“STANDARDIZATION OF IN-VITRO PROPAGATION PROTOCOL … › 6906 ›...

i

“STANDARDIZATION OF IN-VITRO PROPAGATION

PROTOCOL FOR CHIRONJI (BUCHANANIA LANZAN

SPRENG)”

M.Sc. (Ag) Thesis

By

INDRAPAL PATEL

DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND

BIOTECHNOLOGY

COLLEGE OF AGRICULTURE RAIPUR

FACULTY AGRICULTURE

INDIRA GANDHI KRISHI VISHWAVIDYALAYA RAIPUR

(CHHATTISGARH)

2018

ii

“STANDARDIZATION OF IN-VITRO PROPAGATION

PROTOCOL FOR CHIRONJI (BUCHANANIA LANZAN

SPRENG)”

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur

By

INDRAPAL PATEL

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR

THE DEGREE OF

MASTER OF SCIENCE

In

Agriculture

(Plant Molecular Biology and Biotechnology)

Roll No.120115119 ID No.20151622533

JANUARY, 2018

v

ACKNOWLEDGEMENT

My first and foremost gratitude to my beloved parents and my family

members for their encouragement, sincere prayers, expectations and blessings

which have always been the most vital source of inspiration and motivation in my

life.

It is a pleasure to thank those who helped me to complete this work. First

and foremost, I would like to thank my major advisor, Dr. (Smt.) Zenu Jha

Associate Professor, Department of Plant Molecular Biology and Biotechnology;

IGKV, Raipur for his thoughtful guidance, supervision and consistent support to

my studies and research.

I wish to record my sincere thanks to Dr. S. K. Patil Hon’ble Vice

Chancellor, Dr. O. P. Kashyap Dean, Dr. S. S. Rao Director Research Services

and Dr. S. S. Shaw, Director of Instructions, Dr. Madhav Pandey Librarian IGKV,

Raipur for their help both administrative and technical which facilitated my

research work.

I would like to express my cordial appreciation to Dr. S. B. Verulkar,

Professor and Head; Dr. Girish Chandel, Professor; Dr. (Smt.) Shubha Banerjee

Assistant Professor; Dr. (Smt.) Archana S. Prasad Assistant Professor; Dr. (Smt.)

Kanchan Bhan Assistant Professor, Department of Plant Molecular Biology and

Biotechnology, IGKV, Raipur; Dr. (Smt.) Shubha Banerjee Assistant Professor ,

Department of Plant Molecular Biology and Biotechnology, , IGKV, Raipur for

their valuable suggestions and providing necessary facilities in completing the

research work.

I express my deep sense of gratitude and sincere thanks to my advisory

committee member Dr. R, R. Saxsena Professor, Department of Agriculture

Statistics; Dr.A.S.Kotesthane Professor and head Department of plant pathology

and Dr.Dhananjay Sharma Associate professor department of Horticulture

(Vegetable Science), College of Agriculture, Raipur for their valuable suggestions

critical comments and help rendered as and when needed.

My respected seniors Arpita mahobia madam,Triveni madam, Pratik

sir,Deepak Jha sir, Sujata madam , , Ajit Mannade sir, Vikrant sir, Sanjay sir, Ashish

vii

TABLE OF CONTENTS

Chapter Title Page

ACKNOWLEDGEMENT I

TABLE OF CONTENTS Iii

LIST OF TABLES Vi

LIST OF FIGURES Vii

LIST OF PLATES Viii

LIST OF NOTATIONS Ix

LIST OF ABBREVIATIONS X

ABSTRACT Xi

I INTRODUCTION 1

II REVIEW OF LITERATURE 5

2.1 Chironji (Buchanania lanzan Spreng) 5

2.1.1

2.1.2

Botanical description

Uses and economical importance of Chironji

5

6

2.1.3 Constraints in conservation and propagation of Chironji 7

2.1.4 Conservation methods adapted for Chironji 7

2.2

In-vitro propagation of Chironji and other woody tree

species

8

2.2.1 In-vitro propagation of Chironji 9

2.2.2 In-vitro propagation of woody tree species 10

2.2.2.1 Criteria for selection of explants for micro-propagation of

woody tree species

10

2.2.2.1.1 Nodal segments as explants for micro-propagation 10

2.2.2.1.2 Shoot tip as explants for micro-propagation 11

2.2.2.1.3 Inter-cotyledonary region as explants for micro-propagation 11

2.2.2.1.4 Root as explants for micro-propagation 12

2.2.3 Artificial media used for micro-propagation of woody tree

species

13

2.2.4 Effect of growth regulators on organogenesis under in-vitro

conditions

15

2.2.4.1 Effect of growth regulators on in-vitro shoot initiation 15

2.2.4.2 Effect of growth regulators on in-vitro root initiation 17

III MATERIALS AND METHODS 20

3.1 Media preparation 20

3.1.1 Preparation and storage of stock solutions 20

3.1.2 Preparation of Culture Medium 20

3.1.3 Preparation of stock solution of growth regulator 22

3.2 Sterilization 22

3.2.1 Glassware sterilization 22

viii

Chapter Title Page

3.2.2 Sterilization of tissue culture media 23

3.2.3 Sterilization of working area 23

3.2.4 Sterilization of tools 24

3.2.5 Sterilization of laboratory and culture room by weekly

fumigation

24

3.3 Plant materials used and source of explants for in-vitro

propagation of Chironji

24

3.3.1 Source of explants 24

3.3.2 Age of the plant used 24

3.4 Collection, sterilization and preparation of explant for

Inoculation

25

3.4.1 Explant E-1 and E-2 25

3.4.1.1 Collection of E-1 and E-2 25

3.4.1.2 Sterilization of E-1 and E-2 25

3.4.1.3 Preparation of E-1 and E-2 26

3.4.2 Explant E-3 26

3.4.2.1 Collection of E-3 26

3.4.2.2 Sterilization of root to raise Explant E-3 26

3.4.3 Preparation of E-3 26

3.4.4 Explant E-4 26

3.4.4.1 Collection of E-4 27

3.4.4.2 Sterilization of E-4 27

3.4.4.3 Preparation of E-4 27

3.5 Experimental methods 27

3.5.1 Sterilization of explants 27

3.5.2 Inoculation of different types of explants for culture

initiation and multiple shoot initiation

27

3.5.3

3.5.4

3.5.5

3.5.5.1

Cultural conditions and care during inoculation

Sub culture

Rooting

In-vitro Rooting

28

28

28

28

3.5.6 Observations and data collection 29

3.5.6.1 Number of days required for shooting 29

3.5.6.1.1 Number of shoots produced per explants 29

3.5.6.2 Mean length of shoots (cm) 29

3.5.6.3

3.5.6.4

Number of days required for initiation of roots

Mean number of roots

29

29

3.5.7 Statistical analysis of data 30

IV RESULTS AND DISCUSSION 31

4.1. Selection of potential explants for in-vitro micropropagation

of Chironji (Buchanania lanzan).

32

ix

Chapter Title Page

4.2 Standardization of protocol for multiple shoot induction 32

4.2.1 Shoot initiation percentage (%) in different explants of

Chironji

36

4.2.2

4.2.3

4.2.4

4.2.5

4.3

4.3.1

4.3.2

4.3.3

Number of days for initiation of shoots

No of shoots initiated

Shoot length (cm)

Shoot multiplication among different treatment combinations

Standardization of protocol for root initiation %

Root initiation percentage (%)

Number of days for initiation of roots

Total root initiated

Total shoots inoculated

36

38

38

39

42

43

43

44

SUMMARY AND CONCLUSIONS 45

REFERENCES 49

VITA 59

x

LIST OF TABLES

Table Title Page

3.1 Stock solutions of WPM (Lloyd and Mc Cown, 1980) 21

3.2 Stock solutions of MS medium (Murashige and Skoog, 1962) 21

3.3 Stock solutions of hormones 22

3.4 Different explants used for in-vitro propagation of Chironji 25

3.5 Different surface sterilization treatments for Chironji explants 26

3.6 Different treatments used for multiple shoot initiation in Chironji 28

3.7 Different treatments used for in-vitro rooting in Chironji 29

4.1 Culture response of different explants in different treatment

combinations

32

4.2 Influence of different treatments in different explants on shoot

initiation (%) among different replications

33

4.3 Influence of different treatments in different explants on shoot

initiation(%)

33

4.4

Different treatment combinations used on different parts for

shooting attributes

37

4.5 Shoot multiplication among different treatment combinations 39 4.6 Effect of different treatments on root initiation % from the explant

shoot tip of chironji

42

xi

LIST OF FIGURES

Figure Title Page

4.1 Graphical representation of Influence of different treatments

in different explants on shoot initiation(%)

34

4.2 Graphical representation of Influence of different treatments

in different explants on shoot initiation (%)

34

4.3 Graphical representation of Different treatment combinations

used on different parts for shooting attributes

37

4.4 Graphical representation of Shoot multiplication among

different treatment combinations

39

4.5 Graphical representation of Effect of different treatments on

root initiation % from the explant shoot tip of Chironji

43

xii

LIST OF PLATE

Plate Title Page

1 Picture showing culture response of different explant 35

2 Shoot multiplication % among different treatment

combinations

40

3 Showing different treatments on root initiation % 41

xiii

LIST OF NOTATIONS/SYMBOLS

% Percentage

1N One normality

2,4-D 2, 4-dichlorophenoxyacetic acid

BAP 6-benzylamino purine

ABA Abscisic acid

Est (Latin; for instance)

HCl Hydrochloric acid

IAA Indole-3-acetic acid

IBA Indol buteric acid

KN Kinetin

Ls Linsmaier and Skoog (1965) basal medium

Lux Unit of illumination

MS Murashige and Skoog (1962) basal medium

N6 Chu (1978) callus induction media

NAA a-naphthalene acetic acid

NaOCl Sodium hypochloride

Μm Micro meter

cm Centimeter

pH Negative logarithm of hydrogen ion concentration [-log(H+)]

xiv

LIST OF ABBREVIATIONS

µl Microliter

Al Aluminium

Fig. Figure

g/L gram per liter

l Litre(s)

m, Metre

mg Milligram

µM Millimolar

Min Minute

Ml Millilitre

No. Number

SN Serial number

Psi pound per square inch

H Hours

uv Ultraviolet light

v/v Volume by volume

w/v Weight by volume

16

Shoot tip when used a explant showed best response over leaf, root and node explant and

maximum shoot initiation % was obtain in the treatment S1- ½ WPM + BAP (2.0 mg/L) + GA3

(0.5 mg/L) + kn 1.0mg/l.

In-vitro propagation techniques may further be improved for commercial production of

planting materials from elite high yielding lines.

18

nks lseh- vxz “kkdh; dks fy;k] lHkh drZdksa dks futhZohdj.k fd;k x;k x;k A fQj mipkfjr drZdksa

dks lao/kZu cksry ek/;e vkSj uyh ek/;e esa Mkyk x;k ]5&6 lIrkg ckn lw{e “kkd dks nwljs ek/;e esa

Mkyk x;kA vf/kdre “kkd E1S1 esa 28-33 izfr”kr~ o cgq “kkdh; o`f) vf/kdre vkSlr 5-5 izfr”kr

E1S1 esa fn[kk A R6 mipkj ek/;e esa vf/kdre 25 izfr”kr~ o U;wure 0-00 izfr”kr~ R1 esa tM+ks dk

cuuk ik;k x;k A lHkh drZdks esa “kh’kZ “kkd]uksM]tM+ o ;qok ifRr es] “kh’kZ “kkd dk izn”kZu lcls vPNk

jgk vkSj vf/kdre “kkd fudyus ds fy, S1 ½ WPM + BAP (2.0 mg/L) + GA3 (0.5 mg/L) + kn

1.0mg/l. mipkj ek/;e lcls vPNk jgkA bu foVªks rduhd ls vkxs fof”k’V oxZ vkSj vf/kd mit

nsus okyh ikS/k lkekxzkh dk O;kolkf;d mRiknu fd;k tk ldrk gSA

19

CHAPTER - I

INTRODUCTION

Buchanania lanzan Spring (Chirounji),a member of family anacardiaceae is a useful tree species

that has great medicinal value. It is commonly known as Char, Achar, Charoli and Priyal. It is an

excellent fruit tree of agro-forestry and social forestry. It is growing under forest condition at present as

an under exploited fruit crop and gives monitory reward to the tribal community of the count yard

seems to be boon for them. It is valuable species found in dry deciduous forest through out the country

excluding eastern Himalayan forests (Singh,1982), According to Cataloge of Life (Hessler, 2015),

accepted the botanical name of Chironji is Buchanania cochinchinenesis (Lour.) Almeida. The trees are

found in farmlands as well as widely scattered in forests and hence it takes lot of time and patience to

collect significant amount of Chironj to process for selling. Seven species of Buchanania have been

reported in India of which two B. lanzan (Syn. B. latifolia) and B. axillaries (Syn. B. angustifolia) produce

edible fruits. B. lanceolata is an endangered species. It is found in the evergreen forests of Kerala. B.

platyneura is found in Andaman only. Other species of the genus are B. lucida, B. glabra and

B.accuminata.

Economic importance

Fresh fruit are eaten raw having pleasant, sweetish, sub-acid flavor and consumed by local

people and also sold in the village market. Chironji is mainly regarded for its costly, high-priced kernels.

These kernels has almomd like flavor, eaten raw or roasted form, used as cooking spice and dry fruit in

sweets, kheer, meaty korma in India. Chironji seeds are rich in nutrients and medicinal properties.

Chironji is an active source of phenolics, natural antioxidants, fatty acids and minerals. Its seed oil is

used to treat skin diseases, remove spots and blemishes from the face.

Chironji is a source of income for tribal people of Chhattisgarh and other states It is

backbone of their economy. A considerable reduction in the population of Chironji in the forest

and non-forest areas has been recorded (Singh et al.,2002) and facing a severe threat of extinction. Due

to this, Chironji is categorized under the 195 red listed medicinal plant species of Indian origin, that

requires conservation measures as reported by Foundation of Revitalization of Local Health Tradition

20

(FRLHT), Environmental Information System (ENVIS)- Centre on Medicinal Plants, Bangalore, Govt. of

India.

Origin and distribution

It seems to have been originated in the Indian sub-continent. The tree is almost evergreen and

grows naturally in the tropical dry deciduous forests of Northern, Western and Central India, mostly in

the states of Chhattisgarh, Jharkhand, Madhya Pradesh, Uttar Pradesh, Maharashtra, Bihar, Orissa,

Jharkhand, Andhra Pradesh and Gujarat. Chironji is widely distributed in all parts of Chhattisgarh and

rich diversity is available throughout the state. According to botanical classification of different plant

species, 1525 medicinal species are found in the state. These species comes under 911 genera and 196

families, among which 19.3 per cent (294) are tree species. Occurrence of Chironji in Chhattisgarh is

concentrated mainly in Sal region comprising Bastar, Dantewada, Kanker, Kondagoan, Raipur and

Sarguja.

Present status in India

Information regarding the area and production of this fruit in India is not available because it is

not grown on plantation scale and limitation in forest areas. The production in india is mainly

concentrated in the drier states and the produce is collected by the villagers and sold in the local

market. It cultivation may spread to semi-arid areas, resource poor areas and wastelands. The collection

and sell of nationalized forest produce is done by CG MFP Federation only. This agency has network for

the collection of superior quality Non-Nationalized Non-Wood Forest Produce (NWFP). Buchanania

lanzan (Chironji) is non-nationalized Non-Wood Forest Produce in Chhattisgarh. Estimated annual trade

of Chironji is 5000-10000 MT per year at national level (Ghosh and Shrivastava, 2014). Its price varies

and depends on its size. Average price of Chironji ranges from Rs. 500 to 750 per kg (Anon., 2015).

Demand of Bastar Chironji is high in national market and now it becomes a rare commodity and fetches

higher prices more than Rs. 1000 per kg (Dulhani, 2013). According to CGMFPF, Chhattisgarh have

51,200 quintal per year production potential of Chironji valued for Rs. 44.29 cores contributing more

than 50 per cent of national production (Anon, 2015). Chironji kernels also exported to many Asian and

European countries (www.zauba.com/export-Chironji).

Soil and climate

21

Chirounji is very hardy plant and thrives well on rocky and gravelly red soils. Through it is very

hardy tree but plants do not survive under waterlogged conditions. Well drain deep loam soil is ideal. It

prefers tropical and subtropical climate and can withstand drought admirably.

Chhattisgarh State is rich in forest wealth and 44.2 per cent of its geographical area is covered with

forest (Anon., 2015 ).

Problems

The seeds are the major source of regeneration of Chironji in India. The major problem in the

reforestation or domestication of Chironji is the low percentage germination of seeds due to hard seed

coat, recalcitrant in nature and fungal contamination associated with the storage of seeds. Fungal attack

by Fusarium spp., humidity and high temperature are also conducive to fungal contamination. Seeds

lose viability soon even after 3 months of harvesting. The seeds exposed to sunlight fail to germinate

and lose their viability, presence of a hard seed coat which leads to low germinating capability.

Vegetative propagation methods like chip budding (Tewari and Bajpai, 2000) and softwood grafting

(Singh and Singh, 2014) are also standardized and reported in Chironji. But these are less effective due

to loss availability of rootstocks and dependency on seasonal conditions. Moreover, propagation

through root cutting is a very slow process (Singh et al., 2002).

22

In-vitro propagation

In this regard, biotechnology can play an important role and a boon for conservation of these

important plant species. Micro-propagation is the technique of in-vitro multiplication of large number of

plants from its part, whether it is leaves, seeds, shoot tips, nodes and roots etc. In the recent years,

tissue culture has emerged as a promising technique to obtain genetically pure elite populations under

in-vitro conditions and limited space . It is a fast and dependable method for production of a large

number of plantlets in a short time. Moreover, the plant multiplication can continue throughout the

year irrespective of season and the stocks of germplasm can be maintained for many years.

In India, micro-propagation techniques have been standardized for many temperate, tropical

and sub-tropical woody fruit crops by different institutes. Scientists in India and different parts of world

have been trying hard for the micro-propagation of fruit trees and perennial forestry plants through

tissue culture but the success has been limited due to recalcitrance and browning of explants. Previous

work on many forestry and woody tree species provided robust knowledge about need of biotechnology

approaches, factors affecting, constraints and achievements. However, to effectively utilize the

technique in achieving desired objectives, there is the need to understand the critical factors affecting it.

The factors are, however, interrelated and must be employed in appropriate combinations to achieve

the desirable goals.

Keeping in view the above facts, the research was planned under the title “Standardization of in-

vitro propagation protocol for Chironji (Buchanania lanzan Spreng)” to conserve the species from

extinction with the following objectives:

1. Selection of potential explants for in-vitro micropropagation of Chironji (Buchanania lanzan Spreng).

2. Standardization of protocol for multiple shoot induction.

3. Standardization of protocol for root initiation.

23

CHAPTER - II

REVIEW OF LITERATURE

Buchanania lanzan Spreng. (Chironji) is a socio-economically important underutilized

fruit tree species, belonging to family Anacardiaceae. It is locally known as ‘Char’, ‘Achar’,

‘Charoli’ and ‘Chawar’ by tribal people of different Indian states. It is believed to have

originated in the Indian sub-continent (Zeven and de Wet, 1982). The tree is found as natural

wild in the north, west and central India mostly in the states of Madhya Pradesh, Bihar, Orissa,

Andhra Pradesh, Chhattisgarh, Jharkhand, Gujarat, Rajasthan and Maharashtra (Malik et al.,

2010). Recently, it has attracted the attention of research workers due to its high price and very

fast depletion of the species from its natural zone. But very lesser information is available on this

species. In-vitro clonal propagation studies have been reported in economically important fruit

crops, medicinal trees and forestry tress, but not much work has been done on in-vitro

propagation of Chironji in particular. Hence, the present review deals with the information

about Chironji and other woody perennial trees to cover various aspects influencing in-vitro

shoot proliferation, in-vitro rooting and hardening of plantlets on following two points.

2.1 Chironji (Buchanania lanzan Spreng)

2.1.1 Botanical description

It bears fruits each containing a single seed, which is used as an edible nut. It has thickly

leathery leaves which are broadly oblong, with blunt tip and rounded base. Leaves have 10-20

pairs of straight, parallel veins. The tree sheds its leaves for a very short period during May-June

under subtropics. Pyramidal panicles of small bisexual greenish white flowers appear in

auxiliary and terminal panicles during early spring in January- March. A single panicle bears

about 3000-5000 flowers. When buds start growing externally, it takes about 18-28 days to

anthesis. Fruit set is around 3 per cent. Fruits ripen during April and they continue to ripen till

May. At ripening stage pericarp of fruits changes its colour from green to purple. Fruits remain

on the tree for quite longer. Fruits are drupe, ovoid or globose, black, 8-12 mm in diameter

with hard stones. Unripe fruits are green in colour (Singh and Singh, 2014).

2.1.2 Uses and economical importance of Chironji

24

All parts of this plant root, leaves, gum, bark and fruits have various medicinal

applications. Chironji seeds oil is also used to treat skin diseases, remove spots and blemishes

from the face. The methanolic extract of Chironji kernel exhibited anti-inflammatory activity.

Seeds are also medicinally valuable; contribute in ayurvedic and unani medicine as a nervine

tonic, anticough and antileprotic. Cell mediated immunity (CMI) and humoral immunity was

significantly stimulated by Chironji kernel. Methanolic leaves extract of Chironji possess

antidiabetic, antihyperlipidemic and antioxidant activity. Ethanolic and methanolic extract of

Chironji roots has shown good anti-diarrheal activity and significant wound healing activity,

respectively (Khatoon et al., 2015).

Local people also earn money by collecting gum/resins and lac by rearing kussumi

strain of lac on the Chironji tree. During summer, when green fodder becomes unavailable, local

inhabitants use its leaf as green fodder for their animals, especially buffalo, goat and sheep. Its

dried wood is utilized as a fuel. The timber of Chironji is slightly resistant to termite and is

utilized for making furniture, boxes and crates, desks, fine furniture, match boxes, mill work,

moulding, packing cases, stools, tables and agricultural implements. It is a good species for

growing over bare hill slopes (Sharma, 2012).

It is a life support and medicinally important tropical tree species and a significant

source of livelihood for local tribal population. Almost all the parts of this plant are used

for the treatment of various disorders. Bark or leaf paste of Chironji and Diospyros melanoxylon

mixed with a glass of water is given twice daily to treat snakebite (Shukla et al., 2001).

Ointment prepared from the kernel is used to relieve itch and prickly heat. The gum from the

bark is used for treating diarrhoea and pains, while leaves are used for the treatment of wound

and skin diseases (Kala 2009; Puri et al., 2000).

2.1.3 Constraints in conservation and propagation of Chironji

Genetic diversity of Chironji is facing severe genetic erosion as a result of large scale

urbanization and developmental activities undertaken in the tribal inhabited areas of states

holding natural population of this species (Singh, 2007). Local people do not prefer to grow this

species in their fields or home gardens and prefer to exploit the natural wild population for

25

various commercial uses. Consequently, the natural populations existing in the forests and

marginal lands of Chironji are facing a severe threat of extinction.

The major problem in the reforestation or domestication of Chironji is the low percentage

germination of seeds due to hard seed coat, recalcitrant in nature and fungal contamination

associated with the storage of seeds. Moreover, the fungal attack by Fusarium sp. (wilting

disease) is common after sowing the seeds in soil. The seedlings are also attacked by Fusarium

monililforme var. subglutinans Wr. and Rg., F. semitectum Berk & Rav. present in soil. Other

fungi which occur most frequently include Alternaria alternate (Pr.) Keissler, Aspergillus flavus

Link, A. ochraceus Withelm., A. niger Van Tiegh., A. aculeatus Lizuka, A. funiculosus Smith,

Cladosporium Link ex Fr., Chaetomium globosum Kunze and Schm., Curvularia lunata

(Wakker) Boedijn, Macrophomina phaseolina Ashby, Mucor varians Povah, Penicillium

citrinum Thom., Trichothecium roseum Link., Rhizopus arrhizus and Verticillium species

(Sharma et al.,1998).

Humidity and high temperature are also conducive to fungal contamination. The seeds

exposed to sunlight fail to germinate and soon lose their viability (Shende and Rai, 2005).

2.1.4 Conservation methods adapted for Chironji

As far as conservation of genetic diversity of Chironji is concerned, both in-situ and ex-

situ approaches should be used. In the present scenario, most appropriate strategy for Chironji

germplasm conservation is to adopt immediate ex-situ conservation (i.e. field genebank and

cryobanking) complemented with in-situ conservation (In-situ on-farm conservation and in

protected areas such as National Parks) for this species. Ex-situ field genebanks are presently

being established at horticulture research institutes of Indian Council of Agricultural Research at

Godhra, Gujarat and Lucknow, Uttar Pradesh for conservation and developing advance

propagation methods. Collected germplasm has been cryostored as base collection representing

sizable diversity in the form of 127 accessions in the National Cryogene bank at NBPGR, New

Delhi for posterity and future utilization (Malik et al., 2012)

2.2 In-vitro propagation of Chironji and other woody tree species

The in-vitro propagation technique dates back to 1902 when Haberlandt predicted the

26

totipotency of plant cells, i.e., the ability of a plant cell to develop into a complete plant.

Efforts to demonstrate totipotency led to the development of tissue culture. Major

breakthrough in plant tissue culture was seen after Skoog and Miller (1957) putforth the

concept of hormonal control of organ formation and showed that differentiation of roots and

shoots was a function of relative concentration of auxin and cytokinin in the medium. The

early work of Morel (1964) on in-vitro propagation of orchid provided the stimulus for

propagating the ornamental species through tissue culture.

Development of nutrient media for tissue culture of tobacco by Murashige and Skoog

(1962) was a great achievement, which is now being used for most of the species. Murashige

(1974) was instrumental in giving the techniques of in-vitro culture, the status of

viable practical means for rapid and mass propagation of horticultural crops. He also

described the concept of developmental stages of micropropagation.

Main commercial application of tissue culture has been in the production of clonal

plants at very rapid rates compared to that of conventional methods. Numerous herbaceous

plants have been propagated successfully in-vitro. However, the number of woody trees,

shrubs and coniferous trees, which commercially micropropagated is limited, because

of problems like bacterial contamination, vitrification, browning of explants due to

phenolic exudation, acclimatization of plantlet (Krishna and Singh, 2007; Chauhan and

Kanwar, 2012). However, in-vitro clonal propagation studies have been reported in

economically important fruit crops, medicinal trees and forestry tress, but not m uch work has

been done on in-vitro propagation of Chironji in particular. Hence, the present review deals

with the other woody perennial trees to cover various aspects influencing in-vitro shoot

proliferation, in-vitro rooting and hardening of plantlets.

2.2.1 In-vitro propagation of Chironji

Sharma et al. (2005) developed a protocol for somatic embryogenesis and plantlet

regeneration of Chironji (Buchanania lanzan) by immature zygotic embryos cultured on

Murashige and Skoog (MS) medium supplemented with various combinations of 2,4

dichlorophenoxy acetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthalene acetic acid

(NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures

27

grown on MS medium fortified with 4.53 µM 2,4-D, 5.32 µM NAA and 4.48 µM BA. The

medium supplemented with 15 µM abscisic acid (ABA) was most effective for maturation and

germination of somatic embryos.

Shende and Rai (2005) claimed to develop a tissue culture technique for the rapid clonal

multiplication of Chironji. They reported multiple shoot initiation in decoated seeds cultured on

MS medium enriched with various concentrations of auxins and cytokinins alone or in

combination. Murashige-Skoog (MS) medium supplemented with 22.2 µM of BAP and 5.37 µM

of NAA promoted formation of the maximum number of shoots. Furthermore, MS medium

containing 23.3 µM kinetin induced profuse rooting of the initiated shoots.

Niratker (2016) studied in-vitro multiple shoot induction from shoot tips and nodal

segments explants of Chironji in half strength MS medium supplemented with 1 mg/l BAP and

0.5 mg/l IAA with an average number of 3-4 shoots per explants.

28

2.2.2 In-vitro propagation of woody tree species

2.2.2.1 Criteria for selection of explants for micro-propagation of woody tree species

The tissue which is obtained from the plant to culture is called an explant. Based on work

with certain model systems, particularly tobacco, it has often been claimed that a totipotent

explant can be grown from any part of the plant. In many species, explants of various organs vary

in their rates of growth and regeneration, while some do not grow at all. Also, the risk of

microbial contamination is increased with an inappropriate explant. Thus, it is very important

that an appropriate choice of explant be made prior to tissue culture.

The most commonly used tissue explants are the meristematic ends of the plants such as

the stem tip, auxiliary bud tip, and root tip. These tissues have high rates of cell division and

either concentrate or produce the required growth-regulating substances including auxins and

cytokinins (Akin-Idowu et al.,2009). The different explants such as nodal segments, cotyledonary

nodes, hypocotyls, roots, and cotyledons selected also could influence the rate of shoot

regeneration in many trees including different species of Acacia, teak, Eucalyptus, Salix

tetrasperma, V. negundo, P. marsupium, and Albizia lebbeck (Vengadesan et al., 2002; Yasodha

et al., 2004; Anis et al., 2012). The type of genotype is also a key criterion in determining the

material suitable for micropropagation (Kunze, 1994; Tang and Guo, 2001; Tereso et al., 2006;

Cortizo et al., 2009).

2.2.2.1.1 Nodal segments as explants for micro-propagation

Moreover, juvenile plants are an excellent explant source to achieve successful in-vitro

propagation of tropical forest trees, medicinal trees and horticultural woody fruit trees. Nodal

segments have been widely used for in-vitro shoot proliferation of woody tree species such as

Syzygium travanocoricum Gamble (Anand et al., 1999), Bixa orellana L. (Sharon and D’Souza,

2000), Jatropha curcus L. (Datta et al,. 2007), Acacia nilotica L. (Dhabhai et al., 2010),

Populus deltoids (Cavusoglu et al., 2011), Terminalia bellerica Roxb. (Mehta et al., 2012)

Ceridiphyllum japonicum Sieb. EEt Zucc (Fu et al., 2012), Cedrela montana (Diaz-Quichimbo

et al., 2013), Delbergia sissoo Roxb. (Rani et al., 2014), Brahylaena huillensis (Ndakidemi et

al., 2014), Delbergia melanoxylon (Kiondo et al., 2014), Shorea robusta (Singh et al., 2014) and

Gymnema sylvestris (Singh et al., 2015).

29

Fruit trees are also successfully micro-propagated by using nodal segments viz.,

Anacardium occidentale L. (Boggetti et al., 1999), Zizyphus mauritiana cv. Umran (Sudhersan

et al., 2001), Litchi chinensis Sonn. (Kumar et al., 2006), Citrus limon (Rathore et al., 2007),

Emblica officinalis cv. Balwant (Goyal and Bhadauria, 2008), Ficus glomerata Roxb. (Sayeed

Hasan and Khatun, 2010), Vitis vinifera (Kurmi et al., 2011), Aegle marmelos L. (Puhan and

Rath, 2012), Citrus sinensis Osbeck (Kanwar et al., 2015), Citrus reshni Tanaka (Kumar et al.,

2014), Morus indica (Kakarla and Rama, 2014), Punica granatum L. (Singh et al., 2007;

Bharose et al., 2014), Carrizo citrange (Kaur et al., 2015).

2.2.2.1.2 Shoot tip as explants for micro-propagation

The production of plants from auxiliary buds or shoots has proved to be the most

generally applicable and reliable method of true-to-type in-vitro propagation. Micro-propagation

and regeneration of plants under in-vitro conditions were successfully utilized in many woody

fruit trees like Zizyphus mauritiana cv. Umran (Sudhersan et al., 2001), Punica granatum L.

(Murkute et al., 2004; Bharose et al., 2014), Prunus avium L. (F12-1) and Prunus mahaleb L. x

P. avium L. (Maxma 14) rootstocks (Canli and Demir, 2014). In guava, shoot tips explants were

found better than nodal and inter nodal explants (Singh et al., 2015)

2.2.2.1.3 Inter-cotyledonary region as explants for micro-propagation

Intercotyledonary region also known as cotyledonary nodes. These cotyledonary nodes

were the most regenerable explants and successfully utilized in micro-propagation of many

woody tree species such as Anacardium occidentale L. (Boggetti et al.,1999), Pinus pinea L.

(Olivera et al., 2003), Pinus pinaster Ait. (Alvarez et al., 2009), Punica granatum L. (Naik et al.,

2000, Saron and Sinha, 2000 and Bharose et al., 2014) and Milletia pinnata L.(Nagar et al.,

2015).

2.2.2.1.4 Root as explants for micro-propagation

Among the possible initial explants, roots have proven to be highly regenerative explants

for in vitro regeneration in different species, including forest ones. Initiation of shoots from

root explants has been described in several plant species, indicating a possibility of developing

regenerative excised root culture for mass multiplication and their germplasm preservation, viz.,

30

Azadirachta indica (Arora et al., 2010), Shorea robusta (Chaturvedi et al., 2004), Melia

azedarach (Vila et al., 2005), Populus alba (Tsvetkov et al., 2007), Cleome rosea (Simoes et

al., 2009), Swertia chirata (Pant et al., 2010), Passiflora edulis (Viana da Silva et al., 2011),

Albizia lebbeck (Perveen et al., 2011) and Caesalpinia bonduc (Santosh Kumar et al., 2012).

These explants were then dipped in various surface sterilants, i.e., NaOCl (1.0%), HgCl2 (0.1%)

and KCl (1.0%) for different durations and subsequently dipped in 70 per cent ethyl alcohol for

30 Seconds and 2.2.3 Surface sterilization of different explants used for micro-propagation.

Phulwaria et al. (2012) treated nodal segments of Terminalia bellirica with 0.1%

Bavistin for 15 min followed by surface sterilized with 0.1 per cent HgCl2 for 5 min under

aseptic condition and rinsed five to six times with sterile double distilled water and found

maximum contamination free explants.

Abou Dahab et al. (2010) found the best results in sterilization of nodal explants of

Taxodium distichum and Taxodium distichum var. ‘distichum’ with using 20 per cent Clorox for

5 min., followed by 0.2 per cent HgCl2 for 5 min.

Shende and Rai (2005) used 0.5 per cent mercuric chloride for 15 minutes followed by

four washings with sterile distilled water for surface sterilization of seed explants.

Sharma et al. (2005) surface sterilized the Chironji explants in a laminar-flow hood by

immersion for 15 min in 0.2 per cent mercuric chloride, followed by rinsing 3–4 times in sterile

distilled water.

Kumar et al. (2014) developed sterilization protocol for Cleopatra mandarin (Citrus

reshni Tanaka). The seeds was extracted and washed thoroughly by tap water and treated with

0.2 per cent Bavistin. The seed testa was removed under aseptic condition. These decoated seeds

were first quick rinsed with 70 per cent ethanol followed by mercuric chloride 0.1 per cent for

five minutes.

Singh et al. (2015) surface sterilized the different explants, viz., shoot tips (0.5-1 cm), leaves,

nodal and intermodal segments (1-1.5 cm) of guava for micropropagation. The explants were washed

thoroughly under running tap water containing few drops finally rinsed thrice with autoclaved double

distilled water.

31

Singh et al. (2015) were able to established contaminant free culture in Gymnema

sylvestre by washing nodal explants in running tap water, followed by different surface

sterilization treatments using, firstly with 2-3 drops of Tween-80 for 8-10 min followed by the

0.002 per cent bavistin for 2-3 min and then mixture of HgCl2 (0.1%) and sodium hypochlorite

(1%) in equal amount for 8 min. Finally the explants were rinsed 5-6 times with double distilled

sterile water.

2.2.3 Artificial media used for micro-propagation of woody tree species

The basic components of plant tissue culture media are the mineral nutrients. How

rapidly a tissue grows and the extent and quality of morphogenetic responses are strongly

influenced by the type and concentration of nutrients supplied. Nutrient medium is a mixture of

substances in which cells, tissues or organs can grow with or without agar. The nutrient

media consists of macrosalts, microsalts, vitamins, growth regulators and sucrose which are

essential for growth and development of plant. The requirement varies with the explant and

species (Hartmann et al., 1997).

Early research by Gautheret (1939); Heller (1953); White (1942); Hildebrandt et al.,

(1946); Nitsch and Nitsch (1956) culminated in the development of MS medium by Murashige

and Skoog (1962). The organic and mineral compositions of the culture medium are particularly

important to improve differentiation and to optimize explant’s growth. It is well known that the

amount of nutrients present in the culture medium must be sufficient to foster growth throughout

the entire culture period.

The potential benefits of optimizing the nutrient component of culture media for a

particular response are well documented across a wide range of species and applications. Because

there are 13 mineral elements essential for plant growth (Epstein and Bloom, 2005), the

experimental determination of optimal nutrient levels is complex. This complexity illustrates

why the “revised medium” developed by Murashige and Skoog (1962) was an important

development. Although MS medium is not optimal for many tissues, many tissues will grow on

it to some degree; hence, MS medium represents a starting point to begin the process of

improving a response. The significant and distinguishing feature of MS (1962) medium is its high

nitrate, ammonium, and potassium contents.

32

The composition, type, and strength of basal medium also played an important role in

shoot multiplication. Full strength of MS medium was found favorable for multiple shoot

production in Holarrhena antidysentrica (Mallikarjuna and Rajendrudu, 2007), Actinidia

deliciosa (Akbas et al., 2007), Pterocarpus santalinus (Rajeswari and Paliwal, 2008), Acacia

nilotica (Abbas et al., 2010), Albizia lebbeck (Perveen et al., 2011), and Vitex negundo (Ahmad

and Anis, 2011). Modification in the MS medium such as MS salts reduced to one half, one

third, one fifth, or three fourth has been found effective in Acacia senegal (Badji et al., 1993),

Acacia mearnsii (Huang et al., 1994), Anacardium occidentale (Das et al., 1996).

Wang et al. (2005) showed that B5 and WPM media were the optimal basal media for

shoot regeneration from axillaries bud in Camptotheca acuminate.

Thakur and Kanwar (2008) reported that WPM resulted in enhanced auxiliary shoot

proliferation of Pyrus pyrifolia when compared to MS and various modifications, and Gonzalez-

Rodriguez et al. (2010) found that it was true for adventitious shoot regeneration from stem

explants of Tabebuia donnellsmithiirose.

Khan et al. (2011) demonstrated the best shoot induction response in nodal explants of

Salix tetrasperma in WPM medium supplemented with different PGRs. Bhatt and Dhar (2004)

established a higher efficiency of WPM over other types of medium like B5 (Gamborg et al.,

1968) and MS and their different strengths were tried for shoot proliferation in Myrica esculenta.

They observed that neither MS nor B5 gave satisfactory response even when the salt concentration

is reduced to half, and shoot response was severely inhibited.

Cavusoglu et al. (2011) reported direct organogenesis of clones of Populus deltoides

Bartram ex Marsh from nodes in woody plant medium (WPM).

The MS formulation is the most commonly used medium for in-vitro propagation of

pomegranate genoytypes, although some success has been achieved with WPM (Chauhan and

Kanwar, 2012). Recently, Bharose et al. (2014) successfully established in-vitro protocol for

Pomegranate (Punica granatum L.) cv. Bhagava by using MS medium.

Singh et al. (2014) tested (MS, SH, WPM and B5) and reported WPM medium best

suitable medium for the micro propagation of Shorea robusta.

33

2.2.4 Effect of growth regulators on organogenesis under in-vitro conditions

During adventitious organogenesis, new organs (shoots, roots) can develop on explants of

different plant tissues such as leaves, stems, and roots. Leaves and hypocotyls, for instance, do

not have any apparent preexisting meristems, and therefore most plant cells could be considered

totipotent. The huge amount of variability seen in the frequency of organogenesis between

varieties and species suggests that it is the proportion of cells that are receptive to in-vitro culture

conditions that vary. Organogenesis in vitro consists of several factors, such as PGR perception

and transduction, redifferentiation after dedifferentiation of differentiated cells, organization for

specific organ primordial and meristems, etc. The process depends on external and internal

factors, such as exogenously applied PGRs, and the ability of plant tissue to perceive these

PGRs.

2.2.4.1 Effect of growth regulators on in-vitro shoot initiation

The production of shoots that may eventually become new plant is called shoot

proliferation. The formation of adventitious shoots or roots was first determined by Skoog and

Miller (1957) through discovery of the regulation of organ formation (shoots and roots)

by changing the ratio of cytokinin/auxin, when ratio of cytokinin/auxins is high, it

favours the formation of shoot but root formation is inhibited. The reverse favours the root

formation. The most commonly used cytokinins include kinetin, 6-benzyl amino purine (BAP)

and Thiodizuran (TDZ). Several attempts have been made to establish protocols for efficient

plant regeneration and production of number of shoots in woody perennials.

Bains (2000) found that all the inoculated auxiliary buds grew in size and new shoots

began to proliferate from the bases within 15 days of incubation and MS medium supplemented

with BAP (2.0 mg/l) resulted in maximum number of shoots per culture (12.8) that were

supported by maximum length of shoots (5.8 cm) and number of leaves (16.4) on 30th

days.

Sharon and D’Souza, (2000) observed that regenerated shoots of Bixa orellana L. from

shoot apex explants rooted best on MS medium supplemented with 0.01 mg/l α-

naphthaleneacetic acid (NAA), whereas shoots regenerated from nodal explants needed 0.5 mg/l

NAA for rooting.

34

In Citrus aurantifolia, best results for multiple shoot formation, 8 shoots per node, were

obtained with 1 mg/l BAP and 0.5 mg/l kinetin was reported by Al-Khayri and Al-Bahrany

(2001).

Sudhersan et al. (2001) observed in Zizyphus mauritiana cv. Umran, multiple shoots

were obtained from the shoot tip and stem nodal explants cultured on MS medium supplemented

with 0.01-0.1 mg/l BA. Isolated shoot tips elongated in MS medium and produced plantlets with

6-7 nodes within 3 weeks time.

Thengane et al. (2006) developed an efficient protocol for in-vitro micro propagation of

Calophyllum inophyllum (L.) and multiple shoot formation was achieved on WPM supplemented

with BAP (2.22–44.00 μM) and TDZ (0.91-4.54 μM) from the decapitated seedling explants.

The maximum multiple shoots, 20.9 per explants were induced on TDZ (0.91 μM) after two

subcultures. Elongated shoots of size more than 4.0 cm were obtained on all media combinations

with an average of 2.2–8.7 per explants.

In Jatropha curcas L. auxiliary shoot bud proliferation was best initiated on Murashige

and Skoog’s (MS) basal medium supplemented with 22.2 μM N6-benzyl-adenine (BA) and 55.6

μM adenine sulphate, in which cultures produced 6.2 shoots per nodal explant with 2.0 cm

average length after 4-6 weeks (Datta et al., 2007).

Abou Dahab et al. (2010) reported that half strength B5 medium supplemented with 0.4

mg/l BA gave better shootlets multiplication, as compared with half strength MS medium. The

longest shootlets were recorded with woody plant medium (WPM) at full salt strength in

Taxodium distichum and Taxodium distichum var. ‘distichum’.

Phulwaria et al. (2012) standardized in-vitro propagation method for Terminalia bellirica

and found MS medium containing 2.22 μM BAP was best for shoot multiplication in a single

step. After excision of newly formed shoots, mother explants successively transferred to the

same medium produced maximum shoots per explant after IV passage. Further enhancement in

morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on

MS medium supplemented with 2.22 μM BAP, 1.16 μM Kn and 0.57 μM IAA.

35

In Rubia cordifolia L., significant callus (89.3%) and shoot induction (71.8 %) was

observed in MS medium with 4.0 mg/l TDZ. An average of 24.4 shoots having 4.01 cm length

were regenerated in the culture (Khadke et al., 2013).

2.2.4.2 Effect of growth regulators on in-vitro root initiation

Rooting of in-vitro regenerated shoots and transplantation of the plantlets to the field is

the most important, crucial, and essential step, but a difficult task in tissue culture of woody trees

(Murashige, 1974). Generally, rooting in micropropagated shoots can be achieved by two

different methods, i.e., in-vitro and ex-vitro methods. Root induction and elongation are complex

processes that are influenced by a large number of factors, such as genotype, type, and

concentration of PGRs, and culture conditions (Bennett et al., 1994; Mylona and Dolan, 2002).

Sharon and D’Souza (2000) observed that regenerated shoots of Bixa orellana L. from

shoot apex explants rooted best on MS medium supplemented with 0.01 mg/l α-naphthalene

acetic acid (NAA), whereas shoots regenerated from nodal explants needed 0.5 mg/l NAA for

rooting.

In Citrus aurantifolia, root initiation was observed within 3 weeks after transfer to

rooting medium. The best rooting treatment was 1 mg/l IAA since it gave the highest percentage

of root induction. The percentage of rooting ranged from 13 to 56%, the highest percentage was

obtained on MS medium containing 1 mg/l IAA (Al-Khayri and Al-Bahrany, 2001).

Isolated in-vitro shoots of Zizyphus mauritiana cv. Umran were rooted in MS medium

containing 10 mg/l IBA (Sudhersan et al., 2001).

Thengane et al. (2006) reported 52 % rooting with 1-5 roots per rooted plant in

Calophyllum inophyllum (L.). Successfully root induction was observed in elongated shoots in

half WPM and/or full strength WPM supplemented with indole-3- butyric acid (2.46-24.60 μM)

alone or in combination with BAP (2.22 μM).

Datta et al. (2007) reported 52 % root induction in micro-shoots of Jatropha curcas L.

cultured in MS basal medium supplemented with 1.0 μM IBA in 2-3 weeks. Further elongation

36

of roots with average length of 8.7 ± 1.35 cm was obtained in unsupplemented MS basal medium

for 2-3 weeks.

In micro-propagation of Taxodium distichum and Taxodium distichum var. ‘distichum’,

the longest shoots were recorded with woody plant medium (WPM) at full salt strength. Half

strength WPM medium + 1.0 g l-1

activated charcoal (AC) + 0.5 mg/l IBA was the best medium

for in vitro rooting percentage and root number/shoot (Abou Dahab et al., 2010).

Phulwaria et al. (2012) found that half-strength MS medium supplemented with 24.60

μM IBA and 100 mg/l acitvated charcoal was most effective for rooting of the in-vitro

regenerated shoots of Terminalia bellirica . To reduce labour, cost and time, an experiment on

ex-vitro rooting was also carried out and it was observed that highest per cent sho ots rooted ex -

vitro when treated with 24.60 μM IBA for 5 min.

Khadke et al. (2013) observed and reported the highest rooting response (93.7%) in the

medium with 1.0 mg/l IBA with a mean number of 4.9 roots per shoot with mean length of 4.7

cm in Rubia cordifolia L.

37

CHAPTER-III

MATERIALS AND METHODS

The present investigations on “Standardization of in-vitro propagation protocol for

Chironji (Buchanania lanzan Spreng)” was carried out in the laboratory of the Department of

Plant Molecular Biology and Biotechnology, College of Agriculture, Indira Gandhi Krishi

Vishwavidyalaya, Raipur (C.G.) from the year 2016 to 2017. The details of materials used and

methods followed to fulfill the objectives were as under.

3.1 Media preparation

Culture media used to establish Chironji cultures initiation, multiple shoot induction

were MS (Murashige and Skoog, 1962) and WPM (Woody Plant Medium, Lloyd and Mc

Cown, 1980). For direct organogenesis different hormonal combinations with MS and WPM

were tried to optimize the media for culture initiation for direct regeneration, multiple shoot

induction and rooting.

3.1.1 Preparation and storage of stock solutions

MS and WPM media were prepared from stocks solution (Table-3.1 and 3.2).

Modification to the medium was done by adding growth regulators and other organic

additives. Stock solutions of macronutrients, micronutrients, vitamins and growth hormones

were prepared and kept in refrigerator at 4-8°C.

3.1.2 Preparation of Culture Medium

For the preparation of culture media for 1000 ml of media, required amount of stock

solutions were mixed along with growth regulators and sucrose (30g/l) in 800 ml of double

distilled water was added and stirred. The pH of the medium was adjusted to 5.8 by using

either 0.1 N HCl or NaOH with the help of a digital pH meter. Agar (6-8g/l) was weighed

and added into the medium for gelling. Then, final volume was made up to 1000 ml by adding

double distilled water. The media was autoclaved at 121oC at 15 lbs per square inch pressure

for 20 minutes and then allowed to cool to room temperature then poured into sterile bottles

and test tubes in laminar air flow followed by capping using lids and cotton plugs, respectively

and stored in culture rooms(temperature 25±27º C) until further use.

Table 3.1: Stock solutions of WPM (Lloyd and Mc Cown, 1980)

38

Stock Composition Concentration of the

stock solution

Quantity for

1 litre medium

I NH4NO3

X 20 10 ml Ca (NO3)2.4H2O

II K2SO4 X 20 20 ml

III

KH2PO4

X 100 10 ml H2BO3

Na2MoO4.H2O

IV

MgSO4.7 H2O

X 100 10 ml MnSO4.4H2O

ZnSO4.7H2O

CuSO4.5H2O

V FeSO4.7H2O

X 100 10 ml Na2 EDTA

VI

Thiamine HCl

10 ml Nicotinic acid X 100

Pyridoxine HCl

Glycine

VII Myo-Inositol X 50 5 ml

Table 3.2: Stock solutions of MS medium (Murashige and Skoog, 1962)

Stock Composition Concentration of

the stock solution

Quantity for

1 litre medium

I

NH4NO3

X 10 100 ml

KNO3

CaCl2.2H2O

MgSO4.7 H2O

KH2PO4

II

KI

X 100 5 ml

H2BO3

MnSO4.4H2O

ZnSO4.7H2O

CuSO4.5H2O

CoCl2.6H2O

Na2MoO4.2H2O

III FeSO4.7H2O

X 100 5 ml Na2 EDTA

IV Myo-Inositol X 50 5 ml

V

Thiamine HCl

X 100 5 ml Nicotinic acid

Pyridoxine HCl

Glycine

3.1.3 Preparation of stock solution of growth regulator

39

Stock solutions of 6-benzyl amino purine (BAP), kinetin (Kn), gibberellic acid

(GA3), naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) were prepared by dissolving

50mg of hormone first in 5ml of 1N NaOH or 70% ethanol and the final volume was made

up to 50 ml with autoclaved double distilled water to prepare stock solution of 1mg/1ml (Table-

3.3). Then hormone stocks were filtered by using syringe filter (0.25mm, 0.45µm) under

Laminar air flow cabinet and cap were covered by wrapping parafilm and kept in refrigerator at

4-8°C.

Table 3.3: Stock solutions of hormones

Hormone

Required

amount for stock

solution (mg)

Amount of solvent

required to dissolve

Amount of water

added (ml)

BAP 50 5ml 1N NaOH 45

2,4-D 50 5ml 70 % ethanol 45

NAA 50 5ml 1N NaOH 45

Kn 50 5ml 1N NaOH 45

GA3 50 5ml 1N NaOH 45

IBA 50 5ml 70 % ethanol 45

3.2 Sterilization

In tissue culture procedures, one of the vital steps is the complete sterilization of

glassware, growth media, working area and surgical instruments. The explants used must also

be surface sterilized for its successful subsequent growth. The sterilization procedures adapted

during the research were as under.

3.2.1 Glassware sterilization

Glassware sterilization includes the following steps:

i. All the glassware (culture-tubes, petridishes, pipettes, beakers, flasks etc.) used for

experiments were washed thoroughly with household detergent and given several

washings under tap water followed by a rinse with distilled water.

ii. Then, glassware were soaked in chromic acid for overnight. Chromic acid was prepared

by mixing potassium dichromate (K2Cr2O7, 10 %) 100 g in 1000 ml water.

iii. The glassware was then washed thoroughly with running tap water by giving several

40

washings to remove chromic acid solution followed by two or three rinses with distilled

water.

iv. Finally, the glassware was dried overnight followed by autoclaving. Then, the glasswares

were kept in hot air oven at 800

C for drying for 24 hours.

3.2.2 Sterilization of tissue culture media

Plant tissue culture medium, containing a high percentage of sucrose and nutrients

which supports the growth of microorganisms. Therefore, it is necessary to maintain the

aseptic conditions inside the culture vessels, so the culture tubes containing different media

were tightly covered by cotton plugs and wrapped with parafilm of appropriate size. The

media were sterilized by autoclaving at 15 lbs inch per square for 15–20 minutes at 121oC.

After autoclaving, the sterilized media were allowed to cool down at room temperature. The

sterilized media were then kept in culture room until use.

3.2.3 Sterilization of working area

Aseptic transfer techniques are considered to be basic requisite for the induction and

maintenance of clean cultures, free from any microbial contamination. Prior to the inoculation

or sub-culturing of explants into the culture tubes, hands, instruments and working area to be

cleaned and sterilized. Therefore, the inoculation is generally done under Laminar airflow

cabinet and it acts as a sterile area for the tissue culture procedure. The working area of

laminar airflow cabinet was sterilized by:

i. Thoroughly scrubbing all the interior of the cabinet with 70 % ethanol (70 ml ethanol+30

ml water).

ii. Ultra violet light is known to be detrimental for the micro-organism therefore, laminar

air flow chamber was irradiating with UV light for about 1-2 hour prior to start of work

inside Laminar flow. The UV light was switched off at least 30 minutes before

inoculation.

iii. Sterilizing the working bench by scrubbing with ethanol. All the work was carried

out under the gentle flow of micro-filtered air.

3.2.4 Sterilization of tools

41

All the tools (scalpels, forceps, needles, scissors and blades) used during the

aseptic manipulation of explants in culture media were sterilized by autoclaving at 15 lbs inch

per square for 15–20 minutes at 121oC followed by drying in hot air oven at 80

oC for 24 hours.

Before use, different surgical tool were sterilized by putting them on flame of burner. The hot

forceps and other tools were allowed to cool down for some time to room temperature, before

the holding the explants and putting them into different culture media.

3.2.5 Sterilization of laboratory and culture room by weekly fumigation

Every weekend, laboratory, working place and culture room were fumigated by using

potassium permanganate and formaldehyde. Potassium permanganate was taken in a glass bottle

and small amount of formaldehyde put carefully in bottle till fume starts coming out. Face must

be covered during fumigation. Two to three bottles should be kept in a room for fumigation.

After fumigation, put a notice on door of fumigated room to restrict others to enter in the room.

3.3 Plant materials used and source of explants for in-vitro propagation of

Chironji

3.3.1 Source of explants

3.3.2 Age of the plant used:

One to two year old Chironji plants were utilized as a source of explants kept under green

house conditions. Some of the tissue culture raised plants are also used to obtain the explants viz.

shoot tips nodes, leaf and root, respectively. However, Expalnt E1 – shoot tips, E2 –nodes, E3 -leaf

and E4-roots of 1-2cm size were excised from the 1-2 year old plants grown under green house at

PMBB, IGKVV, Raipur.

3.4 Collection, sterilization and preparation of explant for inoculation

Every explant differs from each other which has been used to initiate the cultures (Table-

3.4). So, different procedures were followed for collection and preparation of explants. A

number of chemical sterilants were tried separately or in combinations to see their effect on per

cent contamination and survival after culture for different explants (Table-3.5).

Table 3.4:- Different explants used for in-vitro propagation of Chironji

42

Sr. No. Explant Code Explant Source

1 E1 Shoot tips 1-2 year old plants

2 E2 Nodes 1-2 year old plants

3 E3 Roots 1-2 year old plants

4 E4 Young leaves 1-2 year old plants maintain at green house

3.4.1 Explant E-1 and E-2

3.4.1.1 Collection of E-1 and E-2

Shoot tip of 1-2 cm size were excised from the 1-2 year old plants grown at PMBB,

IGKV, Raipur and 1-2 year old plants of Chironji grown under green house conditions by

sterilized scissor in the morning. These excised shoot tips were collected in a culture bottle

containing tap water.

3.4.1.2 Sterilization of E-1 and E-2

Shoot apices (2-3 cm long), obtained from young and actively growing shoots of 1-2 year

old plants and young seedling plants of Chironji were placed in plastic trays containing tap

water with two to three drops of detergent (Tween-20). The explants were stirred gently and then

washed with running tap water until all the traces of soap were completely removed. Further

processes were carried under Laminar air flow cabinet, the explants were then placed in different

sterilants for different time durations to attain complete asepsis and then rinsed 3 times with

double distilled water and finally with autoclaved double distilled water.

Table 3.5 : Different surface sterilization treatments for Chironji explants

Sr. No. Treatment Code Treatment details

1 SS1 Bavistin (1%) 5 min + HgCl2 (0.1%)3 min.

2 SS2 Bavistin (1%) 5 min + HgCl2 (0.1%) 5 min.



+ Ethanol (70%) 10 sec.



+ Ethanol (70%) 10 sec.

3.4.1.3 Preparation of E-1 and E-2

Sterilized explants were put on autoclaved tissue papers for drying of water which

43

favours growth of micro-organisms. After drying of water, explants were cut into 1.5-2.0 cm

length after trimmed off leaves on it. The exposed cut end was trimmed off to eliminate toxic

effect of sterilant which may have moved into cells during sterilization.

3.4.2 Explant E-3

3.4.2.1 Collection of E-3

Sterilized root of Chironji were used as source of explants. Inter-cotyledonary regions

were excised and collected from in-vitro grown of Chironji.

3.4.2.2 Sterilization of root to raise Explant E-3

Roots excised from in-vitro grown of Chironji were first washed under tap water using

two to three drops of detergent (Tween-20) and then they were taken to laminar air flow cabinet

and sterilized with different sterilants for different time durations, finally rinsed twice with

autoclaved double distilled water.

3.4.3 Preparation of E-3

Sterilized explants sample of E-3 were kept on autoclaved tissue papers for absorbing of

moisture from the surface, which promotes growth of micro-organisms. Then, exposed cut ends

were trimmed off to eliminate toxicity of the sterilant which may have moved into cells during

sterilization.

3.4.4 Explant E-4

3.4.4.1 Collection of E-4

E-4 from young leaves seedlings grown under green house conditions were cut 4-5 cm

long by sterilized scissor. These explants were collected in a culture bottle containing tap

water.

3.4.4.2 Sterilization of E-4

E-4 were washed by tap water with two to three drops of detergent (Tween-20). The

explants were stirred gently and then washed with running tap water until all the traces of soap

were completely removed. Further processes were carried out under laminar air flow cabinet as

similar for shoot tip explants.

44

3.4.4.3 Preparation of E-4

Sterilized explants sample of E-4 were put on autoclaved tissue papers for drying of

water which favours growth of micro-organisms. After drying of water, exposed cut end of

petals were trimmed off to eliminate toxic effect of sterilant which may have moved into cells

during sterilization.

3.5 Experimental methods

3.5.1 Sterilization of explants

The sterilization procedure applied for all types of explants mentioned above was

similar but the duration of bavistin and mercuric chloride treatment varied.

3.5.2 Inoculation of different types of explants for culture initiation and multiple shoot

initiation

Sterilized explants were inoculated into the tubes and culture bottles contains aseptic

different basal medium (MS and WPM) supplemented with different growth hormones alone

or in combination inside the laminar air flow chamber. Each treatment has given a treatment

code (Table-3.6). Three replications were taken for each treatment. After covering lid of bottles

and mouth of tubes by parafilms, cultures were placed in growth chamber.

3.5.3 Cultural conditions and care during inoculation

Maintenance of constant environmental conditions throughout incubation is important.

Before inoculation process, all the required materials were properly sterilized and kept in laminar

air flow. Throughout experiment, growth chamber was kept completely aseptic and most

explants were cultured under cool white light with 1500-3000 Lux light intensity. Temperature

of the room was maintained between 22-28oC with 60-70% relative humidity.

Shoot tips/nodal segments and seeds of Chironji often possess hidden contaminants.

Their impact was controlled by proper sterilization and immediately discarding the

contaminated tubes and bottle from growth chamber and incubator.

Table 3.6 : Different treatments used for multiple shoot initiation in Chironji

45

Treatments Combinations (%)

S1 ½ WPM + BAP (2.0 mg/L) + GA3 (0.5 mg/L) + kn 1.0mg/l.

S2 ½ WPM BAP (2.0 mg/L) + NAA (0.5 mg/L) + kn 1.0 mg/l.

S3 ½ MS BAP (2.0 mg/L) + GA3 (0.5 mg/L)

S4 ½ MS without hormones

3.5.4 Sub culture

Micro shoots formed in the test tubes were taken out 5-6 weeks after inoculation

depending upon the shoot initiation and proliferation potential of the explants. The shoots

were separated by dissecting them in the sterile environment of laminar air flow cabinet

with sterile dissecting needle and forceps. They were placed in the bottles containing fresh

media.

3.5.5 Rooting

3.5.5.1 In-vitro Rooting

The micro shoots of more than 2-3 cm in length from E-2, E-3 and E-4 were taken

out placed in the bottles containing media (MS and WPM) with different concentrations of

IBA and NAA for rooting, alone or in combination (Table-3.7). Each treatment has given a

treatment code. Three replications were taken for each treatment. After covering lid of bottles

and mouth of tubes by parafilms, cultures were placed in growth chamber.

Table 3.7 : Different treatments used for in-vitro rooting in Chironji

Treatments Composition

R1 MS (Without hormone)

R2 1/2 WPM+IBA 1.0 mg/L+1%activated charcoal





3.5.6 Observations and data collection

3.5.6.1 Number of days required for shooting

The number of days taken to show initial differentiation of shoot from the date of

inoculation of different explants was recorded and was expressed as mean number of days.

46

3.5.6.1.1 Number of shoots produced per explants

While sub culturing multiple shoots were separated, counted from explants and

expressed as shoot per explant.

3.5.6.2 Mean length of shoots (cm)

The shoot length was measured from base to the tip of the plantlet at the time of sub-

culture and the average length was expressed in centimeters.

3.5.6.3 Number of days required for initiation of roots

The number of days taken for initiation of roots, after inoculation was recorded.

3.5.6.4 Mean number of roots

The number of roots formed per micro shoot was recorded and average was worked

out.

47

3.5.7 Statistical analysis of data

The experimental data relating to contamination percentage, per cent survival of

plantlets was transformed to arcsine values and analyzed under Completely Randomized

Design. The data were subjected to analysis of variance test (ANOVA) as suggested by

Gomez and Gomez (1983). Critical difference values were tabulated at one per cent probability

wherever ‘F’ test found significant.

48

CHAPTER- IV

RESULTS AND DISCUSSION

In-vitro propagation has become a reliable and routine approach for large- scale rapid

plant multiplication, which is based on plant cell, tissue and organ culture on well defined tissue

culture media under aseptic conditions. A lot of research efforts are being made to develop and

refine in-vitro propagation methods and culture media for large-scale plant multiplication of

several number of plant species. However, many woody and fruit plant species still remain

recalcitrant to in-vitro culture and require highly specific culture conditions for plant growth and

development. Today, the need for appropriate in-vitro plant regeneration methods has become a

necessity to overcome problems facing in-vitro propagation such as somaclonal variation,

recalcitrant rooting in woody species, hyperhydricity, high labour cost, contamination, loss of

material during hardening, quality of plant material and polyphenol. Moreover, the useful

applications of in-vitro propagation in various aspects make this technology more relevant for

example to production of virus-free planting material, cryopreservation of endangered and elite

woody species, applications in tree breeding, afforestation and reforestation.

The present investigation entitled “Standardization of in-vitro propagation protocol for

Chironji (Buchanania lanzan Spreng)” was undertaken to standardize the complete protocols for

selection of explants, multiple shoot induction and root initiation of Chironji, The results and

discussion are presented under the following headings.

1. Selection of potential explants for in-vitro micropropagation of Chironji (Buchanania lanzan

Spreng).

2. Standardization of protocol for multiple shoot induction.

3. Standardization of protocol for root initiation.

49

4.1. Selection of potential explants for in-vitro micropropagation of Chironji

(Buchanania lanzan).

The type of organs or explants chosen affects the successful establishment of the cultures

and their subsequent growth. Not all the tissues or organs of a plant are equally capable of

exhibiting morphogenesis (Hartmann et al., 1997).

Table 4.1: Culture response of different explants in different treatment combinations

Treatment

No. Media Composition

E1-shoot

tip

E2-

node

E3-

root

E4-

leaf

B

mean

S1 ½ WPM + BAP (2.0 mg/L) + GA3 (0.5

mg/L) + kn 1.0mg/l. 34.33 11.33 30.00 18.00 23.42

S2 ½ WPM BAP (2.0 mg/L) + NAA (0.5

mg/L) + kn 1.0 mg/l. 30.00 31.67 17.67 22.33 25.42

S3 ½ MS BAP (2.0 mg/L) + GA3 (0.5

mg/L) 30.00 20.00 16.67 10.67 19.33

S4 ½ MS without hormones 9.67 20.00 15.00 10.67 13.83

SEm± 5.30 3.28 4.04 2.35

A mean 26.00 20.75 19.83 15.41

A X B CD ( at 5 %) 6.28

CV (%) 18.38

As per the table 4.1 it was concluded that among different treatment combinations the

interaction between E1 and S1 has maximum callus response (34.33%) fallowed by E2S2, E1S2

and E1S3, respectively. But incase of their alone A factor (explant) E1 gained highest response

(26.00 %) and factor (treatments) S2 has optimum response (25.42%), respectively.

4.2 Standardization of protocol for multiple shoot induction

The type of organs or explants chosen affects the successful establishment of the cultures

and their subsequent growth. Not all the tissues or organs of a plant are equally capable of

exhibiting morphogenesis (Hartmann et al., 1997).

In the present investigation, different explants were used to find out their responses in

various shooting media treatments and differences in response were observed, A comparison of

in-vitro responses by different explants (E-1, and E-2) is presented in Table- 4.2.

Table 4.2. Influence of different treatments in different explants on shoot initiation (%)

among different replications

50

Treatments

Shoot initiation %

B mean R1 R2 R3 SEm±

E1S1 27.78 35.00 22.22 3.69 28.33

E1S2 16.67 25.00 10.00 4.33 17.22

E1S3 6.67 10.00 13.33 1.92 10.00

E1S4 5.56 11.11 5.56 1.85 7.41

E2S1 6.67 3.33 3.33 1.11 4.44

E2S2 3.33 5.00 4.00 0.48 4.11

E2S3 5.56 5.00 6.67 0.49 5.74

E2S4 8.33 4.00 6.67 1.26 6.33

E3S1 0.00 0.00 0.00 0.00 0.00

E3S2 0.00 0.58 1.00 0.28 0.53

E3S3 1.22 0.96 0.99 0.08 1.06

E3S4 1.36 1.11 1.00 0.10 1.16

E4S1 1.56 1.35 1.88 0.15 1.60

E4S2 0.00 0.00 1.89 0.63 0.63

E4S3 0.00 0.00 0.00 0.00 0.00

E4S4 1.00 1.00 1.21 0.07 1.07

A factor mean 10.07 12.31 8.97 10.45

A x B CD (%)

4.72,6.36

CV (%)

50.75

According to the interaction table the A (Explants) and B (Treatments) 4.3 shoot

initiation (%) ranged between 0.00% to 28.33%.

Table 4.3 Influence of different treatments in different explants on shoot initiation (%)

Treatments EXPLANT TYPE

E1-shoot tip E2-node E3-root E4-leaf SEm± B mean

S1 28.33 4.44 1.16 1.60 6.52 8.88

S2 17.22 4.11 0.53 0.63 3.95 5.62

S3 10.00 5.74 1.06 0.00 2.30 4.20

S4 7.41 5.96 0.00 1.07 1.81 3.61

SEm± 5.40 0.53 0.30 0.39

A mean 15.74 5.06 0.68 0.82 43.99

A X B CD (%) 4.72, 6.36

CV (%) 50.75

51

Fig 4.1: Influence of different treatments in different explants on shoot initiation(%)

Fig 4.2 Influence of different treatments in different explants on shoot initiation (%)

0

5

10

15

20

25

30

35

40 E1

S1

E1S2

E1S3

E1S4

E2S1

E2S2

E2S3

E2S4

E3S1

E3S2

E3S3

E3S4

E4S1

E4S2

E4S3

E4S4

Influence of different treatments in different explants on shoot

initiation (%) among different replications

Shoot initiation % R1



0

5

10

15

20

25

30

S1 S2 S3 S4

Influence of different treatments in different explants on

shoot initiation(%)

EXPLANT TYPE E1-shoot tip

EXPLANT TYPE E2-node

EXPLANT TYPE E3-root

EXPLANT TYPE E4-leaf

52

Plate No. 1: Picture showing culture response of different explant

53

The maximum shoot initiation per centage was gained with E1S1(28.33 %) fallowed by

E1S2 (17.22%). However, minimum per cent of shoot induction response was with the treatment

combination E3S4 i.e 0.00%.shoot initiation per cent significantly differed in different explant

and treatment combinations at 5% level as well as in 1% level in F table value.

4.2.1 Shoot initiation percentage (%) in different explants of Chironji

Among, different explants, maximum response to shoot initiation was observed in

explants E1S1 (28.33 %) followed by explant E1S2 (17.22 %) and explants E1S3 (10.00 %),

respectively (Table-4.2). Similar response percentages were reported by Nagar et al. (2015) in

cotyledonary nodes of Milletia pinnata (L.). They reported 66.66 to 92.33 per cent shooting

initiation from cotyledonary nodes. It might be due to fact that cotyledon provides endogeneous

signal for bud development in absence of shoot tips. Inter-cotyledonary region also known as

cotyledonary nodes. These cotyledonary nodes were the most regenerative explants and

successfully utilized in micro-propagation of many woody tree species such as Anacardium

occidentale L. (Boggetti et al., 1999), Pinus pinea L. (Olivera et al.).

4.2.2 Number of days for initiation of shoots

The days taken for shoot initiation in various treatments varied between 52.33 days and

31.66 days. Significantly minimum days taken for shoot initiation with E1S1 31.66 days, which

was significantly superior among all the treatments fallowed by E1S2 39.67 Days (Table- 4.4).

So, E1S1 gave quickest response for shoot initiation. The differences in response among different

explants might be due to differences in physiological state of explants (Sreelatha et al., 1998).

Similar finding was reported by Shende and Rai (2005) in Chironji. They reported multiple shoot

initiation from seed explants 10-12 days after inocultaion on MS medium supplemented with

22.2 jiM BAP and 2.68 jiM NAA. Nagar et a!. (2015) also reported shoot regeneration from

cotyledonary nodes after one week of culture on BAP supplemented media in Millettia pinnata

(L.).

Table 4.4: Different treatment combinations used on different parts for shooting attributes

54

Treatments Days taken for

shoot initiation

No of shoots

initiated

Shoot length

(cm)

E1S1 31.67 4.76 1.88

E1S2 39.66 2.02 0.94

E1S3 41.67 3.80 0.67

E1S4 52.33 1.50 0.51

SEm± 4.25 0.76 0.30

CV (%) 11.43 17.85 15.94

CD (at 5% & 1%) 12.94,8.89 1.47,1.02. 0.43,0.30

Fig 4.3 Different treatment combinations used on different parts for shooting attributes

0

10

20

30

40

50

60

E1S1 E1S2 E1S3 E1S4

Different treatment combinations used on different parts for

shooting attributes

Days taken for shoot initiation

No of shoots initiated

Shoot length (cm)

55

4.2.3 No of shoots initiated

However, the same treatment had secured highest number of shoots initiated

E1S1(4.76%), and Lowest obtained with E1S4 (1.50%) The maximum of shoot initiated 4.76%

was recorded in explants E1S1 Followed by E1S3 3.80% (Table- 4.4). Nagar et at. (2015) reported

average shoot initiated of 3.02% of micro-shoots regenerated from cotyledonary nodes of

Millettia pinnata (L.). These findings were similar to results of Kumar et al. (2014) in Cleopatra

mandarin, Parveen et a!. (2015) in Aegle marnelos and Niratker (2016) in Buchanania lanzan.

4.2.4 Shoot length (cm)

However, the same treatment had secured highest number of shoots (4.00), and shoot

length (1.88cm), Lowest obtained with E1S4 (1.50 shoots and shoot length 0.51cm), respectively.

The maximum length of shoot 1.88 cm was recorded in explants E1S1 Followed by E1S2

0.94 cm (Table- 4.4). Nagar et at. (2015) reported average shoot length of 1 cm of micro-shoots

regenerated from cotyledonary nodes of Millettia pinnata (L.). These findings were similar to

results of Kumar et al. (2014) in Cleopatra mandarin, Parveen et a!. (2015) in Aegle marnelos

and Niratker (2016) in Buchanania lanzan. They reported 3.8 cm, 2.7 cm and 1.50 cm average

length of shoot obtained from nodal segments, respectively. This might be due to difference in

age as well as source of explant which have different level of physiological activities and

endogenous hormones which leads to differences in response to growing conditions.

The result revealed that, the multiple shoot formation in explants E1S1 was more as

compared to other explants in term of response percentage. The shoot induction and proliferation

depends on the plant growth regulators and types of explants (Mohamed, 1999).

56

4.2.5 Shoot multiplication among different treatment combinations

Among different treatment combinations the interaction between E1 and S1 was found

maximum average number of shoot multiplication (5.5%) fallowed by S2E1(4.5) and S3E1( 4.00),

respectively showed in table 4.5.

Table 4.5 Shoot multiplication among different treatment combinations

Treatments E1-shoot tip E2-node E3-root E4-leaf SEm±

S1 5.5 2.5 3.5 1.0 6.28

S2 4.5 1.0 2.0 1.0 0.52

S3 4.0 2.0 2.5 1.0 0

S4 4.0 1.5 1.0 0.5 0

Fig 4.4. Shoot multiplication among different treatment combinations

4.3 Standardization of protocol for root initiation %

Success in micro-propagation is dependent on the production of roots in micro-shoots. The

maximum root initiation observed 7.69 % under the R2 -1/2 activated charcoal followed the R3-

½ and the maximum root initiation 25 % under the R6- ½ (Table 4.6)

0

1

2

3

4

5

6

S1 S2 S3 S4

Shoot multiplication among different treatment

combinations

E1-shoot tip

E2-node

E3-root

E4-leaf

57

Plate.No.2 Shoot multiplication % among different treatment combinations

58

Plate No. 3 Showing different treatments on root initiation %

59

Table 4.6 Effect of different treatments on root initiation % from the explant shoot tip of

Chironji

Treatments Total Shoots

inoculated

Total root

initiated

Root

initiation %

Days taken for

root initiation

R1- MS (Without hormone) 17 0 0 0

R2 -1/2 WPM+IBA 1.0

mg/L+1%activated charcoal 13 1 7.69 50.22

R3- 1/2 WPM+IBA 2.0


R4- 1/2 WPM+IBA 3.0


R5- 1/2 WPM+IBA 4.0

mg/L+1%activated charcoal 12 1 12 40.66

R6- 1/2 WPM+IBA 5.0

mg/L+1%activated charcoal 16 4 25 38.79

SEm± 0.95 0.56 3.41 7.70

SD 2.33 1.37 8.36 18.87

4.3.1 Root initiation percentage (%)

The response of micro-shoots for rooting was significantly affected by use of activated

charcoal in rooting media. Different levels of IBA were used for in-vitro rooting with and

without activated charcoal in media. From the table 4.6 it was concluded that the Root initiation

percentage was found maximum (25) under treatment 6 R6- 1/2 WPM+IBA 5.0

mg/L+1%activated charcoal followed by treatment 4 R4- 1/2 WPM+IBA 3.0 mg/L+1%activated

charcoal and the minimum total shoots inoculated was found in treatment 1, R1- MS (Without

hormone). Al-Khayari and Al-Bahrany (2001) reported 56 per cent in-vitro rooting in micro-

shoots of Citrus aurantfolia which supports the present findings. Kaur et al, (2015) also reported

53.89 per cent rooting in micro-shoots of Carrizo citrange inoculated in MS medium

supplemented with IBA.

60

Fig 4.5: Effect of different treatments on root initiation % from the explant shoot tip of

Chironji

4.3.2 Number of days for initiation of roots

There was no difference with respect to number of days taken for initiation of roots

among different explants used. In treatment R-6, rooting was observed in micro-shoots of

explant E-1 shoot tip after 38.79 days of inoculation (Table- 4.6). these findings were supported

with results of Chabukswar and Deodhar (2005) in in-vitro rooting of Garcinia indica Chois.

They found root initiation in micro-shoots of Garcinia indica Chois 40-90 per cent after 35 days

of inoculation in different rooting media. Sudhersan et al. (2001) reported root initiation in 30

per cent of the cultures of Zizyphus mauritiana

cv. Umran after 30 days of inoculation in rooting media. These findings are in agreement with

the present results regarding Chironji.

4.3.3 Total root initiated

From the table 4.6 it was concluded that the total number of root initiated was found

maximum (4) under treatment 6 R6- 1/2 WPM+IBA 5.0 mg/L+1%activated charcoal followed by

treatment 4 R4- 1/2 WPM+IBA 3.0 mg/L+1%activated charcoal and the minimum total number

0

10

20

30

40

50

60

R1 R2 R3 R4 R5 R6

Effect of different treatments on root initiation % from the

explant shoot tip of Chironji

Total Shoots inoculated

Total root initiated

Days taken for root initiation

61

of root initiated was found in treatment 1, R1- MS (Without hormone). This results was close

agreements with the findings of Kumar et al. (2014), Parveen et a!. (2015) and Niratker (2016).

4.3.4 Total shoots inoculated

From the table 4.6 it was concluded that the total shoots inoculated was found maximum

(17) under treatment 1 R1- MS (Without hormone) followed by treatment 6 R6- 1/2 WPM+IBA

5.0 mg/L+1% activated charcoal and the minimum total shoots inoculated was found in

treatment 3, R3- 1/2 WPM + IBA 2.0 mg/L + 1% activated charcoal. Similar results were also

found by Hartmann et al., 1997.

62

CHAPTER V

SUMMARY AND CONCLUSIONS

5.1 Summary

Buchanania lanzan Spreng is a tree which produces the seeds commonly known as

Chironji. It is an excellent fruit tree of agro-forestry and social forestry. In the wasteland

development and dry land horticulture, it assumes great significance due to its multifarious uses

and capacity to withstand adverse environmental conditions. Chironji seeds are rich in nutrients

and medicinal properties. The species was in abundance in past, but due to rapid deforestation,

mishandling, felling of trees during fruit collection, consequently overexploiting and lack of

care, the species population is depleting very fast from its natural habitat. The major problem in

the reforestation or domestication of Chironji is the low percentage germination of seeds.

Vegetative propagation methods are also standardized and reported in Chironji, but these are less

effective due to less availability of rootstocks and dependency on seasonal conditions. In this

regard, biotechnology can play an important role and a boon for conservation of these important

plant species. . The tissue culture of perennial and woody species, being difficult to yield quick

results because of their inhehrent slow-growing nature beside intractable regeneration potential.

But of late, the accent has shifted to a good extent to regenerate trees which used to pose

insurmountable challenges in conventional practices of propagation. Previous work on many

forestry and woody tree species provided robust knowledge about need of biotechnology

approaches, factors affecting, constraints and achievements.

The present investigations on “Standardization of in-vitro propagation protocol for

Chironji (Buchanania lanzan Spreng)” was carried out in the laboratory of the Department of

Plant Molecular Biology and Biotechnology, College of Agriculture, Indira Gandhi Krishi

Vishwavidyalaya, Raipur (C.G.) from the year 2015 to 2017. The present investigations were

carried out complete protocols for selection of explants, multiple shoot induction and root

initiation of Chironji.

In the present investigation,from different explants (E-1,E-2,E-3 and E-4) were used for

development of in-vitro regeneration of Chironji. One year old Chironji plants were utilized as a

63

source of explants kept under green house conditions. Some of the tissue culture raised plants are

also used to obtain the explants viz. shoot tips, nodes, leaf and root, respectively. However,

Expalnt E1 – shoot tips, E2 –nodes, E3 -leaf and E4-roots of 1-2cm size were excised from the 1-2

year old plants grown under green house at PMBB, IGKVV, Raipur.

Different surface sterilization treatments were used to get healthy and contamination free

cultures. The highest percentage (85 %) of contaminant free E - 2 explants were established

when they were exposed to treatment SS-6 (Bavistin (1%) 10 min + HgCl2 (0.1%) 15 min. +

Ethanol (70%) 10 sec.). But maximum survival of explants was observed when they were

exposed to treatment SS-4 (Bavistin (1%) 10 min + HgCl2 (0.1%) 5 min. + Ethanol (70%) 10

sec.).

Different explants were used to find out their responses in various shooting media

treatments and differences in response were observed. The result revealed that, the multiple

shoot formation in explants E-1 was (5.5%) as compared to other explants in term of response

percentage. But in term of average number of shoots per explant was more in explants E-2 (

nodes).

Among different shooting treatments, maximum response to shoot initiation was

observed in explants E-1 on treatment S-1 (28.33 %) followed by treatment S-2 (17.23%). BAP

along with lower concentration of GA3 also found effective for multiple shoot initiation in

explant E-1 and E-2. Response percentage of explant E-1 was 34.33 per cent. Maximum days for

shoot initiation from explant E-1 observed in treatment R-6(38.79 days).

Six different rooting treatments were tried for in-vitro root initiation in Chironji micro

shoots obtained from Explant E-1 (Shoot tip). From these treatments, root initiation observed

only in treatment R-6 (1/2WPM + IBA 5.0 mg/l + 1% activated charcoal). In treatment R-6, 25

per cent rooting was observed in micro-shoots isolated from explants E-1 (Shoot tip) of

Chironji. In treatment R-6, rooting was observed in micro-shoots of explant E-1 38.79 days of

inoculation. Significant differences were noticed for survival percentage of plantlets after 15

days of transferring to hardening media.

5.3 Conclusions

64

From the research work done it can be concluded that

1. Shoot tip when used a explant showed best response over leaf, root and node explant.

2. Maximum shoot initiation %was obtain in the treatment S1- ½ WPM + BAP (2.0 mg/L) +

GA3 (0.5 mg/L) + kn 1.0mg/l.

3. Shoot multiplication % was highest (28.33%) in S1-½ WPM + 2 BAP + 1.5 NAA + kn

1.0mg/l.

4. Root initiation percentage was recorded 25% which was highest in treatment R6- 1/2

WPM+IBA 5.0 mg/L+1%activated charcoal.

5.4 Suggestions for future works

The finding of this investigation shall enlighten the other researchers for biotechnological

experiments on buchanania lanzan (Spreng). The following further studies are suggested:

Survey, collection and characterization of Chironji germplasms may be undertaken for

diversity rich area of Chhattisgarh. From these collections, superior and elite types of

Chironji should be used for production of tissue cultured plants.

The developed protocol may be used for the comparison of effect of different genotypes

collected from different locations.

Protocol may be developed by using other explants like leaves and other possible vegetative

parts including antioxidants and activated charcoal in shoot initiation medias.

Successfully regenerated tissue cultured plantlets may be evaluated under field conditions for

growth and yield attributes.

Phytochemical comparison of in-vitro materials could be helpful in getting the idea of

performance of in-vitro developed plants on different media combinations.

Cell hybridizations techniques need to be exploited between toxic and non-toxic derivatives

of buchanania lanzan (Spreng) genotypes to get better active principles and combinations.

The developed plants may be used for alkaloids extraction.

In-vitro propagation techniques may further be improved for commercial production of

planting materials from elite high yielding lines.

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