TThhee eeffffeecctt ooff ggrroowwtthh rreegguullaattoorrss oonn iinn vviittrroo

mmuullttiipplliiccaattiioonn && pprrooppaaggaattiioonn ooff RRaauuwwoollffiiaa sseerrppeennttiinnaa

Under the guidance of

Senior Scientist cum Associate Professor

COLLEGE OF BIOTECHNOLOGY

BIRSA AGRICULTURAL UNIVERSITY

Ranchi-834006

Submitted By:-

Yogesh Bhai Patel B.Sc. in Biotechnology (H), 3RD YEAR

ROLL- DURG BT. NO- 2008/42

REG.No- 1064 OF 2007-2008.

The University of Burdwan

ACKNOWLEDGMENT It bring me a great felicity and proud to gratify every individual who helped me in making the

project a success and giving their invaluable time to me.

I express my deep indebtedness to Dr. Z. A. Haider, Associate Dean, College of Biotechnology,

Birsa Agricultral University, Ranchi-6 for his continous encouragement and providing necessary facilities

to complete this work.

My sincere gratitude also to the Dr. Maduparna Banerjee, Senior Scientist-cum-Associate

Professor,BAU,for her guidance and encouragement throughout my project work.I thank madam for giving me her precious time and help, without which the project would not have reached up to its excellence.

I convey my sincere gratitude and thanks to Dr. Joydev Gangopadhyay, Principle & Director,

DIST, Dugapur, for providing the permission to complete my dissertation. I wish to avail this to express my heartfelt devotion and profound respect to our teachers,

Mr, Abijit Sarkar, HOD of Biotechnology, D.I.S.T (Durgapur) and Dr.Anita Paul for generating great

enthusiasm inside me. I render my thanks to all the faculty of D.I.S.T.

I am thankful to my seniors and friends, who have co-operated me from time to time throughout

my study.

I am also thankful to all other staff members of college of Biotechnology, BAU.

Last but not least, I wish to avail this opportunity to express my heartfelt devotion profound respect

to my worthy novel Parents and family member, whose inspirational value and sacrifice helped me a lot

in achieving the goal.

Above all I offer my heartfelt devotion and much more thanks to the Almighty for his sacred blessing

bestowed on my life.

August 28, 2009 - Y. B. PATEL

INDEX

CONTENT PAGE NO

ACKNOWLEDGMENT

1. INTRODUCTION 1-3

2. OBJECTIVE OF PROJECT 4

3. DESCRIPTION OF Rauwolfia serpentina 5-7

4. REVIEW OF LITERATURE 8-9

5. MATERIALS AND METHODS 10-15

LABORATORY EQUIPMENT

SURFACE STERILIZATON OF EXPLANT

TRASFER OF EXPLANT

MULTIPLICATION OF SHOOTS

ROOTING AND HARDENING

6. OBSERVATION 16-17

7. RESULT AND DISCUSSION 18-20

8. CONCLUSION 21

9. BIBLIOGRAPHY 22

The plant cell, tissue and organ are cultured in a defined medium under strict

aseptic conditions and incubated under artificial environment (in vitro) to induce

growth and development. The developmental fate of the plant tissues to be

manipulated towards desired ends is by inclusion of growth regulators (phyto

hormones) and other chemicals in the medium.

Tissue Culture is considered a cost-effective technology but with a capable impact

which is clearly visible in the form of improved yields, supply of virus-free clones

of true-to-type cultivars with provision of improved germplasm. It also considered

as a„Lab to Land‟ technology.

HABERLANDT(1902) is known as the father of plant tissue

culture. First time he cultured the plant cells in in vitro conditions. In the very first

this technique was used to grow the ornamental plants. This technique was used to

grow the orchid plants. But now it is the most exciting field of modern biology.

Tissue culture has laid a foundation for studying the regulation of cell proliferation,

differentiation and regeneration under controlled condition.

Micropropagation is the regeneration of whole plant to produce a large no. of

progeny plants, using single mother plant by tissue culture methods. It is used to

multiply novel plants, such as those that have been genetically modified or bred

through conventional plant breeding methods. Murashige (1974) has described the

procedure of development of micropropagation in to three developmental stages.

Establishment of explants aseptically.

Multiplication of propagules by repeated subculture on a specific nutrient

medium.

Rooting and hardening of plantlets and planting in to soil.

INTRODUCTION

APPLICATIONS

In commercial applications it may be possible to multiply in vitro plants that

are very difficult to propagate by cuttings or other traditional methods ,e.g.;

orchids and Nepenthes.

Micropropagation is widely used in forestry and in floricultural plants;

micropropagation can also be used to conserve rare or endangered plant

species.

Large numbers of genetically identical clones may be produced through it.

A plant breeder may use tissue culture to screen cells rather than plants for

advantageous characters, e.g. herbicide resistance/tolerance.

Removal of viruses by propagation from meristematic tissues.

Seeds can be germinated with no risk of damping off/predation.

Certain techniques such as meristem tip culture may be employed that can be

used to produce clean plant material from virus stock, such as potatoes and

many species of soft fruit.

High multiplication rate e.g. 106 of plants per year from a single explant. And

very small explants can be used.

Micropropagation using meristem and shoot culture to produce large numbers

of identical individuals.

Large scale growth of plant cells in liquid culture as a source of secondary

products.

Crossing distantly related species by protoplast fusion and regeneration of the

novel hybrid.

Production of diploid plants from haploid cultures to achieve homozygous

lines more rapidly in breeding programs.

DISADVANTAGES

Micropropagation is not always the perfect means of multiplying plants, conditions

that limit its use include:

It is very expensive, and can have a labor cost of more than 70% .

As monoculture is produced after micropropagation, in case of infection whole

crop can get damaged.

An infected plant sample can produce infected progeny. This is uncommon if

the stock plants are carefully screened and vetted to prevent culturing plants

infected with virus or fungus.

Not all plants can be successfully tissue cultured, often because the proper

medium for growth is not known or the plants produce secondary metabolic

chemicals that stunt or kill the explant.

Sometimes plants or cultivars do not come true to type after being tissue

cultured; this is often dependent on the type of explant material utilized during

the initiation phase or the result of the age of the cell or propagule line.

Some plants are very difficult to disinfest of fungal organisms.

The major limitation in the use of micropropagation for many plants is the cost of

production. For this reason, many plant breeders do not utilize micropropagation

because the cost is prohibitive other breeders use it to produce stock plants that are

then used for seed multiplication.

Mechanisation of the process could reduce labour costs, but has proven difficult to

achieve, despite active attempts to develop technological solutions.

Rauwolfia sp. is threatened in India due to indiscriminate collection and over

exploitation of natural resources for commercial purposes to meet the requirements

of pharmaceutical industry.

Since seed germination in Rauwolfia sp. is highly variable. It is reported to vary

from 5 to 30 percent even when only heavy seeds are chosen for sowing purpose.

The problem of poor germination is forcing the farmers to use cuttings for

propagation. The increasing demand of cuttings is again becoming a problem for

natural population. Therefore cuttings and in vitro production is a better option, the

cutting process has also got its limitation, as the production through cuttings is

often seasonal and is subject to seasonal & somatic variations, infections of

bacteria, fungi & insects as well as environmental pollution that can affect the

medicinal value of the harvested plants. It must couple with some modern

technique.

Hence, micropropagation is the most effective method to get many

number of planting materials at any given time throughout the year.

Due to the prevailing reasons there is a huge need for in vitro propagation of

Rauwolfia serpentina to satisfy the growing commercial demand of the plant for

the production of life supporting alkaloids and conservation of this valuable

endangered plant itself. Hence improvements in plant tissue culture techniques for

the mass propagation of R. serpentina are highly desirable.

The present study was undertaken to develop a more efficient protocol for rapid

in vitro propagation & multiplication of Rauwolfia serpentina i.e., the

standardization of low cost media and proportion of phytohormones for induction

of efficient multiplication of apical as well as nodal and axillary tissue of field

grown plant. The present work deals with species Rauwolfia serpentina. The total

work has been carried out at Plant Tissue Culture Laboratory, College of

Biotechnology, BAU campus, Ranchi.

OBJECTIVE OF PROJECT

Scientific Name: Rauwolfia serpentina (L.) Benth. ex Kurz.

(Syn. Ophioxylon sepentinum L.)

English Name: Rauwolfia root, serpentine

Trade Name: Serpentine Roots

Common (Indian) Names:

Assamese: Arachoritita

Bengali: Chandra

Hindi: Chandrabhaga, Chota-chand, Sarpagandha

Malyalma: Churannavilpori, Suvapavalporiyam

Marathi: Harkaya: Harki

Oriya: Patalagarur, Sanochado

Sanskrit: Sarpagandha, Chandrika, Patalguruda

Tamil: Chevanamalpodi

Family: Apocynaceae (Nathan Kline, 1954).

Habitat: Moist forests shady places near rain-forest.

Status: The natural reserves of this plant are declining, especially after reports of

its medicinal properties appeared in literatures. International Union for the

Conservation of Nature and Natural Resources (IUCN) has kept this plant under

endangered status.

Distribution: The snake-weed genus includes about 50 species; this has fairly

wide area of distribution, including the tropical part of the Himalayas, the Indian

peninsula, Sri Lanka, Burma, and Indonesia.

Related Species: Rauwolfia tetraphylla L.(Syn. R. canescens L.; R. heterophylla

Roem. and Schult.). In Hindi, it is named Barachandrika. Found in Bihar, Orissa,

Chhattisgarh, Madhya Pradesh, West Bengal, Andhra Pradesh, Tamil Nadu, and

Kerala states of India.)

DESCRIPTION OF SARPGANDHA

Rauwolfia vomitoria (A. fzel) and R. caffra both are African species, having

medicinal properties similar to R. serpentina but with low total alkaloid content

and also low in serpentine.

Botany: An erect perennial shrub with a long, irregularly, nodular, yellowish, root

stock.

Leaves: In whorls of 3, thin, lanceolate, acute, bright green above and pale

beneath.

Flowers: in irregular corymbose cymes, white, often tinged with violet.

Fruit: Drupe, single or didynamous, shining black, the inflorescenece with red

pedicels and calyx and white corolla.

Flowering Time: March to May in Indian conditions.

Natural Components: The root contains ophioxylin (an alkaloid having orange

colored crystalline principle), resin, starch and wax. The total alkaloid yield is

0.8%. Five crystalline alkaloids isolated are ajmaline, ajmalicine, serpentine,

serpentinine, and yohimbine.

Useful Parts: Roots and leaves.

Medicinal Properties and Uses: According to ayurveda root is bitter, acrid,

heating, sharp, pungent and anthelminic. Drug of Rauwolfia sp. consists of air-

dried roots. Rauwolfia sp. preparations are used as antihypertensive and as

sedative. It is also used for the treatment of various central nervous system

disorders associated with psychosis, schizophrenia, insanity, insomnia, and

epilepsy.

Ayurvedic Preparations: Sarpagandha ghanavati, sarpagandha yoga,

Sarpagandha churna, Mahesvari vati etc.

Cultivation: This plant is under cultivation in India, Sri Lanka, and Java.

Experiments on cultivation are in progress in the United States.

Climate: It grows luxuriantly well where the rainfall is 2500 mm or more. The

areas having more equable climatic variations seem to be more suited than the

areas having higher climatic variations.

Soil: It prefers soil with plenty of humus and rich in nitrogenous and organic

matter with good drainage. Alkaline soils are not suitable for commercial

cultivation.

Propagation: It Can be propagated both through seeds and vegetatively, but

propagation by seed is not preferred.

Seed Rate: 10 kg/ha.

Nutrients: Generally organic cultivation is practiced. Initially before sowing 10–

15 tones of farm yard manures/ha are used.

Maturity Period: 3 Years. At this time the sub aerial parts dry and main root

reach a depth of 0.9 meters.

Average Yield: 2700 to 3300 kg dried roots/ha and 8–10 kg seed.

Medicinal uses

Reserpine is an alkaloid first isolated from R. serpentina which was widely used as

an antihypertensive drug. Other plants of this genus are also used medicinally, both

in conventional western medicine and in Ayurveda, Unani medicine. Alkaloids in

the plants reduce blood pressure, depress activity of central nervous system and act

as hypnotics.

It is widely used in Hypertension, sexual weakness.

Also useful in violent form of insanity and madness.

Used to lower blood pressure.

Used in epilepsy.

To treat anxiety and insomnia.

Used in hypertension.

Roots are used in skin disorder, excessive sweating and itching.

Roots are used in case of snake bite antidepressant.

The root of Rauwolfia sp. was popular from Asian times, both in India and on the

Malay Peninsula, as an antidote to the stings of insects and bites of poisonous

reptiles. It has been also used as anti pyretic, an oxytoxic, a sedative and a

palliative for insanity.

The roots of Rauwolfia serpentina contain the alkaloid reserpine. This

alkaloid was isolated for the first time by Muller, Schllitter and Bein; Bein

demonstrated that reserpine as sedative and hypotensive action one year later.

According to Besset (1958) the roots of R. sepentina contain not less than 21 kinds

of alkaloids.The later work in this concern is as follows. In 1931,Siddique & Siddique found 5 alkaloids that they classify into the

ajamaline group of 3 (ajamaline,ajamalinine and ajamalicine) and the

serpentine groups of 2 yellow, crystalline, stronger basis(serpentine and

serpentinine).

In 1891, Dymock detected the presence of an alkaloid and a yellow resin in the

root of R.sepentina.

In 1993,Chopra and his associates observed the hyposentive, sedative and

hypnotic properties of root of Sarpagandha in experimental animals.

In 1940, Vakil made the first recorded reference to the therapeutic application

of Rauwolfia sp. in case of human hypertension.

In 1941&1942, Chopra and his associates reported on the pharmalogic and

toxic effect of Rauwolfia sp. root extract and of the individual alkaloids.

In 1942, Bhatia demonstrate that R.sepentina act as a drug for treatement of

high blood pressure.

Around the same time, Gupta, Deb and Kahali, reported the application of

Rauwolfia sp. in mental disorder.

REVIEW OF LITERATURE

In 1944, Bhatia and Kapur reported after the administration in animals of the 2

alkaloids isoajamaline and neoajamaline,stimulation followed by depression

of central nervous system and lowering of blood pressure.

In 1952, Muller, schlittler and Bein isolated reserpine, which accounted for

approximately 50% of the activities of Rauwolfia sp. root.

In 1953, Vakil reported a good response to alkaloid reserpine in 72% of the

cases of hypertension, few side effects in 1954; he reviewed the indication,

contradiction, dosing regimens, efficacy and adverse effect of Rauwolfia sp.

and suggested useful drug combination for the management of hypertension.

In 1953, Bein, Muller and associates found resepine to possess marked and

long lasting hypotensive vasodepression and sedative, hypnotic properties.

In 1953, Ford and Moyer after treating 25 cases of essential hypertension with

combined Rauwolfia sp. and hexamethonium therapy were able to report

adequate reduction of pressure levels of large number of cases.

As a result, high volume collection of R. serpentina is required for plant as a

natural resources. A protocol for mass artificial propagation of R. serpentina has

been published in 2002 by Dr. M.A.K.Azad et al, department of botany,

Jahangirnagar university of Bangladesh in which the tissue is taken from the apical

meristem region. The later work in this concern is as follows.

Chakroborty and colleagues (1951), Vlda (1952), Amold and Bock (1953),

Sarre (1953) and Klausgraber (1953), demonstrated the efficiency and safety

of Rauwolfia sp. serpentine.

In 1996, Sudha and Seeni has been achieved micropropagation from explant of

Rauwolfia sp. micrantha Hook F cultures.

In 2001, Sehrawat AR, Sanjogta U, Punia A .formlate in-vitro culture and

multiplication of Rauwolfia serpentina – A threatened medicinal plant.

In2008, Bhatt R, Arif M,Gour A.K, Rao P.B,devised protocol optimization for

in vitro propagation.

2009, Singh et al, Somatic embryogenesis and in vitro regeneration of

medicinal plant Sarpgandha.

►LABORATORY REQUIREMENTS

a) Explant from Rauwolfia sp.

b) Chemicals i. SAAF solution (0.2%) [A fungicide]

ii. Mercuric chloride (HgCl2)

iii. 70% ethanol

iv. Savlon

c) Glasswares‟

i. Culture bottles

ii. Beakers and Petri plates

iii. Measuring cylinders

iv. Pipettes

d) Small apparatus The small apparatus such as forceps of different sizes,

scalpels with sterilized blades, dissection needle, scissors, etc and indepenable

glass markers always present while working.

e) Analytical balance

f) Autoclave

g) Laminar air flow cabinet

h) Water distillation plant

i) Gas stove

j) pH meter

k) Miscellaneous

i. Brown paper

ii. Cotton

iii. Trays

l) Growth regulators

i. Adenine sulphate (AdSO4)

ii. 6-Benzyl amino purine (BAP)

iii. Citric acid (C6H8O7 )

MATERIALS AND METHODS

m) MS Media Murashige and Skoog medium is a plant growth medium which

i used in my project for plant tissue culture. It was invented by plant scientists

Toshio Murashige and Folke K. Skoog during 1962. Its constituent are_

i)Macronutrients & Micronutrients.

Ammonium nitrate (NH4NO3) 1,650 mg/L

Boric acid (H3BO3) 6.2 mg/L

Calcium chloride (CaCl2 · 2H2O) 440 mg/L

Cobalt chloride (CoCl2 · 6H2O) 0.025 mg/L

Magnesium sulfate (MgSO4 · 7H2O) 370 mg/L

Cupric sulfate (CuSO4 · 5H2O) 0.025 mg/L

Potassium phosphate (KH2PO4) 170 mg/L

Ferrous sulfate (FeSO4 · 7H2O) 27.8 mg/L

Potassium nitrate (KNO3) 1,900 mg/L

Manganese sulfate (MnSO4 · 4H2O) 22.3 mg/L

Potassium iodide (KI) 0.83 mg/L

Sodium molybdate (Na2MoO4 · 2H2O) 0.25 mg/L

Zinc sulfate (ZnSO4·7H2O) 8.6 mg/L

Na2EDTA · 2H2O 37.2 mg/L

ii)Common organic additives.

Myoinositol 100 mg/L

Niacin 0.5 mg/L

Pyridoxine · HCl 0.5 mg/L

Thiamine · HCl 0.1 mg/L

Glycine (recrystallized) 2.0 g/L

Edamine (ethane-1,2-diamine) 1.0 g/L

Sucrose 20 g/L

Agar 10 g/L

All the constituent were added to double distilled water and the volume was

adjusted to1 liter. Later growth regulators are added to form various RS

(Rauwolfia serpentina) media formulation for growth.

The pH of the media was adjusted between 5.6 to 5.8, N/10 HCl and N/10

NaOH was used if necessary.

The media was boiled and agar was then added. It was homogenized by

boiling and continuous stirring. Then fill the culture bottles with media.

Step 1► SELECTION OF EXPLANT

First stage begins with the collection of sterile explants.

The explants were collected from the field/nursery of Department of

Horticulture, BAU, Ranchi.

All the explants were taken from this donor Plants for present investigation.

Totipotent explants can be grown from any part of the plant, but explants of

various organs vary in their rates of growth and regeneration and some are

grow more fastly at all e.g. shoot tip, axillary bud and apical bud .

Shoot tips and buds are cut from healthy plants, leaving a short length of

shoot attached.

They should be selected to each yield have several explants of shoot tips

with approximately 1 cm sides.

The oldest shoots should be avoided.

Buds are also very much applicable as it possesses much amount of

proliferating cells and rapidly growing tissue.

Fig: A healthy mother plant.

METHODS

Step 2► SURFACE STERILIZATION OF EXLANT

This part of the procedure was carried out in a sterile working area, or with

meticulous aseptic technique.

The explant material is then surface sterilized, usually in multiple courses of

bleach and alcohol washes and finally rinsed in sterilized water.

Fig: Surface sterilization of explants.

Step 3:► TRANSFER OF EXPLANT TO GROWTH MEDIA.

Now this small portion of surface sterilized plant tissue is placed on a growth

medium, typically a medium containing sucrose as an energy source and one or

more plant growth regulators in specific combination. Usually the medium is

thickened with agar to create a gel which supports the explants during growth.

At first sterilized cotton was divided into three different parts, the first part was

used to sterilize the platform of the laminar flow, the glassware were sterilized

with the second part dipped into ethanol.

The hands were also sterilized with third part, all prior to the inoculation of the

explants.

For cutting the explants inside the laminar airflow chamber, sterilized brown

paper was used.

This shoot tips as well as buds were cut to about 1× 1.5 cm small pieces with

the help of sterilized scalpels and surgical blade on brown paper to make them

exposed for up taking the nutrients from the appropriate media contained in the

bottles.

The bottles were opened at an angle of 450 and 1-2 pieces of explants were

placed on the medium with the upper epidermis pressed gently against the

surface of the agar to make a good contact.

The bottles were capped tightly and transfer to the culture room at 3000 lux of

light for 16 hrs. and at 25 C.

Observations were recorded weekly.

Fig: Transfer of explants into growth media.

Step 4:► MULTIPLICATION OF EXPLANT.

After some days the excised explants which may be shoot tips axillary bud

and meristem is cultured aseptically on nutrient medium and incubated in

culture room for providing a control environmental condition such as

temperature, light and humidity. Under the appropriate condition the

explants will start regeneration by means of multiplication within the

meristematic cells which forms small leaf primordia, shoots and sprouting

apical bud.

Explant tissue should now being to divides its meristematic cells and shows

multiplication which ultimately gives rise to multiple shoot formation.

The shoot primordia grow out into multiple shoots which can be propagated

further by nodal cutting, the axillary bud of each segment will grow out in

culture to form yet another shoots.

Various hormonal regimes (RS media) shows different response kept in

controlled environmental condition.

Following the successful growth of plant tissue, the establishment stage may

be repeated, by taking tissue samples from the plantlets produced in the first

stage.

Step 5:►ROOTING AND HARDENING.

Root growth does not always occur in the earlier stages in the plant cell

culture, and is of course a requirement for successful plant growth in vitro

by transferring the plantlets to a growth medium containing auxin in

subsequent stages.

Since duration of project work was only 30 days, it was not possible to carry

the complete micro propagation stages of my work. But it can be carrying

out further.

“HARDENING” refers to the preparation of the plants for natural growth

environment, in which culture plant is subjected to hardening in sterilized

soil under control humidity condition.

Until this stage, the plantlets have been grown in “ideal conditions”,

designed to encourage rapid growth.

Thus in this way a plantlet is obtained which possesses both shoots and

young leaves having a root system for its autotrophic growth in subsequent

environment.

After hardening plants are ready to grow in the field.

These pictures show typical results, after about 4 weeks on each medium. To

summaries, multiple adventitious buds formed on the medium, leading to many

small shoots on the upper surface where the leaf is not in contact with the medium.

Fig: Freshly transferred explants.

Fig: Nodal cut on RS2 media, Fig: Shoot tips on RS5 media, after

after 14 days of incubation. 21 days of incubation.

OBSERVATION

Fig: Shoot tips on RS10 media, after 28 days of incubation.

Fig: Apical buds on RS14 media, after 21 days of incubation.

Fig: Apical buds on RS12 media, after 28 days of incubation.

►RESPONSE OF SHOOT TIP

For this experiment the MS basal media was supplement with four different

concentration of 6-benzylamino purine (BAP) and other growth regulator are taken

and observation was taken in following interval of time period.

i)Effect of BAP on shoot multiplication.

MS media +

BAP (mg/L)

Response

of shoots/explants.

7 days 14 days 21 days 28 days

1 RS1 No response Respond less Respond Response more

2 RS2 No response Respond Response more Response much

3 RS3 Respond less Respond less No response No response

4 RS4 No response No response No response Respond less

Amongst the different sets of media, the best shoot multiplication was

observed from the RS2 media containing BAP (2mg/L).

ii)Effect of BAP and Adenine sulphate on shoot multiplication:

MS media+ BAP+

AdSO4 (mg/L)

Response

of shoots/explants.

7 days 14 days 21 days 28 days

1.0+50.0 RS5 No response Respond Good response Respond much

1.0+100.0 RS6 No response No response No response Respond

2.0+50.0 RS7 No response No response Respond Response stop

2.0+100.0 RS8 No response No response No response Response

Amongst the different sets of media, a combination of BAP (1mg/L) and

AdSO4 (50 mg/L) was found to be best for shoot multiplication.

RESULT AND DISCUSSION

iii)Effect of BAP, AdSO4 and Citric acid on shoot multiplication.

MS media +

[BAP +AdSO4+

Citric acid]

(mg/L)

Response

of shoot / explants.

7 days 14 days 21 days 28 days

1 +50 +2 RS9 No response No response No response No response

1 +50 +1 RS10 No response Response Good respond Respond much

1 +25 +1 RS11 No response No response Response Respond more

Amongst different sets of media, a combination of BAP (1 mg/L), AdSO4

(50 mg/L) and citric acid (1 mg/L) was found to be best for shooting.

►DISCUSSION

The smaller size of explants were chosen due to fact that smaller size of explants

provide less chance of contamination, as well as it contain large amount of

meristmatic tissue. During initiation the explants did not show any leaching or

browning of tissues.

The explants cultured on MS basal medium supplemented with different

Combinations of BAP and AdSO4 showed varied response for regeneration. Plant

hormones affect gene expression and transcription levels, cellular division and

growth. Plant hormones are in small amounts can promote and influence the

growth, development, and differentiation of cells and tissues. Explants culture on

MS basal medium without any PGR supplementation has no growth. This was

possibly due to significant role of PGR over multiplication.

The plant regeneration via adventitious organ arising directly from explants

requires mainly cytokinin or high cytokinin to low auxin ratio and the suitable

combination of growth regulators. Cytokinins are derived from adenine and

produce two immediate effects on undifferentiated cells; the stimulation of DNA

synthesis and increased cell division (Ting, 1982). Cytokinins also produce a

delayed response in undifferentiated tissue which is the formation of shoot

primordia. In the media supplemented with BAP and small amount of AdSO4

showed significant shoot multiplication. Further it was observed that the

supplement of citric acid (1 mg/L) extensively promote the multiplication and

regeneration. It was also noticed that the high concentration of cytokinin and other

regulator result in moderate or no response.

►RESPONSE ON BUD BREAKING

For this the MS basal media was supplement with four different concentration of

benzyl amino purine (BAP) and adenine sulphate are taken and observation was

recorded in following interval of time period.

i)Effect of BAP, Adenine sulphate on bud breaking.

MS media +

[BAP

+AdSO4]

(mg/L)

Response on bud breaking.

7 days 14 days 21 days 28 days

1 +50 RS12 No response Response Response less Response less

1 +100 RS13 No response No response No response No response

2 +50 RS14 Response Good response Good response Respond much

2 +100 RS15 Response less No response No response No response

Amongst the different sets of media, a combination of BAP (2 mg/L),

AdSO4 (50 mg/L) was found to be best for bud breaking.

►DISCUSSION

Exogenously supplied cytokinin in the nutrient medium placed a major role for the

breaking of apical buds from shoot. Addition of Adenine sulphate in the nutrient

medium induces a bud breaking in most of the cases when supplement with BAP.

The exogenous cytokinin in combination with an AdSO4 does markedly increase in

response with gradual increase in time.

It was observed that minimal amount of adenine sulphate with both 1mg/ L and

2 mg/L of BAP showed positive result. But the higher amount of both growth

regulator leads to subsequent decrease in expression. Further the more productive

result was observed when 2 mg/l of BAP and 50 mg/L of adenine sulphate was

used for bud breaking.

Since duration of my project work was only 30 days, it was not possible to observe

the complete results of my work. However, initiation and multiplication of shoot

tips and bud breaking was observed. The present study describes a well

documented and reliable protocol for R. serpentina from shoot explants with much

higher rate of multiplication. This protocol can be used as a basic tool for

cultivation and study of Sarpagandha plant. The shoot tips (explants) of Rauwolfia

sp. which were cultured on MS media supplemented with BAP,AdSO4 and Citric

acid to standardized the optimum concentration as well as combination for

multiplication of the explants. Four concentration of BAP was used alone and two

conc. with association of two types of PGR viz, adenine sulphate and citric acid.

The duration of incubation was 7, 14,21and 28 days.

Under critical observation, we observe that different concentration and

combination effected the growth and multiplication of explants, at optimum

concentration the percentage of shoot multiplication in Rauwolfia sp. is maximum.

Our conclusion is that micropropagation of plant is affected by combination

of different hormones and their concentration in culture media. Of which the BAP

(1 mg/L), AdSO4(50 mg/L) and citric acid (1 mg/L) was found to be most

favorable for shoot proliferation and multiplication in Rauwolfia serpentina. And

plants need phyto hormones at very specific time during plant growth and at

specific concentration.

CONCLUSION

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