Introduction of Syzygium Alternifolium

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TABLE OF CONTENTS

S.no Contents Page.No

1. Introduction 1-7

2. Material and methods 8-21

3. Results and discussions 22-26

4. References 27-36
CONTENTS

Chapter 1: Introduction

1.1 Introduction

1.2 Introduction of Syzygium alternifolium

1.2.1 Scientific Classification Of S.alternifolium

1.2.2 Common Names

1.2.3 Economic Importance

1.2.4 Chemical Constituents

Chapter 2 : Materials and Methods

2.1 List Of Instrumentation

2.2 List Of Chemicals Used

2.3 Collection Of Plant Material

2.4 Categorisation Of Seeds Based On Their Size

2.5 Preparation Of Explants For Culture

2.6 Invitro Propagation

2.6.1 Preparation Of Nutrient Media

2.6.2 Preparation Of Stock Solutions

2.6.3 Macro Nutrients

2.6.4 Micro Nutrients

2.6.5 Stock Solution For Plant Growth Regulators

2.6.6 Carbon Sources

2.6.7 Gelling agent

2.6.8 Inoculations
2.6.9 Culture Conditions

2.6.10 Surface Sterilization

Chapter 3 : Results and Discussions

3.1 Effect Of Plant Growth Regulators

3.2 Rooting Of Adventitious Shoots

3.3 Data Collection and Statistical Analysis

3.4 Effect Of Auxins

3.5 Effect Of Cytokinins

Chapter 4 : References
LIST OF TABLES

Table Title of the table Page No


No

1. Scientific classification of S.alternifolium 5

2. Common names of S.alternifolium 6

3. List of instrumentation 8

4. List of chemicals used 8

5. Length and width of seeeds 11

6. Effects of auxins on adventitious shoots from the seeds 23

7. Effects of cytokinins and auxins on adventitious shoots 24


from the seeds

8. Effects of growth regulators on shoot length 24

9. Percentage of seed germination in vitro for two weeks 25


LIST OF FIGURES

Figure No Title of figures Page No

1. Morphological appearance of S.alternifolium 10

2. Categorization of seeds based on their size 11

3. S.alternifolium seeds measured with vernier calipers 12

4. Seed cultures in test tubes with medium containing different 26


concentrations of auxins and cytokinins
LIST OF ABBREVIATIONS

2,4D 2,4 Dichloro phenoxy acetic acid

IAA Indole -3-acetic acid

IBA Indole -3-buteric acid

MS Murashige and Skoog

BAP 6, Benzyl amino purine

KIN Kinetin
1.1Introduction:

Plant tissue culture is a broad term that refers to the culture of any part of the
plant (cell, tissue or organs ) in artificial media, in aseptic conditions, and under controlled
environment. The initiation of in vitro studies of plant cell and tissue culture dates back to
1902, when Gottlieb Haberland presented a “totipotency” hypothesis of each cell has all the
genetic information needed to produce a perfect plants. The set of techniques emerged as an
experimental approach to demonstrate the cell theory, which establishes that all living
organisms are constituted of cells, the basic units of structure and reproduction, and also the
totipotency concept, which is defined as the genetic potential of cell to generate an entire
multicellular or organism. Several reports have shown the totipotent ability of plant cells
through which the plant can be regenerated, which in turn Is widely used in several basic
studies such as an micropropagation, Germplasm of conservation, and formation of
genetically modified plants. Micropropagation commercially world wide, although the
capability of plant regeneration Varies significantly varies in different genotypes.

The physiological state of the plant does have an influence on its response
to tissue culture. The mother plant must be healthy and free from obvious signs of disease or
pest. The shoot tip explants being juvenile contain a higher proportion of actively dividing
cells. It is important to use quality mother plant stock to initiate cultures. The cultural
conditions required to initiate and sustain plant cells in culture, or to regenerate intact plants
from cultured cells, are different for each plant species. Each variety or clone of a species
often have a particular set of cultural requirements. Nontraditional inducers such as some
amino acids; light intensity and quality. weak electric current; and some antibiotics, for
example, cefotaxime, avee also been reported to affect in vitro plant regeneration. Rathore
and Goldsworthy passed very weak electric current 1 microamp between the tissue and the
culture medium and noticed dramatic increases in tobacco callus growth. Azmi et al.
Reported the beneficial effects of a mixed light color of LED (red and blue) on in vitro plant
regeneration of Rosa kordesii. This review covers novel findings of how plants adjust
regeneration in terms of the cellular, molecular and physiological aspects and discuss
influence of developmental and environmental factors on plant regeneration efficiency.

Micropropagation or in vitro clonal propagation is one of the most current


extended commercial applications of tissue culture. Plant tissue culture is an excellent tool for

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the asexual multiplication of those species that are naturally reproduced asexually, but it is
also used to overcome some problems of germination of seeds in different plant species; for
example, recalcitrant species are particularly characterized for their short-seed viability
(recalcitrant seeds), and therefore, asexual multiplication is a good alternative. Although
tissue culture can be applied for the micropropagation of almost any plant species, it is
recommended only for those that are economically profitable. Among the plant species that
are currently micropropagated at the commercial level, the ornamentals occupy the first
place.

Plant shoots and roots are able to retain their apical meristem functions
even after a part of their meristems is removed. However, when the whole meristems are
excised, plant cells of differentiated tissues or organs have the ability to produce new shoots
and lateral roots via organogenesis. In vitro plant regeneration by organogenesis is the result
of organ formation through dedifferentiation of differentiated cells and reorganization of cell
division to create particular organ primordia and meristems after the vascular connection
between the explant and the newly regenerating organ.

Somatic embryogenesis is one of the biotechnological techniques for


multiplication of important economic cultivars. This process is a type of plant cell totipotency
in which embryos arise from somatic or vegetative cells if no fertilization takes place. Several
factors such as the origin of the explant, culture medium, and in vitro environmental
conditions affect the success or failure of the somatic embryogenesis response. Somatic cells
undergo embryogenesis stages by developing structures similar to zygotic embryos without
merging of gametes.

For plant crops that are difficult to breed or have a poor genetic basis,
somaclonal variation can be a very useful option for breeders as a new option. Indirect plant
regeneration is carried out by organogenesis or embryogenesis in two steps.In the first step,
callus is induced, followed by the second stage, in which the shoot meristems or somatic
embryos are initiated from the callus tissues, resulting in an organ formation. Choosing the
right explant, medium, phytohormones, genotype, carbohydrate, and gelling agent, as well as
some other agents such as light regime, temperature, and humidity, noticeably affects
organogenesis and embryogenesiss processes. Shoot clumps can be regenerated from shoot
tips or bud stems that have only one bud, various mature somatic tissues, pollen, and

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protoplast. Protoplasts possess the ability to develop new cell wall and to regenerate complete
plants when grown in an appropriate culture medium. Crop improvement could be facilitated
by genome editing in regeneration from protoplastss . By genome editing, it is possible to
modify genome sequences as well as modify the arrangement of gene expression patterns in a
pre-specified area of an organism. Genome editing covers wide spectra of techniques
applying either a site-specific recombinase (SSR) or site-specific nuclease (SSN) system.
Genome editing is speedy with a very low hazard of unforeseen effects, and can be employed
with any crop, even those that have complex genomes and are difficult to breed.

Success in plant cell culture is largely determined by the quality of


nutrient media. A medium containing ‘chemically-defined’ compounds is referred to as a
‘synthetic medium’. One of the earliest plant tissue culture media is the root culture medium
of White (1939). The formulation prepared by Murashige and Skoog (1962) (MS Medium)
and revised by Linsmaier and Skoog (1965), Gamborg et al. (1968) and Schenk and
Hildbrandt (1972) can be regarded as a standard medium. MS medium was designed to test
the effect of organic supplements on tissue cultures. The medium therefore was standardized
with regard to inorganic nutrients and formulated for tobacco pith tissue. B5medium
(Gamborg et al.) formulated for growing soyabean tissues and SH medium for growth of
friable callus serve as feedstock for cell suspension culture and production of protoplasts .

Growth regulators play a key role for developing a specific mode of


growth in the cultured cells or tissues, which may be due to accumulation of specific
biochemical contents in them. The single or combination of different hormones in the
medium causes maintenance of specific and balanced inorganic and organic contents in the
growing tissue. This leads the cells or tissues to develop either into shoots/or roots or even
death . In tissue culture, plant growth regulators are important media components in
determining the development and developmental pathway of the plant cells. Growth
regulators are used in different proportions to break dormancy and enhance shoot formation
since it is well demonstrated that the apical dormancy is under control of these growth
regulators . The cytokinins and auxins are of importance in in-vitro culture as the later are
concerned with root formation, the former is mainly required in the media for shoot
formation and growth of buds . These growth regulators are required in combination in the
media as it is always the manipulation and variation of auxins and cytokinins levels that can

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successfully change the growth behavior of plant cultures.

Cytokinins such as benzyl aminopurine (BAP) and kinetin are known to


reduce the apical meristem domi- nance and induce both axiliary and adventitious shoot
formation from meristematic explants in syzigium alternifolium . However, the application of
higher BAP concentrations inhibits elongation of adventitious meristems and the conversion
into complete plants .

Auxins and other growth regulators such as gibberel- lins play important
roles in the growth and differentiation of cultured cells and tissues [. Auxins such as Naph-
talene acetic acid (NAA) have been reported to promote plant rooting in vitro.

1.2. Syzigium alternifolium

S. alternifolium occurs in the tropical dry deciduous forests of Kurnool,


Kadapa (YSR) and Chittoor districts of Andhra Pradesh, Chengalpattu and North Arcot
districts of Tamil Nadu and Bangalore District in Karnataka in India (Gamble 1957; Chitra
1983; Saldanha 1996; Reddy et al. 2006). Mohan & Lakshmi (2000) reported that S.
alternifolium occurs in the upper plateau, slopes and valley tops with dry, slaty and rocky
conditions at an elevation ranging from 600–1000 m in Sri Venkateswara Wildlife Sanctuary
of Chittoor and Kadapa districts. They stated that the distribution of this species appears to be
related to the geology and rock structure along with elevation and aspect.Reddy & Reddy
(2008) documented that S. alternifolium is an endemic and globally endangered species as
per the criteria of IUCN but not yet included in the IUCN Red List.

S.alternifolium (Wight) Walp belongs to family Myrtaceae most


commonly in Telugu it is called mogi chettu.The trees, 6-20 m tall. Branchlets grayish white
when dry, terete. Petiole 1-2 cm; leaf blade broadly elliptic to narrowly elliptic, 6-12 × 3.5-7
cm, leathery, abaxially slightly pale when dry, adaxially brownish green to blackish brown
and slightly glossy when dry, both surfaces with small glands, secondary veins numerous, 1-2
mm apart, and gradually extending into margin, intramarginal veins 1 mm from margin, base
broadly cuneate to rarely rounded, apex rounded to obtuse and with a short cusp.
Inflorescences axillary on flowering branches or occasionally terminal, paniculate cymes,
upto 11 cm. Hypanthium obconic or long pyriform, 4 mm or 7-8 mm. Calyx lobes
inconspicuous, 0.3-0.7 mm. Petals 4, white or light purple, coherent, ovate and slightly

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rounded, 2.5 mm. Stamens 3-4 mm. Style as long as stamens. Fruit red to black, ellipsoid
topot-shaped, 1-2 cm, 1-seeded; persistent calyx tube 1-1.5 mm.

The genus contains about 500 species mostly in the old world originated
and grown mostly in south-east Asia. The term Syzygium is derived from the Greek word
“Syzygos” which means yoked together preferably indicating to the paired leaves.The
members of genus having anti-bacterial property against bacterias like Salmonella
typhimurium, Psuedomonas, Bacillus, Staphyllococcus and Eschirichia coli (Shafi et al.,
2002; Djoukeng et al., 2005). In traditional medicine S. cumini has been used against
dysentry, to treat inflammation (Chaudhuri et al., 1990) and diabetes mellitus (Bhattari,
1992).

1.2.1. Scientific classification of Syzigium alternifolium:

Table:1

Kingdom Plantae

Phylum Tracheophytes

Division Eudicots

Class Rosids

Order Myrtales

Family Myrtaceae

Genus Syzigium

Species S.alternifolium

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Table:2

1.2.2. Common names of syzigium alternifolium:

English Black berry

Hindi Jamun

Sinhala Mahadan

Marathi Jambool

Telugu Mogi

Tamil Kottai nakam

1.2.3. Economic importance:

S. alternifolium is a fruit tree of great timber, medicinal and economic


importance. Timber is used for making furniture and agricultural implements. The plant tops
are used to cure skin diseases as it has excellent anti-fungal properties (Reddy et al. 1989).
The leaves are used in the treatment of liver cirrhosis, hepatitis, infective hepatitis, liver
enlargement, jaundice and other ailments of liver and gall bladder. Leaves fried in cow ghee
are used as a curry to treat dry cough.

A mixture of leaves and mineral oil is used to maintain dark hair and
also to promote hair growth by external application to the scalp. Tender shoots, fruits and leaf
juice are used to treat dysentery, seeds for diabetes and stem bark for gastric ulcers. Flowers
yield honey and possess antibiotic properties. The ripe fruits are used in making squashes and
jellies. Fruit juice is used to cure stomach-ache and ulcers while the external application of
fruit pulp reduces rheumatic pains (Reddy et al. 1989; Nagaraju & Rao 1990; Rao & Rao
2001; Bakshu 2002; Mohan et al. 2010).

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1.2.4. Chemical constituents:

The plant is rich in compounds comtaining anthocyanin


glucoside,Ellagic acid,Isoquerctin,Kaenferol and Myrecetin.The seeds are clived
to contain alkaloid,Jambolin or Antimellin which halt the diastatic conversion of
starch into sugars.

objectives of present study:


1.collection and establishment of seed cultures of syzigiumAlternifolium in M.Smedium.

2.Observation on effects of auxin and cytokinin containing culture medium.

3.The number of adventious shoots per seed and shoot length and their number ,the data is
collected and statistically analyzed.

4.plantlets which are acclimatized in medium containing test tubes are transfer to plastic pots
containing sterilized vermiculture.

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2. Materials and methods:

2.1.List of instrumentation:

TABLE:3

s.no Instruments used Make


1. Analytical balance Denver electronics
2. P.H meter Systronics,india
3. Magnetic stirrer Systronics ,india
4. Laminar air flow Bionics BST/HAO- 1124
HEPA FILTER L.F
5. Hot air oven Bionics BST/HAO-1124
model hot air oven
6. Vernier calipers cisco
7. Fluoroscent bulbs Philips
india(P)Ltd;Mumbai,india
8. Autoclave Oswal, india

2.2.List of chemicals:

TABLE:4

S.no Chemicals used


1. Ammonium nitrate
2. Pottasium nitrate
3. Calcium chloride
4. Magnesium sulphate
5. Mono potassium phosphate
6. Potassium iodide
7. Boric acid

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8. Manganese sulphate
9. Zinc sulphate
10. Sodium molybdate
11. Copper sulphate
12. Cobalt chloride
13. Glycine
14. Nicotinic acid
15. Pyridoxine Hcl
16. Ferrous sulphate
17. Sodium EDTA

2.3.Collection of Plant material:

Polyembryonic ripened fruits of syzigium


alternifolium(Wight)walp,endemic to sesachelam biosphere reserves were collected in the
months of July and august,taken from the trees which are situated in palakondalu hills of
Kadapa district of Andhra Pradesh.Intially the fruits of S.alternifolium taken in the polythene
zip bags in the forest and then carry to the tissue culture laboratory Department Of Botany in
Yogi Vemana University.

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Fig :1 Morphological appearance of Syzigium alternifolium

Fig 1 A&B) Vegetative characters of Syzygium alternifolium C) Flower before pollination D)


Flower after pollinatiom E) Fruits

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2.4 Categerization of seeds based on their size:

The seed size of the fruit may vary from one to another,so that
based on the size ,we categorized the seeds into four sizes i.e Very Small, Small,Medium&
Large . The length and the width of each size of seed is measured with the help of vernier
calipers. S. alternifolium fruits were allowed to dry under sunlight for two hours followed by
shade drying in the laboratory to remove moisture and prevent from fungal
contamination.These dried fruits were stored in a dessicator glass container until further use.

Fig: 2 Different sizes of Seeds A) Very Small B) Small C) medium D)Large

Table:5

Seed size Length (cm) Width (cm)

Very small 1.17 1.01

Small 1.29 1.03

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Medium 1.4 1.09

Large 1.84 1.28

* Values are mean of ten replicates.


Fig:3 S.alternifolium seeds measuring with vernier calipers:

Fig A) Calculating Seed Length By Using Vernier Calipers B) Calculating Seed Width By
Using Vernier Calipers

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2.5 Preparation of explants for culture:

For knowing that which size of the seed having more viable
capability on culture medium.The dried pulp or seed coat of S.alternifolium is removed with
the help of foreceps, now the seeds appear in a lush green in colour.

These processed dicotyledenous seeds are explants for tissue


culture.These seeds were firstly immersed in the distilled water ,cleaned the surface of the
seeds by rinsing and then followed by Hgcl2 for 2minutes.again the seeds were sterilized
with distilled water. seeds are now explants for vitro culture.

Plant tissue culture:

1.Plant species:

Syzigium alternifolium

2.Chemicals and glass ware:

Liquid soap(labolene) : National scientific products,Gntr

Mercuric chloride hgcl2 : Himedia,Mumbai ,india

PGRs and vitamins : Himedia ,Mumbai,india

Macro and micro salts :Himedia,Mumbai,india

Sucrose :Himedia,Mumbai,india

Agar Agar :National scientific products,GNTR

Glass ware :Borosil,india

3.Apparatus:

Autoclave :Oswal,india

Laminar air flow : clean air systems,india

P.Hmeter : systronics,india

Fluorescent tubes : Philips india(p)Ltd,Mumbai,india

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Hot air oven :Yorco,india

Apparatus used to raise cultures:

a.Borosil and corning test tubes

b.Conical flasks

4.For the preparation of the medium:

a.Micro pipettes of variable volumes

b.Borosil conical flasks of capacity 1000ml,500ml,250ml and 100ml

c.Measuring cylinders of capacity 500ml,250ml,100ml and 50ml

d.Borosil beakers of capacity 500ml and 250 ml.

2.6 INVITRO PROPAGATION OF Syzigium alternifolium:

Plant tissue culture is the science of growing plant cells tissues are
organs isolated from the mother plant on artificial medium under control and aseptic
conditions,It includes techniques and methods appropritate to research into several practical
objectives. In tissue culture,All experiments are designed according to the method of
Muraschige and Skoog (1962).

Cleaning and maintainence of glass ware is te first step to begin with


the invitro studies.Since tissue culture experiments aare conducted in control and aseptic
conditions eveyry article used in experiment should be sterile.Major problem in tissue culture
is contamination.Since glassware ios the main source of contamination,It has to be throughly
washed.During washing all the required was soaked overnight in 40% chromic acid solution
followed by rinsing under exact flow of tap water.Later they washed with 5% laboline
solution and rinsed with tap water until soap traces were removed .Oncwe again all the
glassware was rinsed with distilled water and oven dried at 150°C for 1hour.Contaminated
culture tubes were autoclaved at 121°C at 15lb/m2 foe 30 minutes before washing.

2.6.1 PREPARATION OF NUTRIENT MEDIA:

The success in cell,Tissue and organ culture technology is related to


the selection of development of the culture media,As no single media will support thr growth

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of all tissues. The principal components of most tissue culture media can be categorised into
inorganic nutrients(macro and micro),Organic nutrients(vitamis and aminoacids),Carbon
source,Plant growth regulators and gelling agent.The hormones from HIMEDIA
company,Bombay.Nutrient used in the Present study were Muraschige and Skoog(MS)
medium (Muraschige and Skoog,1962).

2.6.2 PREPARATION OF MS STOCK SOLUTIONSAND VOLUME TAKEN FROM


THE STOCKS TO PREPARE THE MEDIUM(GAMBORG(1982,1991)Dodd’s and
Roberts(1982):

STOCK A(Macro nutrients):10x conc.

1. Ammonium nitrate (NH4NO3) - 1.65gm/l


2. Potassium nitrate (KNO3) - 1.9gm/l
3. Calcium chloride (CaCl2) - 0.44gm/l
4. Magnesium sulphate (Mgso4) - 0.37gm/l
5. Mono potassium phosphate (Kbpo4) - 0.17gm/l

STOCK B (Micro nutrients):1000x conc.

1. Potassium iodide (KI) - 0.86gm/l


2. Boric acid (H3Bo3) - 6.2gm/l
3. Manganese sulphate(Mnso4.2H2O) - 22.3gm/l
4. Zinc sulphate (Znso4.7H20) - 8.6gm/l
5. Sodium molybdate (Na2Mo4.2H2O) - 0.25gm/l
6. Copper sulphate (Cuso4.5H20) - 0.025gm/l
7.Cobalt chloride (CoCl2.6H20) - 0.25gm/l

STOCK C (Vitamins):1000x conc.

1. Glycine - 2gm/l
2. Nicotinic acid - 0.5gm/l
3. Thyamine - 0.4gm/l
4. Pyridoxin Hcl - 0.5gm/l

15
STOCK D (Iron source):

1. Ferrous sulphate (Feso4.7H20) - 0.05gm/10ml


2. Sodium EDTA (Na2EDTA.2H2O) - 0.074gm/10ml
Myoinositol - 0.1gm/l
Sucrose - 30gm/l
Agar - 8gm/l

NOTE:

⚫ If stock concentration is 10x - 100ml/l


⚫ If stock concentration is 100x - 10ml/l
⚫ If stock concentration is 1000x - 1ml/l

PROTOCOL FOR 1LITRE CULTURE MEDIA PREPARATION :

Stock A - 100ml

Stock B - 1ml

Stock C - 1ml

Stock D - 5ml

Myoinositol - 100mg in 100ml distilled water

Sucrose - 30g in 100mlk distilled water

Set or adjust pH 5.7 - 5.8 then make up the media to 1litre with distilled water
then add 8gm of agar.Boil the media until agar disolved clearly.After that add plant growth
regulators and autoclave.

PREPARATION OF 2-4-D (2,4 DICHLORO PHENOXY ACETIC ACID) FOR 1


LITRE:

To prepare 1mg/1ml stock solution of 2-4-D take 100mg of 2-4-D in a


testtube and add 25ml of ethanol to dissolve the powder heat gently if required once
completely dissolved gradually diluted.The solution with 100ml of disstilled water store

16
diluted.The solution with 100ml of disstilled water store the stock solution in the refrigerator
at 4°C.

Preparation of BAP Stock Solution :

To prepare 100ml of stock solution 100mg of 6,benzyl amino purine was


dissolved in 2ml of 70%ethanol and make upto 100ml with distilled water.The ssolution was
stored in refregirator at 4°C.

Preparation Of IBA Stock Solution :

To prepare 100ml of stock solution 100mg of indole 3 acetic cid was dissolved
in 2ml of 70% ethanol and made upto 100ml distilled water.The solution was stored in
refrigerator at 4°C.

HALF STRENGTH MEDIA PREPARATION:

Stock A - 25ml

Stock B - 0.25ml

Stock C - 0.25ml

Stock D - 1.25ml

Myoinositol - 25mg

Sucrose - 7.5gm

Agar - 4gm

CaCl2 - 0.03gm/750ml (In Stock A)

NOTE:

⚫ If stock concentratin is 10x - 0.44gm/l


⚫ If stock concentration is 1x - 0.04gm/l
⚫ If stock concentration id 1x - 0.02gm/50ml

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2.6.3 MACRO NUTRIENTS:

Analytical grade(AR) chemicals,Sterile,Double distilled water were


used to prepare media and stock solutions.When a small amount of medium was required or
when amedium was required in frequently,Freshly weighed macro nutrients were dissolved
one by one as 10times the concentration(10x)of the final volume.A separate solution of
calcium chloride was prepared to avoid precipitation problems.

2.6.4 MICRO NUTRIENTS:

Stock solutions of micro nutients stock 1(40x),Stock 2(40x) and


vitamin and aminoacid stock(40x)were prepared by dissolving the constituents individually in
500ml flask over a magnet stirrer and finally stored in 100ml volumetric flask at 4°C.The
stock solution of iron(40x)was prepared by dissolving Na2EDTA in 50ml boiling
doubledistilled water and to this added Feso4.7H2O gradually.The mixture was kept on a
magnetic stirrer in hot conditions for 1hour untikl the colour of the solution changes to
golden yellow.Volumes was made upto 100ml and stored in amber coloured bottle at room
temperature.

2.6.5 STOCK SOLUTIONS FOR PLANT GROWTH REGULATORS:

Different types of plant growth regulators such as auxins,Cytokinins


were prepared at 0.01xconcentration using suitable solvent and made to 100ml using sterile
distilled water.Different solvents used for dissolving various plant growth regulators were
given in table1.All the stock solutions were stored in a refrigirator at 4°C.It was made as a
customeric practice that stocks once were not used after 12 weeks.

2.6.6 CARBON SOURCES :

These include lactose,Galactose,Maltose and starch as they were


reported to be less effective than either sucrise or glucose,The latter was similarly more
effective than fructose considering that glucose is utilising by the cells in the
beginning,Followed by fructose.It was frequently demonstrated that autoclaved sucrose was
better for growth than filter sterilised sucrose.Autoclaving seems to hydrolyze sucrose into
more efficiently utilizable sugars such as fructose.Sucrose was reported to act as a

18
morphogenetic trigger in the formation of auxillary buds and branching of adventitious roots.

2.6.7 GELLING AGENT:

Bacteriological grade agar at 0.8% was used in almost all experiments


except rooting experiments where 0.6% was added.To prepare 1litre medium,Required
quality of macro nutrients (100ml)and micro nutirnts (1ml)were taken in a clean sterile
1000ml beaker containing 400ml of double distilled water.Harmonal stocks were added in
required concentrations either individually or in various combinations.Freshly weighed
sucrose (30g) was added,Dissolved and made into 1000ml with double distilled water.The pH
of the media was adjusted between 5.6 - 5.8 using 0.1NHcl or 0.1NaoH prior to agar addition
(8gm).The medium was the boiled and dispensed into culture tubes (Borosil,India) 25x
150ml(15ml/tube) and air tied with alluminium foil.All the culture tubes containing media
was sterilized at 121°C,15lbs for 15minutes in an autoclave. After sterilization the tubes were
incubated vertically for organogenesis and slants were for prepared callus initiation.After
cooling the medium was ready for inoculation.The prepared medium was used only after
3days to identify and avoid microbial contamination if any.

2.6.8 INOCULATIONS:

Prior to inoculation,The laminar air flow cabinet was sprayed with 70%
ethanol and all the required paraphernalia were transferred into it.Sterilization of the chamber
was done by switching on the ultra violet(UV) lamp half an hour before flows atbthe velocity
of 27± m/s.Inoculation done near the spirit lamp,Which was kept inside the chamber.Hand
sand inoculating area were swabbed with alcohol frequently to minimise the
concentration.During inoculations before and after every use the instruments were dipped in
ethyl alcohol and flamed

2.6.9 CULTURE CONDITIONS:

All the cultures were incubated in culture room at 25±2°C with arelative
humidity of 50 - 60% at 50 - 1200μmol m-2 sec-1.Light intensity for 16hour photo
period(preece and sutter,1991).To provide the slight inmtensity two 4 feet (1.2m)cool- white
fluroscent lamps spacing 50cm apart were provided for each shelf.

19
2.6.10 SURFACE STERILIZATION:

The plant material exposed to environment would be infested


superficially with many micro organisms like bacteria fungi etc.,due to their brief life cycle
they grow luxiriantly on the nuitrient medium and growth of the explants will be
hampered.So to avoid these problems,Explants were subjected to surface sterilization.

FLOW CHART

Direct operation to prepare 1litre medium

Ca.400ml DDW

30gms Sucrose was dissolved

100ml Macro nutrient stock was added

2.9ml Calcium Chloride stock was added

1ml Micro nutrient stock was added

1ml Potassium iodide was added

5ml Iron stock was added

20
1ml Vitamin stock was added

Volume was adjusted to 800ml

pH was adjusted to 5.8 and made and upto 1litre

8gm Agar was added and melted

Dispensed into culture vessels

Autoclave

21
3.Results and Discussions:

3.1 EFFECT OF GROWTH REGULATORS:

Three sets of experiments were carried out to test the effect of auxins and
cytokinins on morphogenic potential of dicotyledonous seeds
In the first set different auxins indole-3-acetic acid and 2,4 Dichlorophenoxy
acidic acid at three different concentrations of 2-4-D (0.25,0.5,1.0mg) were incorporated in
the basal medium.The controls receives no auxins
In the second set cytokinins (Benzyl amino purine)were tested at 2
concentrations (1.0,0.5mg)te control receive no cytokinins
2,4 D at a concentration of 0.25mg was choosen because it yielded the maximal
number of shoots with the best quality.It was combined with cytokinins (BAP) in the
concentration ranging from 0.5 to 1.0mg basal medium (BM) supplemented with 2-4 D
which is 0.25 concentration served as a control.

3.2 ROOTING OF ADVENTITIOUS SHOOTS :


Individual adventitious shoots isolated from dicotyledons seed cultures growing
on 0.25mg 2,4D and 0.5mg IAA wee used for rooting.The bases of excised shoots were
dippeed in different concentration of pre autoclaved IAA or Indole 3 butyric acid for rooting
there after they were implemented on solidfied half strength ms medium lacking auxins the
shoots which were not pulsed with auxins served as control.

3.3 DATA COLLECTION AND STATISTICAL ANALYSIS:


Data were collected for four weeks in the seond and third sets of experiments
culture were examined for number of adventitious shoots per seed and shoot length.12
Explants were cultured per treatment and all experiments were repated two times the data
were statistically analysed using standard error of mean.
The dicotyledonous seeds cultured on a growth regulator free medium i.e.,
(control) germinated after 14days all dicotyledonous seeds produced two to three adventitious
shoots with a common root.Similar results were obtained in highly polyembryoinc citrus
(Maheshwari and Rangaswamy 1958).About 80% of dicotyledonous seeds produced a single
shoot and root from their embryonal access upon germination,While the others showed
formation of either single shoot or root or multiple adventitious shoots or roots.According to
Pijl(1934) germination of polyembryonic eugnia Syzygium seeds is often uneven or
incomplete.Many seeds from roots or shoots only,Which Pijl attributed to incompete
development of embryos.These may be due to an imbalancing endogenous growth regulators.

22
3.4 EFFECTS OF AUXINS:
The morphogenic response was noticd in dicotyledonous seeds within 2weeks of
incubation on auxin containing medium.The degree of morphogenic response of seeds is
varied greatly with the type and concentration of auxin. 2,4D or IAA in concentration of
0.25mg induced single shoot and multiple adventitious roots from the embryonal
access,While the (higher concentration like 1.0mg) resulted in profuse root formation and
inhibited shoot formation.Sometimes a white nodular and friable callus with roots was also
induced from the embryonal access as well as cotyledonous surface.Media with 2,4D induced
a loose friable white embryogenic callus from the embryonal acess and the surface of
cotyledons.

3.5 EFFECTS OF CYTOKININS:


Addition of a cytokinin to the medium induced development of adventitious shoots
from the embryonal axes of dicotyledonous seeds within 2weeks of incubation.The BAP (Benzyl
amino purine ) induced healthy adventitious shoots.The number of shoots increased with increasing
concentration of BAP which is having concentration of 0.5mg.The BAP has been shiown in tissue
culture of other tree species of myrtaceae(Yadav et al.1990),Speer(1993).

Table :6 Effect of auxins on adventitious shoots from the embryonal axes of dicotyledonous
seeds of Syzygium alternifolium grown in vitro for 4 weels Means ± se of three replicants

AUXINS Concentration(mg/l) Dicotyledonous seeds Shoot Length(cm)


Shoot Number

0.25 13.0±1.2 3.2±1.1


2,4D 0.5 17.4±1.4 2.1±1.3
1.0 16.2±1.1 2.2±1.3
0.25 11.2±1.1 3.9±1.1
IAA 0.5 10.3±0.8 2.9±1.4
1.0 13±1.4 3.1±1.2

23
Table :7 Effect of cytokinins and auxins on adventitious shoot formation from
embryonal acess of dicotylydonous seeds of Syzygium alternifolium grown invitro for 4
weeks Mean ± of three Replicates

CYTOKININS+AUXINS Dicotyledonous Shoot Shoot Length(cm)


Number

BAP(0.5)+2,4D(0.25) 1.5±0.6 1.1±0.9

BAP(0.5)+2,4D(0.5) 1.9±0.9 1.7±1.1

BAP(1.0)+2,4D(0.5) 2.1±1.1 1.9±1.3

Table : 8 Effect Of Growth Regulators On Shoot Length After 5 weeks Mean ± Se Of Two
Regulators

CYTOKININS + AUXINS CONCENTRATION (mg/l) DICOTYLEDONOUS


SHOOT LENGTH(cm)

0.25 1.4±0.5
IAA 0.5 1.1±0.7
1.0 0.9±0.2
0.5+0.25 0.9±0.3
BAP 0.5+0.5 0.7±0.4
+2,4D
1.0+0.5 1.0±0.9

24
Table ;9
Percentage of Seed Germination Of Syzygium alternifolium invitro for 2weeks

Seed Sizes NO. Of Seeds Kept No. Of Seeds % Of Germination


For Germination Geerminated

Very small 10 1 10

Small 10 4 40

Medium 10 8 80

Large 10 9 90

25
Fig:4 Seeds of S.alternifolium cultured in test tubes with medium containing different
concentrations of Auxins and cytokinins for shoot generation

(A)Seed on culture medium before sprouting (Band c)Seed is getting cleaved and small
roots are formed (D,EandF) Adventitious shoot formation from embryonal axes of
dicotyledonous seed cultered on basal medium containing 0.25 mg2,4-D and 0.5 mg IAA

(G,H and I) Growth regulators effect on shoot length.

26
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