure of all

photosystem 2 reaction centres in a dark-adapted state Fm’ maximal chlorophyll fluorescence with maximal closure of all

photosystem 2 reaction centres in a light-adapted state

vi

Fo minimal chlorophyll fluorescence with photosystem 2 centres maximally open in the dark-adapted state

Fo’ minimal chlorophyll fluorescence with photosystem 2 centres maximally open in the dark-adapted state

FRA in vitro treatment using high far-red light, and agar-solidified medium FRD in vitro treatment using high far-red light, and AC-containing medium FRG in vitro treatment using high far-red light, and Gelrite-solidified medium FRT in vitro treatment using high far-red light, and AC-free medium Fs chlorophyll fluorescence at steady state in the dark-adapted state Fs’ chlorophyll fluorescence at steady state in a light-adapted state Fv maximal variable chlorophyll fluorescence Fv/Fm maximal quantum yield of primary photochemistry FW fresh weight Fx, FA, FB a series of electron carriers, with 3 different Fe-S centres, in photosystem

1 G-3-P glyceraldehyde 3-phosphate HAUPT filtered solution of 1 g gelatine and 2 g phenol crystalline in 15 ml glycerine HIR high irradiation response IAA (3-indolyl)acetic acid IBA indolbutyric acid inc. increase kDa kilodalton LCA leaf chamber analyser LH* excited light harvesting molecule LHC1 light harvesting complex (light-harvesting chlorophyll a/b proteins)

associated with photosystem 1 LHC2 light harvesting complex (light-harvesting chlorophyll a/b proteins)

associated with photosystem 2 MDA monohydroascorbate N photon flux, equivalent to photon fluence rate (mol/m2/s) NAA α-napthaleneacetic acid NAD nicotinamide-adenine-dinucleotide NAD-ME NAD-malic enzyme NADP nicotinamide-adenine-dinucleotide phosphate NADPH nicotinamide-adenine-dinucleotide, reduced form NADP-ME NADP-malic enzyme OAA oxaloacetate PAM pulse amplitude modulation PAR photosynthetic active radiation PC plastocyanin PCR photosynthetic carbon reduction PEP phosphoenol pyruvate PEP-case phosphoenol pyruvate carboxylase PEP-CK PEP-carboxykinase Pfr far-red light absorbing (active) form of phytochrome Ph pheophytine P-I photosynthesis – irradiance (intensity of light) Pn net photosynthesis rate

vii

PPDK pyruvate Pi dikinase PPFD photosynthetic photon flux density Pr red light absorbing (inactive) form of phytochrome PS photosystem PS1 photosystem 1 PS2 photosystem 2 PVPP polyvinyl polypyrrolidone QA & QB two quinone molecules, accepting electron from pheophytine in

photosystem 2 centres QA

- quinone, reduced form qE energy dependent quenching of chlorophyll fluorescence, which is

associated with energization of the thylakoid membrane and eliminated by uncoupling agents.

qN non-photochemical quenching of chlorophyll fluorescence qP photochemical quenching of chlorophyll fluorescence R/FR ratio of red to far-red photon flux [Σλ(655-665) / (Σλ(725 735)] RA in vitro treatment using high red light, and agar-solidified medium RC reaction center RD in vitro treatment using high red light, and AC-containing medium red. reduction RG in vitro treatment using high red light, and Gelrite-solidified medium RH relative humidity RT in vitro treatment using high red light, and AC-free medium Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase RuBP rubilose-1,5-biphosphate SOD superoxide dismutase TCD thermal conductivity detector TE total energy flux absorbed by photosynthetic parameters TF1 energy flux reaching reaction centres of photosystems TF2 energy flux corresponding to the electron transport beyond quinone UV ultraviolet Vt relative variable chlorophyll fluorescence XOD xanthine oxidase

CHAPTER 1

GENERAL INTRODUCTION

Chapter 1

2

Nowadays, along with the growing importance of micropropagation to plant

breeding and production, the demands for good-quality culture derived plants also

increases. Apart from true-to-type and phytosanitary aspects, more and more concern

is being paid to the physiological quality of micropropagated plants and, significant

efforts have been spent for the description of their good attributes. However, while

their genetic stability and pathological status can be monitored and guaranteed by

certification schemes, assessment of microplants’ physiological competence remains

still problematic (van der Linde, 2000). Although it is well defined that a physiological

healthy micropropagated plant should be able to grow without any lag after planting ex

vitro, the description of its good physiological quality is still a challenge to both

producers and purchasers. Thus, for correct prognosis of microplants’ behaviour upon

transfer to ex vitro conditions, much more effort need to be made to identify reliable

parameters as well as to develop appropriate methods for their observation. This is of

value not only for evaluation of micropropagated plants’ quality but also for

production of physiologically competent microplants, as according to various authors

microplants’ ex vitro performance can be improved by modifying the culture growth

conditions in vitro. For instance, the poor water retention capacity of the leaves which

developed under in vitro conditions can be improved by reduction of the relative

humidity in the culture vessels through bottom cooling (Maene and Debergh, 1987).

Also, the role of the environmental elements both inside and outside a container have

been well documented, and the advantage of autotrophic culture recognised (Kozai et

al., 1992; Kubota et al., 1997b; Nguyen and Kozai, 1998). More recently, many

workers have demonstrated that the tissue cultured plants’ abnormalities, both

morphological and physiological, can be reduced by different in vitro treatments, such

as changing the culture medium, adjusting light conditions, optimising temperature

regime, application of chemicals and so on (Debergh et al., 2000; Langens-Gerrits et

al., 2000; Uosukaimen et al., 2000; de Klerk, 2000).

In the present thesis, a comparative study of the behaviour of micropropagated

plants of a C3 vs. a C4 and a CAM species in the last in vitro stage and during the ex

vitro acclimatization period is presented. From each of these three photosynthetic

groups, a species was used as the study’s model plants: Prunus avium (C3), sugarcane

Chapter 1

3

(C4) and pineapple (CAM species). These thee plants were selected due to their both

economical and social importance worldwide, also in Viet Nam, and because they

have attracted great attention from tissue culturists. Although different procedures for

their micropropagation have been developed, problems still exist with their

microplants’ quality, such as hyperhydricity (especially in the case of Prunus), and

poor ex vitro growth (pineapple in particular).

Our observations were concentrated on parameters related to the plantlets’

growth, photomorphogenesis, photosynthesis, photoinhibition and photoprotection,

with a focus on differences among the three model species, and between

micropropagated plants of each species derived from different in vitro light quality and

medium conditions. The study’s objectives include (1) contributing a step forward in

development of methods and markers for the still problematic task of evaluating the

physiological quality of tissue cultured derived plants, and (2) optimising the

micropropagation procedures of the model plants.

CHAPTER 2

LITERATURE REVIEW

Chapter 2

5

2.1. Light, and the plant growth and development

Light is arguably one of the most important signals which strongly regulate and

stimulate the growth and development processes of plants. It is recorded that a large

portion (ca. 80%) of the total radiant energy falling upon plant leaves is absorbed

(Weier et al., 1982). The harvested light is also large in wavelength range; from red

(780 – 660 nm), to orange-red (660 – 650 nm), blue-green (500 – 470 nm), blue (470 –

430 nm) and violet light (430 – 390 nm) can be trapped. In addition, some long-wave

infrared radiation (heat radiation) and ultra-violet radiation are also absorbed (Larcher,

1980). This great absorbance ability is attributed to highly diverse photoreceptors

existing abundantly in leaf tissues, of which the most important include photosynthetic

light harvesting pigments, photomorphogenic pigments (phytochromes), blue- light

harves te rs (c ryptochromes) and UV- receptors . Through absorbing and

transferring/converting irradiation energy, the photoreceptors modulate various light-

dependent and light-induced reactions of plants. In the present work however, the

focus is restricted mainly to the processes of photosynthesis, photomorphogenesis and

their accompanying pigments as well as the plant responses to over-irradiation –

photoprotection.

2.1.1. Light and photosynthesis of plants

2.1.1.1. Photosynthetic systems and light reactions

The photosynthetic light harvesting systems of higher plants contain chlorophyll

a, chlorophyll b and carotenoids (both carotenes and xanthophylls), with the ratio of

chl a/b in most plants’ leaves around 3.5 (Ting, 1982). The experiments with the green

alga Chlorella by Emerson and Arnold (1932), followed by the work of Gaffron and

Wohl (1936), led to the concept of the photosynthetic unit which comprises a large

number of pigment molecules functioning as light harvesters and a reaction centre

containing few pigment molecules where the primary photochemical reactions occur

(Govindjee, 2000). It is now known that there are 2 kinds of photosynthetic systems,

photosystem 1 (PS1) and photosystem 2 (PS2), located in the thylakoid membranes,

with several hundreds of each kind in each thylakoid (Weier et al., 1982). While the

Chapter 2

6

reaction centre of PS1 is optimally excited by light of about 700 nm and hence its

name P700, that of PS2 absorbs light with wavelength of around 680 nm and is called

P680. Both PS1 and PS2 reaction centres contain chlorophyll a, but exhibit special

absorbance properties because of their association to specific proteins in the thylakoid

membranes. In addition, the two photosystems differ from each other also in the chl

a/b ratio and in their overall absorbance properties; in comparison to PS2, PS1 has a

higher proportion of chlorophyll a relative to chlorophyll b, and is sensitive to longer

wavelength light.

According to Gomez and Chitnis (2000), plant photosynthetic light harvesters are

organised in core (proximal) and peripheral (distal) antenna. While the core antenna

are ultimately associated with the reaction centre and are necessary for functional

electron transfer, the peripheral ones are external to the core complex and not

necessary for primary photochemical charge separation. Each core complex contains

chlorophyll a (about 90 molecules for PS1 and 50 for PS2) and carotene, which are

both bound to polypeptides of different kinds in the thylakoid membranes, such as

PsaA and PsaB in PS1, and CP43 and CP47 in PS2. In contrast to the core antenna,

peripheral antenna contain both chlorophyll a & b, and some xanthophylls occur

instead of carotene. The distal antenna pigment molecules are also associated to

different integral membrane proteins, mainly PsbS, LHCI (light-harvesting chl a/b

proteins of PS1) and LHCII (light-harvesting chl a/b proteins of PS2) (Horton and

Ruban, 1994). The occurrence of carotenoids, both carotenes and xanthophylls, not

only increases the absorbance efficiency of the photosystems [as they both harvest

blue-green light which is poorly absorbed by chlorophylls (Owens, 1994)] but also

plays an important role in photoprotection of plants. Also, both the size of peripheral

antenna and their association with the core complexes are believed to have

significance in modulation of photosynthetic efficiency and photoprotective responses.

The role of light harvesting systems in photoprotection of plants will be further

discussed in section 2.1.3.2.

In PS1, light energy trapped by antenna pigments is transferred to the specific chl

a molecule (P700) in the reaction centre (RC) where it is stored as a stable charge

Chapter 2

7

separation. The excited electron of P700 is then donated to Ao (another specific chl a

molecule), and eventually to NADP +. Similarly, in PS2, P680 in the RC, after

obtaining energy from antenna molecules, transfers its excited electron to a lower-

energy recipient, pheophytine (Ph), followed by a series of oxidant/reductant pairs, and

finally to P700+ recovering the missing electron of this chlorophyll molecule. P680+ in

turn recovers its missing electron through oxidation of water to proton and oxygen.

The recovered P680 and P700 then, in their ground (So) state, are ready for the next

cycle of electron acceptance/donation. By moving electrons through the redox chain,

PS2 also pumps protons through the thylakoid membranes to produce a proton

gradient which drives synthesis of ATP. A schematic view of electron transport in the

Z scheme (nowadays also presented as N scheme) is illustrated in fig. 2.1.

Fig.2.1: Schematic view of electron transport in the Z scheme (after Mathews and

van Holde, 1990). Ph (pheophytin), QA and QB, (primary and secondary quinone electron acceptor), Cyt bf

(cytochrome bf), PC (plastocyanin), Ao (a monomeric chl a molecule), A1 (phylloquinone) Fx, FA, FB (a series of e- carriers with 3 different Fe-S centres), Fdx (ferredoxin).

The final products of light reactions, NADPH and ATP, are rich in energy and

serve as energy sources for assimilation of CO2 in the dark reactions of photosynthesis.

The light reactions can be summarized as below (Mathews and van Holde, 1990):

12 H2O + 12 NADP + 18 ADP + 18 iP + light ⇒ 12 NADPH + 6 O2 + 18 ATP

(iP = inorganic phosphate molecule)

Photosystem 2 Photosystem 1

P680*

Ph QA QB

QH2

Cyt bf PC

Ao

A1 Fx

FA/FB

FFd

NADPH

P680

P700

P700*

H2O

e-

e-

Protons pumped intoThylakoid lumen

1/2 O2

e-

hν

hν

0.8

0

-0.4

-0.8

-0.12

NADP

0.4

Chapter 2

8

2.1.1.2. Calvin-Benson cycle and C3 photosynthesis

The Calvin-Benson cycle or C3-pathway (Fig. 2.2) is the main route by which

atmospheric CO2 is ultimately assimilated in all higher plants (MacDonald and

Buchanan, 1990). In C3-photosynthesis, the enzyme ribulose-1,5-biphosphate

carboxylase/oxygenase (Rubisco) catalyses primary fixation of exogenous CO2 into a

molecule of ribulose-1,5-biphosphate (RuBP), a 5-carbon sugar, forming 2 molecules

of a 3-carbon compound, 3-phosphoglycerate (3PG) which are then phosphorylated by

ATP to 1,3 biphosphoglycerate and subsequently reduced by NADPH to

glyceraldehyde 3-phosphate (G-3-P). Running through the cycle 6 rounds (6 molecules

of CO2 have to pass the route for every new hexose molecule), 12 molecules of

glyceraldehyde 3-phosphate are formed, of which 2 are used for production of a

hexose molecule and the remaining 10 for regeneration of 6 molecules of RuBP

(Mathews and van Holde, 1990). The overall dark reaction can be written as follows:

6 CO2 + 18 ATP + 12 NADPH ⇒ C6H12O6 + 18 ADP + 18 iP + 12 NADP + 6 H2O

and the sum of both light and dark reactions:

6 CO2 + 12 H2O + light ⇒ C6H12O6 + 6 O2 + 6 H2O

The plants of which

photosynthetic CO2 assimilation

occurs through the Calvin-Benson

cycle alone are called C3 plants.

The C3 leaf mesophyll cells are

well differentiated into palisade

and spongy types (Weier et al.,

1982). The palisade parenchyma,

just below the upper epidermis,

consists of one or several layers of

narrow cells arranged closely to

one another and at right angles to

the leaf surface. In contrast , the

Fig.2 .2: Schematic view of the Calvin-Benson cycle (after Taiz and Zeiger, 2002)

CO2 + H2O

Carboxylation

3-phosphoglycerate

ATP +

NADPH

ADP + Pi + NADP+

Reduction

Regeneration

Ribulose-1,5- bisphosphate

ADP

ATP

Glyceraldehyde 3- phosphate

Sucrose, starch

Rubisco

Chapter 2

9

spongy parenchyma cells are irregular in shape and loosely arranged under the

palisade layer. In C3 leaves, both light and dark reactions of photosynthesis occur in

the same place, in both palisade and spongy mesophyll cells.

2.1.1.3. Low efficiency of Rubisco and other photosynthesis pathways

The enzyme Rubisco (EC. 4.1.1.39) is well known as the most abundant enzyme

on the Earth, accounting for about 50% of the total soluble proteins in leaves

(Bhagwat, 2000). This is not only because of its importance but also due to its

inefficiency. Having affinity to both CO2 and O2 at the same active site (Bowes et al.,

1971), Rubisco catalyses both carboxylation and oxygenation reactions as below:

Carboxylation: RuBP + CO2 ⇒ 2 G-3-P

Oxygenation: RuBP + O2 ⇒ G-3-P + Phosphoglycolate

Thus, when the oxygenation reaction occurs, the formation of phosphoglycolate

leads to the wasting process known as photorespiration, resulting in a reduction in the

plant photosynthesis efficiency (Ting, 1982, Padmasree and Raghavendra, 2000). At

25oC and with normal atmospheric gas conditions (the CO2 concentration varies

around 0.25% and the O2 level is about 20%), the rate of CO2 loss due to

photorespiration equals ca. 20% of the gross rate of photosynthetic CO2 assimilation

(Singh, 2000). However, under the conditions of higher temperatures, high light

intensity or reduced relative CO2 level, Rubisco affinity to CO2 significantly decreases

(Weier et al., 1982) resulting in greater photorespiration rate and lower photosynthetic

efficiency.

C4 photosynthesis

To overcome this problem some plants, living in tropical and subtropical regions,

have developed a subservient C4-cycle (also called the Hatch-Slack pathway), for

enrichment of CO2 in the immediate environment of Rubisco so that the oxygenase

reaction does not have a chance to occur (fig. 2.3a). These plants are classified in the

C4 photosynthetic group, and are characterised by the specific Kranz leaf anatomy

Chapter 2

10

(Singh, 2000). In C4 leaves, mesophyll tissue is not differentiated into palisade and

spongy types as in C3 plants, but forms 2 concentric layers of chloroplast containing

cells arranged around the veins: (1) the bundle sheath (BS) cells surrounding the

vascular bundles, and (2) the mesophyll cells surrounding the BS. Compared to the

mesophyll cells, the BS have thickened walls, without or with very few intercellular

spaces and possess numerous large, starch containing chloroplasts. The 2 types of cells

also differ from each other in their enzyme component (Singh, 2000) and in

chlorophyll a/b ratio (Ting, 1982). While all of the phosphoenolpyruvate carboxylase

(PEP-case) in the leaf occurs in mesophyll cells, most of the Calvin cycle enzymes and

all Rubisco are located in BS. Also, the BS cells have higher chl a/b ratio compared to

the mesophyll ones. This specific enzyme location and leaf structure allows C4 plants

to minimize photorespiration by spatially separating the reactions of photosynthesis

between mesophyll and BS cells. In mesophyll cells, atmospheric CO2 is initially fixed

onto phosphoenolpyruvate (PEP) forming a 4-carbon compound, oxaloacetate (hence

the name C4) by the enzyme PEP-case, which lacks affinity to O2 and has a high

carboxylase efficiency. Oxaloacetate (OAA) is very unstable and quickly converted,

depending upon the plant species, to malate or aspartate which are transported to BS

cells where they are decarboxylated and, CO2 released next to Rubisco is assimilated

through the Calvin cycle as in C3 plants. Pyruvate or alanine produced in BS in the

decarboxylation reactions of malate or aspartate are transported back to mesophyll

cells to serve as source of PEP for exogenous CO2 fixation.

Depending on the C4 acids decarboxylase enzymes in their BS cells, C4 plants

are classified into 3 subgroups: NADP-ME, NAD-ME and PEP-CK C4 plants (Singh,

2000).

• NADP-ME C4 plants use NADP-malic enzyme (NADP-ME) for decarboxylation

of malate in chloroplasts of their BS cells. Chloroplasts in BS cells of these C4 type

plants are agranal and in centrifugal location, with deficient PS2 and have high chl

a/b ratio (up to 10), while the chloroplasts of mesophyll cells are typically granal

and with much lower ratio of chl a/b (around 2.5) (Singh, 2000; Buchanan et al.,

2000). The decarboxylation reaction of malate is as below:

Chapter 2

11

Malate + NADP+ + NADP-ME ⇒ Pyruvate + CO2 + NADPH

• NAD-ME C4 plants. In these plants instead of malate, aspartate is converted from

OAA and transported to BS cells, where it is finally converted to malate, which is

then decarboxylated by NAD-malic enzyme (NAD-ME) located in mitochondria of

BS cells. The BS cells of these plants are organised in one layer and contain

centripetal, clearly granal chloroplasts. They are also characterised wi th occurrence

of a large number of mitochondria (Singh, 2000; Buchanan et al., 2000). The

reaction catalysed by NAD-ME is as below:

Malate + NAD+ + NAD-ME ⇒ Pyruvate + CO2 + NADH

Fig 2.3: C4 carbohydrate biosynthetic pathway in C4 (a) and CAM (b) plants

[after Hallick, 2001 (website 1)]

• PEP-CK C4 plants. In these plants aspartate is also transported from mesophyll

to BS cells, but after being converted to OAA is decarboxylated by the enzyme

PEP-carboxykinase (PEP-CK). In these plants, the BS cells are arranged in just one

layer and their chloroplasts are fully stack structured and arranged centrifugally

(Hatch, 1992; Buchanan et al., 2000). The reaction can be summarized as below:

a b

Chapter 2

12

OAA + ATP + PEP-CK ⇒ PEP + CO2 + ADP

CAM photosynthesis

Another kind of photosynthetic adaptation which helps plants to minimize

photorespiration and water loss is the Crassulacean Acid Metabolism (CAM). This

metabolic pathway was first studied in succulent members of Crassulacea family (and

hence the name, CAM) and has now been recorded in more than 2000 obligate CAM

species belonging to 335 genera in 39 families, and in many other facultative CAM

plants (Reddy and Das, 2000). Characterised by specially well developed succulent

parenchyma tissue with large vacuoles and reduced intercellular spaces, CAM plants

exhibit a high resistance to water loss (Weier et al., 1982). Further, these plants can

both reduce water loss and prevent photorespiration by closing their stomates during

day and opening them at night for CO2 trapping. At night, when there is no ATP and

NADH available for CO2 assimilation, exogenous CO2 is first fixed by PEP

carboxylase as in C4 photosynthesis (fig.2.3b). In contrast to C4 plants however, in

CAM leaves malate converted from oxaloacetate (OAA) is stored in the vacuoles and

serves as a CO2 source for photosynthetic carbon reduction in the Calvin-Benson cycle

during the day. Depending on which enzyme the plants use for decarboxylation of

stored malic acid and the primary carbohydrate reservoir used in their daily cycle,

obligate CAM plants are divided into 2 types (Kore-eda et al., 1996; Reddy and Das,

2000): (1) Malic enzyme, and (2) PEP-CK type CAM plants. Plants in the first

subgroup use polysaccharides (e.g. starch/glucan) as the primary carbohydrate storage,

and NADP-ME or NAD-ME enzyme as the major decarboxylase and pyruvate Pi

dikinase (PPDK) for converting pyruvate to PEP. In the second type plants, soluble

hexose (glucose or fructose) and the enzyme PEP carboxykinase (PEP-CK) are used

instead.

C3-CAM intermediate photosynthesis

In addition to obligatory CAM there is another plant group with the striking

feature of switching from CAM to C3 photosynthesis and vice versa (Orsenigo et al.,

Chapter 2

13

1995; Kore-eda et al., 1996). In their review on CAM photosynthesis, Reddy and Das

(2000) divided C3-CAM photosynthesis into 3 different modes: (1) idling facultative

CAM plants show low diurnal acid fluctuation with no gas exchange and are

hypothesized to recycle respiratory CO2; (2) cycling facultative CAM plants exhibit

organic acid fluctuation but with little or no exogenous nocturnal CO2 fixation; and (3)

those CAM plants that shift from C3 to CAM photosynthesis during ontogeny or in

response to environmental changes such as water deficit, salinity or photoperiod. Also

according to these authors it is clear that the shift from C3 to CAM photosynthesis is

usually accompanied by several changes in physiology, biochemistry and gene

expression. Of various environmental elements, water stress and salinity are

considered to have great significance in CAM induction (Ting et al., 1994; Kore-eda et

al., 1996). The role of some growth regulators in CAM induction have also been

recorded (Reddy and Das, 2000).

C3-C4 intermediate plants

There are also plants with predominance of C3 (Calvin-Benson cycle) yet with

significantly reduced photorespiration (Padmasree and Raghavendra, 2000). These

plants have the same maximum activities of the enzymes of the photorespiration cycle

as C3 plants, but are characterised with the confining of glycine decarboxylase to their

bundle sheath cells. Therefore, glycine can not be decarboxylated in mesophyll cells of

C3-C4 intermediate plants, and the site of CO2 release during the photorespiration (in

mitochondria on the inner wall of the BS) is in close association with chloroplasts

through which CO2 must pass to exit the leaf. This enhances the potential for recapture

of the photorespiratory CO2 of C3-C4 intermediate plants (Padmasree and

Raghavendra, 2000) and correspondingly raises the plant’s photosynthetic efficiency.

The advantages and disadvantages of C3, C4 and CAM photosynthesis

Because there is a rapid increase of oxygenase activity of Rubisco compared to

its carboxylase activity under the conditions of high temperatures and/or high light

intensity, C4 photosynthesis helps C4 plants to avoid high rates of wasteful

Chapter 2

14

photorespiration processes. In addition, as CO2 enrichment in the Rubisco immediate

environment allows C4 plants to sustain higher photosynthesis rates and to reduce

photorespiration, C4 plants also exhibit higher light saturation point, higher

photosynthesis rate and very low CO2 compensation point in comparison to C3 plants

(Larcher, 1980; Ting, 1982; Weier et al., 1982). It is however recorded that the

quantum efficiency, i.e. the ratio of oxygen molecules evolved to photons absorbed

measured under limited light, is similar for both C3 and C4 plants under normal

atmospheric gas conditions and temperature of 20 – 25oC (Singh, 2000). Moreover,

under certain conditions (e.g. low O2 to CO2 ratio, or temperate temperatures) C3

plants may perform even better, and the main reasons for this, as proposed by Singh

(2000), include: (1) the greater reduction in catalytic activity of some enzymes in C4

plants, especially those of the photosynthetic carbon reduction (PCR) cycle at low

temperatures; (2) the adverse effects of low temperatures on the metabolite transport

which is an important element in C4 photosynthesis, (3) the higher Rubisco levels in

C3 plants, and (4) both PEP carboxylase and pyruvate dikinase of C4 pathway are cold

labile. Thus, certainly C4 photosynthesis has advantages only in tropical climates with

high temperature and high light intensity; under these conditions, C4 plants exhibit

both higher growth rate and dry matter production (Hatch, 1992). It is also noted that

in comparison to C3 plants, those with C4 photosynthesis are more efficient in water

and nitrogen use, and this is attributed to lower levels of Rubisco in their leaves

(Singh, 2000). All these explain for the prevalence of C4 plants in the tropics and the

predominance of C3 plants in temperate zones (Stryer, 1988; Ehleringer et al., 1997).

Having stomates closed during the day, CAM plants can greatly reduce the

transpiration rate to 50 – 150 g of water transpired per 1 g CO2 taken up (the figure

averages 500 g for C3 and 1000 g for C4 leaves) (Ting, 1982). With very low

transpiration rate and high water use efficiency, CAM plants have advantages specially

in arid and semi-arid regions. Regarding the cost of net photosynthesis and the average

productivity, CAM plants often rank between C3 and C4 plants (Reddy and Das,

2000). Nevertheless, having the lowest rates of both photosynthesis and instantaneous

growth in comparison to C3 and C4 plants, CAM plants show reduced competitiveness

in resource-rich environments.

Chapter 2

15

2.1.2. Light and photomorphogenesis of plants

Besides photosynthesis, light also affects the higher plant growth and

development through modulating a variety of other processes and phenomena, such as

the plant’s ability to measure the photoperiod (photoperiodism), the bending of plant

organs towards directional cues (phototropism) and light-directly dependent growth

and development processes. Although some authors (Kendrick and Kronenberg, 1994)

classify all the above mentioned as photomorphogenic responses, in the present work

however for convenience the concept of Ting (1982) is accepted. As defined by this

author, photomorphogenesis is the light-directed control of plant growth and

development which is independent from photosynthesis and depends on both light

wavelength and the total irradiation, and thus does not include photoperiodism and

phototropism. In contrast to photoperiodism and the induction-reversion phenomena

(also caused by phytochromes) that are reversible and independent on the total

irradiation, photomorphogenic responses require continuous irradiation and are not

reciprocal. They are therefore also classified as high irradiation responses (HIR) (Ting,

1982; Quail, 1994; Bjorn, 1994). The most studied and frequently recorded HIR are

inhibition of internode elongation, expansion of cotyledons, biosynthesis of

anthocyanes and etiolation (Ting, 1982). Working with mustard seedlings Mohr and

Schopfer (1978) recorded also such phenomena as development of primary leaves,

mature leaf primordia formation, xylem elements differentiation, differentiation of

stomata within the epidermis of cotyledons, changes in intensity of cell respiration,

increase in synthesis of carotenoids and ethylene, increased capacity for chlorophyll

synthesis, modulation of the enzyme synthesis in cotyledons and so on.

The major photoreceptors responsible for photomorphogenesis belong to the

phytochrome family, which comprises 5 distinct types identified in higher plants under

the names of phytochromes A, B, C, D and E. Each phytochrome may have specific

regulatory roles but is presumed to have the same absorbance spectrum, and consists

of a linear tetrapyrole chromophore covalently attached to a protein with a molecular

weight of 120 – 130 kDa forming a chromoprotein complex existing in 2 different

forms (Ting, 1982; Quail, 1994). These 2 forms, Pr and Pfr, can be converted from one

Chapter 2

16

to another upon light exposure. The inactive form (Pr) absorbs red light with the peak

at around 660 nm, while the active form (Pfr) absorbs far-red light with the peak at

about 730 nm. In plants, phytochromes are initially synthesized and accumulated

under the Pr form, which upon exposure to sun light is quickly transferred to Pfr form.

According to Ting (1982), the Pfr form is extraordinarily unstable and can quickly be

degraded, and hence the 2 forms exist in a photoequilibrium between Pr synthesis and

Pfr breakdown as below:

Biological responses

Synthesis Pr Pfr Destruction Dark reversion (in some plants only)

At photoequilibrium the ratio of Pr/Pfr is proportional to the red light to far-red

light photon ratio, ∑ −

∑ −=ζ735725N665655

N (where N655 – 665 and N725 - 735 are photon flux at

wavelength λ = 655 - 665 and 725 – 735 nm, respectively).

It is also noted that in addition to the peak in the red region, some high irradiance

responses have an action spectrum not covered by the absorbance spectrum of

phytochromes. The inhibition of hypocotyl lengthening for example was recorded to

have a second peak in the blue light region (Senger and Schmidt, 1994). Also, the

regeneration of anthocyanes was observed with the action spectrum extending to both

blue and UV-light area (Mancinelli, 1980). This indicates the participation of

photoreceptors other than phytochromes in photomorphogenesis. Nevertheless, current

understanding on both blue light receptors (cryptochrome) and UV- harvesters remains

very limited.

Chapter 2

17

2.1.3. Photoinhibition and photoprotection of plants

2.1.3.1. Photodamage caused by the excess of light energy absorbed

Photoinhibition, as defined by Osmond (1994) is the light-dependent inhibition

of the light-dependent reactions of photosynthesis, caused by the excess of photons

absorbed by the photosynthetic pigments in plants. The short-term response of plants

to light intensity is described by the photosynthesis-irradiance (P-I) curve, illustrated

in fig. 2.4. When exposed to low radiant intensity, plants exhibit the highest

photosynthetic efficiency, indicated by their maximum quantum yield of

photosynthesis, i.e. most of the photons absorbed are utilized for photochemical

reactions. At higher light intensity, when the rate of photon absorption exceeds that of

photon utilisation in photosynthesis, the excess energy absorbed if not dissipated as

heat can cause damage to photosystems.

Fig. 2.4: The P-I curve showing the relationship

between the rate of photon absorption and the rate of photon utilisation in photosynthesis. Photon absorbance (---) is linear with light intensity while photon utilisation for CO2 fixation (___) exhibits saturation kinetics. The shaded area represents the amount of light absorption which is in excess of the plant photosynthetic capacity (after Osmond, 1994 and Owens, 1994).

Although recently gathered evidence indicate that PS1 can also be damaged by

over-irradiation (Ohad et al., 2000), this photosystem was long considered immune to

photodamage, and therefore most studies on this aspect were focused on PS2. The

subject of photoinhibition, including both causal factors and molecular mechanisms,

has been well reviewed in various works, such as those of Styring and Jegerschöld

(1994), Telfer and Barber (1994), Asada (1994), Whitmarsh et al. (1994), Ohad et al.

(2000) etc. Based on the results obtained with isolated PS2 reaction centres, Telfer and

Barber (1994) proposed the occurrence of 2 photoinhibition mechanisms: donor side

Incident light intensity

Phot

osyn

thes

is

Excess light

Chapter 2

18

and acceptor side mechanisms. Under constant high light intensity, the plastoquinone

pool in the electron transport chain (see fig. 2.1) can be fully reduced resulting in

generation of the triplet state of P680 (3chl or 3P680), which if not promptly scavenged

will react with O2 to form singlet excited oxygen (1O2). Due to its high reactivity,

singlet oxygen if not promptly scavenged will destroy P680 leading to degradation of

D1, one of the 2 core PS2 reaction centre polypeptides binding in vivo all the co-

factors required for electron transport from the Mn cluster of the water splitting system

to the plastoquinone pool. The other core protein (D2) of PS2 is more stable and less

affected (Andersson et al., 1994). This mechanism of photoinhibition occurs at the

acceptor side of PS2 and only under aerobic conditions. The second photoinhibition

mechanism happens when electron donation from water can not keep up with electron

withdrawal on the acceptor side (see fig. 2.1), leading to longer life time of P680+, and

this radical, with a redox potential higher than 1V, would be able to extract electrons

from its surrounding environment causing damage to both the chromophore and

proteins of PS2. This donor side mechanism of photoinhibition occurs under both

aerobic and anaerobic conditions.

According to Asada (1994), in addition to 1O2 and 3chl, other reactive radicals,

including superoxide anion radicals (O2-), hydroxyl radical (•OH), hydrogen peroxide

(H2O2), monodehydroascorbate (MDA) and other organic radicals (R•) are also

formed. The reduced oxygen species (O2-, H2O2, and •OH) and singlet excited oxygen

(1O2), also referred to as active oxygen species (AOS), as well as other reactive

radicals if not promptly scavenged at their production site will interact with target

molecules, mostly D1 protein and enzymes of the Calvin cycle, causing damage to

PS2 resulting in photoinhibition. Osmond (1994) divided photoinhibition into 2 types

based on their relaxation times: dynamic and chronic types. While dynamic

photoinhibition is rapidly relaxing, quickly reversible and occurs most rapidly in sun

plants, chronic photoinhibition is slowly reversible, predominating in shade plants and

often occurs following sustained exposure to excess sun light resulting in

photodamage of photoapparatura. Dynamic photoinhibition therefore is also

considered a photosynthetic regulation strategy of plants (Osmond, 1994).

Chapter 2

19

It is worthy to note that photoinhibition can be caused by various factors other

than over-irradiation; any perturbation that causes a decrease in the rate of

photosynthesis will lead to an increase in excess light absorption and thereby may

induce photodamage. For example, mild water stress causes stomatal closure and

hence reduces the leaf internal CO2 level leading to depression of photon utilisation

rate. Or, as already mentioned above (see 2.1.1), both stress temperature and reduced

CO2 concentration may have adverse effects on the Rubisco carboxylase efficiency,

and thus lower the photon-utilising capacity of plants. Also, biotic stresses and

nutrition deficiencies may inhibit plant photosynthesis by affecting both capacity and

efficiency of their photosynthesis systems (Powles, 1984; Cormic, 1994; Ort et al.,

1994; Krause, 1994; Baker et al., 1994; Ball, 1994; Rao and Terry, 2000).

Regardless of all the above mentioned, one will make a mistake to consider

photoinhibition as entirely adverse. As discussed by many, photoinhibition, especially

the chronic types, should also be viewed as one of the mechanisms of plants to adjust

photosynthetically to stress conditions, especially those causing decrease in

photosynthetic capacity (Krause, 1994; Osmond, 1994; Anderson, 2000). The

ineffective PS2 centres may have a protective effect on neighbouring active units. The

fast turnover of D1 protein, and to lesser extent of D2 polypeptide, allows plants to

prevent net loss of PS2 up to the light saturation point under non-stressed conditions.

Above the light saturation point D1 turnover greatly declines, and thus with sustained

high intensity light, non-functional PS2 still contain D1/D2 protein binding all the

redox factors and accumulate in stacked granal membrane regions. Only when

photoinhibited plants are exposed to low irradiance, D1 protein turnover is restored,

and the undegraded non-functional PS2 migrate to the stroma-exposed membranes

where their function is restored. In comparison to shade plants, sun and high-light

plants maintain greater D1 turnover and increased synthesis of ATP and NADPH; sun

plants are also characterised by greater capacity for nonphotochemical energy

dissipation due to higher xanthophyll cycle pools and activities, and all these make

them less vulnerable to high light intensity (Anderson, 2000).

Chapter 2

20

2.1.3.2. Photosynthetic photoprotection and acclimatization of plants

Plants can cope with light stress to their photosynthetic apparatus via repair of

damaged photosystems, and through various protective and acclimative mechanisms.

They respond to over-irradiation at different levels in order to minimise the potential

damage. At the whole plant level, leaf orientation can be adjusted in order to control

the amount of light absorbed; at the cell level, the chloroplast orientation changes in

order to alter the amount of light intercepted; and at chloroplast and molecular level

various molecular processes are involved both to protect and to repair photosystems.

As discussed by Osmond (1994) and Strasser et al. (2000), light energy absorbed

by photosynthetic pigments may undergo one of the following fates (fig. 2.5).

Fig. 2.5: Different fates of the light energy absorbed. TF, total energy flux absorbed; LH* excited light harvesting molecules, mainly chlorophylls; TF1, energy flux that reaches reaction centres (RC); TF2, energy flux corresponding to the electron transport beyond QA

-; DE, dissipated energy flux, including heat dissipation, fluorescence emission and energy used in other processes (after Strasser et al. 2000)

One of the molecular mechanisms of photoprotection evolves the conversion of

excess excitation energy into heat and thereby decreases the amount of energy

reaching PS2 reaction centres (Whitmarsh et al., 1994). This photoprotective

mechanism can be explained through studying non-photochemical quenching (qN)

of chlorophyll fluorescence occurring in light-harvesting complexes of PS2. The

major part of qN is attributed to the energy-dependent quenching, qE (also called

∆pH-dependent non-photochemical quenching) which relaxes with kinetics similar to

LH*

TF

RC QA- QA

Ne- TF1

TF2

DE

Chapter 2

21

those of ∆pH, and plays the central role in the overall regulation of photosynthesis. As

the yield of qE is demonstrated to be strongly correlated with the conversion of the

carotenoid violaxanthin to zeaxanthin via the xanthophyll cycle (Demmig et al., 1987)

it is proposed that carotenoids are of great importance in non-photochemical

quenching. Results obtained by Mathis et al. (1979) and Hermant et al. (1993) implied

that zeaxanthin has a direct role in quenching of chlorophyll excited states either

through transient charge separation between the antenna chlorophylls and zeaxanthin

or via singlet-triplet fusion, leading to an increase in dissipation of excess energy in

the form of heat. This topic has been well discussed by Owens (1994), and Horton and

Ruban (1994). There is also another mechanism of non-photochemical quenching

independent of the xanthophyll cycle, which as proposed by Horton and co-workers

(Horton and Ruban 1994) is related to the aggregation of light harvesting complexes of

PS2. Structural changes due to LHCII aggregation can alter the distance between

carotenoid-chl a pairs or can affect the asymmetry of the carotenoid-binding

environment, resulting in some alteration of energy transfer between zeaxanthin and

chl a, and thereby modulating qE quenching. The application of chlorophyll

fluorescence measurement for studying photosynthesis and photoprotection of plants

will be further discussed in section 2.3.

Another proposed molecular mechanism protecting against photodamage

involves the role of β-carotene and cytochrome b559 in discharging potentially

damaging radicals. Working with isolated PS2 reaction centres, Telfer and Barber

(1994) have come to the conclusion that β-carotene directly quenches 1O2 and

suggested a protective role of this carotenoid against photoxidative damage. Regarding

the role of cytochrome b559 however, as according to Whitmarsh et al. (1994),

although proofs of both cation- and anion-quenching by b559 are available whether

this helps to protect plants from photodamage remains to be proven. There are also

many enzymatic mechanisms of scavenging photogenerated reactive molecules at

different sites, and some of them are discussed below.

Chapter 2

22

Superoxide dismutase and scavenging of superoxide

It is known that if not scavenged promptly at the site where superoxide anions

(O2-) are produced, this radical will interact with transition metal ions (metal-catalysed

Haber-Weiss reactions) producing the most reactive species of oxygen-hydroxyl

readical (•OH) (Asada, 1994). The enzyme superoxide dismutase (SOD) (EC 1.15.11)

exists in chloroplasts in 3 forms, which corresponding to their prosthetic metals are

called CuZn-, Mn- and Fe- SOD (Asada, 1994). In most higher plants, SOD in the

chloroplast stroma exist in CuZn form, though in several plants Fe-SOD may also

occur. In contrast, the Mn-form is mainly bounded to the thylakoid membranes. The

enzyme SOD catalyses the disproportionation of superoxide forming hydrogen

peroxide and oxygen so that the production of the most reactive hydroxyl radical

(•OH) is suppressed.

2O2- + 2H2O ⇒ H2O2 + O2

Scavenging of hydrogen peroxide by catalase and peroxidases

Similarly, if not removed promptly enough from chloroplasts, hydrogen peroxide

can also react with transition metals leading to formation of •OH, and thus cause

photodamage to the photoapparatus. In addition, it is also proposed that H2O2 can

inactivate several stromal enzymes of the CO2 fixation cycle; working with isolated

chloroplasts, Kaiser found that at the concentration of 10 µM, H2O2 could cause half

inhibition of CO2 fixation (Kaiser, 1976). In leaf cells, H2O2 produced by glycolate

oxidase (in photorespiration) is scavenged by catalase (EC 1.11.1.6) exclusively

existing in peroxisomes.

2H2O2 ⇒ H2O + O2

This enzyme however does not occur in chloroplasts, and hence H2O2 produced there

is not scavenged by catalase but through its reduction to water by a peroxidase reaction

using different photoreductants as electron donors (Asada and Badger, 1984). For

Chapter 2

23

example, ascorbate peroxidase (EC 1.11.1.7) present in both stroma and thylakoids of

chloroplasts catalyses the following reaction (Asada, 1994).

H2O2 + 2 ascorbate ⇒ 2 Η2Ο + 2 ΜDA (monodehydroascorbate)

Scavenging of MDA radicals

As mentioned above, for the reduction of H2O2 in the chloroplasts by ascorbate

peroxidase, ascorbate is essential. Normally, its concentration in the chloroplast is over

10 mM, but would rapidly drop within 100 s. For recovering of the ascorbate source,

MDA is directly reduced by FAD (flavin adenine dinucleotide) enzyme in the

following reaction (Asada, 1994):

2 MDA + NAD (P)H ⇒ 2 ascorbate + NAD (P)

In addition, MDA can also be photoreduced to ascorbate in thylakoids mediated

by ferredoxin or spontaneously (Asada 1994). Also according to this author, the

scavenging systems of superoxide, hydrogen peroxidase and MDA radicals existing in

both thylakoid and stroma may also have a role in down-regulation of PS2.

Other scavenging agents

Besides enzymatic, there is also non-enzymetic scavenging of AOS by

antioxidant molecules. In addition to carotenoids, which function in quenching 1O2 and 3chl was already mentioned earlier, other non-enzymatic antioxidants such as vitamin

C, glutathione and vitamine E (lipophilic α-tocopherol), as well as some phenolic and

flavonoid compounds have also been recorded to have a role in scavenging AOS or in

preventing free radical production (Foyer et al., 1994; Mckershie and Leshem, 1994).

Photorespiration as a photoprotective strategy

Photorespiration (also called C2 cycle) is also considered important in the

protection of photosynthetic systems against photon inactivation (Padmasree and

Raghavendra, 2000). According to Heber et al. (1996), non-photochemical quenching

is insufficient to protect the chloroplast electron transport chain against

Chapter 2

24

photoinactivation, and in C3 plant especially, photoprotective photorespiration is

effective in lowering the energy flux reaching PS centres by dissipating excessive

absorbed light energy through the glycolate oxidation pathway (Osmond, 1981) or by

decreasing the limitation of Pi supply for photosynthesis by the additional recycling of

Pi through the photorespiratory pathway (Guo et al., 1995). Having the highest

photosynthesis capacity, C4 plants have also the lowest photorespiration rate. Also, the

specific location of the C2 cycle enzymes and leaf anatomy significantly enhance the

potential for recapture of the photorespired CO2 in C3-C4 intermediate plants in

comparison to C3 leaves (Padmasree and Raghavendra, 2000).

Long- and short-term acclimative strategies to over-irradiation

Besides the photoprotection mechanisms, plants have developed various

acclimative responses. These responses reside from differences in the structure of

photosynthetic apparatus and leaf anatomy between plants and leaves grown under

different conditions. Shade and low-light plants for example have more chl b and more

light-harvesting chlorophyll a/b proteins of PS1 and PS2, while sun plants are

characterised by a greater amount of cytochrome bf complex, ATP synthase,

plastoquinone pool, plastocyanin, ferredoxin and carbon fixation enzymes (Anderson,

2000). As already mentioned, the size of the photosystems is of importance in

regulating energy distribution between the photosystems, and in balancing stress-

altered energy consumption with energy supply. This is supported by data obtained by

Anderson et al. (1988) showing the decrease in the amount of LHCI and LHCII with

increasing light, and the change of the PS2/PS1 ratio when light intensity is variable.

The sun and high-light plants for example are richer in PS2 units with smaller light-

harvesting antenna relative to PS1; the ratio of PS2/PS1 is around 1.8 – 2.3 in sun

plants and ca.1 – 1.3 in shade plants. Also, in response to increased photon flux

density, the concentration of some photosynthetic enzymes per unit area and of

electron transport carriers grows, the stomatal conductance increases while the ration

of chlorophyll to protein in leaf reduces. The topic of photosynthetic acclimatization to

increases in photon flux density was reviewed by many authors, such as Bjorkman

(1981), Anderson and Osmond (1987), Pearcy (1994), and Anderson (2000).

Chapter 2

25

2.2. Effects of special in vitro culture conditions on the in vitro cultured plant growth and development

Normally, in vitro propagated plants are provided with abnormal growth

conditions compared to the ex vitro environment. As described by Debergh et al.

(1992), Aitken-Christie et al. (1995), Jeong et al. (1995), Fujiwara and Kozai (1995a),

Zobayed et al. (1999a) and Cui et al. (2000) the in vitro environment is characterised

by high relative humidity, constant temperature, low photosynthetic photon flux (PPF),

large diurnal fluctuation in CO2 concentration, limited gas exchange, high

concentration of sugar, salts and growth regulating substances, and accumulation of

toxic components. Any of these factors is known to have effects on plantlets’ quality,

and consequently on their performance upon transfer to the ex vitro environment. Both

structural and functional abnormalities attributed to the unique growth conditions in

vitro have been observed in micropropagated plants, among them hyperhydricity,

bushiness, multi-apexing and apex necrosis, reduced amounts or altered structure of

epicuticular wax, poor cuticle development, improperly functional stomata, excessive

water loss, poor photosynthetic capacity, low chlorophyll and carotenoid

concentrations, change in leaf anatomy and stomata density, low transpiration rate,

high dark respiration, variation in size and shape, occurrence of somaclonal variation,

and so on (Ziv, 1991; Zimmerman et al., 1991).

In general, it is accepted that the most important abnormalities hindering ex vitro

acclimatization of micropropagated plants are related to their low water retention

capacity and poor photosynthetic behaviour, which can somehow be “corrected”

through adjusting different environmental parameters in vitro (Debergh, 1991). High

RH in the culture vessels for example if known to be responsible for the abnormal

function of stomates in vitro, which in turn is the prime cause of rapid wilting and

desiccation after transplanting (Ali-Ahmad et al., 1998). A reduction of the inside

container RH can therefore improve microplants’ water retention, resulting in their

better performance upon transplanting to ex vitro conditions. Through reducing the

inside vessel RH by using a bottom cooling system, Capellades (1989) obtained more

‘normally’ structured stomates in cultured roses. Also, when RH is reduced by an

Chapter 2

26

increase in the vessel ventilation, leaf resistance to desiccation is increased and

stomatal regulation improved (Smith et al., 1990, Ghashghaie et al., 1992). Other

means for reduction of RH in culture continers, such as changing the container size

and forms, adjusting gelling agent concentration and type, application of an

autoclavable paper under the perforated polycarbonate lids, using microperforated

polyethylene films and etc., may also have value in improving the stomatal function

and water retention of in vitro leaves (Capellades et al., 1990a; Debergh, 1991).

The photosynthetic ability of plantlets in vitro can also be enhanced through

stimulating photoautotrophy (Zobayed et al., 1999a; Kozai and Iwanami, 1988).

Nevertheless, a disagreement still exists on the importance of in vitro photoautotrophy

vs. mixotrophy on the ex vitro acclimatization of tissue culture derived plants. While

Kozai and co-workers (Kozai, 1991; Kubota et al., 1997a) stressed that plantlets

grown photoautotrophically in vitro survive and perform better upon transfer to the ex

vitro conditions, Debergh and co-workers revealed that in vitro photoautotrophy is

certainly not a must, and that the highest survival rate was obtained with mixotrophic

microplants, which quickly regained photoautotrophy ex vitro under appropriate

conditions during the last stages in vitro and over the initial stages ex vitro (Capellades

et al., 1990b; Debergh, 1991; Van Huylenbroeck, 1997). Moreover, Serret et al.

(2001a & b) noticed negative effects of in vitro photoautotrophy on plantlet

acclimatization capacity; when grown in vitro under increased PPFD and omitted from

sugar supply Gardenia micropropagated plants suffered more from photoinhibition,

both while in vitro and during the ex vitro acclimatization. The topic of in vitro

environmental control and plantlet growth and development has been extensively

reviewed by many, such as Debergh and Maene (1984), Debergh (1991), Debergh et

al. (1992), Jeong et al. (1995), Fujiwara and Kozai (1995a), Kozai (1996), Nguyen

and Kozai (1998) and Zobayed et al. (2000). Hereafter, some elements of discussion

are presented on the photosynthetic behaviour, photoinhibition and

photomorphogenesis of tissue cultured plants, and the related environmental factors.

Chapter 2

27

2.2.1. Photosynthetic behaviour of plantlets in vitro

Experimental data obtained in various plant species and varieties reveal the low

photosynthetic capacity of plantlets under conventional micropropagation conditions

[Capellades et al., 1991 (in rose); Heo et al., 1996 (Cymbidium); Kozai et al., 1996

(sweet potato); Seko and Kozai, 1996 (turfgrass); Kubota et al., 1997a (tomato); Van

Huylenbroek, 1997 (Calathea and Spathiphyllum); Niu et al., 1998 (potato); Heo and

Kozai, 1999 (sweet potato); Nguyen et al., 1999b (coffee); Zobayed et al., 1999b

(cauliflower); Nguyen et al., 1999a (coffee, banana); Ermayanti et al., 1999 (Garcinia

mangostana and Acacia mangium)], and causes have been identified as low

photosynthetic photon flux (PPF), low CO2 concentration during the photoperiod,

limited gas exchange, high relative humidity, presence of sugar in the culture medium,

etc.

2.2.1.1. Effects of CO2 and O2 levels inside the vessel

It is recorded in various cases that under non-ventilation conditions, within 2-3 h

after the start of the photoperiod, the concentration of CO2 inside a tissue culture

vessel containing chlorophyllous shoots/plantlets declines from 3000 – 9000 µmol/mol

to less than 100 µmol/mol, much lower than the normal atmospheric concentration (ca.

340 µmol/mol) and close to the CO2 compensation point of C3 plants (Fujiwara et al.,

1987; Desjardin, 1995). A clear diurnal fluctuation of CO2 was also observed by

Debergh et al. (1992), although in their specific experimental conditions, the inside

culture vessel CO2 concentration remained always higher than the normal atmospheric

level. Obviously, the fluctuation in CO2 level indicates the photosynthetic competence

of plantlets in vitro (De Riek, 1995), while its low concentration during the

photoperiod explains why raising the photosynthetic photon flux (PPF) alone in many

cases does not result in higher photosynthetic rate of tissue cultured plants, why

plantlets in vitro have a low light saturation point (about 90 µmol/m2/s) (Kozai et al.,

1996), and why increasing the CO2 level inside the culture vessel alone (Arai et al.

1989; Heo et al., 1996; Heo and Kozai, 1999; Ermaynti et al., 1999; Zobayed et al.,

1999b) or in combination with increased PPF (Kozai and Iwanami, 1988; Aitken-

Chapter 2

28

Christie et al., 1995; Solarova et al., 1996; Urban et al., 2001) could lead to an

increase in the net photosynthetic rate. Experimental results reveal that the CO2

saturation point of plantlets in vitro depends on PPF, temperature and other

environmental elements (Niu et al., 1995; Kozai et al., 1998), and that further

increasing the CO2 concentration above the saturation point could lead to a reduction

in the net photosynthetic rate of microplants (Nguyen et al., 1999b; Davies and

Santamaria, 2000).

Although the oxygen level inside the culture container has been reported to be

variable depending on the ventilation type (Zobayed et al., 1999b), it often ranges

between 18 and 22 % in conventional micropropagation, high enough for

photorespiration reactions to occur (Nguyen and Kozai, 1998), and therefore it also

contributes to the low net photosynthesis of in vitro cultured plants. Indeed, when

reducing the inside container O2 level, Shimada et al. (1988) obtained a higher net

photosynthetic rate in micropropagated Primula malacoides (a C3 plant). The relative

changes in O2 and CO2 concentration inside the culture vessel were also documented

to be influenced by the type of carbon fixation system of the plantlet, e.g. CAM vs. C3

photosynthetic pathway (Fujiwara and Kozai, 1995a).

2.2.1.2. Effects of light conditions

Normally, conventional micropropagation is conducted at rather low PPF, often

between 50 – 100 µmol/m2s (Heo and Kozai, 1999), and never exceeds 250 µmol/m2s

(Kozai et al., 1998). In most cases, this PPF already exceeds the plantlet light

saturation point, however with increasing concentration of CO2 inside the container the

light saturation point of plantlets in vitro also rises (Niu et al., 1995; Nguyen et al.,

1999b). The P-I curves of different plants in vitro obtained by Niu and Kozai (1997),

Ohyama and Kozai (1998), and Kozai et al. (1998) as well as the Kautsky curves of

tissue cultured roses recorded by Capellades (1989) indicate a similarity between the

photosynthetic responses of plantlets in vitro and of those growing in the greenhouse

or in the field. Once the light intensity exceeds a certain level, tissue cultured plants

also suffer from photoinhibition (Nguyen et al., 1999b). In addition to PPF,

Chapter 2

29

photoperiod and lighting cycle also influence the photosynthetic capacity (Niu et al.,

1995). The effect of the lighting cycle however is proposed to be indirect, and can be

attributed to changes in CO2 availability and relative humidity inside the culture vessel

caused by variable lighting cycles (Fujiwara and Kozai, 1995 a&b).

2.2.1.3. Effects of sugar in the culture medium

Although there is a discrepancy among the results on the impact of exogenous

sugar on the photosynthesis of micropropagated plants and on their ex vitro

acclimatization, it is believed that both the type and concentration of sugar in the

culture medium affect growth and photosynthesis of in vitro cultured plants. In

strawberry Borkowska (2000) recorded a higher photosynthetic activity of shoots

grown in a culture medium supplemented with glucose than those grown on sucrose

containing medium, and proposed that this was due to the faster utilisation of glucose

resulting in complete depletion of sugar in the medium at the end of the culture period.

Regarding the effects of sugar concentration, Tichá et al. (1998) revealed that 3%

sucrose in the medium increased the photosynthetic potential of tobacco plantlets

grown in vitro, while Capellades and co-workers (Capellades, 1989; Capellades et al.,

1991) observed a decrease in the photosynthetic rate of in vitro cultured roses with

increasing sucrose concentrations in the culture medium, and Vorackova et al. (1998)

found that both too much or too little sucrose inhibit photosynthesis of wheat and rape

plantlets in vitro and hindered their subsequent acclimatization in the greenhouse. The

effects of sugars on the photosynthetic capacity of plantlets in vitro was also studied

by Kozai and co-workers, who also noticed a discrepancy among the results obtained;

e.g. with sweet potato a reduction in net photosynthetic rate was recorded when sugar

was added to the culture medium, while in coffee the effects of exogenous sugar

depended on both PPF and CO2 concentrations (Kozai et al., 1999). Similarly, Le et al.

(2001) remarked that the effects of exogenous sucrose (3%) on photosynthesis of in

vitro tomato plants were dependent on light intensity and CO2 level inside the

container, and this can be one of the reasons leading to the discrepancy among the data

stated by different authors. An explanation for adverse effects of sugar oversupply on

the photosynthesis of in vitro cultured plants, as proposed by Vorackova et al. (1998)

Chapter 2

30

and Le et al. (2001), was the accumulation of glucose, fructose and sucrose in shoots,

which in turn caused feedback-inhibition of photosynthetic enzymes and their down-

regulation as well. This is in consistence with data published by other authors

demonstrating that the adverse effects of exogenous sugar on the microplant

photosynthesis is related to a reduction in their Rubisco activity, as argued by Aitken-

Christie et al. (1995) and Desjardins (1995), and to the accumulation of carbohydrates

in shoots in vitro, as observed by Capellades (1989) and Van Huylenbroeck (1997).

2.2.1.4. Relative humidity and temperature inside the vessel

In addition to the above mentioned parameters, photosynthetic ability of plantlets

in vitro is also influenced, either directly or indirectly, by other environmental

parameters, namely temperature and humidity inside the culture vessel, gas exchange

rate, plant species and variety etc. In traditional micropropagation, the temperature is

often maintained at 20 – 25oC, and the relative humidity inside the container is higher

than 95% (Fujiwara and Kozai, 1995a). As discussed by Niu et al., (1995), this

temperature range frequently covers the optimum temperature for photosynthesis of in

vitro cultured plants, except those of tropical origin may require higher temperatures

(George and Sherrington, 1984).

High relative humidity (RH) inside the container has also been blamed for bad

quality of in vitro cultured plants (Maene and Debergh, 1987; Debergh, 1991; Kozai,

1996; de Klerk, 2000). Besides its effects on the stomata function and cuticle

development, the RH has also been recorded to have influence on the photosynthetic

behaviour of plantlets in vitro. Working with potato, Tanaka et al. (1992) noticed a

reduction in the net photosynthetic rate when RH decreased. In cultured roses

however, Capellades (1989) observed that the effect of RH depended on the

concentration of sugar in the medium: under low sucrose level (3%) net photosynthetic

rate increased with increasing RH while at higher sucrose (5%) the net CO2 uptake rate

scored lowest under 100% RH.

Chapter 2

31

2.2.2. Photoinhibition and photorespiration of plantlets in vitro

Data obtained with various plants, such as Primula malacoides (Shimada et al.,

1988), Caladium bicolor and Dendrobium phelaenopsis (Doi et al., 1989), coffee

(Nguyen et al., 1999b), Cymbidium (Kozai et al., 1990), demonstrate some similarity

between the photosynthetic mechanisms of in vitro plants and those of plants grown in

the field or greenhouse. A decline in photochemical efficiency of in vitro cultured

coffee plantlets was attributed to photoinhibition caused by increasing the light

intensity above a certain level (Nguyen et al., 1999b). Photoinhibition was also

recorded in tobacco plantlets grown photoautotropically at PPF of 200 µmol/m2s by

Ticha et al. (1998) with a reduction in chlorophyll content, photochemical efficiency,

D2/LHCII and CP47/LHCII ratios. These authors also revealed an increase in the

content of xanthophyll cycle pigments in photoinhibited plantlets, and argued that this

was due to the activation of xanthophyll cycle photoprotective mechanisms.

Under high O2 concentration, frequently recorded in the culture vessel headspace,

micropropagated plants do photorespire at variable levels depending on their

photochemical mechanisms (Fujiwara and Kozai, 1995a). In 1988 Shimada et al.

recorded an increase in the net photosynthetic rate of Primula malacoides plantlets (C3

plant) with decreasing O2 concentrations inside the vessel and proposed this was due

to reduced photorespiration. By comparing CO2 and O2 evolution in containers

containing C3 (Caladium bicolor) and CAM (Dendrobium phalaenopsis) plantlets,

Fujiwara and Kozai (1995a) found that the change in O2 concentration depended on

the type of carbon assimilation, and thus plantlets in vitro react similarly as those

growing ex vitro to changes in environmental conditions. This is in agreement with the

findings of Szendrak et al. (1994) that environmental factors resembling the natural

conditions favoured in vitro propagation of Orchis morio, and that their in vitro

plantlets reacted in a circadian way. Nevertheless, not much effort has been spent on

the comparative study of photosynthetic behaviour of C3 vs. CAM and C4 plantlets in

vitro.

Chapter 2

32

2.2.3. Photomorphogenesis of in vitro propagated plants

As for plants growing ex vitro the light environment [near ultraviolet (300 – 380

nm), blue (430 – 490 nm), red (640 – 700) and far-red (700 – 760 nm)] is important for

photomorphogenesis of in vitro cultured plantlets, and thus their quality can be

improved through adjusting the light quality (mainly red/far-red and blue/red ratio) by

using appropriate light sources (Fujiwara and Kozai, 1995a).

Changes in microplant morphogenesis and quality caused by variable light

qualities have often been documented. Working with Azorina vadalii (Wats.) Feer,

Moreira da Silva and Debergh (1997) found that an appropriate red/far-red ratio (0.6 in

this case) improved not only the multiplication rate but also the plantlet quality. Also

according to these authors, a reduced red/far-red ratio stimulated both internode

elongation and leaf expansion, while the number of internodes as well as fresh and dry

weight of shootlets remained unchanged. Kozai and colleagues also noticed the

importance of red and far-red light on microplant morphogenesis, and suggested that

red and far-red light emitting diodes have advantages over fluorescent lamps in

regulating plantlet height and quality (Fujiwara and Kozai, 1995a; Kozai, 1996). In in

vitro cultured potato plantlets, Miyashita et al. (1997) observed a positive reaction of

shoot length to red light intensity (under 100 µmol/m2s of the total PPF with negligible

far-red) while the dry weight and leaf area remained unaffected. The same authors also

stated that when the red/PPF was between 0.1 – 0.5 the shoot length of potato plantlets

in vitro increased with increasing far-red/PPF ratio. However, when red/PPF equalling

1, the length of both shoots and internodes was greatest while plantlet dry weight, leaf

area and Pn lowest (Miyashita et al., 1995). The positive effects of far-red light on the

plantlet internode elongation, as observed by Kirdmanee (1995) in Eucalyptus, was

correlated with the increased cell length of stem epidermis. Formation and

development of both adventitious and axillary shoots as well as roots were also found

to be influenced by light quality, e.g. the number of adventitious bulbs (Hyacinthus) or

corms (Freesia) increased under red light irradiation (Bach et al., 2000). Similarly, a

higher number of auxiliary shoots was scored in far-red enriched light (Moreira da

Chapter 2

33

Silva and Debergh, 1997), and better rooting of Azalea microcuttings was obtained

with far-red light supplementation (Read and Economou, 1982).

In addition to red and far-red, blue and UV light also have value in regulating

plantlet height and quality. Appelgren (1991) recorded a strong inhibition of stem

elongation in Pelargonium by blue light and proposed there are 2 different

mechanisms regulating stem elongation depending on the blue/red and blue/far-red

ratios. Moreira da Silva and Debergh (1997) also stated that blue light suppressed

internode elongation and leaf expansion of cultured Azorina vadalii (Wats.) Feer. The

effect of blue light on leaf expansion and shoot elongation however can be species

dependent. For example, in contrast to the data recorded by Appelgren (1991) and

Moreira da Silva and Debergh (1997), Sœb∅ et al. (1995) observed a positive effect of

blue light on leaf expansion in micropropagated birch, and Michalczuk and

Michalczuk (2000) obtained the longest shoots of petunia with the largest leaf area

under blue light. Working with different bulbous plants, Bach et al. (2000) found that

both blue and green lights increased fresh weight of shoots (Hyacinthus, Cyclamen,

Freesia) or bulbs (Lilium), while far-red and darkness inhibited the development of

plantlets.

Light quality also affects the content of carotenoids and chlorophylls of in vitro

cultured plants/shoots. Bach et al. (2000) stated that in some ornamentals blue light

enhanced the total amount of both anthocyanins and chlorophylls, while UV promoted

chlorophyll a and b formation. Also, according to Moreira da Silva and Debergh

(1997), in Azorina vadalii reduced red/far-red ratio led to a reduction in chl a and total

carotenoids concentration. Similarly, in in vitro cultured potato plantlets, Miyashita et

al. (1997) recorded an increase in chlorophyll content when the red/PPF ratio

increased (under 100 µmol/m2s of the total PPF with negligible far-red).

The morphogenic effects of light quality can be deduced to its impacts on the

synthesis or availability of endogenous growth regulators and different enzymes in

cultures. Evidences on the relation between the level of gibberellins, cytokinins, auxin

IAA and some enzymes were obtained in cultures (reviewed by George, 1993/1996).

Chapter 2

34

In addition, phytochromes are said to have role in regulating the expression of some

gene in plants (Batschauer et al., 1994).

2.3. Adaptation of tissue culture derived plants to ex vitro conditions

Upon transplanting to ex vitro microplants must adapt to new growth conditions,

and their capacity to “correct” the structural and functional abnormalities acquired

during the in vitro growing stages is crucial for their survival and growth. As already

mentioned in the previous section, the most important abnormalities hindering their

acclimatization to ex vitro growth conditions are related to their low photosynthetic

and low water retention capacities. Correspondingly, the ability of transplants to

develop normally structured leaves, with normally functioning photosynthetic

apparatus and stomata, able to switch from mixo- or heterotrophic to photoautotrophic

growth and to control transpiration, is the key factor determining the success rate of

their reestablishment ex vitro. The time period required for full adaptation of

micropropagated plants to environmental conditions in a greenhouse or in the field

depends largely on both the plant quality and the ex vitro environment. The success of

the acclimatization however depends significantly on the human interventions through

adjusting different environmental variables, and thus this transition period is best

termed “acclimatization” (Debergh, 1991).

2.3.1. Factors affecting ex vitro acclimatization

While in vitro, high relative humidity and low PPF are important factors

responsible for the poor quality of micropropagated plants (Debergh, 1991; Debergh et

al., 1992; Jeong et al., 1995; Kozai, 1996), low humidity and high light intensity

compared to the in vitro environment have been considered the most important among

various stress conditions hindering the transplant adaptation to the ex vitro

environment (Kirdmanee et al., 1994). It is documented that when provided with

appropriate humidity and light conditions, transplants more readily correct their

abnormalities and adapt to the normal ex vitro environment. For instance, maintaining

plants at high relative humidity upon transplanting and gradual decreasing this

Chapter 2

35

environmental variable has been proved effective in improving plant survival and

growth, and several systems have been developed for this purpose (Debergh, 1991,

Isutsa et al., 1994). Another example was given by Kirdmanee et al. (1995) who

found that one-day of acclimatization ex vitro under either high PPF (1000 µmol/m2s)

with high RH (85%) or under low PPF (10 µmol/m2s) with low RH (55%) reduced the

extent of both leaf and chl damage resulting in higher survival and better growth of

Eucalyptus transplants. Also, Kirdmanee (1996) stressed that a short acclimatization ex

vitro to low RH (70%) followed by 60 % RH could induce stomatal closure

contributing to subsequent higher photosynthetic rate, high survival and better growth

of eucalyptus micropropagated plants.

In general, microplants acclimate to low irradiance while in vitro (see section

2.2), and hence for the best ex vitro acclimatization results, the irradiation around

transplants should be increased gradually otherwise their photosynthetic apparatura

will suffer from photodamage. Van Huylenbroeck et al. (1995) found that when light

intensity in the greenhouse suddenly shifted from 100 – 300 µmol/m2s, Spathyphillum

transplants showed partial photoinhibition and reduced net photosynthesis (up to

50%). Photodamage was also recorded in transplants of Liquidambar styraciflua L.

under 315 µmol/m2s by Lee et al. (1985), and in Rosa hybrida plantlets under 200

µmol/m2s by Sallanon et al. (1998).

Besides light intensity and RH, CO2 concentration, temperature and planting

substrate also have effects on the microplant ex vitro acclimatization. Ex vitro, CO2

enrichment also often has positive effects on the acclimatization of transplants, and as

noted by Desjardins et al. (1990) this is attributed to its ability to promote the

development of photoautotrophy. Working with Gerbera jamesonii, Van

Huylenbroeck and Debergh (1992) also recorded that high CO2 concentration (900

µmol/mol) hastened the acclimatization of microplants, and the effects were

strengthened by increasing light intensities (95 µmol/m2s). These findings consist with

the results obtained by Pospisilova et al. (1999) that CO2 enrichment (1000 µmol/mol)

Chapter 2

36

can improve acclimatization of tobacco transplants by improving photosynthesis and

reducing photoinhibition in transplants, but also by inducing closure of stomata.

Temperature and planting substrate are two other important factors influencing

the ex vitro acclimatization of micropropagated plantlets (Debergh, 1991). Certainly,

optimal temperatures (varying according to the plant species) and a suitable substrate

with appropriate humidity can largely improve the survival and growth of transplants.

For instance, micropropagated Rosa hybrida plantlets transplanted to low water

potential medium exhibited poor growth compared to those planted on medium with

higher water potential (Sallanon et al., 1998). In case of pineapple also, Pham Thi Sen

and co-workers (unpublished) noticed significant differences in both survival and

growth of transplants when comparing different substrates.

2.3.2. The plantlets adaptation ability and methods for its quantification

The adaptation process of microplants to the ex vitro environment involves

various protective responses against potential damage caused by stress conditions, and

include different acclimative mechanisms in order to regain photoautotrophy and to

control transpiration. The success rate of the acclimatization as argued by Debergh

(1991) depends largely on the intrinsic quality of microplants, and their behaviour

upon transfer to ex vitro conditions can be predicted and followed by quantification of

variables or characters related to their photoprotection, photosynthesis and

transpiration. Some of these variables and their use in studying the acclimatization of

micropropagated plants are discussed below.

2.3.2.1. Water retention capacity and leaf structure

No doubt that poor water retention capacity is one of the most frequently

observed abnormalities of tissue culture derived plants hindering their reestablishment

ex vitro (Maene and Debergh, 1987; Davies and Santamaria, 2000). Although together

with abnormally functioning stomata, defective cuticle and reduced epicuticular wax

are also frequently observed on in vitro formed leaves (Brainerd and Fuchigami, 1981;

Sutter, 1988; Capellades et al., 1990a), through monitoring the water loss of apple

Chapter 2

37

microcutting leaves by covering either upper or lower sides of leaves with paraffin,

Brainerd and Fuchigami (1981) came to the conclusion that poor water retention is

likely caused by malfunctioning of stomata and not by a poor cuticle. A similar

statement was made by Santamaria and Kerstiens (1994) and Kerstiens (1996) that in

vitro epicuticular wax and cuticle may be thinner but have little effects on regulation

of the leaf water loss. Thus, stomata number, size, structure and functions rather than

cuticle and epidermis should be studied in relation to plant water retention capacity

and adaptation ability, and indeed the works of Capellades (1989), Van Huylenbroeck

and Debergh (1992) and Pospisilova et al. (1999) have proved the usefulness of these

parameters. Normally, the rate of water transpiration decreases during acclimatization

together with the development of properly functioning stomata.

In addition, many other leaf structural features are also useful for the assessment

of the “normality” of tissue culture derived plants. Various anatomical abnormalities

contributing to the plantlet low photosynthetic capacity, such as poorly developed

palissade layers (Grout, 1975; Brainerd and Fuchigami, 1981), large intercellular

spaces in mesophyll layers (Paques and Boxus, 1987; Taji et al., 1996), changes in

amount of chloroplasts, chlorophyll and carotenoids (Franck et al., 1998a; Capellades

et al., 1990a), reduced palisade parenchyma, poorly developed vascular system (Taji et

al., 1996) etc., have been recorded in micropropagated plants. Moreover, many

symptoms of abnormality were observed at larger extent in hyperhydric shoots than in

“normal” ones (Franck et al., 1998a &b). It is thus expected that better acclimatized

transplants should have more normal leaf structures.

2.3.2.2. Net CO 2 uptake and carboxylating enzymes

While there is not an unanimously point of view on the importance of in vitro

photoautotrophy, it is well accepted that the capacity of transplants to regain

photoautotrophy and to improve their photosynthetic ability under ex vitro conditions

defines to a large extent the success rate of the transplant acclimatization (Van

Huylenbroeck and Debergh, 1996), and the net photosynthetic rate is therefore one of

the most important physiological indicators for evaluating transplants’ reestablishment

Chapter 2

38

ex vitro. Directly after transplanting, micropropagated Calathea plantlets continue to

grow heterotrophically and suffer from light stress, but with the time passing their net

photosynthesis increases and the plants gradually recover from the stress (Van

Huylenbroeck, 1997). Data also frequently document that promotion of photosynthetic

capacity soon after transplanting results in good growth and survival of transplants

(e.g. Capellades et al. 1990b; Kirdmanee et al., 1994).

Besides Rubisco, PEP-case, the primary carboxylating enzyme in C4 and CAM

photosynthesis, catalysing the irreversible fixation of CO2 (as HCO3-) onto PEP

(Section 2.1), can also serve as a reliable indicator for the plant photosynthetic

competence. In addition, the relative activities of Rubisco and PEP-case enable the

estimation of the relative importance of heterotrophic and autotrophic carbon fixation

in in vitro cultured material (Kwa et al., 1997; Triques et al., 1997). Nevertheless, not

many data are available on these enzymes during acclimatization of micropropagated

plants.

2.3.2.3. Chlorophyll fluorescence

As already mentioned earlier, fluorescence emission of a chlorophyll molecule is

one of its de-excitation pathways from the excited state (chl*) to the ground state (chl)

(fig. 2.5), and the total yield of chl fluorescence (ΦF) is reversibly proportional to the

sum of photochemical yield (Φp) and heat dissipation. Dogmatically it is accepted that

an excited PS2 with an open RC (its QA in unreduced state) shows the lowest

fluorescence emission, and that an exited PS2 with a closed RC (its QA in reduced

state) exhibits the highest fluorescence emission. Upon illumination of a dark adapted

leaf sample (with all the RC open) with a saturating light pulse, the fluorescence

emission quickly rises (normally within 500 ms – 10 s) from its minimum value (Fo) to

a maximum value (Fm), then declines and finally reaches a steady state (Fs). Similarly,

corresponding values (Fo’; Fm’ and Fs’) are obtained when the leaf sample is light-

adapted. Therefore by studying and comparing the fluorescence transition of a leaf

sample in its different states (dark-adapted vs. light adapted or light 1 vs. light 2) one

can estimate the capability of that sample to adapt from dark to certain light conditions

Chapter 2

39

or from one light condition to another. The fluorescence transition hence is a signal

extremely rich in both quality and quantity of different information. For better

understanding of chl fluorescence kinetics and of how their analysis and interpretation

can provide information on the photosynthetic capacity and physiological state of a

plant, one can refer to Cormic (1994) and Osmond (1994), Strasser et al. (2000). In

their work, Strasser et al. (2000) also described in detail how to derive quantum yield

of primary photochemistry (Φp), non-photochemical quenching (qN), photochemical

quenching (qP) and relative variable fluorescence (Vt) from Fo, Fm, Ft, and Fs (t is for

the time moment t). A schematic view of measurements of chl fluorescence from a leaf

sample in dark- or light-adapted state is presented in fig. 2.6.

Fig. 2.6: Schematic view of measurements of chl fluorescence from a leaf sample in its dark- or light-adapted state

In short, the following information can be derived from chl a fluorescence

transient studies :

1) The higger the value of minimal fluorescence Fo or Fo’ the higher the level of

photodamage (Cormic, 1994; Osmond, 1994).

Saturation pulse

DARK ADAPTED LIGHT ADAPTED

Fm

Fo

Fv

Fm’

Fo’

Saturation pulse

Fs Fs’

Decrease due to non-photochemical quenching

Decrease due to photochemical quenching

Chapter 2

40

2) The quantum yield of primary photochemistry (Φ), i.e. the ratio of the

photons used for photochemical reactions to the total photons absorbed

(TF1/TF, see Fig. 2.5), can take all values from zero to unity, and the higher this

value the better the overall photosynthetic efficiency.

m

t

FF

1−=Φ , 0 ≤ Φ ≤1

The maximal quantum yield of a leaf sample in its dark-adapted state is

always close to 0.8 for healthy leaves independently from the plant species. A lower

maximal quantum yield indicates that part of the PCII reaction centres are

photodamaged (Osmond, 1994).

m

v

m

om

m

omax F

FF

FFFF

1 =−

=−=Φ

3) The relative variable fluorescence (Vt) is an indication for the fraction of

closed RC (Strasser et al., 2000):

om

0tt FF

FFV

−−

= , 0 ≤ Vt ≤1

4) The photochemical quenching (qP) quantifies the decrease in chl fluorescence

caused by the increase in overall photochemical capacity (increase in the

proportion of open RC); and the bigger the value of (1-qP = V) the larger the

extent of chronic photoinhibition (Cormic, 1994; Strasser et al., 2000):

om

m

FF

FFV1qP

−

−=−= , 0 ≤ qP ≤1

5) The non-photochemical quenching (qN) quantifies the decrease in chl

fluorescence due to causes different to that of photochemical quenching. The

bigger the qN the more excess energy is dissipated in the form of heat and the

less photodamage occurs (Osmond, 1994).

Chapter 2

41

darkv

lightv

)F(

)F(1qN −= , 0 ≤ qN ≤1

Early reports confirm the usefulness of chl a fluorescence study in quantification of the

plantlet physiological status and of the stress conditions as well. Positive correlation

between the photosynthetic capacity of cultured roses and direct measurements of chl a

fluorescence was recorded by Capellades (1989). Similarly, Van Huylenbroeck (1997)

found a linear relation between the net photosynthetic rate and photochemical

quenching in Calathea and Spathiphyllum transplants, and came to the conclusion that

chlorophyll a fluorescence is a fast and reliable measuring technique to obtain

information about the plant quality. By studying chl a fluorescence Van Huylenbroeck

and Debergh (1992) could also deduce the successions of stress situation during the

acclimatization; these authors noticed two prominent changes in fluorescence values

coinciding with the two periods of stress: rooting phase and transfer to normal relative

humidity ex vitro. In addition, the decrease in Fv/Fm was also observed to be

positively correlated with the light intensity, and reduced with time course during the

acclimatization period (Van Huylenbroeck et al., 1997 & 2000).

2.3.2.4. Protective enzymes

One of the most important biochemical mechanisms of the plant reactions to

adverse changes in growth conditions involves enzymatic defence against the harmful

molecules generated under stress conditions (section 2.1). Correspondingly, activities

of protective enzymes, including peroxidases, catalase and SOD, can serve as reliable

variables for quantification of the transplant adaptation capacity. This indeed has been

confirmed by the works of Franck et al. (1995) and Van Huylenbroeck et al. (1997,

1998 & 2000). During ex vitro acclimatization of Calathea and Spathiphyllum

plantlets, the activities of catalase and SOD increased and reached their maximal

values after a certain time period (Van Huylenbroeck et al., 1998). Changes in

activities of different peroxidases were also observed (Van Huylenbroeck et al., 1997).

Furthermore, the changes seemed to be affected by the growth conditions; e.g. the

changing pattern of both SOD and catalase activities were dependent upon the light

Chapter 2

42

intensity (Van Huylenbroeck et al, 2000). Working with Prunus avium, Franck et al.

(1995) notified differences in the activities of SOD, catalase and peroxidases in

hyperhydric shoots compared to the normal ones and concluded that the hyperhydric

shoots were unable to recover their enzymatic defence system.

As argued by Debergh (1991) however, for assessment of the micropropagated

plant quality defferences between “good” and “bad” plants should be described. This

methodology is also used in the present work, and further discussed in chapter 4-

“Experimental results and discussions”.

CHAPTER 3

GENERAL EXPERIMENTAL MATERIALS AND METHODS

Chapter 3

44

3.1. Plant materials

3.1.1. Model plants

For each of the three photosynthetic groups of C3, C4 or CAM plants a

representative was selected as model plants of this study:

• C3 group: Prunus avium, a valuable temperate plant, which is also grown in the

Northern mountainous areas of Viet Nam.

• C4 group: Sugarcane (Saccharum officinarum) a NADP-ME C4 plant (Singh,

2000) and one of the most important industrial plants worldwide, also having

both economic and social values in Viet Nam. The sugarcane used, named K84

– 200, was introduced from Thailand.

• CAM group: Pineapple (Ananas comosus), a obligatory CAM plant (Reddy and

Das, 2000) and a valuable tropical fruit important in Viet Nam for both in-

country consumption and exportation. The variety used was Cayen Phu Ho,

originated in Viet Nam.

3.1.2. Stock plant sources

In vitro pathogen ‘tested’ shoot cultures of Prunus avium were obtained from

Prof. Thomas Gaspar, Université de Liège, Belgium, while those of sugarcane and

pineapple were provided by the Department of Biotechnology, National Agricultural

Science Institute of Viet Nam, Ha Noi. For maintenance and production of in vitro

shootlets necessary for the experimental works, cultures were propagated on

appropriate medium for each plant; PR-1 for Prunus, PN-1 for pineapple and SG-1 for

sugarcane (table 3.1), and incubated under the growth conditions as defined in 3.2.1.1.

These media were selected based on the results obtained in our previous experiments,

where they gave reasonable multiplication rates and visually normal microshoots.

Chapter 3

45

3.2. Experimental and growth conditions

Except otherwise stated, all the experiments were carried out in the Department

of Plant Production, Faculty of Agricultural and Applied Biological Sciences,

Universiteit Gent, Belgium.

3.2.1. In vitro conditions

3.2.1.1. General in vitro culture conditions

All in vitro cultures were grown in 320-ml glass jars (Meli Jars, De Proft et al.,

1985) with transparent polypropylene screw on lid and 100 ml medium per jar. They

were incubated at 21 ± 2oC, under 16 h photoperiod with a total photosynthetic active

radiation (PAR) of 35 - 50 µmol m-2s-1 at shelf level provided by white 40W liteguard

fluorescent lamps (Osram Ltd. UK.). For Prunus cultures, the relative humidity (RH)

in the containers was maintained around 85% by bottom cooling (Vanderschaeghe &

Debergh, 1987), while for sugarcane and pineapple RH was not controlled. The culture

media, glassware and instruments were sterilised at 121oC, 98 kPa for 30 min. All the

subculture manipulations were carried out under aseptic conditions, in a laminar flow

cabinet.

3.2.1.2. Production of microplants of different physiological quality

To produce microplants of different physiological quality, different medium and

light quality treatments were applied during the in vitro rooting stage. Uniform

microshoots (2-3 cm height) of the model plants were rooted on different rooting

media and incubated under different light qualities. For Prunus two kinds of medium,

solidified with either 6 g/l agar (type MC29 from Amersham) or 2 g/l Gelrite (cat. n°

G1101 from Merck) were used (medium PR-2 and PR-3, table 3.1). In the case of

sugarcane and pineapple, two types of rooting medium, either with (1 g/l) or without

activated charcoal (OCB n°1242), were applied (table 3.1; medium PN-2 & PN-3 for

pineapple, and SG-2 & SG-3 for sugarcane).

Regarding light quality, the following 4 radiation proportion treatments were

used:

Chapter 3

46

(1) Control light (C): R/Fr = 8.78, and B/R = 0.26

(2) High red light (R): R/Fr = 20.33, and B/R = 0.22

(3) High far-red light (FR): R/Fr = 0.23, and B/R = 0.33

(4) High blue light (B): R/Fr = 7.41, and B/R = 2.39

whereas R/FR = ΣNλ(655 – 665) / ΣNλ(725 – 735), and B/R = ΣNλ(400 – 500) / ΣNλ(600 – 700).

Table 3.1: Codes and medium compositions used for in vitro propagation and rooting

Prunus Pineapple Sugarcane

PR-1 PR-2 PR-3 PN-1 PN-2 PN-3 SG-1 SG-2 SG-3

Macro-elements MS* MS* MS* MS 1/3 MS 1/3 MS MS MS MS Micro-elements MS MS MS MS MS MS MS MS MS Vitamins MS MS MS MS MS MS MS MS MS Ca-gluconate.H2O (g/l) 1 1 1 - - - - - - Na-FeEDTA (mg/l) 35 35 35 35 35 35 35 35 35 myo-inositol (mg/l) - - - - - - 100 100 100 Activated charcoal (g/l) - - - - 1 - 1 1 - IBA (mg/l) 0.5 0.5 0.5 0.5 0.2 0.2 - 0.3 0.3 NAA (mg/l) - - - - 0.2 0.2 - 0.3 0.3 BAP (mg/l) 0.5 - - 1.0 - - 0.2 - - Sucrose (g/l) 20 20 20 20 20 20 20 20 20 Agar (g/l) 7 6 - 6 7 7 6 7.0 7.0 Gelrite (g/l) - - 2 - - - - - - pH 5.7 5.7 5.7 5.7 5.7 5.7 5.7 5.7 5.7

MS stands for Murashige & Skoog (1962) full macro elements (CaCl2 332.02; KH2PO4 170; HNO3 1900; MgSO4 180.45; NH4NO3 1650 mg/l), micro-elements (CoCl2. 6H2O 0.025; CuSO4.5H2O 0.025; FeNaEDTA 36.7; H3BO3 6.2; KI 0.83; MnSO4.H2O 16.9; Na2MoO4.2H2O 0.25; ZnSO4.7H2O 8.6 mg/l) and vitamins (glycine 2; nicotinic acide 0.5; pyridoxine HCl 0.5; thiamine HCl 0.1 mg/l); MS* stands for MS macro-elements with CaCl2 omitted; activated charcoal was UCB n°1242, agar type was MC29 from Amersham, and Gelrite type was G1101 from Merck.

For all the treatments, the photoperiod was 16 h and the total PAR was 40 – 50

µmol m-2s-1 at shelf level. Light was provided by the following lamps: white (40W

liteguard fluorescent lamps supplied by Osram Ltd. UK.), blue (Philips TLD

40W/03RS), far red (Sylvania F 40 W/5310) and red (Philips TLD 36W/15). The

combination of 2 coloured lamps and 2 white ones was applied for each shelf.

Chapter 3

47

3.2.2. Greenhouse conditions

For reestablishment to ex vitro conditions, microplants were transferred to a

regular unfertilized peat mixture (provided by Structural TM, n° 9A1) initially in

seedling trays (50 x 30 cm) with 80 holes (of 5 cm diameter) and subsequently to

larger pots at appropriate stages. The transplants were maintained for the first month in

a growth chamber, where the temperature varied between 18 – 22°C, under 16h

photoperiod provided by 40W liteguard fluorescent lamps (Osram Ltd. UK), with the

total PAR of 35 – 45 µmol/m2/s. For the first week, the RH was kept high (80 – 85%)

using an ultrasonic humidifier provided by BURG, Austria. After a month, they were

transferred to glasshouse at 15oC - 25oC, under normal daylight conditions during

February – May of year 2001, eventually supplemented to 16-hour photoperiod by

mercury vapour lamps (HQI-T 400 W/D, Osram Germany) when needed. They were

watered and fertilized as required, using the NPK fertiliser Nitrophoska, Blaun

Special, provided by COMP Benelux (BASF).

3.3. Observation methods

3.3.1. Plant growth parameters

• Number of roots per plant: total number of primary roots per plant;

• Number of nodes per plant: total node number up to the newest opened leaf;

• Leaf length: the length of the blade of the second youngest fully expanded leaf;

• Fresh weight and dry matter accumulation: intact plants were weighted (FW);

they were put in an oven at 80oC for 48 h and their dry weight recorded (DW).

The dry matter content (DM) was calculated using the following formula:

100FWDW

DM x= (%)

Dry matter accumulation was recorded by comparing DW at different stages of plant

development.

Chapter 3

48

3.3.2. Stomatal density and size

A translucent nail polish imprint of the abaxial surface of the second youngest

fully expended leaf was examined under a microscope for studying the stomata density

and length (Fig. 3.1).

The average of 20 randomly

chosen stomata from each

microscopic field of 1 cm2 (at 100x

magnification) was regarded as the

length of the stomata existing in the

microscopic field examined. The

mean of 3 randomly selected

microscopic fields of each sampled

leaf was used as the value for each

plant.

3.3.3. Study of leaf anatomy

We studied the anatomy of leaf blades using the second youngest fully expanded

leaf according to the following methods (a combination of techniques and

modifications of Johansen, 1940 and Ruzin, 1999).

1/ Fixation: Leaf blade sections of 1- 4 mm. Fix for 24 h at room temperature in FAA

solution [90 ml aq. ethanol 70 %; 5 ml glacial acetic acid and 5 ml formaldehyde

(35 – 37 %].

2/ Dehydration: Discard the fixative and immerse the sample in the dehydration

solution I (80 ml aq. ethanol 70%, 15 ml tertiary-butanol and 5 ml distilled water),

incubate for 24 h at room temperatures. Replace the solution I for dehydration

solution II (65 ml aq. ethanol 70% and 35 ml tertiary-butanol), incubate for another

24 h at room temperature. Repeat with the dehydration solution III (45 ml aq.

ethanol 70% and 55 ml tertiary-butanol), IV (25 ml aq. ethanol 70% and 75 ml

tertiary-butanol) and V (pure tertiary-butanol).

Fig. 3.1: Measurement of stomatal length (a)

a

Chapter 3

49

3/ Infiltration and embedding: Add solid paraffin pieces in the dehydration solution V

containing the sample, incubate for 24 h at room temperature. Put in an oven at

50°C overnight. Replace the mix of paraffin and solution V with pure paraffin

previously melted at 50°C and, incubate for another 24 h at 50°C. Pour altogether in

a small veil (make sure the sample is well ‘wrapped’ by the paraffin) and let it to

cool down. In this state, the sample could be stored for some time.

4/ Slicing: Trim off the paraffin in order to make a small block of paraffin with the

sample inside. Make slices of 5 - 12 µm using a microtome, put the slices on a glass

slide previously covered with HAUPT (1g gelatin and 2g phenol crystalline in 15

ml glycerine, filtered). Put the glass slide on a hot plate at 50°C for the paraffin to

melt (just melted enough).

5/ Staining: Wash off the paraffin by dipping the slide in a series of solutions as

follows: xylol 10 min, again xylol 10 min, xylol and aq. ethanol (1:1) 5 min, 95%

ethanol 3 min, 85% aq. ethanol 3 min, 70% aq. ethanol 3 min. Stain with safranine

(4 g safranine, 100 ml aq. ethanol 95%, 100 ml distilled water, 4 g Na-acetate, 8 ml

formaldehyde) for 2 h for sugarcane and pineapple, and overnight for cherry leaf

samples. Wash off the safranine with running tap water followed by distilled water.

Put the slide in ethanol containing 4-5 drops of ammoniac per 100 ml for not longer

than 2 min. Transfer the slide into 95% ethanol and butanol normal (1:1) for 5 min

followed by pure butanol normal for another 5 min. Stain in fastgreen (200 mg

fastgreen in 100 ml butanol normal) for 5 min. Wash off the fastgreen by

transferring the slide into the following solutions: butanol 5 min, butanol and xylol

(1:1) 5 min, xylol 5 min and again xylol 15 min. Mount with Canada balsam and

cover with a cover glass. The slide was then ready for microscopic examination and

photography.

3.3.4. Observation of gas evolution in the culture vessel headspace

The level of some gas components in the culture vessel headspace, including

ethylene, oxygen and carbon dioxide were determined by gas chromatography

(Demeester et al., 1995). The level of ethylene (in ppm) was measured using a flame

Chapter 3

50

ionisation detector (FID) provided by Biorad RSL N.V., Belgium, in 0.3 ml samples.

The concentrations of CO2 and O2 (in %) were determined with a thermal conductivity

detector (TCD), also supplied by Biorad, in 0.3 ml samples. To reduce most the gas

exchange, the culture jars were tightly closed using a clamping device, and for taking

gas samples a sampling device was also attached as described by Demeester et al.

(1995) (see also fig. 3.2).

Fig 3 .2: Atightly closed jar with a clamping device (a) and a gas sampling device (b) used for study of the evolution of gasses in the culture headspace. A rubber ring (c) was inserted between the lid and the container rim.

3.3.5. Determination of photosynthetic pigment contents

Both chlorophyll contents and total carotenoids concentration were determined

using a method modified from Wellburn (1994). Grind 250 mg of fresh second

youngest fully expended leaves in 15 ml of 80% acetone, mix well with a mixer and

leave in a freezer overnight for precipitation. Read the absorbance of the liquid phase

at 470, 646.8 and 663.2 nm using a 1 cm-path-length glass cuvette. The concentrations

of chlorophyll a and b and total carotenoids (in µg/ml extract) were calculated using

the following formulas:

Ca = 12.25 A663.2 - 2.79 A646.8 (µg/ml)

Cb = 21.50 A646.8 - 5.10 A663.2 (µg/ml)

Ccar = (1000 A470 - 1.82 Ca - 85.02 Cb) : 198 (µg/ml)

3.3.6. Measurement of net photosynthetic rate

The net photosynthetic rate of plantlets was determined using a Portable Plant

Photosynthesis System, the Leaf Chamber Analyser type ADC LCA4 (400-J1500/C)

a

b

c

Chapter 3

51

supplied by Analytical Development Company, United Kingdom, at 60 – 80 µmol.m-2s-1,

and the leaf temperature of 26 – 28oC. The relative humidity was uncontrolled. The

average figure of three measurements on the second youngest fully expended leaf was

used as the net photosynthetic rate of each sampled plant.

3.3.7. Determination of PEP-case activity

For all enzymes and protein extractions, the second youngest fully expanded

leaves of plantlets were collected and immediately frozen in liquid nitrogen and stored

in a deep freezer (-80oC) until the extraction was carried out. The protein content and

enzyme activities were assessed immediately after extraction. All the manipulations

were carried out at 4oC.

The activity of PEP-carboxylase (EC. 4.1.1.31) was assayed by coupling the

formation of oxaloacetate to the oxidation of NADH by exogenous NADH-malate

dehydrogenase (in excess) at room temperature, pH 7.8 according the following

method:

1/ Extraction of the enzyme (after Kumar et al., 1988): Grind the frozen material

equivalent to 100 mg of fresh leaves in liquid nitrogen. Add 1.5 ml of extraction

buffer pH 7.8 [50 mM Tris-HCl, 1 mM dithiothreitol, 2 mM Na2-EDTA, 6 mM Na-

diethyldiothyldithio-carbamate, 25 mM Na-ascorbate, 1 % (w/v) soluble polyvinyl

pyrrolindone (MW 40000)] and centrifuge at 26000 g for 15 min. The supernatant

was used for PEP carboxylase activity determination.

2/ Assessment of the ezyme activity (after Jones et al., 1978): The PEP carboxylase

activity in the extract was assayed by following the oxidation of NADH in a 1 cm-

path-length cuvette at 340 nm for 5 min at an interval of 12 min. The assay buffer

(AB) contained 50 mM Tris-HCl (pH 7.8), 1 mM DTT, 10 mM NaHCO3, 5 mM

MgCl2, 0.15 mM NaDH, and 2 mM PEP. The reaction was started by adding 100 µl

enzyme extract (previously diluted sufficiently to give ∆A340/min ranging between

0.01 and 0.2) into a 1ml cuvette containing 900 µl of AB and 5 µg malate

Chapter 3

52

dehydrogenase. The ∆A340/min/mg soluble proteins were used for comparison of

PEP-carboxylase activities in the extracts of plantlets from different treatments.

3.3.8. Determination of SOD- activity

Total SOD- activity in leaf tissues was assayed using a compilation and

modification of some methods as follows:

1/ Extraction of the enzyme (after Giannopolitis and Ries, 1977): Grind an amount of

frozen leaf material equivalent to 100 mg fresh leaves in liquid nitrogen. Add 1.5 ml

of extraction buffer, pH 7.8 containing 0.1 M K-phosphate and 0.1 mM EDTA.

Centrifuge the homogenate twice at 13000 g for 10 min. The supernatant was used

as crude extract for assessment of total SOD-activity in leaf tissue.

2/ Assessment of the enzyme activity (McCord and Fridovich, 1969): Total activity of

SOD in the enzyme extract was assayed by measuring its ability to inhibit the

photochemical reduction of ferricytochrome C through following the optical

intensity of the assay mixture consisting of ferricytochrome C, xanthine and

sufficient xanthine oxidase to produce a rate of reduction of ferricyt. C at 550 nm of

0.025 absorbance units per min at room temperature. The assay buffer (AB)

contained 50 mM K-phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.01 mM

ferricytochrome C, 0.1 mM xanthine. The reaction was performed as follows: to the

4.5 cuvette (1 cm-path-length) containing 2.98 ml AB add 10 µl xanthine oxidase

(from the 20 units/ml stock solution) and follow the absorbance at 550 nm for 2 min

at 12s intervals to establish a baseline slope. Then, add 10 µl enzyme extract and

continue to measure the absorbance for another 4 min. Under these conditions, one

unit of SOD- activity was defined as 50% decrease in the rate of cyt. C reduction.

Thus, for calculation of the units of SOD-activity in the extract (ASOD), the percent

decrease in the rate of the absorbance increase of the reaction assay (compared to

the baseline slope) was calculated and divided by 50 and by the mg of proteins in 10

µl extract (a), using the following formula:

Chapter 3

53

∆Acontrol - ∆Asample

ASOD = 100 : 50 : a (units/ mg soluble proteins ) ∆Acontrol

3.3.9. Determination of catalase activity

The enzyme catalase was extracted using the same method as for SOD. The

enzyme activity was then determined according to the method of Aebi (1984) with

some modifications by following the decomposition of peroxide at room temperature

and pH 7.0. The assay mixture contained 1900 µl of 50 mM K-phosphate buffer, pH

7.0 and 100 µl enzyme extract (1 cm-path-length 3 ml cuvette). The reaction was

started by adding 1000 µl of 30 mM peroxide in 50 mM K- phosphate buffer, pH 7.0

as substrate. The rate of peroxide decomposition was followed by reading the

absorbance at 240 nm for 2 min at a 6s interval. The catalase-activity in the enzyme

extract (k) was then calculated using the following formula:

k = (2.3 : ∆t) x log (A1/A2) x 30 (unit ml-1s-1)

where ∆t was the time interval between the two measurements [= t2 - t1 (s) ]; A1 and A2

were the absorbance at t1 and t2 respectively, and 30 was the dilution factor. From the

values obtained, the enzyme activity was calculated and expressed in unit/mg proteins.

3.3.10. Determination of total soluble proteins

The total soluble proteins in the extracts were determined using Bradford reagent

(100 mg of Coomassie Brilliant Blue G-250 previously dissolved in 50 ml 95%

ethanol; 100 ml of 85% phosphoric acid; final volume to 1000 ml with distilled water)

as described by Bradford (1976). To a 1 cm-path-length 4.5 ml cuvette add 0.1 ml

extract and 3 ml Bradford reagent. Mix well by inversion. Wait at least 5 min, and not

longer than an hour, read the absorbance at 595 nm against a blank containing 0.1 ml

of the extraction buffer instead of the extract. Calculate the protein concentration

based on the absorbance of a standard protein (bovine serum albumin 1g/ml).

Chapter 3

54

3.3.11. Chlorophyll fluorescence study

The chlorophyll fluorescence of the abaxial surface of the second youngest fully

expanded leaf was measured using one of the modulated systems with a pulse

amplitude modulation (PAM) fluorometer (model PAM-2000) supplied by Heinz

Walz GmbH, Effeltrich, Germany. After 15 min dark adaptation, Fo and Fm were

measured by application of a saturating flash. Then, total photochemical yield, Fm',

Fo', Ft, qP and qN were followed for 5 min in continuous presence of actinic light

while saturating pulses were given at 20s intervals (Schreiber et al., 1986).

3.4. Experimental design and data analysis

All experiments were designed as completely randomised blocks. The replicate

numbers varied between parameters observed, as defined in chapter 4. All data were

subjected to appropriate statistical tests as described in each experimental work

(Chapter 4).

CHAPTER 4

EXPERIMENTAL RESULTS AND DISCUSSION

4.1. Growth and photomorphogenesis of micropropagated plants during

the acclimatization period; differences between plantlets produced

under different in vitro light quality and culture medium conditions,

and between C3, C4 and CAM plants

4.2. Photosynthesis of micropropagated plants; differences between

plantlets produced under different in vitro light quality and medium

conditions, and between C3, C4 and CAM plants.

4.3. Photoinhibition and photoprotection of micropropagated plants

during the acclimatization period; differences between plantlets of

different physiological quality, and between C3, C4 and CAM plants.

Chapter 4.1

56

4.1. Growth and photomorphogenesis of micropropagated plants

during the acclimatization period; differences between

plantlets produced under different in vitro light quality and

culture medium conditions, and between C3, C4 and CAM

plants

4.1.1. Specific introduction and objectives

As already mentioned in previous sections, good physiological quality is the

prerequisite for successful re-establishment of tissue cultured derived plants ex vitro.

While it is well defined that a physiologically competent microplant should grow

without any lag upon transfer to the ex vitro conditions, quantification of the plantlet

physiological status is still a problem, and as argued by Debergh (1991), comparison

of presumably “good” vs. “bad” plantlets is often carried out for this purpose. Using

this method, in the present work, a comparative study on the growth and

morphogenesis during the ex vitro acclimatization period of microplants of different

physiological qualities from three model species was undertaken. For sugarcane and

pineapple, plantlets rooted on activated charcoal (AC) free and AC containing medium

were used, while for Prunus, microplants rooted on agar-solidified medium were

compared with those on Gelrite-solidified medium. In addition, 4 light treatments with

variable blue, red and far-red proportions were also applied during the in vitro rooting

stage.

Normally, the quality of tissue cultured plantlets is affected by the growth

conditions in vitro, among them light regime and culture medium. It is revealed that of

various plants physiologically “good” microplants can be obtained by addition or

omission of certain substance(s) from the culture medium (George, 1993/1996) and/or

by adjusting the light conditions (Fujiwara and Kozai, 1995a).

As for culture medium components, AC can be added to improve the quality of

microplants. Possessing strong adsorptive properties, AC can adsorb both gasses and

dissolved solid chemicals. In tissue culture it adsorbs both inhibitory and promoting

Chapter 4.1

57

substances, including toxic components produced as a result of autoclaving or exuded

by cultured tissues, such as phenols (Fridborg et al., 1978; Weatherhead et al., 1979);

polyphenols (Bon et al., 1988); 5-hydroxymethyl-furfural (George, 1993/1996);

growth regulators [e.g. IAA and IBA (Nissen and Sutter, 1988 & 1990), NAA

(Fridborg and Eriksson, 1975, Steinitz and Yahel, 1982), BAP (Takayama and

Misawa, 1980; Maene and Debergh, 1985)]. Activated charcoal can also prevent the

build up of polyphenols by stopping their steady synthesis [e.g in Sequoiadendron

giganteum (Bon et al., 1988)], and reduce browning [e.g. in Palmae (Fisher and Tsai,

1978; Tisserat, 1979)]. In azalea, supplementing 2.5 g/l AC resulted in a larger number

of well-developed plantlets, and consequently their performance after transplanting ex

vitro improved (Preil and Engelhardt, 1977). Also, as remarked by some authors, the

use of AC not only increases the percent plantlets rooting in vitro but also improves

the plant root quality. For instance, higher proportions of plantlets rooted in vitro were

obtained in orchid with 2 g/l AC by Fu (1978) and in Pinus sylvestris with 0.01% AC

by Gronroos and Von Arnold (1985). Also, long and fine roots with more branches

were obtained in Gladiolus (Lilien–Kipnis and Kochba, 1987) through supplement of

5g/l AC. From our early experiments on sugarcane and pineapple it was also revealed

that addition of AC (1g/l) improved the plantlet rooting in vitro and their subsequent

performance ex vitro (unpublished). Positive effects of AC on the plant rooting in

vitro, as observed by Krikorian and Kann (1987) in Sapium sebiferum, is attributed to

its ability to adsorb auxin slowly and thus reduces the detrimental effects of these

growth regulators after root formation have been initiated. According to Ernst (1974)

and George (1993/1996), darkening of medium and adsorption of inhibitory

substances can be another reason.

Regarding gelling agents, there are also data demonstrating their influence on

the quality of micropropagated plants (Debergh et al., 1981; Fujiwara and Kozai,

1995a; George, 1993/1996; Beruto, 1997; Cassells and Collins, 2000). Having specific

characteristics (melting point at ca. 100oC, solidifying point at around 45oC covering

all feasible incubation temperatures, undigested by plant enzymes, does not react much

with medium components) agar is the most widely used gelling agent for plant tissue

culture. This product however has also disadvantages such as high cost, low aeration

Chapter 4.1

58

and poor transparency (Brochard, 1991), and thus for overcoming these problems

Gelrite or other gelling agents are used instead. Gelrite also has the setting point at 35

– 50oC, but in comparison to agar, it is rapidly gelling and more transparent.

Furthermore, water in Gelrite is only slightly bound, and hence diffusion of water-

soluble substances through Gelrite is quicker than through agar (George, 1993/1996).

A negative attribute of Gelrite is that it liquifies rapidly when pH is dropping.

The influence of agar or Gelrite on micropropagated plants depends largely on

the plant species; in various cases agar is considered superior (Zimmerman et al.,

1991; Bonga and Von Aderkas, 1992), while in numerous others Gelrite is preferred

for production of good quality plantlets (Huang et al., 1995). In addition, agar from

different brands may have different characteristics and so their effects on the plant

quality varry (Beruto et al., 1999, Cassells and Collins, 2000). Nevertheless, Gelrite

seems to induce hyperhydricity more often than agar; e.g. in apple (Pasqualetto et al.,

1986 & 1988), in Clianthus formosus (Taji and Williams, 1989), in Pinus radiata

(Nairn, 1988), and in Olearia microdisca, Prostanthera calycina, Prostanthera

eurybioides & Swainsona viridis (Taji et al., 1996). On in vitro Prunus shoots, severe

symptoms of hyperhydricity (translucent stems and leaves, wrinkled, curled and

thicker leaves etc.) were identified from day 7 on when cultured on medium solidified

with 2.5 g/l of Gelrite (Frank et al. 1995).

Light regime, as already discussed in Chapter 2, significantly affects not only

the plantlets’ physiological functions, but also their morphological features. With

many plants it is revealed that the photon ratios of red/far-red and blue/red can be of

significant importance for photomorphogenesis and quality of microplants (Moreira da

Silva and Debergh, 1997; Fujiwara and Kozai 1995a), and thus for regulating the

tissue cultured plant growth and development different types of light sources can be

used to emit different ratios of blue, red and far-red irradiation (Jeong et al., 1995).

Chapter 4.1

59

4.1.2. Specific materials and methods

Shootlets of 2-3 cm height of the three model plants representing the three

photosynthetic groups (C3, C4 and CAM), including Prunus avium, Saccharum

officinarum and Ananas comosus obtained under the conditions as described in 3.1.2,

were used as materials for this experiment. For producing plantlets of different

physiological qualities, shootlets were rooted in vitro on different media (agar-

solidified vs. Gelrite-solidified for Prunus, and AC-free vs. AC-containing for

pineapple and sugarcane) and incubated under 4 different spectrum qualities (control,

high blue, high red and high-far red lights) as described in 3.2.1. For simplicity,

hereafter in vitro treatments and microplants obtained correspondingly from each

treatment are coded as presented in table 4.1.

Table 4.1: Codes of in vitro treatments during the rooting stage, and for the plants derived from the corresponding treatment

Light (*) Medium

Control (C) (R/Fr = 8.78 B/R = 0.26)

High red (R) (R/Fr = 20.33 B/R = 0.22)

High far-red (FR) (R/Fr = 0.23 B/R = 0.33

High blue (B) (R/Fr = 7.41 B/R = 2.39)

Prunus Agar (6 g/l) CA RA FRA BA Gelrite (2 g/l) CG RG FRG BG

Pineapple & AC (1g/l) CD RD FRD BD

Sugarcane AC (0 g/l) CT RT FRT BT (∗) [R/FR = ΣNλ(655 – 665) / ΣNλ(725 – 735)

, and B/R = ΣNλ(400 – 500) / ΣNλ(600 – 700)]

Every treatment comprised 20 replicates, each in turn was a culture jar containing

uniform shootlets (2-3 cm high) as duplicates. The number of duplicates per jar was 10

for sugarcane and 6 for pineapple and Prunus. For establishment in the greenhouse, 80

randomly selected 1.5 month old in vitro rooted plantlets were used. The growing

conditions in the greenhouse were as described in 3.2.2.

The experiment was designed as a completely randomised block.

Morphogenesis and growth parameters of plants were scored as below:

Chapter 4.1

60

- The proportion of plants rooting in vitro, and the number of roots per plant were

scored at the end of the in vitro rooting stage, just prior to transplanting to the

greenhouse (day 0). All duplicates in the 20 replicates (jars) were sampled.

- The length of the second youngest fully expended leaf, and the number of

leaves per plant were scored on day 0, 30, 60, 90 and 120 after transplanting.

From each treatment, 10 randomly chosen plants were sampled as 10 replicates.

- The plant fresh and dry weights were scored on day 0, 30, 60, 90 and 120. From

each treatment, 8 randomly chosen plants were sampled as 8 replicates.

- The stomatal density and size, and the leaf structure were studied on day 0, 16,

30, 60, 90 and 120 after transplanting. From each treatment, 8 randomly chosen

plants were sampled as 8 replicates.

- The survival rate was scored a month after transfer to the greenhouse, day 30

The observation methods for each parameter were described in chapter 3 (3.3).

4.1.3. Results

4.1.3.1. Rooting of plantlets in vitro

As seen in table 4.2 and fig. 4.1, the 3 model plants reacted differently to variable

medium compositions and light conditions during the in vitro rooting stage.

Fig. 4.1: Microplants of Prunus from CG and CA treatments (a), of pineapple (b) and sugarcane (c) from CD and CT treatments (table 4.1), just prior to transfer to the greenhouse conditions (1.5 month old in vitro).

Chapter 4.1

61

Prunus

Significant differences in rooting were recorded among Prunus microplants

produced on different culture media. On agar-solidified medium all microplants

rooted, while 20-30% on Gelrite medium did not. The use of Gelrite as gelling agent

also reduced the number of roots per plant (from ca. 11 to about 3) and the secondary

root number. Root length however was insignificantly affected. No significant

variation could be identified between the light treatments (table 4.2 & fig. 4.1a).

Table 4.2: Rooting of plantlets in vitro

PRUNUS

SUGARCANE and PINEAPPLE

Root

number

% Average

root

SUGARCANE

PINEAPPLE

(*) per

plant plants rooted

length (mm)

(*)

Root number

% plants rooted

Average root length

(mm)

Root number

% plants rooted

Average root length

(mm)

CA 11.0 a 100 a 71.5 a CD 6.2 a 100 a 21.1 a 4.1 b 100 89.3 a

CG 3.2 b 81.5 b 62.6 a CT 6.3 a 60.2 b 6.3 b 9.2 a 100 52.5 b

BA 11.1 a 100 a 68.2 a BD 7.1 a 100 a 19.7 a 3.2 b 100 82.7 a

BG 4.2 b 73.4 b 61.7 a BT 5.6 a 51.9 b 4.8 b 8.6 a 100 42.8 b

RA 11.4 a 100 a 72.2 a RD 7.9 a 100 a 18.9 a 3.5 b 100 81.3 a

RG 2.9 b 82.6 b 68.1 a RT 6.2 a 46.8 b 6.5 b 10.1 a 100 39.4 b

FRA 12.2 a 100 a 72.3 a FRD 6.7 a 100 a 21.0 a 3.8 b 100 90.2 a

FRG 3.5 b 72.1 b 71.6 a FRT 5.5 a 46.9 b 5.1 b 7.9 a 100 41.7 b

(*) Refer to table 4.1 for the codes of treatments Data were scored just prior to transfer of plantlets to the greenhouse (day 0) and subjected to Kruskal-Wallis test (n = 20). Medians were separated by Dunn's Multiple Comparison critical value (Zar, 1984). Values in the same column with a common letter are not significantly different, P = 0.05.

Sugarcane

In the case of sugarcane, 100% of microplants cultured on AC-containing

medium formed roots in vitro. The figure was significantly less, only between 50 -

60%, for AC-free medium. Omission of AC also significantly reduced the root length,

from around 2 cm to about 0.5 cm while the per plant root number was unaffected

Chapter 4.1

62

(table 4.2 & fig. 4.1c). As in the case of Prunus, light quality did not influence the in

vitro rooting of sugarcane microplants.

Pineapple

On either medium, with or without AC, and under any light regime tested, 100%

of the pineapple microplants produced roots in vitro. Significant differences were

noted in both the per plant root number and average root length. Addition of AC

significantly reduced the per plant root number, from 8-10 to 3-4, while increased the

root length from 40 -50 mm to 80 - 90 mm. On AC medium, roots were also finer and

more branchy (fig. 4.1b). The difference was similar for all light treatments indicating

that the 4 tested light regimes had the same effects on the in vitro rooting of pineapple

microplants (table 4.2 & fig. 4.1b).

4.1.3.2. Leaves

For all the three model plants, prior to transfer to the greenhouse, no significant

differences were observed in the per plant leaf number among treatments (table 4.3).

This was also true for all the scoring times during the ex vitro acclimatization period,

and thus data will not be further discussed. In contrast to the leaf number, as discussed

below variation in the leaf length was observed between certain treatments.

Prunus

In vitro, differences in leaf length of Prunus microplants were not significant

(table 4.3). During the time course of ex vitro acclimatization also, despite the poorer

performance of the plantlets derived from Gelrite solidified medium (see later), their

leaf length remained unaffected.

Sugarcane

In contrast to Prunus, variation in leaf length of sugarcane microplants was

observed while still in vitro. Under the light regimes tested, microplants produced on

AC-free medium had significantly shorter leaves compared to those on AC-containing

Chapter 4.1

63

medium, and this difference remained significant during the whole ex vitro

acclimatization period. The impact of light quality was insignificant.

Table 4.3: Some growth parameters of plantlets during the ex vitro acclimatization period.

Leaf

number/ Leaf length (mm) Fresh weight (mg) Dry weight (mg) % dry matter

(*) plant Day 0 Day 90 Day 0 Day 90 Day 0 Day 90 Day 0

Prunus CA 11 a 12 a 102 a 329.5 b 3860.0 a 74.12 b 1115.9 a 22.5 a CG 12 a 10 a 100 a 316.2 b 2017.5 b 56.37 c 728.9 b 17.8 b BA 11 a 12 a 97 a 331.5 b 4348.0 a 70.26 b 978.5 a 21.2 a BG 10 a 10 a 104 a 298.6 b 2590.7 b 52.11 c 726.8 b 17.5 b RA 10 a 11 a 112 a 388.3 a 4208.6 a 82.35 a 1009.0 a 21.2 a RG 11 a 10 a 92 a 309.7 b 2617.8 b 56.71 c 768.7 b 18.3 b FRA 10 a 13 a 103 a 321.6 b 4180.2 a 68.52 b 996.5 a 21.3 a FRG 11 a 12 a 106 a 301.5 b 2369.3 b 53.23 c 750.1 b 17.7 b

Sugarcane CD 6 a 21 a 256 ab 269.67 b 1302.5 b 27.01 b 168.5 b 10.0 a CT 6 a 15 b 230 c 179.80 c 1126.7 c 18.85 c 164.8 b 10.5 a BD 6 a 20 a 240 b 251.85 b 1239.8 b 24.65 b 169.7 b 9.9 a BT 6 a 12 b 218 c 157.60 c 1098.9 c 16.75 c 158.4 c 10.6 a RD 5 a 17 a 268 a 305.80 a 1610.7 a 30.03 a 190.9 a 9.8 a RT 5 a 12 b 229 c 163.85 c 1206.2 bc 17.90 c 156.4 c 10.9 a FRD 6 a 22 a 273 a 295.00 a 1654.9 a 29.45 a 191.4 a 10.0 a FRT 6 a 16 b 235 bc 181.30 c 1236.4 bc 20.00 c 159.9 c 11.0 a

Pineapple CD 12 a 115 a 133 a 917.9 a 6374.7 a 75.71 a 522.0 a 8.2 a CT 13 a 91 ab 128 a 867.3 ab 6502.6 a 70.14 a 512.7 a 8.1 a BD 13 a 115 a 115 a 912.7 a 7050.1 a 73.22 a 556.0 a 8.0 a BT 12 a 76 c 119 a 876.5 ab 6524.3 a 71.82 a 513.4 a 8.2 a RD 13 a 125 a 133 a 899.3 a 7255.5 a 71.06 a 561.7 a 7.9 a RT 13 a 82 bc 128 a 845.5 b 6934.3 a 69.95 a 534.3 a 8.3 a FRD 13 a 105 a 133 a 902.4 a 7162.6 a 76.36 a 581.1 a 8.5 a FRT 15 a 87 bc 122 a 867.7 ab 6878.0 a 74.56 a 554.3 a 8.6 a (*) Refer to table 4.1 for the codes of treatments and plants Data were scored just prior to transfer of plantlets to the greenhouse (day 0) and subjected to Kruskal-Wallis test (n = 10 for leaf length and number, n = 8 for plant weight). Medians were separated by Dunn's Multiple Comparison critical value (Zar, 1984). Values in the same column with a common letter are not significantly different, P = 0.05.

Chapter 4.1

64

Pineapple

Among in vitro produced pineapple plantlets, those produced on AC free

medium under blue enriched light (BT) had the shortest leaves, followed by RT and

FRT microplants (table 4.3). In general, on AC-free medium pineapple plantlets

produced significantly shorter leaves compared to those on AC-containing medium.

During the course of the ex vitro acclimatization, however, this difference declined,

and from day 90 on all pineapple plantlets had equally long leaves (table 4.3).

4.1.3. 3. Plant weight and dry matter content

Prunus

At the end of the in vitro rooting stage, Prunus plantlets grown on different

media had the same fresh weight, but those grown on the Gelrite solidified medium

had significantly lower dry weight (table 4.3). Replacement of agar by Gelrite reduced

the dry matter proportion of plants, from 21.2 – 22.5% to 17.5 – 18.2%. Compared to

control light, high red light increased the difference between the 2 medium treatments

(agar vs. Gelrite solidified medium), and RA plants had both highest fresh and dry

weights. Light quality had insignificant effects on the percentage of plant dry matter

content.

Figure 4.2: Prunus plantlets from CG and CA treatments (table 4.1) after 120 days in the greenhouse.

Chapter 4.1

65

During the time course of the ex vitro acclimatization, plants from agar-

solidified media expressed a better growth and always had higher fresh and dry

weight. Moreover, plantlets from Gelrite solidified medium expressed highly non-

uniform performance ex vitro (fig. 4.2).

The difference between light treatments decreased and became insignificant

from day 30 on, e.g. data of day 90 presented in t able 4.3.

Sugarcane

Before transfer to the greenhouse, among sugarcane microplants those cultured

on AC free medium under blue enriched light (BT) had the lowest fresh and dry

weight, while those on AC containing medium under high red (RD) or enriched far-red

light (FRD) had the highest weight. On both media, red and far-red enriched light

increased plant weight, both fresh and dry. Under all 4 light regimes, AC increased

both fresh and dry weight. No significant differences were observed in plant dry matter

percentage, which ranged between 8.3 % and 11% (table 4.3).

Fig. 4.3: Sugarcane plantlets from RD and RT treatments (table 4.1) after 60 days in the greenhouse

Chapter 4.1

66

Also during the whole course of the ex vitro acclimatization, sugarcane plantlets

derived from AC-containing medium had a significantly higher weight compared to

those from AC-free medium. Sugarcane plantlets from RD and FRD treatments were

best in terms of plant weight and overall look, followed by CD & BD, while BT were

the poorest ones (table 4.3, fig. 4.3).

Pineapple

In contrast to the other two plant species, in vitro, pineapple plantlets showed

no significant differences in dry weight. There was also no variation in the proportion

of dry matter content, which varied between 7.9 - 8.5%.

Fig. 4.4: Pineapple plantlets from CD and CT treatments (table 4.1) after 120 days in the greenhouse

For pineapple, during the course of the ex vitro acclimatization period, the

differences between treatments in both leaf length and fresh weight declined, and from

day 90 on all plantlets of this CAM species looked the same and had equal weights

(table 4.3, fig. 4.4).

4.1.3.4. Stomatal density and size

Data on stomatal density and size of plantlets prior to their transfer to the

greenhouse are summarised in table 4.4 and fig. 4.5.

Chapter 4.1

67

Table 4.4: Stomatal size and density of 1.5 month old in vitro plants (prior to transfer to greenhouse conditions)

PRUNUS

SUGARCANE and PINEAPPLE

Size (µm)

Number (per 1 mm2)

SUGARCANE

PINEAPPLE

(*) Stomate

(**) Stomate Hydathode

(***)

(*) Size (µm)

Number (per mm2)

Size (µm)

Number (per mm2)

CA 27.7 a 108.5 c 5.3 c CD 24.2 a 112.5 b 30.5 a 44.0 a

CG 28.1 a 89.9 c 12.3 c CT 24.3 a 114.5 b 30.1 a 45.6 a

BA 28.7 a 179.3 a 39.1 b BD 23.5 a 102.3 b 31.0 a 45.6 a

BG 28.5 a 144.2 b 182.1 a BT 22.9 a 120.0 b 29.8 a 53.1 a

RA 27.4 a 110.1 c 3.8 c RD 22.7 a 161.6 a 29.3 a 47.3 a

RG 26.4 a 89.2 c 6.3 c RT 25.1 a 127.0 b 30.3 a 51.3 a

FRA 27.5 a 98.3 c 2.1 c FRD 24.4 a 159.7 a 30.2 a 55.0 a

FRG 26.8 a 95.4 c 13.5 bc FRT 23.1 a 125.3 b 30.2 a 51.3 a

(*) Refer to table 4.1 for the codes of treatments and plants (**) we only considered normally looking stomata with normal size; (***) and collapsed stomata. Data were subjected to Kruskal-Wallis K-test (n = 8), and medians were separated by Dunn's Multiple Comparison critical value (Zar, 1984). Values in the same column with a common letter are not significantly different, P = 0.05.

Prunus

In contrast to sugarcane and pineapple (see latte), Prunus microplant leaves

contained besides normally-sized stomata, structures with smaller and variable sizes

(fig. 4.5a). Some of these structures looked like hydathodes or collapsed stomata, as

described by Capellades (1989, 1990a) (fig. 4.5a, BG3), but there were also small

ellipsoidal stomata (fig. 4.5a, BG 1 & BG2). These ‘abnormal’ structures were present

in the greatest density on microplants grown in vitro under high blue light, with a

density of about 180/mm2 for BG and around 40/mm2 for BA plantlets. The increased

blue photon ratio also resulted in a larger number of both stomata and hydathodes per

area unit. During ex vitro acclimatization, from day 30 on, both hydathode and

stomatal number of plantlets from blue enriched light reduced; only an insignificant

number of hydathodes persisted while the stomatal density reached that figure for

other treatments. Under control, high red and far-red light, plantlets cultured on either

Chapter 4.1

68

media had the same density of stomata and hydathodes, which also remained stable

during the ex vitro acclimatization. The stomatal size of Prunus plantlets was not

affected by the treatments.

a) Prunus plants from BG, CA and RA treatments had different stomatal density (BG1, CA & RA, at 100x magnification), and hydathodes and stomata with variable sizes on BG plants at 400x magnification (BG2 & BG3) .

b) Stomata on pineapple (PN) and sugarcane (SG) leaf abaxial surface (at 100x magnification).

Fig. 4.5: Imprints of the leaf abaxial surface of 1.5 month old in vitro plantlets, just prior to their transfer to the greenhouse. (Refer to table 4.1 for the legends of plants and treatments)

Sugarcane

In the case of sugarcane, significant differences were recorded in stomatal

density, but not in their size. While in vitro, sugarcane microplants from RD and FRD

treatments had the highest stomatal density of about 160 stomata/mm2. Plantlets from

other treatments had insignificantly different stomatal number, varying within 102 –

127/mm2. Upon transplanting, the stomatal density of RD and FRD reduced and after

30 days in the greenhouse plantlets from all 8 treatments had equal stomatal density of

Chapter 4.1

69

100-120/mm2. Compared to Prunus and pineapple, sugarcane plantlets had the

smallest stomatal size, which did not change after transplanting (table 4.4, fig. 4.5b).

Pineapple

As for pineapple, both while in vitro and during the ex vitro acclimatization

period, there was no significant difference between all the treatments; plantlets

cultured on either AC-free or AC-containing medium under any light condition had the

same stomatal density and size, varying between 44 - 55/mm2 and 29 - 31 µm,

respectively (table 4.4), and remained constant over the experimental period.

Compared to Prunus and sugarcane, pineapple plantlets had the least stomatal number

on their abaxial leaf surface.

4.1.3.5. Leaf mesophyll structure

As seen in fig. 4.6, while still in vitro, plantlets already had differentiated leaf

mesophyll structures, and distinctions between C3, C4 and CAM plants could be

identified similar to those described for these plants grown outdoors.

In contrast to data published previously by Brainerd et al. (1981) and Paques

and Boxus (1987) demonstrating abnormalities in leaf mesophyll structure of in vitro

cultured plants, we did not observe significant differences between among treatments

between different observation times.

Prunus

In C3 plantlets (Prunus) from day 0 on, both palisade and spongy mesophyll

layers could be identified, and were similar to those of outdoor growing C3 plants (fig.

4.6a, b &c). Also, the leaf veins had the same structure as described for ex vitro

cultivated C3 plants (fig. 4.6d & e).

Sugarcane

In sugarcane leaves, while still in vitro, Kranz anatomy was already developed

(fig. 4.6f & g), with large bundle sheath cells surrounding the leaf veins.

Chapter 4.1

70

Pineapple

CAM microplants of 1.5 month old, also had leaf mesophyll differentiated into

palisade and spongy layers with large air spaces in the latter (fig. 4.6h & i). Both the

leaf vein and mesophyll structure of pineapple plantlets looked like that of C3 plants

grown outdoors (fig. 4.6h, i, k & n).

Fig. 4.6: Leaf mesophyll structure of the different plants used in our experiment: Prunus, day 0, 16 & 60, at 100x magnification (a, b & c); leaf vein of Prunus at 400x magnification, day 16 and 60 (d & e); Sugarcane, day 0 at 100x maginification (f), and Kranz anatomy of sugarcane at 200x magnification, day 0 (g); Pineapple, day 0 & 60, at 100x (h & i); and day 0 & 60 at 400x magnification (k & n).

4.1.3.6. Survival of plantlets

The survival rate of plantlets scored 30 days after transfer to the greenhouse is

presented in table 4.5. Regardless of variable in vitro growth conditions 100% of the

pineapple plantlets survived. For sugarcane, a small proportion (3.8 – 1.2%) of the

plantlets died upon transfer to the ex vitro conditions, and no significant differences

Chapter 4.1

71

were detected between treatments. In contrast, for Prunus, significant variation was

recorded among medium treatments. Replacement of agar by Gelrite reduced the

Prunus plantlet survival rate from about 95-97% to 60 – 65%. The differences among

light treatments were insignificant for all three plant species.

Table 4.5: Survival rate (%) of plantlets after 30 days in the greenhouse

(*) Prunus

(*) Sugarcane Pineapple

CA 97.5 a CD 97.5 a 100 CG 69.3 b CT 98.8 a 100 BA 95.5 a BD 98.8 a 100 BG 59.5 b BT 96.2 a 100 RA 95.0 a RD 100 a 100 RG 64.3 b RT 96.2 a 100 FRA 96.2 a FRD 97.5 a 100 FRG 66.8 b FRT 96.2 a 100

(*) Refer to table 4.1 Data were subjected to ChiSqure test, ni = 80, P = 0.05

4.1.4. Discussion

From the abovementioned results it is clear that there was variation in growth and

morphogenesis of plantlets derived from different in vitro treatments. We also detected

differences between C3, C4 and CAM plants.

4.1.4.1. Effects of AC and Gelrite

In general, for sugarcane and pineapple, addition of AC (1g/l) to the culture

medium improved the microplant rooting in vitro and some other growth parameters

during the acclimatization period. This is in agreement with data stated previously by

Fu (1978) with orchids, Gronroos and Von Arnold (1985) for Pinus sylvestris. The

effects of AC however could be species dependent. For both sugarcane and pineapple,

addition of AC (1 g/l) resulted in longer and more branchy roots, but increase of the

per plant root number was recorded only for pineapple while a higher percentage of

microplants rooting in vitro could only be obtained for sugarcane. Differences between

sugarcane and pineapple behaviour to AC addition were noted in the plant weight, leaf

length, survival rate and ex vitro performance. For pineapple, during the time course of

Chapter 4.1

72

ex vitro acclimatization, variation among plantlets from different in vitro treatments

declined, and from day 90 on plantlets from all treatments had a uniform overlook, the

same leaf length and weight (both fresh and dry). In contrast, for sugarcane, variation

in growth and morphogenesis between treatments remained, and in general plantlets

produced in vitro on AC-containing medium showed better growth expressed in their

higher fresh and dry weight, longer leaves as well as better overall look (fig. 4.3, table

4.3). This agrees with the statement by Preil and Engelhadt (1977) that addition of AC

improved the quality of in vitro propagated azalea plants and their performance ex

vitro. In contrast to the plantlet rooting and weight, stomatal density and size of both

pineapple and sugarcane plantlets, as well as their survival rate were unaffected by AC

supplement.

As for Prunus, replacement of agar by Gelrite in the in vitro rooting medium

significantly influenced the microplant root quality, weight and survival rate (table 4.3

& 4.4). Although the plant leaf length as well as the density and size of stomata and

hydathodes were not affected, Gelrite replacement reduced not only the root number

per plant but also the percentage of plantlets rooting in vitro. Moreover, despite having

the same fresh weight as plantlets derived from agar medium, those from Gelrite

medium had lower dry weight. This was already observed by Frank et al. (1995, 1998a

&b), and could be due to hyperdydricity caused by the replacement of agar by Gelrite,

which resulted in more watery plants as expressed in lower dry matter content (table

4.3). Because of their poorer quality Prunus plantlets derived from Gelrite solidified

medium expressed a significantly lower survival rate (table 4.5) and a highly non-

uniform performance during the ex vitro acclimatization.

4.1.4.2. Effects of light quality

With all three model plants, compared to the control light, the photon ratios

tested did not have significant effects on the plant rooting in vitro. No variation in root

number and length, and in the percentage of microplants rooted in vitro could be

identified among the light treatments. This is contrary to the reports of Read and

Economou (1982) that far-red light supplementation resulted in better rooting of azalea

Chapter 4.1

73

microcuttings in vitro, and implies that the effects of the photon ratio on the rooting of

tissue cultured plants can be species dependent.

Data demonstrating the species dependence of the effects of light quality on

microplant morphology and growth are also available. For example Moreira da Silva

and Debergh (1997) noticed that under blue enriched light, in vitro cultured Azorina

vidalii (Wats.) Feer produced smaller leaves, while the leaf number remained constant

between different blue/red and red/far-red light ratios. As remarked by Soebø et al

(1995) with micropropagated birch, however, blue light had a positive effect on leaf

expansion. The ratios of red/PPF or far-red/PPF were also stated to have effects on

shoot elongation and on plantlet fresh and dry weight (Kirdmanee, 1995; Moreira da

Silva and Debergh, 1997; Miyashita et al., 1997 & 1995). In the present work, light

quality affected some morphological characteristics of plantlets, both while in vitro

and during the ex vitro acclimatization period, but the impact also depended on the

plant species; no significant effect was observed on pineapple and the largest

influences were recorded on Prunus plantlets.

In the case of Prunus, both leaf number and length were unaffected by light

quality, but the higher red photon ratio increased both fresh and dry weight of in vitro

plantlets on agar-solidified medium (RA treatment). During ex vitro acclimatization,

from day 30 on, however, differences between RA and other Prunus plantlets no

longer existed. Also on Prunus microplants, high blue light had a remarkable effect on

stomatal and hydathode density; under enriched blue light both hydathodes and

stomata were present in a larger amount, while stomatal size remained unaffected.

Prunus was the only model plant characterized with occurrence of hydathodes and

variable-stomatal size. The occurrence of hydathodes in significant number was

previously reported by Capellades (1989) on leaves of tissue cultured roses, belonging

also to the family of the Rosaceae as Prunus.

In the case of sugarcane, stomatal size was unaffected by the light treatments, but

high red and high far-red photon ratios significantly increased the stomatal density

while in vitro. Sugarcane was the only model plant had the plant weight affected by

Chapter 4.1

74

light quality; plantlets from RD and FRD treatments had highest fresh and dry weight,

both while in vitro and during the acclimatization period. This is consistent with the

data obtained for PEP-case activity (4.2.3.6). On sugarcane plantlets, no effects of blue

light could be observed.

4.1.4.3. Leaf mesophyll structure

Regarding leaf anatomy, abnormalities have often been recorded in tissue culture

derived plants. For instance, leaves with reduced number of palisade layers were noted

in cultured ’Pixy’ plum by Brainerd et al. (1981), and with increased mesophyll air

spaces in rootstock apple by Paques and Boxus (1987). Alterations in other leaf

anatomical characteristics were also recorded, such as thinner cuticle of in vitro rose

leaves (Capellades et al., 1990a), abnormal organisation in protoplast grana and stroma

in cultured Liquidamber styraciflua and roses (Wetzstein and Sommer, 1982;

Capellades, 1989), higher stomata density in Liquidamber styraciflua (Wetzstein and

Sommer, 1983), and etc. However, not much data are available on the comparison of

C3, C4 and CAM mesophyll structure.

In the present work, however, data obtained with sugarcane, Prunus and

pineapple demonstrate that while still in vitro, mesophyll of leaves from C3, C4 and

CAM plants already had differentiated structure similar to that described for their

'counterparts' growing outdoors. Of C3 leaves (Prunus), both palisade and spongy

mesophylls could be clearly identified. A similar structure was observed in leaves of

CAM plantlets (pineapple), but with large air space in the spongy layer, a common

characteristic of CAM plants in the field. Also, leaf veins of both Prunus and

pineapple plantlets expressed a similar structure to that of C3 plants growing in the

field. In contrast, in C4 leaves (sugarcane) Kranz-anatomy was characterized with

large bundle sheath cells surrounding the veins. For all the 3 plants, no significant

differences could be detected among treatments and between scoring times.

Chapter 4.1

75

4.1.4.4. Plant survival and overall performance

In terms of survival and overall performance ex vitro, among the three model

plants, pineapple microplants were least affected by in vitro treatments. Although

variation was observed in some parameters while still in vitro, upon transfer to the

greenhouse regardless of in vitro growing conditions 100% plantlets of this CAM

species survived, and from day 30 on they were uniform for all the parameters

observed as well as in the plant overall look. This indicates capacity of the pineapple

plantlets to “correct” the epigenetic abnormalities that developed in vitro.

In contrast, for sugarcane and Prunus, significant variation in plantlet fresh and

dry weights, as well as in their overall look was recorded among treatments. The lower

survival rate and poorer performance of Prunus plantlets rooted in vitro on Gelrite-

solidified medium indicates their low acclimatization ability to the growth conditions

ex vitro. Similarly, the increased and persisting variation between sugarcane plantlets

derived from different medium treatments upon transfer to greenhouse conditions

implies that C4 plantlets derived from AC-free medium also had a low capacity to

“correct” their epigenetic abnormalities as compared to those from AC-containing

medium.

From all the above mentioned it is clear that in vitro growth conditions

influenced the growth and morphology of micropropagated plants. The effects

however were dependent on the model plants, and the abnormalities that plantlets

possessed while in vitro may affect their subsequent ex vitro acclimatization.

According to the data on the survival rate and growth performance ex vitro, addition of

AC (1g/l) to the in vitro rooting medium improved the quality of both sugarcane and

pineapple micropropagated plants, while for Prunus agar is the better gelling agent as

compared to gelrite. As compared to C3 and C4 plantlets, CAM plantlets were least

affected by in vitro treatments, and displayed the best ability to acclimatize to the

growing conditions in the greenhouse.

Chapter 4.2

76

4.2. Photosynthesis of micropropagated plants; differences

between plantlets produced under different in vitro light

quality and culture medium conditions, and between C3, C4

and CAM plants


Upon transfer to ex vitro conditions, tissue culture derived plants are deprived of

sugar and other nutritious medium components, and thus to grow and develop they

must switch from hetero- or mixo-trophy to photoautotrophy. Consequently, the

plantlet’s ability to regain photoautotrophy and to improve their photosynthetic

competence under ex vitro conditions is an important indicator of their physiological

quality. Indeed, earlier published data confirm the usefulness of photosynthesis related

parameters, both while still in vitro and during the ex vitro acclimatization period, in

assessment of the microplant’s physiological status, such as gas exchange, net CO2

uptake, overall photochemical quantum yield, maximal quantum yield and activity of

carboxylating enzymes (Van Huylenbroeck, 1997; Capellades et al., 1990b;

Kirdmanee et al., 1994; De Riek, 1995 etc.). Again, as already mentioned (see 4.1),

comparison between putative “good” and “bad” plants is often made, and this is also

applied in the present work.

The objective of this experimental work was to assess the ability of the

micropropagated plant’s to adapt to ex vitro growth conditions by comparatively

studying the photosynthetic behaviour of C3 relative to C4 and CAM micropropagated

plants of different physiological status.


Except for the photosynthetic rate, of which measurement was carried out using

different plant materials and under different conditions (see later), the experiment was

designed as described for the previous one, using the same plant materials and under

the same conditions (4.1.2). For Prunus, CA, CG, RA, RG, FRA, FRG, BA and BG

Chapter 4.2

77

treatments, and for sugarcane and pineapple, CD, CT, RD, RT, FRD, FRT, BD and BT

treatments, were used (table 4.1; chapter 4.1).

Some photosynthesis related parameters were recorded as below:

- The evolution of ethylene, oxygen and carbon dioxide in the culture container

headspace of Prunus, sugarcane and pineapple cultures were followed at the

end of every week during the in vitro rooting period of 7 weeks. Each week, the

levels of C2H4, CO2, and O2 were recorded at both the start of the photoperiod

(Co), and 6 h later (C6), and the difference, ∆ = C6 – Co, was calculated. Five

jars were sampled from each treatment as 5 replicates.

- PEP-case activity of Prunus, pineapple and sugarcane plantlets was determined

on days 0, 4, 8, 16, 30, 60 and 120 after transplanting to ex vitro conditions.

From each treatment 3 randomly selected plants were sampled as 3 replicates.

- The maximal quantum yield (Fv/Fm), the overall photoquantum yield (Φp) and

photochemical quenching of chl fluorescence (qP) of Prunus and pineapple

plantlets were defined on days 0, 2, 4, 6, 8, 16, 28, 36, 50, 66, 80, 96 and 120

after transplanting to ex vitro conditions. Of each treatment 5 randomly chosen

plants were sampled as 5 replicates. Having too narrow leaves, sugarcane

plantlets could not be sampled.

For quantification of the plant net photosynthetic rate, uniform shoots of 2-3 cm

high of sugarcane and pineapple were rooted either on AC-free or on AC-containing (1

g/l) medium, and incubated in the growth room of the Department of Biotechnology,

National Agricultural Science Institute of Viet Nam, Ha Noi, at 21 ± 2oC and under

16-h photoperiod with a total photosynthetic photon flux (PPF) of 40 – 50 µmol m-2s-1

at shelf level, provided by white 40W liteguard fluorescent lamps (Rang Dong Ltd.,

Viet Nam). For re-establishment ex vitro, 1.5-month old in vitro rooted plantlets were

transferred to the greenhouse of the Biology Faculty, National University of Ha Noi,

Viet Nam with temperatures varying between 18 – 28oC under normal daylight

conditions (during September – December, 2001). As there was no bottom cooling

Chapter 4.2

78

system for RH controlling, Prunus could not be cultured for net photosynthesis

assessment.

The net photosynthetic rate was recorded on days 0, 2, 4, 6, 8, 10, 12, 16, 20, 26,

30, 36, 50, 66, 80 96 and 120 after transplanting of plantlets to the greenhouse. From

each treatment, 10 randomly chosen individual plantlets were sampled as 10 replicates;

and from each plant the mean of 3 measurements of the second youngest fully

expanded leaf was taken.

The observation methods for each parameter were described earlier in chapter 3

(section 3.3).

4.2.3. Results and discussions

4.2.3.1. Evolution of gasses in the culture container headspace

Among various factors influencing the quality of in vitro cultured plants, the

composition of the headspace is known to be of significant importance, and efforts

have been undertaken to quantify and control its composition (reviewed by Demeester

et al.,1995; Matthijs et al., 1993; De Riek et al., 1991). Previous experimental data

demonstrated that both the headspace composition and its effects on plantlet quality

were dependent upon numerous factors, namely the plant (species, genotype,

developing stage, photosynthetic capacity), the culture medium composition, vessel

type (volume, shape, gas permeability, closure device and thickness of the film) and

other environmental conditions (temperature, pressure, light, relative humidity).

For example, it was stated in some cases that during the photoperiod the level of

CO2 in the culture headspace declined below the normal atmospheric level (Fujiwara

et al., 1987; Desjardin, 1995). In contrast, some other authors reported that the

concentration of CO2 was always higher than the normal atmospheric level (De Proft

et al., 1985; Debergh et al., 1992). Rather high concentrations of CO2 were reported in

some papers, such as 6.58% (Matthijs et al., 1993) or even up to 42% (Demeester et

al., 1995) in Prunus shootlet cultures, and exceeded 13% in Magnolia shoot cultures

(De Proft et al., 1985).

Chapter 4.2

79

According to Nguyen and Kozai (1998) the oxygen concentration in the culture

headspace often ranges between 18 and 22%, but in some cases it was stated to be

variable depending on the ventilation types (Zobayed et al., 1999b; Demeester et al.,

1995), and might decline to about 6.5% (Matthijs et al., 1993) or even below 2%

(Demeester et al., 1995) in Prunus shoot cultures in closely tight containers.

Similarly, studies on the evolution of ethylene were conducted, and the results

obtained also varied between plant species and culture conditions. In the case of

Magnolia shoots, after 9 weeks in culture, a level of more than 2.54 ppm was

measured by De Proft et al. (1985). In other cases it was recorded to be variable

depending, e.g. on the container closure type (Matthijs et al., 1993, for Prunus), the

plant development stage (Huang et al., 2000, for Sequoia sempervirens), or the plant

genotype (Fal et al., 1999, with Dianthus caryophyllus L.).

Under the conditions of our present experiment, variation among the subject

plants was obtained in the evolution of CO2, O2 and C2H4 in the culture headspace

during the in vitro rooting period. Differences were also recorded between treatments.

4.2.3.1.1. CO2

For the 3 subject plants, the level of CO2 in the culture headspace of the container

was always higher than that under normal atmospheric conditions, and plants differed

from one another in the patterns of CO2 evolution. When measured at the start of the

photoperiod, as shown in fig. 4.7a, at the beginning of the in vitro rooting stage the

level of CO2 was low (around 0.2%), and no significant differences could be detected

between the plant species. With the passing of time, however, variation between plants

increased, and at the end of the in vitro rooting stage (week 7), the CO2 concentration

was highest in the Prunus culture headspace (exceeded 18%), followed by sugarcane

(about 5.5%) and pineapple (around 0.4%). For Prunus, a prominent increase was

recorded during week 4, while for sugarcane the level of CO2 gradually increased

during the whole in vitro rooting stage. In the case of pineapple, the CO2 concentration

only slightly increased, from 0.2 at the beginning to 0.4% by the end of the culture

period.

Chapter 4.2

80

The differences between CO2 concentrations recorded at the start of the

photoperiod (Co) and 6 h later (C6) (∆ = C6 – Co) also depended on the plant species

(fig. 4.7b). During the first week, regardless of the plant species the level of CO2

remained constant over 6 h under the photoperiod (∆ ≅ 0). From week 2 on, significant

variation among the model plants was observed.

Fig. 4.7: a: evolution of CO2 over the in vitro rooting stage (CO2 concentrations at the onset of the photoperiod)

b: changes of CO2 concentration 6 h after the photoperiod started (∆CΟ2) [Measurements of CA Prunus, CD pineapple and CD sugarcane plantlets are presented (Refer to table 4.1 for the codes of plants)]. Vertical bars represent the standard errors, n = 5.

For Prunus, from week 2 – 5, the level of CO2 declined notably after 6 hours in

the photoperiod, wi th the ∆ values most negative at the end of week 3 (- 4.09%). This

agrees with remarks by Fujiwara et al. (1987) and Desjardin (1995) that the in side

container concentration decreased during the photoperiod. The negative ∆ values

recorded in the Prunus headspace may indicate the photosynthetic competence of this

C3 plant’s shoots during the first 5 weeks in culture. The positive ∆ values obtained in

the last 2 weeks could be attributed to increased respiration (when a root system

developed) and . At the end of the culture period (week 6 and 7) necrosis and decay

were observed in Prunus plantlets, and this agrees with the observations by Demeester

et al. (1995) and Matthijs et al. (1993), also with Prunus shoot cultures in tightly

closed containers. These decays and necrosis could also contribute to the increase of

CO2 level in culture containers.

∆ C02 (%)

-4.5

-3.0

-1.5

0.0

1.5

Time in culture (week)

b

1 2 3 4 5 6 7

Pineapple Sugarcane Prunus

CO2 (%)

-4.0 0.0 4.0 8.0

12.0 16.0 20.0

1 2 3 4 5 6 7 Time in culture (week)

a


Chapter 4.2

81

Fig. 4.8: Evolution of CO2, effects of the presence or absence of AC on sugarcane (a & c), and on pineapple (b & d); and effects of the gelling agent (e) and light quality (f) on Prunus. Vertical bars represent the standard errors, n = 5.

Smaller ∆ values recorded in the pineapple and sugarcane headspace (0.03 – 0.4)

however do not necessarily indicate lower photosynthetic activity of these plantlets.

Having well differentiated leaf mesophyll structures (section 4.1.3.5) and high PEP-

CO2 (%)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

1 2

3

4 5 6 7


b CO2 (%)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

1 2 3 4 5 6 7 Time in cultute (week)

a

CO2 (%)

-2.00

2.00

6.00

10.00

14.00

18.00

22.00


Agar Gelrite

e

∆ CO2 (%)

-0.50

-0.40

-0.30

-0.20

-0.10

0.00


d

1 2 3 4 5 6 7

AC, 1 g/l

AC, 0 g/l

∆ CO2 (%)

-0.08 -0.04 0.00 0.04 0.08 0.12 0.16


c

1 2 3 4 5 6 7

AC, 1 g/l

AC, 0 g/l

CO2 (%)

-2.00

2.00

6.00

10.00

14.00

18.00

22.00

1 2 3 4 5 6 7Time in culture (week)

CA

BA

RA

FRA

f

Chapter 4.2

82

case activity (see 4.2.3.6), these CAM and C4 plantlets could fix CO2 using C4

photosynthetic pathway, and /or reuse CO2 produced by photorespiration.

The impact of medium and light treatments on CO2 evolution was also species

dependent. For sugarcane and pineapple, omission of AC significantly increased the

evolution of CO2 in the container headspace; effects however were larger from week 5

on for sugarcane (fig. 4.8a ) and from week 3 on for pineapple (fig. 4.8b).

Significant variation among the medium treatments was also noted in the ∆

values; omission of AC increased the ∆ of weeks 4, 6 and 7 for pineapple (fig. 4.8d)

and of weeks 5 and 6 for sugarcane (fig. 4.8c). The replacement of agar by Gelrite

reduced the CO2 evolution in the container headspace of Prunus shoots from week 5

on (fig. 4.8e).

Light quality had no significant impact on the CO2 evolution in sugarcane and

pineapple headspace (data not presented). In the case of Prunus, effects of high red

and far-red lights were also insignificant, while high blue photon ratio (BA treatment)

reduced the CO2 level from week 3 on (fig. 4.8f).

4.2.3.1.2. O2

The evolution of O2 in the culture container headspace also varied between plant

species and treatments. For pineapple, during the whole culture period, the level of O2

did not vary much from that under normal atmospheric conditions, and ranged

between 21.2% and 24.04 %. In contrast, for Prunus and sugarcane, the concentration

declined markedly by the end of the culture period (fig. 4.9a).

In the case of Prunus, from week 4 on the O2 concentration at the start of the

photoperiod significantly decreased, and by week 7 it dropped below 11%. In the case

of sugarcane, the level of O2 slightly reduced from the beginning, and lowered to about

14% at the end of the rooting stage. For this C4 plant, the ∆ values were negligible

indicating that the level of O2 in the culture headspace of this plants did not change

during photoperiod. For pineapple, 6 hours after the photoperiod started, the level of

Chapter 4.2

83

O2 slightly increased, with ∆ values varying between 0.17 and 0.52%. Prominent

changes in O2 concentrations during the photoperiod were recorded for Prunus (fig.

4.9b). The augmentation of O2 concentration during the photoperiod and its reduction

during circadian cycle recorded in Prunus and sugarcane headspaces might indicate a

high dark respiration of their shootlets in culture.

Fig. 4.9: a: evolution of O2 over the in vitro rooting stage (O2 concentrations at the onset of the photoperiod) b: changes of O2 concentration 6 h after the photoperiod started ( ∆Ο2) [Measurements of Prunus CA, pineapple CD, and sugarcane CD plantlets are presented (Refer to table 4.1 for the codes of plants)]. Vertical bars represent the standard errors, n = 5.

The effects of medium treatments on O2 evolution were insignificant for

pineapple, no difference were detected between AC-free and AC containing medium

(fig. 4.10a). In contrast, for sugarcane, omission of AC reduced the level of O2 from

week 3 on, and by the end of the culture period lowered it to about 7%, compared to

13.82% when AC (1 g/l) was added (fig. 4.10b). For this C4 plant, during the first 2

weeks of the culture period, omission of AC also increased the absolute ∆ values,

(between –0.38 and –0.35%) (fig. 4.10c).

The effects of the gelling agent on the evolution of O2 in the Prunus culture head

space is presented in fig. 4.10d & e. As seen, significant differences were recorded at

the end of the culture period, when replacement of agar by Gelrite resulted in an

increase in the level of O2 to 13.72% (the figure was 11% for agar). Replacement of

O2 (%)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

1 2 3 4 5 6 7


a


∆ o2(%)

-0.5

0.0

0.5

1.0

1.5

2.0


b


1 2 3 4 5 6 7

Chapter 4.2

84

agar by Gelrite also affected changes in O2 concentration over the 6 hours after the

photoperiod started, resulting in more negative ∆ values during weeks 6 and 7, and

smaller positive values for weeks 4 & 5 (fig. 4.10e).

The effects of light qualities on the evolution of O2 were insignificant for the 3

subject plants, and thus data are not presented.

Fig. 4.10: Evolution of O2, effects of the presence or absence of AC on pineapple (a), and on sugarcane (b & c); and effects of the gelling agent on Prunus (d & e). Vertical bars represent the standard errors, n = 5.

O2 (%)

0.00

5.00

10.00

15.00

20.00

25.00

30.00


a

O2 (%)

0.00 5.00

10.00 15.00 20.00 25.00 30.00


d ∆ O2 (%)

-0.45

-0.30

-0.15

0.00

0.15

0.30


c

1 2 3 4 5 6 7

AC, 1 g/l

AC, 0 g/l

∆ O2 (%)

-2.00

-1.00

0.00

1.00

2.00

3.00


e

1 2 3 4 5 6 7

Agar

Gelrite

O2(%)

0.00

5.00

10.00

15.00

20.00

25.00

30.00

1 2 3 4 5 6 7Time in culture (week)

b

Chapter 4.2

85

4.2.3.1.3. C2H4

C2H4 accumulated in the culture headspace of the model plants to variable levels

depending on the plant species (fig. 4.11a & b). At the end of the culture period the

highest value (about 0.85 ppm) was scored in Prunus, followed by sugarcane (0.68

ppm) and pineapple cultures (0.32 ppm). For Prunus, the level of C2H4 reached its

maximal value (0.98 ppm) after 2 –3 weeks in culture (4.11a), and this was in

accordance with the results obtained by Demeester et al. (1995), also with Prunus

shoot cultures. A constant rate of C2H4 evolution was recorded by Righetti et al.

(1988, 1990) in the headspace of Prunus shoot cultures over 30 days in culture, and

this was also the case for pineapple in our present experiment. For this CAM plant, the

level of ethylene slightly increased at a constant rate from 0.07 to 0.32 ppm by week 7

(fig. 4.11a). In contrast, in the case of sugarcane, when AC-containing medium was

used, over the first 5 weeks the level of C2H4 constantly increased, and then remained

almost unchanged (fig. 4.11a). In general, C2H4 evolution did not display any diurnal

fluctuations, except for Prunus during week 2 & 3, insignificant ∆ values were scored

(fig. 4.11b).

Fig. 4.11: a: Evolution of C2H4 over the in vitro rooting stage (C2H4 concentrations at

the onset of the photoperiod) b: Changes of O2 concentration 6 h after the photoperiod started ( ∆C2H4)

[Measurements of Prunus CA, pineapple CD, and sugarcane CD plantlets are presented (Refer to table 4.1 for the codes of plants)]. Vertical bars represent the standard errors, n = 5.

∆ C2H4 (ppm)

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2


b

1 2 3 4 5 6 7


C2H4 (ppm)

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4


a Pineapple Sugarcane Prunus

Chapter 4.2

86

As seen in fig. 4.12a & b, omission of AC resulted in an increased accumulation

of ethylene in both sugarcane and pineapple headspace. In contrast to the findings of

Mensuali-Sodi et al. (1992), who claimed that Gelrite released less C2H4 than agar, in

our present experiment, it is clearly indicated that the C2H4 level increased in the

Prunus headspace when Gelrite was used as the gelling agent in the in vitro rooting

medium. This can be explained by the fact that the C2H4 evolution is clearly influenced

by various other factors than the gelling agent (reviewed by Matthijs et al., 1995).

Fig. 4.12: Evolution of C2H4, effects of the presence or absence of AC on sugarcane (a), and on pineapple (b); and effects of the gelling agent on Prunus (c). Vertical bars represent the standard errors, n = 5.

Regarding the effects of C2H4 on the culture growth, discrepancies exist among

the data available in the literature, and both stimulatory and inhibitory effects were

recorded (Abeles et al., 1992; Gonza'lez et al., 1997, Matthijs et al., 1995). Also, while

in Sequoia sempervirens cultures no clear effects were noted (Huang et al., 2000), with

Dianthus caryophyllus L. shoots, accumulation of C2H4 was remarked to be associated

with the occurrence of hyperhydricity (Fal et al., 1999). In our present experimental

C2H4 (ppm)

-0.40

0.20

0.80

1.40

2.00

2.60


a C2H4 (ppm)

0.00

0.20

0.40

0.60

0.80

1.00


b

C2H4 (ppm)

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40


c

Chapter 4.2

87

conditions, higher C2H4 accumulation was found in Prunus headspace when Gelrite

solidified medium was used. It is however not clear whether this has a relationship

with the poorer growth of plantlets derived from Gelrite solidified medium as observed

in the previous experimental work (see 4.1).

The effects of light quality treatments were insignificant for the three model

plants (data not presented).

4.2.3.2. Net photosynthetic rates of plantlets

As recorded in the present work, plantlets from different photosynthetic groups

(C4 or CAM) displayed different net photosynthetic rates (Pn) under the weaning

conditions. During the time course of the acclimatization period (fig. 4.13a), sugarcane

plantlets had a significantly higher Pn (varying between 1.8 and 2.5 µmol/m2/s) as

compared to pineapple (just within 0.8 and 1.7 µmol/m2/s). Nevertheless, the changing

pattern of Pn during this stage was similar for both plant species. Immediately after

transplantation Pn reduced, and after 10-16 days in the greenhouse minimal values

were recorded (1.8 for sugarcane and 0.9 for pineapple).

Omission of AC from in vitro rooting medium led to a significant reduction in Pn

of both pineapple and sugarcane plantlets. Furthermore, plantlets derived from AC-

containing medium displayed a recovery in Pn earlier than those from AC-free

medium (fig. 4.13b & c). Thus, addition of AC improved the capacity of plantlets to

recover from the stresses ex vitro. The effects of medium treatment were larger and

persisted longer for sugarcane. By day 120 after transfer, sugarcane plantlets from AC

containing medium still displayed a higher Pn compared to those from AC free

medium (fig. 4.13b), while from day 36 on, pineapple plantlets from different medium

treatments already gave equal Pn (fig. 4.13c). The faster recovery of pineapple

plantlets indicates their better acclimatization ability, and this is consistent with the

data on photomorphogenesis observed earlier (4.1). As compared to data recorded for

some plants, such as Spathiphyllum and Calathea (Van Huylenbroeck, 1997), or

Rehmannia glutinosa (Seon et al. 2000), where Pn minimal values were recorded on

Chapter 4.2

88

day 3 or 6 after transplanting, pineapple and sugarcane plantlets in our experiment

displayed a longer and deeper decline in Pn.

Fig. 4.13. Net photosynthetic rate of

plantlets during the weaning stage, comparison between C4 and CAM plantlets from AC-containing medium (a), and effects of the presence or absence of AC on sugarcane (b) and pineapple (c) Vertical bars represent the standard errors, n = 3.

4.2.3.3. Maximal quantum yield of plantlets

The ratio of variable to maximal chlorophyll fluorescence (Fv/Fm) is often used

for the definition of the plant photosynthetic efficiency (Van Huylenbroeck, 1997; Van

Huylenbroeck et al., 1992 & 2000; Herrera et al., 2000; Ayari et al., 2000 a & b;

Parker and Mohammed, 2000; Pospisilova et al., 2000; Seon et al., 2000; Taub et al.,

2000; Hofman et al., 2002). The findings by Van Huylenbroeck et al. (2000) showed

that directly after transfer to the ex vitro conditions Calathea micropropagated plants

displayed a decrease in Fv/Fm ratio, which afterwards recovered along with the plant

developing adaptive/protective capacity, and that Fv/Fm was influenced by the light

intensity ex vitro. Similarly, Seon et al. (2000) stated that regardless of the in vitro

growing conditions over 6 days after transplantation Rehmannia glutinosa plantlets

a

c

Pn ( µ mol/m2/s)

0.70

0.95

1.20

1.45

1.70

1.95

2.20

2.45

2.70

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

a Pn ( µ mol/m2/s)

1.40

1.60

1.80

2.00

2.20

2.40

2.60

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

b

Pn ( µ mol/m2/s)

0.70

0.90

1.10

1.30

1.50

1.70

1.90

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

c

Chapter 4.2

89

exhibited a decline in both Pn and Fv/Fm. In contrast, with tobacco tissue culture

derived plants, Pospisilova et al. (2000) recorded a constant Fv/Fm ratio prior and

after ex vitro establishment, and concluded that no photodamage occurred during the

acclimatization period. These discrepancies among data published demonstrate that

besides the growing conditions, the Fv/Fm ratio of plantlets depends largely on the

plant species.

Fig. 4.14: Maximal quantum yield of plantlets during the weaning stage, comparison between CA-Prunus and CD-pineapple plantlets (a) and effects of the gelling agent (agar vs. Gelrite) on Prunus (b) Vertical bars represent the standard errors, n = 5.

In the present work, significant differences in Fv/Fm were remarked between

Prunus and pineapple plantlets (fig. 4.14a). As seen, prior to transplanting the Fv/Fm

ratio was around 0.8 for plantlets from both species (0.81 for pineapple and 0.79 for

Prunus), implying that in vitro leaves of microplants from both species were in a

normal physiological state. Right after transplanting, a decrease in Fv/Fm was

observed for both Prunus and pineapple, and reached the minimal value of 0.78 on day

2 in the case of pineapple, or of 0.76 on day 8 in the case of Prunus. In agreement with

the data obtained for Pn, pineapple plantlets exhibited a faster recovery in Fv/Fm

compared to Prunus; after ex vitro establishment the time period required for

pineapple plantlets to regain normal Fv/Fm ratio was 6 days, and the figure was 16

days for Prunus. Also, from day 6 on pineapple plantlets always had normal Fv/Fm

(varying between 0.79 and 0.82). In contrast, 36 days after transfer Prunus plantlets

displayed a second decline in Fv/Fm which coincided with the time of transfer of

a

Fv/Fm

0.60 0.65 0.70 0.75 0.80 0.85 0.90

0 10 20 30 40 50 60 70 80 90 100 110 120 Day after transfer

a Fv/Fm

0.60 0.65 0.70 0.75 0.80 0.85 0.90


b

Chapter 4.2

90

plantlets from growth chamber to the greenhouse with natural light conditions.

Furthermore, towards the end of the experimental period, for these CAM plantlets, the

ratio of Fv/Fm remained still lower than 0.75, indicating that a part of their PS’

reaction centres were photodamaged, and that Prunus plantlets suffered to a larger

extent from ex vitro stress conditions as compared to pineapple plantlets.

In the case of pineapple, effects of the in vitro medium and light treatments were

insignificant. As for Prunus, in vitro light quality treatments also had no impact, but

replacement of agar by Gelrite influenced the Fv/Fm ratio (fig. 4.14b). The differences

between plantlets obtained from agar and Gelrite medium was small over 8 days after

transplanting. After day 8, while Fv/Fm of the plantlets derived from agar-solidified

increased that of plantlets from Gelrite-solidified medium continued to decrease. Thus,

in comparison to agar, Gelrite reduced the Prunus plantlet potential to adapt to the ex

vitro conditions.

4.2.3.4. Overall photoquantum yield

Fig. 4.15: Overall photoquantum yield of plantlets during the weaning stage,

comparison between CA-Prunus and CD-pineapple plantlets (a) and effects of the gelling agent (agar vs. Gelrite) on Prunus (b). Vertical bars represent the standard errors, n = 5.

As seen in fig. 4.15a, while still in vitro (day 0) and during most of the ex vitro

acclimatization period pineapple plantlets had better overall photosynthetic efficiency

as compared to Prunus plantlets. For both plant species, the overall quantum yield

Yield

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

a Yield

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

b

Chapter 4.2

91

(Φp) was low (varying between 0.25 – 0.50 for pineapple, and 0.19 – 0.44 for Prunus),

indicating that only small part of the absorbed irradiation energy was used in

photochemistry.

In contrast to Fv/Fm and Pn, for both plant species, Φp did not decline after

transplanting, but showed a constantly increasing trend, implying increasing overall

photosynthetic efficiency of plantlets during the ex vitro acclimatization.

For pineapple, no variation in Φp could be measured among treatments. In the

case of Prunus, significant differences were obtained between medium treatments (fig.

4.15b). The replacement of agar by Gelrite reduced the overall photoquantum yield of

Prunus plantlets, from 0.20 to 0.17, while still in vitro, and the effects persisted during

most of the acclimatization period. Thus, when compared to Gelrite, the use of agar as

the gelling agent in the in vitro rooting medium gave better plantlets in term of yield,

Fv/Fm and Pn.

4.2.3.5. Photochemical quenching

Photochemical quenching (qP) of chlorophyll fluorescence is also regarded as

one of the reliable parameters for quantification of photosynthetic activity of plants

(Van Huylenbroeck, 1997; Ayari et al., 2000a&b; Strasser et al., 2000). Working with

micropropagated Spathiphyllum and Calathea plants, Van Huylenbroeck (1997)

recorded a relatively high qP at the end of the in vitro period of autotrophic

Spathiphyllum, and very low qP in mixotrophic Calathea plantlets.

In our present experiment, at transplanting, relatively low qP values were

obtained for both pineapple (0.35) and Prunus (0.47). In contrast to the yield and

Fv/Fm, the photochemical quenching (qP) of Prunus plantlets was higher than that of

pineapple (fig. 4.16a), indicating that the decrease in chl fluorescence caused by the

increase in overall photochemical capacity was greater for Prunus. Also, in contrast to

the yield, Pn and Fv/Fm, for both Prunus and pineapple, no decline in qP was noticed

after transplanting, and this was the case for Calathea plantlets, as observed earlier by

Van Huylenbroeck (1997). Thus for both CAM and C3 plants, after transfer to

b

Chapter 4.2

92

greenhouse conditions, the proportion of open reaction centres increased, the chronic

photoinhibition level reduced, and plantlets gradually switched to autotrophy.

Fig. 4.16: Photochemical quenching of plantlets during the weaning stage, comparison between CA-Prunus and CD-pineapple plantlets (a) and the effects of the gelling agent (agar vs. Gelrite) on Prunus (b). Vertical bars represent standard errors, n = 5.

The impact of in vitro light quality treatments on qP was insignificant for both

Prunus and pineapple. Also, the effects of AC omission from the in vitro rooting

medium were negligible for pineapple. In the case of Prunus, as compared to agar, use

of Gelrite as the gelling agent reduced qP of plantlets while still in vitro, and the

effects persisted upon transfer to the greenhouse conditions (4.16b).

4.2.3.6. Specific activity of PEP-carboxylase

In term of the specific activity of PEP-case, plantlets of C3, C4 and CAM plant

species also differed from one another (fig. 4.17a), and this agrees with the findings of

Van Huylenbroeck (1997) with Calathea and Spathiphyllum.

Both while still in vitro and during the ex vitro acclimatization, sugarcane

plantlets had the highest PEP-case activity (varying between 5.90 and 8.72 nmol HCO-

3/mg proteins/min), followed by pineapple (among 0.45 and 2.43 nmol HCO-3/mg

proteins/min) and Prunus (only about 0.20 – 0.33 nmol HCO-3/mg proteins/min). The

higher PEP-case activities in CAM and C4 plantlets at transplanting indicate their

capacity to fix CO2 primarily by this enzyme, or to assimilate carbon through the C4

a

b

qP

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00


a qP

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00


b

Chapter 4.2

93

pathway while still in vitro. A decrease in PEP-case activity was recorded over first 4

days in the greenhouse for sugarcane plantlets, when they perhaps suffered from stress

conditions. With pineapple, no decline was observed, indicating the high capacity of

these CAM plantlets to acclimatize to ex vitro conditions. In the case of Prunus, PEP-

case activity remained constant and low during the whole acclimatization period.

Fig. 4.17: Specific activity of PEP-case during the weaning stage, comparison between CA-Prunus, CD-pineapple and CD-sugarcane plantlets (a); effects of the presence or absence of AC on sugarcane (b) and pineapple (c). Vertical bars represent the standard errors, n = 3.

The effects of the in vitro medium treatments were also species dependent. As

for pineapple (fig. 4.17c), variation between medium treatments could only be detected

during 30-60 days after transplanting, while in the case of sugarcane, plantlets derived

from AC containing medium always displayed a higher PEP-case activity, even while

still in vitro (fig. 4.17b). Also, omission of AC from the in vitro rooting medium

prolonged the declination period of PEP-case activity to 16 days, compared to only 4

days when AC containing medium was used. In Prunus the effects of in vitro medium

treatment were unclear; this could be due to too small values of this enzyme’s activity

in these CAM plantlets.

a

c

nmol HCO3-/mg proteins/min

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

10.00

0 4 8 16 30 60 90 120 Day after transfer

a


0.00 0.50 1.00 1.50 2.00 2.50 3.00

0 4 8 16 30 60 120 Day after transfer

c


0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

10.00


b

Chapter 4.2

94

The impact of in vitro light quality treatments could only be detected for

sugarcane. In these C4 plantlets, blue enriched light significantly reduced PEP-case

specific activity, while both red and far-red enriched lights increased the activity of

this enzyme (fig. 4.18). Compared to the results on morphogenesis and development

obtained in the previous experiment (4.1), the higher PEP-case activities were found in

plantlets that were better in terms of ex vitro growth and performance.

Fig. 4.18: Specific activity of PEP-case, impact of the light quality on sugarcane. Vertical bars represent the standard errors, n = 3.

4.2.4. Conclusions

From the data obtained in the present experiment, it is clear that plantlets of

different species responded differently to variable growth conditions in vitro and

exhibited different photosynthetic capacity during the acclimatization period.

Consistent with the data obtained for growth and photomorphogenesis (4.1), in

vitro light quality treatments had little or no effects on the plantlets photosynthetic

behaviour. Regarding gas evolution in the headspace, significant effects were detected

only for Prunus with blue enriched light, where CO2 accumulated at lower levels

compared to the control and other light conditions (fig. 4.8f,b). In terms of PEP-case,

sugarcane was the only plant influenced by in vitro light quality treatments. Relative to

the control light, blue enriched light reduced while high red or high far-red light


0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 4 8 16 30 60

Day after transfer

Control Blue

Red Far-red

Chapter 4.2

95

treatments increased the specific activity of this enzyme both while in vitro and during

the acclimatization period (fig. 4.18). The light treatments had no significant effect on

Pn, Fv/Fm, qP, and Φp of plantlets.

Addition of AC (1 g/l) to the in vitro rooting medium influenced the plantlet

photosynthetic behaviour. Increase in both Pn (fig. 4.13) and in PEP-case activity (fig.

4.18) was detected for both pineapple and sugarcane when AC-containing medium

was used. The effects however, were larger and persisted longer for sugarcane. When

considering other parameters, including Fv/Fm, qP and Φp, AC did not have any

significant effect. The effects of AC on the culture headspace gas evolution was also

species dependent. In the case of sugarcane the concentration of both CO2 and

ethylene were reduced, while that of O2 increased when AC-containing medium was

used. As for pineapple, no differences in the O2 evolution could be measured between

AC-containing and AC-free medium treatments, but addition of AC had negative

effects on CO2 and positive impact on ethylene accumulation.

In general, as compared to Gelrite, agar was the better gelling agent for the

Prunus rooting medium; plantlets derived from agar-solidified medium had better

photosynthetic capacity reflected in higher Fv/Fm, yield, and qP. Also, the evolution

of CO2 was increased while that of O2 and C2H4 reduced when agar-solidified medium

were used.

Variation in capacities of photosynthesis over the acclimatization period and in

abilities to adapt to changed growth conditions ex vitro was recorded between C3, C4

and CAM plantlets. As compared to Prunus, pineapple plantlets gave higher Fv/Fm

and Φp, and lower qP. The specific activity of C4-pathway CO2 primary fixation

enzyme (PEP-case) was very small for Prunus, which as a C3 plant does not need this

enzyme for primary fixation of external CO2. Despite that CAM plantlets were least

affected by in vitro treatments and exhibited the best capacity to “correct” the

abnormalities that developed in vitro (4.1), the net photosynthetic rate as well as the

PEP-case specific activity of these plantlets were lower than that of C4 plants, both

while in vitro and during the acclimatization period (fig. 4.13 & 4.17). Thus, for Pn

Chapter 4.2

96

and PEP-case activity to be used in assessing the plantlet’s photosynthetic activity they

must be considered in relationship to their photosynthetic group or plant

species/genotype, or comparative study between “bad” and “good” plantlets of the

same species should be undertaken.

Regardless of plant species and in vitro treatments, upon transfer to the ex vitro

conditions micropropagated plants did suffer from stress conditions. The extent

however was dependent upon both the plant species and physiological quality.

Immediately after transplanting, decreases were observed in Pn, Fv/Fm and PEP-case

activity. A more pronounced decline in terms of both value and persistence in time,

were recorded in plantlets derived from AC-free medium (in the case of pineapple and

sugarcane) or from Gelrite-solidified medium (in the case of Prunus). Thus, for

pineapple and sugarcane, addition of AC (1 g/l) to the in vitro rooting medium did

improve the plantlet quality, and for Prunus agar appeared to be a better gelling agent.

The low values of Φp and qP obtained for both pineapple and Prunus plantlets, at

transplanting (day 0) indicate small proportions of absorbed energy used in

photochemistry, while high values of Fv/Fm (around 0.8) recorded on that day imply

that leaves of plantlets were in a ‘normal’ physiological state. In contrast to data

obtained by some authors (e.g. Van Huylenbroeck, 1997; and Pospisilova et al, 2000;

Hofman et al., 2002) our results do not confirm the argument that Fv/Fm is a reliable

parameter for quantification of the plantlets’ photosynthetic capacity. Over the time

course of the acclimatization period, the Fv/Fm ratio either declined or remained

unchanged (fig. 4.15), while Pn, qP, Φp and PEP-case, after the declination period (if

any), recovered and increased. Thus, in addition to Pn, chl photochemical quenching

and the overall photoquantum yield rather than the Fv/Fm ratio should be used for

assessment of the plant photosynthetic competence.

Photosynthesis of plantlets while still in vitro can also be studied by following

the evolution of gasses, especially of CO2 in the culture headspace (De Riek, 1995). In

our present experiment, on the basic of the data on gas evolution, some remarks can

also be made regarding the plantlets’ photosynthesis and respiration. For example,

decrease in CO2 concentration within 6 h after the start of the photoperiod may

Chapter 4.2

97

indicate the in vitro plants’ photosynthetic competence. However, as in the case of Pn

and PEP-case, data should be considered in relation to the plant species and the growth

conditions, because CAM and C4 plants can reuse respired CO2, and besides

photosynthesis and photorespiration, the level of the headspace components depends

also upon other factors, namely culture medium compositions, temperature, light, gas

permeability, pressure and etc.

Chapter 4.3

98

4.3. Photoinhibition and photoprotection of micropropagated plants

during the acclimatization period; differences between

microplants of different physiological quality, and between C3,

C4 and CAM plants


While in vitro micropropagated plants acclimate to special environment (2.2),

upon transfer to soil they more than often suffer from stress conditions, especially low

relative humidity and high irradiation intensity. Correspondingly the plants’ capacity

to repair, and to protect their photosystems from photodamage is considered one of the

key factors determining the successful rate of their ex vitro acclimatization.

As discussed earlier (2.1.3), plants can cope with light stress conditions through

developing various protective and acclimatization strategies and mechanisms,

including adjusting their photoreception system structure, and developing fluorescence

quenching and protective enzyme systems. As argued by Strasser et al. (2000), chl-

fluorescence kinetics is a signal rich in information on the plant's physiological status.

For example, the chl a minimal fluorescence (Fo) is known to be positively correlated

to the level of photodamage, and the non-photochemical quenching (qN) quantifies the

portion of excess energy dissipated in the form of heat. Among protective enzymes,

the anti-oxidative ones, including catalase and SOD are often used for assessing

plants’ capacity to defend themselves from stress conditions (Van Huylenbroeck,

1997). The concentration of photosynthetic pigments, especially the ratio of chl a/b

and the carotenoid content in leaves can also be of significant importance for assessing

the plant photoprotection capacity (2.1.1.1. & 2.1.3.2).

As presented in the previous experimental sections (4.1 and 4.2), regardless of in

vitro treatments and the model plant species, upon transplanting to greenhouse

conditions microplants suffered from photoinhibition. However, the level of

photodamage as well as the capacity of plantlets to recover from stress, depended on

both the plant species and in vitro growth conditions. According to the data obtained in

Chapter 4.3

99

4.1 and 4.2, the in vitro light quality treatments had little or unclear effects on most of

the parameters observed, and thus in the present work light treatments were omitted,

and only control light was applied during the in vitro rooting stage. In contrast, the in

vitro rooting medium significantly impacted plantlets’ quality and their ex vitro

behaviour. Based on their growing performance, photomorphogenesis and

photosynthesis (4.1 and 4.2), Prunus plantlets derived from agar-solidified rooting

medium were better compared to those from Gelrite-solidified medium in terms of

survival, overall performance, weight, photoquantum yield and qP. Similarly, in the

case of pineapple and sugarcane, plantlets rooted in vitro on AC-containing medium (1

g/l) behaved better than those from AC-free medium, regarding Pn, PEP-case activity,

survival and overall performance. Therefore in order to assess microplant's capacity to

protect from photoinhibition during the ex vitro acclimatization period, in the present

experiment a comparative study was undertaken between plantlets derived from

different medium treatments: agar- vs. Gelrite-solidified media for Prunus, and AC-

free vs. AC-containing media for both sugarcane and pineapple.


For Prunus microplants derived from CA and CG in vitro treatments were

compared, while for sugarcane and pineapple, plantlets from CD and CT in vitro

treatments were used (table 4.1, 4.1.2)

Some parameters related to the plantlets’ photoinhibition and photoprotection

were recorded as below:

- The photochemical quenching (qN) and the minimal (Fo) and maximal values

(Fm and Fm') of chl a fluorescence of pineapple and Prunus plantlets were

followed on day 0, 2, 4, 6, 8, 16, 28, 36, 50, 66, 80, 96 and 120 after

transplanting to the greenhouse. Of each treatment 5 randomly chosen plants

were sampled as 5 replicates. Having too narrow leaves, sugarcane plantlets

could not be sampled.

Chapter 4.3

100

- The total specific activities of catalase and SOD of Prunus, pineapple and

sugarcane plantlets were assessed on day 0, 4, 8, 16, 30, 60 and 120 after

transplanting. From each treatment 3 randomly selected plants were sampled as

3 replicates.

- The concentration of chl a, chl b, and total carotenoid were determined on day

0, 4, 16, 30, 60, 90 and 120 after transfer to the greenhouse, and the level of chl

a+b as well as the ratio off chl a/b and (chl a+b)/carotenoid were calculated. Of

each treatment 3 plantlets were sampled as 3 replicates.

The observation methods of each parameter were as described in chapter 3 (3.3).

4.3.3. Results and discussion

The results presented bellow demonstrate that upon transfer to ex vitro growth

conditions micropropagated plants, depending on the in vitro medium treatment and

plant species, suffered to variable extents from stress conditions. Significant variation

in Fo, Fm, Fm', qN, pigment contents, and total activity of SOD and catalase was

remarked both among model plants and between treatments.

4.3.3.1. Minimal chl a fluorescence (Fo)

During most of the acclimatization period, compared to pineapples, Prunus

plantlets had higher Fo values (fig. 4.19a). In the case of Prunus, Fo reduced over the

first 4 days following transplanting (from 0.35 to 0.29), and then increased to reach a

peak on day 36 (ca. 0.42). This peak coincided with the reduction in the overall

quantum yield recorded earlier in chapter 4.2 (fig. 4.15), demonstrating an increased

photodamage leve l suffered by these C3 plantlets. An increase in Fo was also recorded

by Van Huynlenbroeck (1997) with micropropagated Spathiphyllum plantlets

acclimatized under high light intensity ex vitro. In contrast, for pineapple plantlets Fo

remained unchanged during the first 4 days, then constantly reduced towards the end

of the acclimatization period. A sharp reduction was recorded at the end of the

experimental period for both pineapple and Prunus demonstrating that by that time

plantlets already recovered from stress and encountered less photodamage.

Chapter 4.3

101

Fig. 4.19: Fo of plantlets during the weaning stage, comparison between Prunus plantlets derived from CA and pineapple plants from CD treatments (a), and between Prunus plantlets derived from media wi th different gelling agents: agar vs. Gelrite (b).

Vertical bars represent standard errors, n = 5.

The effects of medium treatment on Fo of pineapple were insignificant (data not

presented). In contrast, for Prunus, as seen in fig. 4.19b, plantlets derived from

Gelrite-solidified medium had significantly higher Fo values compared to those from

agar-solidified medium. In addition, upon transplanting no decrease in Fo was noted

when Gelrite was used as the gelling agent of in vitro rooting medium (fig.4.19b). This

together with high values of Fo indicate that a higher level of photodamage occurred in

Prunus plantlets rooted in vitro on Gelrite-solidified medium compared to those on

agar-solidified medium.

4.3.3.2. Fm and Fm' of plantlets

Data on Fm and Fm' are presented in fig. 4.20. As seen, upon transfer to the

greenhouse, pineapple plantlets exhibited a decrease while Prunus showed an increase

in both Fm and Fm’ (fig. 4.20 a & b). During most of the experimental period, Prunus

plantlets had higher values of both Fm and Fm’. Peaks were recorded on day 28 – 36

and 80 for Prunus and day 16 and 80 for pineapple. Sharp reduction in both Fm and

Fm’ were recorded towards the end of the experimental period when plantlets were

already more or less acclimatized to ex vitro environmental conditions, and less

photodamage occurred to their photosystems.

Fo

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

a Fo

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

b

Chapter 4.3

102

Fig. 4.20: Fm and Fm’ of plantlets during the weaning stage, comparison between CA Prunus and CD pineapple plants (a & b), and between Prunus plantlets derived from media with different gelling agents: agar vs. Gelrite (c & d). Vertical bars represent standard errors, n = 5.

Similar to Fo, no significant variation could be observed between pineapple

plantlets derived from different rooting media (data not presented). For Prunus,

variation between medium treatments was noticed in both Fm and Fm' (fig. 4.20c &

d). As seen, at transplanting and early ex vitro (up to day 50), Prunus plants derived

from Gelrite medium had significantly higher Fm' than those from agar medium,

implying a higher level of photoinhibition encountered by the formers. The differences

in Fm were less clear, indicating that the variation in the maximal quantum yield (see

4.2.3.3) between Prunus plantlets from different medium treatments was due at larger

extent to variation in Fo (fig. 4.19) than in Fm.

ab

c

b

d

Fm

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00


a Fm'

0.30

0.50

0.70

0.90

1.10

1.30


b

Fm

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00


c Fm'

0.30

0.50

0.70

0.90

1.10

1.30


d

Chapter 4.3

103

4.3.3.3. Non-photochemical quenching

Quantifying the amount of excess energy dissipated in the form of heat

(Osmond, 1994), qN is often used for studying the plant photoinhibition and

photoprotection. For Calathea and Spathiphyllum during the first 1 - 2 weeks upon

transfer to the greenhouse an increase in qN was recorded followed by a decrease (Van

Huylenbroeck, 1997). Comparing the two aforementioned plant species, higher qN

values were recorded for Calathea plantlets, which were characterized with

heterotrophic growth while in vitro. In our present experiment, after transplanting, qN

of both, Prunus and pineapple plantlets, increased. The changes however, were larger

and persisted longer for pineapple; over the first 8 days following transfer qN

increased from 0.38 to 0.62, then reduced and reached a steady state from day 66 on.

In the case of Prunus, during the first 4 days after transplanting qN slightly increased

from 0.60 to 0.67, and then remained almost stable until the end of the experimental

period (fig. 4.21a).

Fig. 4.21: qN of plantlets during the weaning stage, comparison between CA-Prunus plantlets CD-pineapple plants (a & b) Vertical bars represent standard errors, n = 5.

For pineapple, variation between plants derived from different media, AC-

containing or AC-free medium was insignificant (data not presented). In the case of

Prunus, plantlets rooted in vitro on agar-solidified medium had significantly higher qN

values compared to those from Gelrite medium (fig. 4.21b). This demonstrates that the

former had a higher capacity to dissipate excess irradiation energy in the form of heat.

qN

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

a qN

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0 10 20 30 40 50 60 70 80 90 100 110 120

Day after transfer

b

Chapter 4.3

104

4.3.3.4. Catalase activity

The activity of catalase is also considered to be a reliable variable for the

assessment of the plants’ photoprotection capacity. For example, in Calathea

propagated plantlets an increase in this enzyme’s activity over 2 weeks following

transplanting was noted to be reversibly correlated to the light stress level (Van

Huylenbroeck et al., 2000). In our present experiment, over the first 5 weeks after

transfer to the greenhouse, depending on their quality and the plant species, plantlets

expressed either an increase or decrease in total catalase activity.

Fig. 4.22: Total catalase activity of plantlets during the weaning stage, comparison between CA-Prunus relative to CD-sugarcane and CD-pineapple plantlets (a), and effects of the presence or absence of AC on pineapple (b) and sugarcane (c) and of the gelling agent (agar vs. Gelrite) on Prunus (d). Vertical bars represent standard errors, n = 3.

For sugarcane and pineapple plantlets derived from AC-containing medium and

for Prunus plants from agar-solidified medium, catalase activity increased during the

b

c d

Units/ g proteins/min

0.00 4.00 8.00

12.00 16.00 20.00 24.00

0 4 8 16 30 60 90 120Day after transfer

a Units/g proteins/min

0.00 4.00 8.00

12.00 16.00 20.00 24.00

0 4 8 16 30 60 120Day after transfer

b

Units/g proteins/min

0.00 2.00 4.00 6.00 8.00

10.00 12.00


c Units/g proteins/min

0.00 4.00 8.00

12.00 16.00 20.00 24.00


d

Chapter 4.3

105

first 36 days following transplanting, then a steady state was reached (fig. 4.22a).

Variation among plant species could only be detected from day 8 on. At the steady

state, the catalase activity was 17.78 units/g proteins/min for Prunus, 7.4 units/g

proteins/min for pineapple and 4.2 units/g proteins/min for sugarcane.

The effects of medium treatments were species dependent. For pineapple, at

transplanting plantlets from AC-free medium had significantly higher catalase activity

compared to those from AC-containing medium, but with time passing during the ex

vitro acclimatization period, differences between medium treatments disappeared

(4.22b). In contrast, in the case of sugarcane the influences of medium treatments

increased towards the end of the experimental period, with higher activity recorded for

plantlets from AC-free medium (fig. 4.22c).

Working with Prunus, Frank et al. (1995) noticed that shoots cultured on

Gelrite-solidified medium suffered from hyperhydricity and were unable to recover

their enzymatic defence systems reflected in low catalase and SOD activity compared

to the normal (apparently non hyperhydric) shoots cultured on agar-solidified medium.

On the contrary, in our present experiment Prunus plantlets rooted in vitro on Gelrite-

solidified medium had higher catalase activity both while still in vitro (day 0) and

towards the end of the experimental period (fig. 4.22d). This could be explained by the

fact that at transplanting the worst Prunus plantlets that suffered most from

hyperhydricity, due to their poor or non-developed root systems, were already

discarded. As already mentioned in 4.3, only rooted plantlets were used for

reestablishment ex vitro, and consequently, Prunus plantlets from Gelrite-solidified

medium used in this experiment were still good enough to be able to develop their

protective enzyme system while still in vitro. Nevertheless, immediately after

transplanting, suffering from severe stress these plants could not recover their catalase

enzyme system, and exhibited a reduction of this enzyme’s total activity over the first

8 days in the greenhouse. Towards the end of the experimental period, when already

somewhat recovered, they again had increased catalase activity. Some similarity was

detected for pineapple plantlets from AC-free medium, but these CAM plantlets

displayed a better recovery capacity (fig. 4.22b).

Chapter 4.3

106

4.3.3.5. Total SOD-activity

Having the capacity to scavenge O2-, a highly reactive oxygen radical

photogenerated under stress light conditions (2.1.3.2), SOD is another enzyme

frequently used to study the plant’s photoprotection capacity. In many cases, the

changes in total SOD-activity were recorded to be related to the level of stress (Moran

et al., 1994; Hernandaz et al., 1994; Tsang et al., 1991; Malan et al., 1990; etc.)

Fig. 4.23: Total SOD-activity of plantlets during the weaning stage, comparison between CA-Prunus relative to CD-sugarcane and CD-pineapple plantlets (a), and effects of the presence or absence of AC on pineapple (b) and sugarcane (c) and of the gelling agent (agar vs. Gelrite) on Prunus (d). Vertical bars represent standard errors, n = 3.

In our present experiment, variation was noticed both among model plant

species and between plantlets from different in vitro rooting media. At transplanting,

the total SOD-activity was 0.49 units/mg proteins for Prunus, 1.12 for pineapple, and

0.072 for sugarcane. The highest activity of SOD in pineapple indicates the highest

a

d

b

c

Units/mg proteins

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

0 4 8 16 30 60 90 120Day after transfer

a

Units/mg proteins

0.00

0.02

0.04

0.06

0.08

0.10


c Units/mg proteins

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80


d

Units/mg proteins

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25


b

Chapter 4.3

107

capacity of these CAM plantlets to protect themselves from stress through scavenging

of superoxide. Regardless of the plant species, over the first weeks after transplanting

the total activity of SOD decreased. The reduction level however was largest for

Prunus, (from 0.49 to 0.12 units/mg proteins), followed by that for sugarcane (from

0.072 to 0.051 mg/proteins) and pineapple (from 1.45 to 1.2 units/mg proteins).

Different levels of decrease in SOD-activity were also recorded over the first 2-3

weeks following transfer of micropropagated plants of Calathea and Spathiphyllum to

the ex vitro conditions (Van Huylenbroeck, 1997). This author also recorded that the

changes of SOD-activity during the acclimatization period depended on the light stress

level.

Similar to catalase, and again in contrast to the data obtained by Frank et al.

(1995), Prunus plantlets derived from Gelrite-solidified medium had higher SOD-

activity compared to those from agar-solidified medium (fig. 4.23d). The explanation

could be the same as for catalase: the most hyperhydric plantlets were already

discarded before ex vitro reestablishment. For pineapple and sugarcane, variation

between medium treatments persisted just until day 16 after transplanting. From that

day on, no differences could be noted between plantlets from different in vitro rooting

media. It is noteworthy that, in pineapple plantlets rooted in vitro on AC-free medium,

the total SOD activity did not reduce but increased over 4 days after transplanting (fig.

4.23b). This could imply a high capacity of these plants to scavenge superoxide and

recover quickly from stress conditions ex vitro.

4.3.3.6. Pigment concentration

As seen in fig. 4.24, there were distinctions between C3, C4 and CAM plants

regarding their photosynthetic pigment contents. Among the three species, Prunus had

the highest total concentrations of both chlorophylls (fig. 4.24a) and carotenoid (fig.

4.24c) followed by sugarcane and pineapple. Regardless of plant species, upon transfer

to the soil, the content of chl per mg fresh leaves decreased. The extent of the decrease

in chl-content was highest for Prunus and lowest for pineapple. Reduction in total chl-

concentration was previously reported by Van Huylenbroeck (1997) for Spathiphyllum

Chapter 4.3

108

plantlets acclimatized to high light intensity ex vitro indicating photodamage to the

photosystems. Following the decrease period, recovery was observed, and towards the

end of the experimental period a steady state was obtained for all the three plants.

Fig. 4.24: Pigment content of plantlets during the we aning stage, comparison between CA-Prunus relative to CD-sugarcane and CD-pineapple plantlets. Standard errors were smaller than the data labels, n = 3.

Changes in the total carotenoid concentration depended on plant species, for

Prunus and sugarcane it increased, while for pineapple it reduced over 4 days

following transplanting (fig. 4.24c). Towards the end of the experimental period, it

increased for Prunus, reduced for pineapple and remained unchanged for sugarcane.

In contrast to the total chl- and carotenoid-content, the highest ratio of chl a/b

was measured in pineapple plantlets (ca. 2.5 –3 ) followed by that of sugarcane (ca. 2 –

2.8) and Prunus (between 1 – 1.8) (fig. 4.24b). Correspondingly, as compared to

Chl a+b (mg/g fresh weight)

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00


a Chl a/b

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50


b

Carotenoids (mg/g fresh weight)

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70


c (Chl a+b) / carotenoids

0.00 2.00 4.00 6.00 8.00

10.00 12.00


d

Chapter 4.3

109

Prunus plantlets, the model CAM and C4 plantlets differed less from sun plants. An

possible explanation for this can be their the tropical origin. In agreement with data

published by Van Huylenbroeck (1997) with Calathea, following transfer to the

greenhouse, the ratio of total chl to carotennoid concentration declined to variable

levels depending on the plant species (4.24d); with the sharpest decline detected for

Prunus followed by that of sugarcane and pineapple.

Fig. 4.25: Pigment contents during the weaning stage, comparison between pineapple plantlets from different medium treatments Vertical bars represent standard errors, n = 3.

Having a sharp reduction in both the total chl content and in the ratio of chl to

carotenoid as compared to pineapple and sugarcane, Prunus plantlets seemed to suffer

more from stress. Nevertheless, these C3 plantlets developed photoprotective

mechanisms through producing more carotenoids for non-photochemical quenching of

chl-fluorescence. This agrees with data obtained on qN (fig. 4.21a), Fo (fig. 4.19a), Fm

(fig. 4.20a) and Fm' (fig. 4.20b). In contrast, pineapple and sugarcane plantlets reduced

photodamage by adjusting the size of their photosystems, reflected in high chl a/b

Chl a (mg/g fresh weight)

0.00

0.20

0.40 0.60

0.80

1.00

1.20


a Chl b (mg/g fresh weight)

0.00 0.10 0.20 0.30 0.40 0.50 0.60


b


0.00 0.05 0.10 0.15 0.20 0.25 0.30


c

Chapter 4.3

110

ratio. The importance of carotenoids and the size of photosystems in chl fluorescence

quenching and photoprotection was already discussed in chapter 2, section 2.1.3.2.

Significant differences between medium treatments were also observed for the

three plant species. In agreement with data presented earlier in 4.1 and 4.2, towards the

end of the experimental period variation among pineapple plantlets rooted in vitro on

different media decreased and disappeared (fig. 4.25). This indicates a high ability of

these CAM micropropagated plants to ‘correct’ abnormalities obtained under special

in vitro growth conditions.

Fig.4.26: Pigment contents during the weaning stage, comparison between sugarcane plantlets from different medium treatments. Vertical bars represent standard

errors, n = 3.

In contrast, persistent and even increasing differences between medium

treatments were observed for sugarcane (fig. 4.26) and Prunus (fig. 4.27) implying

lower potential of sugarcane plantlets from AC-free medium and of Prunus plants

from Gelrite-solidified medium to adapt to changed growth condition ex vitro. In

comparison to Prunus plantlets from agar-solidified medium, those rooted in vitro on

Gelrite medium had low content of chl a, chl b and carotenoid. Similarly, leaves of

Chl b (mg/g fresh weight)

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80


b


0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40


c

Chl a (mg/g fresh weight)

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60


a

Chapter 4.3

111

sugarcane plantlets produced from AC-containing medium were richer in

photosynthetic pigments compared to those from AC-free medium. This is consistent

with data obtained earlier (4.1 & 4.2) that for sugarcane addition of AC (1 g/l) to the in

vitro rooting medium improved the plantlet’s quality, and use of agar as the gelling

agent was better for Prunus.

4.3.4. Conclusions

In agreement with data obtained in the previous experiments (4.1 & 4.2) upon

transfer to the soil, micropropagated plants depending on both their quality and the

plant species suffered to variable extents from stress conditions and exhibited different

capacities to protect their photosystems from photodamage.

Fig. 4.27: Pigments contents during the weaning stage, comparison between Prunus plantlets from different medium treatments. Vertical bars represent standard errors, n = 3.

Among the three model plants, pineapple appeared to be least affected by in

vitro medium treatments. Regarding chl a fluorescence values (Fo, Fm and Fm' and

qN) no significant variation was recorded among pineapple plantlets derived from


0.00

0.20

0.40

0.60

0.80


c

Chl b (mg/g fresh weight)

0.50 0.75 1.00 1.25 1.50 1.75 2.00


b Chl a (mg/g fresh weight)

1.25 1.35 1.45 1.55 1.65 1.75 1.85 1.95 2.05 2.15


a

Chapter 4.3

112

different in vitro rooting media. In addition, differences in catalase (fig. 4.22b) and

SOD (fig. 4.23b) activities, and in pigment contents (fig. 4.25) between medium

treatments were small and occurred just shortly after transfer. In contrast, for

sugarcane and especially Prunus, distinctions between plantlets derived from different

media were bigger and persisted longer during ex vitro acclimatization period. Prunus

plantlets rooted in vitro on Gelrite-solidified medium appeared to be affected to a

larger extent by stress conditions ex vitro; they had higher Fo, Fm, Fm values, and

lower qN and pigment contents compared to those from agar-solidified medium.

Similarly, in the case of sugarcane, plantlets produced from AC-containing medium

encountered less photoinhibitions problems; they had always higher chl a and b

concentrations.

In order to cope with stress ex vitro, micropropagated plantlets of the three

model plants did develop certain protective mechanisms. Prunus plantlets produced

more carotenoids for non-photochemical quenching of chl fluorescence, while

pineapple and sugarcane plantlets regulated the amount of absorbed light energy

through reducing their photosystem size. In addition, catalase and SOD were both

important for plantlets to protect themselves from photoinhibition: towards the end of

the experimental period, sugarcane plantlets derived from AC-free medium and

Prunus plantlets from Gelrite-solidified medium still suffered from stress (4.1 and 4.2)

and consequently had to produce more catalase and SOD for scavenging potentially

harmful photogenerated products (H2O2 and O2-, correspondingly).

CHAPTER 5

GENERAL DISCUSSION AND CONCLUSIONS

Chapter 5

114

Though micropropagation has long enjoyed great attention, until recently not

much effort has been paid to the evaluation of the physiological competence of tissue

culture derived plants. With growing demand for good quality micropropagated plants,

increasing attempts have recently been made to develop methods and markers for

quality evaluation in micropropagation (Cassells et al., 2000). For various reasons,

however, this issue remains still problematic (Van der Linde, 2000), and as argued by

Debergh (1991) differences between putative “good” and “bad” micropropagated

plants should be described for this purpose. By characterising differences between

plantlets of different quality (from different in vitro treatments) of three model plant

species from C3, C4 and CAM photosynthetic groups, we attempted to get some

insight into the use of different physiological parameters, and to assess their reliability,

for characterisation of the physiological quality of micropropagated plants. In total, 8

treatments (combinations of agar vs. Gelrite; with or without activated charcoal; high

blue light, high red light or high far-red light) were applied for each model plant.

From the results presented here it is clear that the quality of micropropagated

plants, and consequently their performance upon transplanting, depended largely on

the in vitro growing environment. Significant differences were detected both between

plant species and between plantlets of the same species derived from different growth

conditions during the in vitro rooting stage. Depending on the plant species, variation

among treatments reduced or increased during the ex vitro acclimatization, implying

different abilities of plantlets’ to “correct” the abnormalities that developed while they

were in vitro, and to adapt to changed growth conditions in the greenhouse.

5.1. In vitro

In accordance with data published by other authors (Soebø et al., 1995;

Kirdmanee, 1995; Miyashita et al., 1995 & 1997; Moreira da Silva and Debergh,

1997) the responses of plantlets to variable light quality conditions in vitro depended

upon the plant species. In general, the photon ratios and light qualities tested in the

present work had no significant influence on pineapple, while their effects on Prunus

and sugarcane could be detected in certain parameters related to growth, morphology

Chapter 5

115

and physiological functions. For Prunus, high red photon ratio increased both fresh

and dry weight (table 4.3) while blue enriched light raised the density of both

hydathodes and stomata on the abaxial leaf surface (table 4.4). The high blue light

treatment also reduced the CO2 accumulation in the culture headspace of C3 plant

during the in vitro rooting stage (fig. 4.8f). Whether this is related to the occurrence of

a large amount of hydathodes and stomata with variable sizes on the leaf surface of

this plant remained still unclear. In contrast, on sugarcane, a high blue photon ratio did

not have significant effects while enriched red and far-red lights increased the plant

weight, both dry and fresh, the leaf length (table 4.3) and stomata density (table 4.4).

For this C4 species, light quality also influenced the specific activity of PEP-case, the

C4 primary carboxylating enzyme. At the end of the in vitro rooting stage high

activities of this enzyme were measured in plantlets having high fresh and dry weight

(rooted under high red or high far-red lights) (fig. 4.18). This correlation between PEP-

case activity and growth demonstrates the functioning of the C4 photosynthetic cycle

in C4 plantlets in vitro.

In contrast to the light quality, medium treatments had significant influences on

most of the parameters observed. On Prunus, the gelling agent of the in vitro rooting

medium also impacted the plantlet’s quality and growth. In agreement with data

published previously by Frank et al. (1995) Gelrite, as the gelling agent, caused

hyperhydricity, reflected in reduced dry matter content and increased non-uniformity

between plants. Compared to agar, Gelrite reduced the percentage of plantlets rooted

in vitro, the root number per plant (table 4.2), and the plant fresh and dry weight (table

4.3). Lower values of Fv/Fm, Φp, qP, qN and chl-concentration and higher

measurements of Fo and Fm’ recorded for Gelrite-solidified medium indicate poorer

photosynthetic competence, higher photoinhibition level of Prunus plantlets cultured

on Gelrite-solidified compared to those on agar-solidified medium. Neve rtheless, in

order to cope with stress problems plantlets developed both catalase and SOD enzyme

systems for scavenging potentially harmful photogenerated O2- and H2O2. At the end

of the in vitro rooting stage higher activities of these two enzymes were measured for

Gelrite-solidified medium.

Chapter 5

116

For sugarcane, AC improved both the percentage of plants rooted in vitro and the

root length (table 4.2). For this C4 plant, heavier plantlets (table 4.3), with higher

values of net photosynthesis rate (fig. 4.13b), PEP-case and SOD-activity (fig. 4.17b &

4.23c) and chl-concentration (fig. 4.25a 1b), but lower values for catalase-activity (fig.

4.22c) and less total carotenoid-content (fig. 4.25c), were recorded when AC was

added to the rooting medium. In the case of pineapple, addition of AC (1 g/l) increased

the plant’s root number, length and branching (table 4.2), leaf length (table 4.3), net

photosynthetic rate (fig. 4.13c), specific PEP-case (fig. 4.17) and total SOD-activity

(fig. 4.23b), while it reduced the total catalase activity (fig. 4.22b). Thus, for the model

C4 and CAM plants, addition of AC to the in vitro rooting medium improved the

plantlet’s photosynthetic and photoprotection capacity while still in vitro.

Influences of the in vitro rooting medium on the plantlets’ physiological

competence were also reflected in variation in the evolution of gasses in the culture

headspace among the medium treatments. In the case of Prunus, compared to agar,

Gelrite as the gelling agent reduced the CO2 evolution, but increased the levels of both

O2 and C2H4. For sugarcane and pineapple, omission of AC increased the evolution of

both CO2 and C2H4 in the culture headspace. The O2 level in sugarcane headspace

however was reduced by when AC-containing medium was used. Circadian

fluctuation in O2 and CO2 reflected in either negative or positive ∆ values also

demonstrate the plantlet’s photosynthetic and respiration ability, and this is consistent

with measurements of Pn, qP, photoquantum yield, PEP-case and observations on leaf

mesophyll structure.

While still in vitro, as observed at the end of the in vitro rooting stage, mesophyll

of C3, C4 and CAM leaves had already well differentiated structures similar to those

described for their ‘counterparts’ growing outdoors. Also, high Fv/Fm values (around

8) recorded on day 0 indicate normal status of microplants’ leaves prior to ex vitro

reestablishment. Nevertheless, they had low chl a/b ratio and were characterised with

low photosynthetic competence reflected in low values of Pn, Φp, qP recorded at

transplanting for all the medium treatments.

Chapter 5

117

5.2. Ex vitro

Regarding light photon ratio, sugarcane was the only plant on which effects of

light quality treatments during the in vitro rooting stage persisted significant over the

whole acclimatization period. Sugarcane plantlets derived from in vitro high red or

high-far red light treatments always performed better than those from control and blue

lights in terms of both fresh and dry weights (table 4.3) and of PEP-case specific

activity (fig. 4.18). Thus, for this C4 plant, enrichment of either red or far-red photon

ratio significantly improved the quality of micropropagated plants. For Prunus and

pineapple, the impact of light quality treatments in vitro reduced and shortly after

transfer to the soil became in significant.

Regarding the in vitro rooting medium, as mentioned above, at the end of the in vitro

culture period, plantlets of sugarcane and pineapple rooted in vitro on the AC-

containing medium had better physiological competence compared to those on AC-

free medium, and Prunus plantlets derived from agar-solidified medium were better

than those from Gelrite-solidified medium in terms of Pn, Φp, Fv/Fm, qP, PEP-case

activity, Fo, Fm, and chlorophyll-content. Therefore for convenience, below pineapple

and sugarcane plantlets rooted in vitro on AC-containing medium under normal light

condition are referred to as “class 1”, while those on AC-free medium under normal

light as “class 2” plants. Similarly, Prunus plantlets from agar-solidified medium

under normal light are considered as “class 1” and those from Gelrite-solidified

medium under normal light as “class 2”. Below, some elements of the discussion are

made on the ability of plantlets to acclimatize to the changed growing conditions ex

vitro by comparing the behaviour of class 1 plantlets from each species relative to their

class 2 ‘counterparts’, and between C3, C4 and CAM plants. A summary of data on

parameters related to the plantlets’ growth, photosynthesis and photoprotection and

their changing pattern over the acclimatization period is summarized in table 5.1.

Chapter 5

118

Table 5.1: Data summary table

(*): Plantlets of class 1 or 2; (**): decline or recover period following transplanting; (d.) : day; (inc.): increase; (red. ): reduction

++++++

d. 0 – 10d. 0 – 12

+++

++++++++

d. 0 – 12d. 0 – 20

+++++

12Pn (µmol/m2/s)

--

d. 0 – 4 d. 0 – 4

++

+++++

d. 0 – 4d. 0 – 16

+++

+++++++

d. 0 – 16d. 0 – 4

+++++

12

Ch a+b(mg/g fresh weight)

++red.-inc.+++++incr.-red.++-Inc. -red+1Ch a/b(mg/g fresh weight)

++

d. 0 – 4d. 0 – 4

++++

+++++

increaseincrease

++++

+++++

increaseincrease

++++

12

Carotenoid(mg/g fresh weight)

++++++++++

d. 0 – 16 increase

+++++++

++++

d. 0 – 4d. 0 – 8

-+

+++

d. 0 – 16d. 0 – 16

+++++

12

SOD(units/mg proteins))

++++

increase increase

++

++++

increaseincrease

+++

12qP

RecoverDecilneRecoverDeclineRecoverDecline

++++++

++

++

-+

++++

++++++++

++++

++++

increased. 0 – 4

inc.– red.inc.– red.

d. 0 – 8d. 0 – 8

d. 4 – 120d. 4 – 120

increaseincrease

d. 0 - 6d. 0 - 6

+++-

Ex vitro (**)

+++

++++

++++

++

++

++++++

++++

+++

In vitro

PINEAPPLE

++++ +

++++

++++

12

12

12

12

12

12

12

12

SUGARCANEPRUNUS

increaseincrease

d. 0 – 4d. 0 - 16

+++

Ex vitro (**)

++

++++

+++

In vitro

--

--

--

PEP-case(nmol/mg pritein/min)

++

d. 0 – 8 d. 0 – 36

++++Fv/Fm

+++++++++

++++++++

++++

+++

++++

+++++

Ex vitro (**)

increased. 0 – 8

+++

Catalase(units/g proteins/min)

increaseincrease

++++

qN

increaseincrease

++Fm

d. 0 – 4inc. – red.

+++++Fo

increase increase

+++

+++Φp

+++Growth

In vitro(*)

++++++

d. 0 – 10d. 0 – 12

+++

++++++++

d. 0 – 12d. 0 – 20

+++++

12Pn (µmol/m2/s)

--

d. 0 – 4 d. 0 – 4

++

+++++

d. 0 – 4d. 0 – 16

+++

+++++++

d. 0 – 16d. 0 – 4

+++++

12

Ch a+b(mg/g fresh weight)

++red.-inc.+++++incr.-red.++-Inc. -red+1Ch a/b(mg/g fresh weight)

++

d. 0 – 4d. 0 – 4

++++

+++++

increaseincrease

++++

+++++

increaseincrease

++++

12

Carotenoid(mg/g fresh weight)

++++++++++

d. 0 – 16 increase

+++++++

++++

d. 0 – 4d. 0 – 8

-+

+++

d. 0 – 16d. 0 – 16

+++++

12

SOD(units/mg proteins))

++++

increase increase

++

++++

increaseincrease

+++

12qP

RecoverDecilneRecoverDeclineRecoverDecline

++++++

++

++

-+

++++

++++++++

++++

++++

increased. 0 – 4

inc.– red.inc.– red.

d. 0 – 8d. 0 – 8

d. 4 – 120d. 4 – 120

increaseincrease

d. 0 - 6d. 0 - 6

+++-

Ex vitro (**)

+++

++++

++++

++

++

++++++

++++

+++

In vitro

PINEAPPLE

++++ +

++++

++++

12

12

12

12

12

12

12

12

SUGARCANEPRUNUS

increaseincrease

d. 0 – 4d. 0 - 16

+++

Ex vitro (**)

++

++++

+++

In vitro

--

--

--

PEP-case(nmol/mg pritein/min)

++

d. 0 – 8 d. 0 – 36

++++Fv/Fm

+++++++++

++++++++

++++

+++

++++

+++++

Ex vitro (**)

increased. 0 – 8

+++

Catalase(units/g proteins/min)

increaseincrease

++++

qN

increaseincrease

++Fm

d. 0 – 4inc. – red.

+++++Fo

increase increase

+++

+++Φp

+++Growth

In vitro(*)

Chapter 5

119

As seen upon transfer to soil, depending on the plant species and physiological

competence, plantlets suffered to variable extent from stress conditions, reflected in

different degrees of decrease in Pn, Fv/Fm, PEP-case activity and chl-content. To

adapt to the changing growing conditions, plantlets developed different acclimating

and protection systems. Consequently the differences between class 1 and class 2

plantlets from each species changed over the acclimatization period, and the pattern of

changes could be an important indicator for their adaptation capacity. For each plant

species, plantlets from class 2 encountered more problems of photoinhibition; they

exhibited both a larger and longer-persisting decrease in Pn, Fv/Fm, PEP-case activity

and chl-content as compared to those from class 1. Also, higher values of Fo, Fm, Fm’

were recorded in plantlets from class 2 implying higher photodamange caused to these

plants’ photosystems compared to plants from class 1.

Among the three plant species, pineapple seemed to have the highest capacity to

“correct” its abnormalities that developed under in vitro conditions and to adapt to the

ex vitro environment. Variation between class 1 and class 2 plantlets of this CAM-

species reduced upon transplanting and disappeared after a certain time in the

greenhouse. As a consequence, towards the end of the experimental period, all

pineapple plantlets were uniform regarding all the parameters observed. This high

adaptation ability of CAM plantlets was attributed to the development of their

photoprotective mechanisms and systems. In comparison to C3 and C4 plants, CAM

had the highest ability to scavenge O2-, and to regulate the amount of absorbed

irradiation energy through adjusting their photosystems’ size. Among the 3 plants,

CAM ranked first in terms of both the total SOD activity (fig. 4.23) and the ratio of chl

a/b (fig.4.24). Moreover, CAM plantlets from class 2 were the only ones characterised

with an increase in total SOD-activity over the first week after transplanting (fig.

4.23b). Due to their high photoprotection capacity, CAM plants encountered less

photodamage, reflected in low values of Fo, Fm, and Fm’ and higher photochemical

yield as compared to C3 plants.

In contrast to pineapple, for sugarcane, differences between class 1 and class 2

plantlets did not decrease, but increased upon transplanting, and towards the end of the

Chapter 5

120

experimental period, plantlets from class 2 still expressed an non-uniform performance

and poor growth compared to those from class 1. Variation in the plant weight (table

4.3), net photosynthetic rate (fig. 4.13b), PEP-case activity (fig. 4.17b) and chl-content

(fig. 4.25a &b) also increased during the ex vitro acclimatization, with higher values

detected in class 1 plantlets. All these indicate a lower capacity of C4-plantlets from

class 2 to “correct” their abnormalities which developed while in vitro and to adapt to

the different growing conditions in the greenhouse. In order to cope with stress

conditions, C4 plantlets also developed protective systems. As compared to the other

two plants, C4 plantlets had the lowest, but constantly increasing, total catalase

activity over the whole experimental period (fig. 4.22). Upon transplanting, the total

SOD activity of C4 plantlets exhibited a faster recovery than C3 and CAM plants.

Among the three species, C4 ranked medium in terms of total concentrations of chl

and carotenoid and the chl a/b ratio.

In the case of Prunus, differences between class 1 and class 2 plantlets also

persisted over the experimental period. Class 2 plantlets exhibited a highly non-

uniform growth and performance ex vitro. Furthermore, 30 days after transplanting

around 30% of them died. Their bad performance and low survival indicate their poor

capacity to adapt to changed growth conditions ex vitro, and this is consistent with

data related to photosynthesis, photoinhibition and photoprotection. Lower values of

qP, Fv/Fm, Φp, and pigment content recorded in these plantlets during most of the

acclimatization period implies also their poor photosynthetic ability compared to the

class 1 plants. Prunus plantlets from class 2 also experienced more photoinhibition

than those from class 1, reflected in a deeper decline and slower recovery of the

abovementioned parameters (4.2), and in their higher values of Fo, Fm, Fm’ (4.3).

Among the three model plants, Prunus had the highest total catalase activity (fig 4.22).

Higher activities of both catalase and SOD recorded in C3 plantlets from class 2

relative to class 1 during most of the acclimatization period demonstrate the potential

of Prunus plantlets to develop photoprotective enzyme systems when they encounter

stress conditions. In the terms of pigment content, while C4 and CAM plants regulated

the amount of absorbed photon energy through reducing their photosystems’ size, C3

plantlets produced more carotenoids for non-photochemical quenching of chl

Chapter 5

121

fluorescence through dissipating excess energy in the form of heat. This was reflected

in an increase in both high carotenoid level and qN-values recorded for Prunus, and in

low chl a/b ratio observed in pineapple and sugarcane.

The usefulness of such parameters as pigment contents, PEP-case activity, Pn,

protective enzymes and chl fluorescence study in evaluating the physiological quality

of micropropagated plants was confirmed in our present work. Nevertheless, as plants

depending on the species, or even genotype, behave and react to environmental

changes differently, any parameter should be considered in relation to the plant

species, and comparative studies between “bad” and “good” plantlets from the same

species/variety should be undertaken. For the three model plants studied in our present

work, there were positive relationships between PEP-case activity, Pn, qP, Φp and the

plantlets growth, both while in vitro and during the acclimatization period; plantlets

from class 1 gave higher values of PEP-case activity, Pn, qP, and Φp compared to

plants from class 2. Also, the extent of decrease or increase in these parameters upon

transplanting were related to the photoinhibition level caused to plantlets, and so the

values of Fo, Fm and Fm’. Plantlets with better growth (from class 1) exhibited a

faster recovery in PEP-case activity, Pn, qP, and Φp and gave lower values of Fo, Fm

and Fm’. Photoprotection of plantlets can also be studied through recording values of

qN, activity of catalase and SOD enzyme systems, and changes in pigments content. In

general, plants of better quality (from class 1) had high values of qN, and experienced

less decline in pigment content upon transplanting. High qN-values were also recorded

in plantlets with high concentration of total carotenoid, whose role in non-

photochemical quenching has been well recognised (2.1.3.2). In plantlets with less

capacity to dissipate the excess energy in the form of heat (Prunus plantlets from class

2), perhaps more AOS were produced and, and thus these plants had to recover most

their catalase and SOD enzyme systems. Prunus plantlets from class 2 had much

higher total activity of both catalase and SOD as compared to those from class 1.

In conclusion, as compared to plants growing outdoors, micropropagated plants

of C3, C4 and CAM plants had similar differentiated mesophyll structure, similar

stomata density and size. They, nevertheless were abnormal in having low chl a/b

Chapter 5

122

ratio, low photosynthetic competence and low photoprotection ability. Abnormalities

presented to variable extents depended on both the plant species and in vitro growth

conditions. For the model CAM and C4 plants, addition of AC (1 g/l) to the in vitro

rooting medium improved the quality of micropropagated plants, while for the studied

C3 species use of agar as the gelling agent of the in vitro rooting medium resulted in

better plantlets in terms of Pn, Φp, qP, PEP-case, Fo, Fm, SOD, catalase and pigment

levels. Upon transfer to soil, plantlets suffered from stress, but could also develop

photoprotection mechanisms. For all the 3 species, upon transplanting plantlets from

class 1 (with better quality as classified according to the above mentioned parameters)

encountered a lower level of photoinhibition and exhibited a faster recovery and better

growth compared to those from class 2. This demonstrates the usefulness of the chl

fluorescence study, measurement of PEP-case, SOD and catalase activity and pigment

levels in evaluating the physiological quality of micropropagated plants.

Further research needs to be carried out with other plant species and observation

should also be made earlier during the in vitro culture period, in order to find out how

early the quality of final products of a micropropagation procedure can be assessed.

This would be of great importance for any plant micropropagation program. For this

end however, observation methods of parameters as well as relevant equipment need

to be improved so that narrow leaves can also be sampled and sampling time reduced.

123

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INTERNET WEBSITES

1. Hallick R. B., 2001. http://www.blc.arizona.edu/courses/181gh/rick/photosynthesis/C4.html

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