The effect of growth regulators on in vitro

multiplication & propagation of Rauwolfia serpentina

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
 INTRODUCTION

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.
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.
 OBJECTIVE OF PROJECT

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.
 DESCRIPTION OF SARPGANDHA

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.)
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.
 REVIEW OF LITERATURE

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.
 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.
 MATERIALS AND METHODS

►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 )
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.
 METHODS

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.

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.
 OBSERVATION

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.
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.

 RESULT AND DISCUSSION

►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.

Response

MS media + of shoots/explants.

BAP (mg/L) 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:

Response

MS media+ BAP+ of shoots/explants.

AdSO4 (mg/L)
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.
 iii)Effect of BAP, AdSO4 and Citric acid on shoot multiplication.
MS media + Response
[BAP +AdSO4+
Citric acid] of shoot / explants.

(mg/L) 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 + Response on bud breaking.
[BAP
+AdSO4]
7 days 14 days 21 days 28 days
(mg/L)

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.
 CONCLUSION

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.
 BIBILIOGRAPHY

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http://en.wikipedia.org/wiki/Rauwolfia sp.
http://www.biotechnologyonline.gov.au/pdf/biotech/plant_tissue_culture_in_class.