Introduction of Syzygium Alternifolium
Introduction of Syzygium Alternifolium
Introduction of Syzygium Alternifolium
1. Introduction 1-7
4. References 27-36
CONTENTS
Chapter 1: Introduction
1.1 Introduction
2.6.8 Inoculations
2.6.9 Culture Conditions
Chapter 4 : References
LIST OF TABLES
3. List of instrumentation 8
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.
1
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.
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
2
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.
3
successfully change the growth behavior of plant cultures.
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.
4
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).
Table:1
Kingdom Plantae
Phylum Tracheophytes
Division Eudicots
Class Rosids
Order Myrtales
Family Myrtaceae
Genus Syzigium
Species S.alternifolium
5
Table:2
Hindi Jamun
Sinhala Mahadan
Marathi Jambool
Telugu Mogi
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:
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
2.2.List of chemicals:
TABLE:4
8
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
9
Fig :1 Morphological appearance of Syzigium alternifolium
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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.
Table:5
11
Medium 1.4 1.09
Fig A) Calculating Seed Length By Using Vernier Calipers B) Calculating Seed Width By
Using Vernier Calipers
12
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.
1.Plant species:
Syzigium alternifolium
Sucrose :Himedia,Mumbai,india
3.Apparatus:
Autoclave :Oswal,india
P.Hmeter : systronics,india
13
Hot air oven :Yorco,india
b.Conical flasks
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).
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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).
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):
NOTE:
Stock A - 100ml
Stock B - 1ml
Stock C - 1ml
Stock D - 5ml
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.
16
diluted.The solution with 100ml of disstilled water store the stock solution in the refrigerator
at 4°C.
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.
Stock A - 25ml
Stock B - 0.25ml
Stock C - 0.25ml
Stock D - 1.25ml
Myoinositol - 25mg
Sucrose - 7.5gm
Agar - 4gm
NOTE:
17
2.6.3 MACRO NUTRIENTS:
18
morphogenetic trigger in the formation of auxillary buds and branching of adventitious roots.
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
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:
FLOW CHART
Ca.400ml DDW
20
1ml Vitamin stock was added
Autoclave
21
3.Results and Discussions:
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.
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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.
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
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
Table : 8 Effect Of Growth Regulators On Shoot Length After 5 weeks Mean ± Se Of Two
Regulators
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
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
26
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