The effect of growth regulators on in vitro multiplication & propagation of Rauwolfia serpentina

Under the guidance of
Senior Scientist cum Associate Professor



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

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




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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.


 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.

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.


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 airdried 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 Sarpagandha churna, Mahesvari vati etc. ghanavati, sarpagandha yoga,

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.

 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. ii. iii. iv. SAAF solution (0.2%) [A fungicide] Mercuric chloride (HgCl2) 70% ethanol Savlon Culture bottles Beakers and Petri plates Measuring cylinders Pipettes

c) Glasswares‟
i. ii. iii. iv.

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) Boric acid (H3BO3) Calcium chloride (CaCl2 · 2H2O) Cobalt chloride (CoCl2 · 6H2O) Magnesium sulfate (MgSO4 · 7H2O) Cupric sulfate (CuSO4 · 5H2O) Potassium phosphate (KH2PO4) Ferrous sulfate (FeSO4 · 7H2O) Potassium nitrate (KNO3) Manganese sulfate (MnSO4 · 4H2O) Potassium iodide (KI) Sodium molybdate (Na2MoO4 · 2H2O) Zinc sulfate (ZnSO4·7H2O) Na2EDTA · 2H2O

1,650 mg/L 6.2 mg/L 440 mg/L 0.025 mg/L 370 mg/L 0.025 mg/L 170 mg/L 27.8 mg/L 1,900 mg/L 22.3 mg/L 0.83 mg/L 0.25 mg/L 8.6 mg/L 37.2 mg/L

ii)Common organic additives.
       

Myoinositol Niacin Pyridoxine · HCl Thiamine · HCl Glycine (recrystallized) Edamine (ethane-1,2-diamine) Sucrose Agar

100 mg/L 0.5 mg/L 0.5 mg/L 0.1 mg/L 2.0 g/L 1.0 g/L 20 g/L 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.

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, after 14 days of incubation.

Fig: Shoot tips on RS5 media, after 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.


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 + BAP (mg/L) 1 2 3 4 RS1 RS2 RS3 RS4 of shoots/explants. 7 days No response No response Respond less No response 14 days Respond less Respond Respond less No response 21 days Respond Response more No response No response 28 days Response more Response much 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+ AdSO4 (mg/L) 1.0+50.0 RS5 of shoots/explants. 7 days No response No response No response No response 14 days Respond No response No response No response 21 days Good response No response Respond No response 28 days Respond much Respond Response stop Response

1.0+100.0 RS6 2.0+50.0 RS7

2.0+100.0 RS8

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+ of shoot / explants. Citric acid] (mg/L) 1 +50 +2 RS9 1 +50 +1 RS10 1 +25 +1 RS11 7 days No response No response No response 14 days No response Response No response 21 days No response Good respond Response 28 days No response Respond much 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 (mg/L) 1 +50 1 +100 2 +50 2 +100 RS12 RS13 RS14 RS15 No response No response Response Response less Response No response Good response No response Response less No response Good response No response

28 days

Response less No response Respond much 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.

a) Bhatia BB, Kapur RD. (1944). Pharmacological actions of alkaloids of Rauwolfia serpentina Benth. Part I, Neo-ajmaline and iso-ajamaline. Indian J Med Res, 32:177-82. b) Bhojwani, S.S and Rajdan, M.K. (1983). Plant Tissue Culture: Theory and practice. Elsevier, Amsterdam. c) Chopra RN, Bose BC, Gupta JC, Chopra IC. (1942). Alkaloids of R. serpentina: a comperative study of their pharmacological action and their role in experimental hypertension. Indian J Med Res, 30:319-24. d) Gupta JC, Kahali BS, Dutta A. (1944). The hypnotic action of resin fraction isolated from root of Rauwolfia serpentina (Benth) obtained from Dehradun. Indian J Med Res, 32:183-8. e) Gupta, P.K. (2002). Tissue Culture and Micro Propagaton, Elements of Biotechnology, Rastogi publication, Pp 277-280. f) Kukreja AK, Mathur AK, Ahuja PS, Thakur RS (1989). Tissue Culture and Biotechnology of Medicinal and Aromatic Plants.ICSIR, Lucknow. g) Mitra GC, Kaul KN (1964). In vitro culture of root and stem callus of Rauwolfia serpentina Benth. for reserpine. Indian J Exp Biol, 2: 49-5. h) Murashige,T. and Skoog, F. (1962). A revised Medium for rapid growth and bioassays with tobacco tissue culture, Physiology Plant 15:473-479. i) Razdan, M.K. (2000). Introduction to Plant Tissue Culture, Second Edition, Oxford and IBH Publishing Co.Pvt.Ltd, New Delhi. j) Roy SK, Hossain MZ, Islam MS(1994). Mass propagation of Rauwolfia serpentine by in vitro shoot tip culture. Plant Tissue Culture, 4(2): 69 -75. k) Vasil, I.K. and Torpe, T.A. (1994). Plant cell and tissue culture, Kaluwer academic publishers, London. sp.

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