Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to...

Biochemical changes in micropropagated grape

(Vitis vinifera L.) plantlets due to arbuscular-

mycorrhizal fungi (AMF) inoculation

during ex vitro acclimatization

Hare Krishna *, S.K. Singh, R.R. Sharma,R.N. Khawale, Minakshi Grover, V.B. Patel

Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute,

New Delhi 110012, India

Received 28 August 2004; received in revised form 21 April 2005; accepted 9 May 2005

Abstract

Improved establishment of mycorrhizal tissue culture derived plantlets during acclimatization

(Stage IV) is commonly attributed to the enhanced vegetative growth as a resultant of different

morphological and in vivo changes. These changes are early and better cuticle development, high

biomass accumulation, enhanced physiological changes, improved mineral nutrition, especially

phosphorus and micronutrients, etc. However, improvement in establishment of micropropagated

plantlets during acclimatization may not only be limited to these mechanisms. In the present

investigation, biochemical status of micropropagated grape plantlets in response to six single and a

mixed strains of arbuscular-mycorrhizal fungi (AMF) during hardening were studied under glass-

house conditions. The histochemical studies revealed that the mycorrhizal inoculation resulted in

accumulation of different biochemicals in the plant system such as chlorophyll, carotenoids, proline,

phenol and enzymes like polyphenol oxidase (PPO) and nitrate reductase (NR). The mycorrhizal

plantlets showed enhanced survival and improved tolerance against stresses experienced during

weaning phase. The mycorrhizal plants also exhibited improved physiological and nutritional status

and had higher relative water content (RWC) and photosynthetic rate. These plantlets also

accumulated higher N, P, Mg and Fe concentrations, which may primarily be as a result of

biochemical changes brought about by mycorrhizal association. Mycorrhizal plantlets also showed

www.elsevier.com/locate/scihorti

Scientia Horticulturae 106 (2005) 554–567

* Corresponding author.

E-mail address: [email protected] (H. Krishna).

0304-4238/$ – see front matter # 2005 Published by Elsevier B.V.

doi:10.1016/j.scienta.2005.05.009

better hardening under glasshouse conditions. The result suggests that the biochemical changes

brought about by mycorrhization were helpful in mitigating different stresses experienced by the

tissue culture plants during hardening, which determine their performance later in field.

# 2005 Published by Elsevier B.V.

Keywords: Enzymes; Hardening; Mycorrhizal inoculation; Stress; Plant survival; Tissue cultured grape plants

1. Introduction

In certain plant species, establishment of tissue culture raised plants under glasshouse

and later in field is often very poor. This is mainly attributed to the inability of such plants to

tolerate different types of stresses such as transplant shock, excessive water loss, pathogen

attack, poor photosynthesis, etc. Under such stressful conditions, several plants processes

are altered or severely affected such as CO2 assimilation, chlorophyll biosynthesis and

water relations. In addition, changes in biochemical status and altered enzymatic activities

were also encountered during stress.

In endurance for different types of stressful conditions, different physiological and

biochemical processes like photosynthesis, transpiration, biosynthesis of proline and

phenol and altered polyphenol oxidase (PPO) and nitrate reductase (NR) activities are of

great importance. Mycorrhizal inoculation of in vitro propagated transplants has proven to

be effective in respect of tolerance to different stresses, improvement in vegetative growth

and mineral nutrient status (Gianinazzi et al., 1989). Earlier, researchers have demonstrated

improved adaptation of AMF colonized plants in fields and attributed it mainly to reduced

water stress (Johnson and Crews, 1979; Menge et al., 1978) and salt tolerance (Jain et al.,

1989). But the improved establishment of mycorrhizal micropropagated plants can not

only be limited to these mechanisms as there are huge possibilities of vital changes in

biochemical status of plantlets to enable them to sustain different stresses during

hardening. Hence, it become inevitable to study whether the enhanced survival of

micropropagated plantlets as influenced by mycorrhiza is because of the aforementioned

mechanisms or whether some supplementary mechanism is also in operation during

hardening. Thus, understanding the physio-chemical processes that underlie the adaptive

mechanism induced by mycorrhiza to various stresses encountered in tissue-cultured plants

during acclimation would be of immense value from research as well as academic point of

view. Possingham and Groot Obbink (1971) reported the beneficial effect of

mycorrhization in grape, while Menge et al. (1983) studied the interaction between

mycorrhizal fungi and soil fumigation. Later, Schubert and Cravero (1985) isolated natural

AMF infection in grapevine roots. It is well established that different mycorrhizal species

impart varied response on growth and nutrient uptake (Karagiannidis et al., 1995). This

property of AM fungi in now exploited in inoculating plants to be transferred to

micronutrient deficient soils (Bavaresco and Fogher, 1992, 1996). These beneficial effects

brought about due to an array of physiological and biochemical changes imparted in the

tissue culture raised plants as found in Chile ancho pepper plants (Estrada-Luna and

Davies, 2003). However, to the best of our knowledge no detailed report has been pub-

lished on biochemical changes as a result of AMF inoculation during hardening of

H. Krishna et al. / Scientia Horticulturae 106 (2005) 554–567 555

micropropagated grape plants. Therefore, the present investigation was conducted to note

the biochemical changes brought about by arbuscular-mycorrhizal fungi (AMF)

inoculation in order to explore the possibility of existence of other mechanism for

enhanced plant survival during glasshouse hardening and establishment upon field

transplant.

2. Materials and methods

2.1. Plant material

Micropropagated plantlets of a grape (Vitis vinifera L.) variety Pusa Navrang were

procured from the Central Tissue Culture Laboratory, IARI, New Delhi. In vitro cultures of

the above variety was established using nodal segments as explants collected from the 12-

year-old, field-grown vines (Singh et al., 2002). Mass multiplication and maintenance of

the plantlets were done employing two node repetitive micro-cutting techniques developed

by Singh et al. (2004). These in vitro raised plantlets were obtained after 30 days of rooting

on agar solidified medium. The plantlets at this stage were 15–18 cm long with 2–3 rootlets

(6–8 cm) and 5–7 small leaves.

2.2. Arbuscular-mycorrhizal fungi inoculum production and application

The arbuscular-mycorrhizal fungi (AMF) strains used were Acaulospora laevis (T1), A.

scrobiculata (T2), Entrophospora colombiana (T3), Gigaspora gigantea (T4), Glomus

manihotis (T5), Scutellospora heterogama (T6) and a mixed AMF strain (T7) comprising G.

manihotis, Glomus mosseae and G. gigantea procured from the Division of Microbiology,

IARI, New Delhi. Soil based AMF cultures were multiplied on Rhodes grass (Chloris

guyana) as a host plant and maintained in plastic pots (5 kg) filled with autoclaved

(1.05 kg cm�2 for 2 h) potting mixture of soil, sand and well rotten farm yard manure (FYM)

in the proportion of 2:2:1 under glasshouse conditions. In order to ensure sufficient root

colonization, rhizosphere soil of Rhodes grass containing mycelia, spores, arbuscules,

vesicles and root segments were used as inoculum. The rooted grape plantlets (30-day-old

tissue) were washed with sterile tap water to dislodge the adhering agar–agar and thereafter

transferred to plastic pots containing sterilized potting mixture (soil, sand and FYM; 2:2:1)

along with approximately 20 g AMF inoculum placed immediately below the roots. This

amount of inoculum had 130–225 AMF spores depending upon the Mycorrhizae species. The

control treatment had only sterile potting mixture. The plantlets were irrigated immediately

after transplanting with sterile tap water and later on regular interval to prevent desiccation.

2.3. Growth conditions

After transplanting, the inoculated and non-inoculated or control (referred as T0)

plantlets were acclimatized (60 days) in a controlled glasshouse with day-night

temperatures ranging from 27 � 1 8C. Relative humidity and day length were maintained

to 80–85% and 16 h with cool white fluorescent lamps at 630 mmol m�2 s�1, respectively.

H. Krishna et al. / Scientia Horticulturae 106 (2005) 554–567556

2.4. Assessment of survival and root colonization (%)

Ex vitro survival (%) and percent root colonization were measured 60 days after

inoculation. Fresh root segments were stained with 0.01% Trypan blue in lactic acid

(Phillips and Hayman, 1970; Koske and Gemma, 1989). A portion of roots was taken and

extent of root colonization was assessed in 10 root segments, averaged and expressed as

percent root colonization.

2.5. Determination of growth, physiological and nutritional parameters

Growth parameters, viz., plant height, root length, leaf no. and area were recorded 60

days after inoculation. Leaf area was measured by leaf area meter (Li-Cor). Physiological

parameters such as leaf relative water content (RWC) and photosynthetic rate were

determined. Photosynthetic rate of the intact mature leaves were determined by a portable

infrared gas analyzer (Li-Cor-6200). The readings were taken thrice at an interval of

15 min and the average values were worked out. Relative water content was determined in

leaves by the method suggested by Weatherley (1950). Fully expanded leaves were

collected and 8 mm discs were made and fresh weight of these were estimated and then

floated over distilled water in Petri dish for 6 h. At the end of this period, individual disc

were surface dried and saturated weight was recorded. Thereafter the samples were dried in

an oven (70 8C) for 24 h and dry weight was recorded. RWC was then calculated by the

suggested formula. For nutrient analysis, the foliar part (shoot) was sampled and subjected

to oven drying (65 8C) for 48 h. The samples were then ground and used for nutrient

analysis. Nitrogen was estimated using Kjeltec 2300 after digesting the samples in

digestion system (Foss-Tecator). Phosphorus was estimated by vando-molybdate colour

reaction method. Magnesium and iron were estimated by atomic absorption spectro-

photometer (GBC-Avanta PM) using nitrous oxide–acetylene and air–acetylene flame,

respectively.

2.6. Determination of biochemical status

After 60 days of acclimatization, histochemical analyses were made for leaf chlorophyll

and carotenoid contents as per the method suggested by Barnes et al. (1992). Fully mature

weighed leaf samples were immersed in dimethyl sulphoxide (DMSO) and incubated at

70 8C for 4 h. The absorbance of the solution was then read at 645, 663 and 480 nm using

spectrophotometer. The proline content in mature leaves was determined by rapid

colorimetric method proposed by Bates et al. (1973). Fresh plant material was

homogenized in sulphosalicylic acid and filtered (Whatman No. 2). Filtrate was reacted

(100 8C) with acid ninhydrin reagent and glacial acetic acid in a test tube for 1 h, and then

kept in an ice bath. The reaction was extracted with toluene and the chromophores

containing toluene and was aspirated from the aqueous phase, warmed to room

temperature and then absorbance was read at 520 nm. Total phenol in leaf samples was

assayed by the method proposed by Malik and Singh (1980). Shoot tips along with a pair of

freshly emerged leaves were taken instead of buds due to scarcity of material. Foliar

samples (approximately 500 mg) were extracted with 80% ethanol and the supernatant


collected were evaporated to dryness. Residues were dissolved in distilled water and to this

fresh Folin-Ciocalteau reagent and Na2CO3 (sodium carbonate) solution (20%) were

added, mixed thoroughly and placed in a hot water both (58 8C) exactly for 1 min. It was

then cooled to room temperature and then absorbance was measured at 650 nm.

The activity of the enzyme nitrate reductase (NR, E.C.1.6.6.1) in leaves was determined

by the methods described by Evans and Nansan (1953) and Hageman and Hucklesby

(1971). Fresh leaf samples were sliced (8–10 mm in diameter) and then thoroughly mixed

and placed in culture tubes containing ice-cold incubation medium consisting of each of

phosphate buffer (0.2 M, pH 7.5), potassium nitrate (0.2 M) solution and n-propanol. Tubes

were removed from the ice bath and evacuated with the help of vacuum pump for about 1–

2 min. After infiltration, tubes were incubated at 30 8C for 30 min in dark. Reaction was

terminated by keeping the tubes in boiling water for 2 min followed by cooling at room

temperature. Then, sulphanilamide solution was added to each tube and mixed well. Nitrite

formed by the catalytic action of the NR enzyme, was then estimated colorimetrically by

reading absorbance at 540 nm.

For estimation of enzyme polyphenol oxidase (E.C.1.14.18.1), foliar samples were

prepared according to the method suggested by Lerner et al. (1971) with slight

modifications. Polyphenol oxidase enzyme was extracted at 4 8C by macerating chopped

sample in pestle and mortar with 100 mM phosphate buffer of pH 7.3 containing sodium

ascorbate. Thereafter, the extract was filtered and treated for 20 min with 1.5% Triton X-

100 solution prepared in 100 mM phosphate buffer (pH 7.3). Final volume of extract was

made with 100 mM phosphate buffer of pH 7.3 containing 10.0 mM sodium ascorbate. It

was then centrifuged at 15,000 rpm for 1 h at 4 8C. The aliquot so obtained was used as

an enzyme source. Both catecholase and cresolase activities were measured employing

the method suggested by Sanchez-Ferrer et al. (1988) and Valero et al. (1989).

Catecholase activity was determined by using 4-methyl catechol 30 mM, in 10.0 mM

sodium acetate buffer (pH 4.5) as substrate. To crude polyphenol oxidase extract,

100 mM phosphate buffer (pH 7.3) was added. To this mixture, 30 mM 4-methyl

catechol made in 10 mM sodium acetate buffer (pH 4.5) was added at zero time. The

increase in absorption was measured spectrophotometrically by appearance of

corresponding 4-methyl o-benzoquinone at 400 nm at 30 8C. Then optical density

(OD) was recorded after 8 min. The enzyme activity was represented as D400 g�1 min�1

(change in absorbance per gram of fresh weight per minute). For measuring cresolase

activity, 0.5 mM 4-methylphenol ( p-cresol) made in 10.0 mM phosphate buffer of pH

7.0, was used as substrate as reported by Sanchez-Ferrer et al. (1988). To crude enzyme

extract, phosphate buffer (pH 7.0) was added. To this reaction mixture, 0.5 mM p-cresol

made in 10 mM phosphate buffer (pH 7.0) was added at zero time. Cresolase activity was

measured spectrophotometrically at 400 nm after 8 min. The cresolase activity was

represented as DA 400 g�1 min�1.

2.7. Experimental design

The experiments were laid out in a complete randomized block design with 15

replications. The percentage data were subjected to arcsinffiffiffiffi

%p

transformation before

subjecting them for ANOVA analysis.


3. Results

With respect to ex vitro survival mixed AMF strains gave the best result followed by A.

laevis. All the mycorrhizal treatments showed about two or more times higher ex vitro

survival than the control plantlets (Table 1). There was a significant variation with regard to

root colonization by the different AMF strains tried. A. laevis and mixed strains were found

to be significantly superior over others.

Perusal of the data presented in Table 1 on physiological status of plantlets, clearly

revealed that mycorrhizal treatments enhanced the relative water content of inoculated

plantlets compared to control. Significant differences in relative water content of leaves

were observed amongst the different treatments as AMF inoculated plantlets registered

about 11.28% gain over control. E. colombiana gave the best result with regard to relative

water content (97.25), while minimum in G. gigantea (93.45) inoculated plantlets.

The mycorrhizal inoculation of micropropagated grape plantlets significantly enhanced

their photosynthetic rate (Table 1). It was enhanced by several folds in inoculated plantlets

compared to control. Among the different treatments tried, G. manihotis inoculation

resulted in highest photosynthetic rate (6.95) of treated plantlets followed by A. laevis


Table 1

Effect of arbuscular-mycorrhizal fungi (AMF) inoculation on root colonization, survival and physiological status

of micropropagated grapevine plants (after 60 days acclimatization)

Treatment Ex vitro

survival (%)

Root

colonization (%)

Relative water

content (%)

Photosynthetic

rate (mmol m�2 s�1)

T0 45.30 9.83 g 85.62 e 2.25 d

T1 91.10 ab 97.97 a 94.16 cd 6.39 ab

T2 81.23 c 90.93 c 95.93 b 4.05 c

T3 83.17 b 83.97 e 97.25 a 3.97 c

T4 84.53 b 82.53 f 93.45 d 5.86 b

T5 82.67 b 86.40 d 96.48 ab 6.95 a

T6 87.00 b 93.00 b 95.26 bc 4.17 c

T7 91.60 a 98.13 a 94.73 c 6.32 ab

Column values followed by the same letter are not significantly different (P < 0.05).

Table 2

Influence of arbuscular-mycorrhizal fungi (AMF) inoculation on some growth parameters of micropropagated

grape plantlets (after 60 days acclimatization)

Treatment Plant height (cm) Root length (cm) Leaf no. Leaf area (cm2)

T0 18.40 f 24.20 h 24.00 f 30.12 g

T1 36.21 a 27.40 g 41.00 c 51.97 b

T2 26.65 e 30.47 f 28.00 e 32.87 f

T3 32.73 c 41.53 b 43.00 b 34.54 e

T4 29.03 d 35.70 e 34.67 d 36.14 d

T5 28.17 de 36.50 d 33.00 de 42.37 c

T6 34.98 b 38.40 c 47.00 a 34.64 e

T7 36.73 a 45.63 a 43.00 b 54.73 a


(6.39) and mixed strains (6.32). Irrespective of the strains and the parameters studied, all

the mycorrhizal treatments were significantly superior over control.

It was revealed from Table 2 that maximum plant height, root length and leaf area were

noted in mixed strains inoculated plantlets, while with respect to leaf no. S. heterogama

proved to be best mycorrhizal strain. Comparative growth of grapevine due to AMF

inoculation is presented in Fig. 1.

Mycorrhizal treatments helped in alleviation of different stresses experienced by the

micropropagated plantlets during hardening stage. Amongst the seven AMF treatments

tried, Acaulospora scrobiculata was proved to be best by increasing the tissue chlorophyll

content followed by S. heterogama. Irrespective of the strains used, the carotenoids were

significantly enhanced by the mycorrhization (Table 3). Maximum carotenoid content was


Fig. 1. Comparative growth of tissue culture raised plants of cv. Pusa Navrang due to mycorrhization (after 60

days acclimatization).

Table 3

Biochemical status of in vitro raised grape plantlets as influenced by arbuscular-mycorrhizal fungi (AMF)

inoculation (after 60 days acclimatization)

Treatment Total

chlorophyll

(mg g�1)

Total

carotenoids

Proline

(mg g�1)

Total

phenol

(mg g�1)

NR

activity

(nmol g�1 h�1)

Catecholase

activity

(D400 g�1 min�1)

Cresolase

activity

(DA 400 g�1 h�1)

T0 2.28 c 0.136 g 10.22 h 4.02 h 4.46 h 366.67 f 342.00 f

T1 2.60 b 0.159 d 39.48 a 40.27 a 30.16 a 389.67 e 367.00 e

T2 2.96 a 0.154 f 35.42 c 25.14 g 16.14 e 393.00 e 379.33 d

T3 2.65 b 0.166 c 38.20 b 28.33 d 14.13 g 505.00 c 365.00 e

T4 2.63 b 0.167 c 25.34 f 29.72 c 18.72 d 395.67 e 383.67 c

T5 2.70 a 0.169 b 29.33 e 28.18 e 19.14 c 705.67 a 390.33 b

T6 2.87 a 0.156 e 23.31 g 25.23 f 15.80 f 431.33 d 376.32 d

T7 2.64 b 0.178 a 32.97 d 32.44 b 25.03 b 624.00 b 397.00 a


exhibited by plantlets inoculated with mixed AMF strain (0.178) while, minimum in

plantlets inoculated with A. scrobiculata (0.154). Levels of proline in inoculated plantlets

were found two to three times higher than the non-inoculated control plantlets. There was

an increase of 386% in A. laevis inoculated plantlets. Data presented in Table 3 clearly

indicates that nitrate reductase (NR) activity differed significantly among the treatments in

the present study. Nitrate reductase activity was three to six times higher in inoculated than

non-inoculated plants. Maximum NR activity (30.16) was recorded in A. laevis inoculated

grape plantlets while, minimum (14.13) in those with inoculated with E. colombiana.

Mycorrhizal inoculation during acclimatization increased the phenol content in shoots by

around seven times as noted after 60 days. Plantlets inoculated with A. laevis recorded

maximum phenol content in shoots followed by mixed AMF strains. AMF inoculated

plantlets exhibited higher level of catecholase and cresolase activities. The catecholase and

cresolase activities were 134 and 111% higher in treated plantlets than the non-mycorrhizal

control.

Macronutrients like N, P and Mg were significantly enhanced by mycorrhizal

inoculation. Mixed AMF strains inoculated grape plantlets accumulated more than two

times P compared to control, while plantlets inoculated with E. colombiana recorded

maximum nitrogen content followed by A. laevis. With regard to Mg content in shoots,

plantlets inoculated with G. manihotis gave the best result.

4. Discussion

In our study the AM fungi inoculation significantly increased the number of surviving

plantlets, a result which is in line with the earlier findings of Gaur and Adholeya (1999),

Estrada-Luna and Davies (2001) and Marin et al. (2003) in micropropagated Syngonium,

prickly pear and persimmon plantlets, respectively. It is evident from the data that the

percentage of root colonization of host plantlets varied among the different AMF

treatments tried (Table 1). The differences in AMF colonization frequency could be

attributed to the differences in mycorrhizal dependency among the host plants and to

abiotic factors (Yano Melo et al., 1999). Maximum root colonization was recorded in

plantlets inoculated with mixed AMF strains followed by A. laevis. Superiority of mixed

culture may be attributed to that of existing compatible AMF communities. Furthermore,

when plants are colonized by more than one AMF isolates, preference of host for specific

isolates of the community is noted (Johnson et al., 1991).

Significant increase was observed in all the growth parameters studied. This result is in

conformity with findings of Azcon-Aguilar et al. (1994) and Estrada-Luna and Davies

(2001, 2003) for cherimoya, prickly pear cactus and Chile ancho pepper, respectively.

Recently, Zemke et al. (2003) obtained similar findings in grape rootstocks.

All the AMF treatments were significantly superior over non-inoculated control for

most of the parameters studied. However, different AMF strains varied in their efficacy to

increase the synthesis of different biochemicals, thereby improving the plantlet survival.

These differences may depend on the genetically controlled physiological characters of the

fungus, which play a role in the uptake of nutrients from the soil and also their transfer to

the host root cells (Schubert et al., 1990).


Significant differences in relative water content of leaves were observed amongst the

different treatments tried. The increased RWC in mycorrhizal plantlets might be due to an

improvement of the water uptake by mycorrhizal root system through extra-radical phase

(Ruiz-Lozano and Azcon, 1995). In addition, increased water transport could also be

attributed to improvement in P nutrition. This result is in conformity with the earlier

findings of Graham and Syvertsen (1984). In a study with micropropagated mycorrhizal

strawberry plantlets, Hernandez-Sebastia et al. (1999) suggested that the higher

concentration of water soluble compounds in plant tissues could be a reason for higher

RWC of whole plantlets.

Carbon dioxide assimilation is one of the key processes, which is severely hampered

when plants are subjected to moisture deficit stress. The enhanced photosynthetic rates in

inoculated grape plantlets suggest that mycorrhizal plantlets may be able to assimilate

more CO2 and thereby accumulate more biomass. This implies more vegetative growth and

ultimately leading to increased plantlet survival even under unfavourable ex vitro

environmental conditions. Earlier, Mathur and Vyas (1995) observed a significant increase

in photosynthetic rate of AMF inoculated Ziziphus nummularia seedlings. The improved

physiological status owing to rapid AMF colonization helps plants in early recovery during

the course of acclimatization and attains vigorous growth leading to higher biomass

accumulation in mycorrhizal plants compared to control post-acclimatization (Estrada-

Luna and Davies, 2003).

The mycorrhizal inoculation of micropropagated grape plantlets significantly enhanced

chlorophyll content in leaves. The enhanced chlorophyll level might be responsible for

increased photosynthesis in inoculated plantlets as observed in the present study (Table 3).

This can further be attributed to increased Mg and Fe uptake, which are essential for

chlorophyll bio-synthesis (Fig. 2C and D). Other pigments like carotenoids function as

light-harvesting pigments which contribute to the process of photosynthesis. In addition,

they serve as antioxidants by scavenging the free radicals in plant system and protecting

them from being damaged by oxidative stress and diseases. Significant variations among

the different AMF treatments for carotenoid content were observed and all the AMF

treatments were significantly superior over control. The increased carotenoid content in

mycorrhizal plantlets implies their greater ability to resist stressful ex vitro environmental

conditions as compared with non-inoculated control plantlets.

One of the best known response of plants to salt, drought and other stresses is the

accumulation of soluble, low molecular mass solutes such as proline (Paleg et al., 1984).

Levels of proline in inoculated plantlets were found two to three times higher than the non-

inoculated control plantlets. Higher level of proline in AMF inoculated plantlets is

desirable as proline protects the different enzyme systems against dehydration caused by

moisture stress (Paleg et al., 1984). Furthermore, the higher proline accumulation favours

the plants in maintaining the osmotic balance and preventing dehydration of tissue, thereby

helping them to grow normal even under stressful conditions.

Nitrate reductase (NR) belongs to oxido-reductase group of enzymes, which catalyzes

the reduction of NO3� to NO2

�. Studies have indicated that NR activity falls quite sharply

during stress and leads to poor nitrogen assimilation and consequently resulting in poor

growth and survival of plants during transplanting. Perusal of data presented in Table 3

clearly indicates that NR activity differed significantly among the treatments in the



Fig. 2. Nutritional status of micropropagated grape plantlets as affected by mycorrhization (after 60 days

acclimatization).

present study. Nitrate reductase activity was three to six times higher in inoculated than

non-inoculated plants. The enhanced activity of NR in AMF inoculated plants of onion and

lettuce were earlier reported by Ruiz-Lozano and Azcon (1996) and Azcon and Tobar

(1998). The enhanced level of enzyme nitrate reductase as a result of mycorrhizal

inoculation could be due to improved P uptake by successful AMF symbiosis (Fig. 2B).

This observation is in conformity with the earlier findings of Cliquet and Stewart (1993).

Higher nitrogen content in foliar parts of treated plantlets (Fig. 2A) could be attributed to

increased NR activity. Similar result was obtained by Mathur and Vyas (1995), who found

increased soluble protein levels in AMF inoculated seedlings of Ziziphus nummularia.

During hardening, the tissue culture derived plants are not only in stress but also an array

of pathogen spores are in the process of disease causation. Phenols and enzymes such as

polyphenol oxidase are important components of plant defense mechanism against the

diseases. Phenolic compounds occur naturally in plant system and owing to their anti-

microbial properties inhibit fungal spore germination and toxin production by pathogens

(Vidhyasekaran, 1973). Tang et al. (2000) reported that there was a significant increase in

the level of phenolic compounds in the bark of poplar plants inoculated with G. mosseae

showing resistance. The increased level of total phenols in the present investigation

suggests increased resistance in inoculated plantlets against diseases, which led to

increased plantlet survival under glasshouse as well as in field conditions. The higher level

of phenols observed in inoculated plantlets could also be attributed to the increased PPO

activity in plants (Mathur and Vyas, 1997; Tang et al., 2000), which is an oxidizing enzyme

of polyphenols converting them into quinones, which are toxic to pathogens. AMF

inoculated plantlets exhibited higher level of catecholase and cresolase activities. There

was a marked increase in the levels of catecholase and cresolase activities in treated

plantlets than the non-mycorrhizal control. This result is in agreement with the findings of

Tang et al. (2000), Nelson and Achar (2001) and Panwar and Vyas (2002), where they

observed increased PPO activity in AMF inoculated plants. The activity of catecholase was

higher than cresolase, which could be attributed to the longer lag period and greater

instability of cresolase (Sanchez-Ferrer et al., 1988; Valero et al., 1989).

In the present investigation, the mortality of plantlets subjected to hardening was

primarily because of rot causing organisms. The incidence was very high in control

plantlets, while very low mortality owing to rotting was observed in inoculated plantlets.

These observations could be attributed to direct effect of phenol and PPO activities on

inoculated plantlets against rot causing micro-organisms. Additionally, it could be

attributed to high percentage of root colonization in mycorrhizal plantlets (Table 1), which,

implies that sites for entrance of microorganisms into the roots are already occupied, i.e.

the basis of potential resistance against pathogenic microorganisms (Gianinazzi et al.,

1990). Yao et al. (2002) also observed that the AM fungi inoculation reduced the extent of

disease caused by Rhizoctonia solani in micropropagated potato plantlets.

The results with root colonization of tissue cultured grape plantlets inoculated with

six individual and mixed arbuscular-mycorrhizal strains have provided conclusive

evidences that AMF are potential inoculants for averting transplantation shock

experienced by such plantlets during acclimatization under glasshouse conditions.

This study suggests that such an association brought about gamut of changes especially

in biochemical status of plants like enhancement in the levels of chlorophyll, carotenoids,


proline, phenol and enzymes like polyphenol oxidase and nitrate reductase, which are

necessary to mitigate adverse effects of transplanting shock and enhancing ex vitro

survival.

Acknowledgements

Authors are grateful to the Indian Council of Agricultural Research (ICAR), New Delhi

for providing the financial assistance in the form of World Bank funded National

Agricultural Technology Project. The senior author is thankful to ICAR, New Delhi for the

grant of Junior Research Fellowship.

References

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