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In vitro propagation and conservation of Swertia bimaculata
- 1. In vitro propagation and conservation of Swertia bimaculata Subash kafle Central Department of Botany M.Sc 2nd Sem Botany Date 2074-01-17
- 3. Introduction plant description Swertia bimaculata is a herb plant belong to family Gentianceae. It distributed from sub tropical to sub alpine zone. Global status of swertia Wordwide 175 species , 29 species are recorded from Nepal 40 from India Swertia graciliescens is endamic to Nepal (S pant ,2014). Swertia species are medicinally important. Swertia bimaculata contain bioactive compound such as Gentiananine and xanthones, an antioxidant that can combat cancer and also help to reduce cholesterol level and hardening of arteries. Indiscriminate harvesting has enormously reduced its population strength in the normal habitat. . .
- 4. A maximum literature reveals the published report on micropropagation are available for S japonica ,S pseudo chinensis and S chirata but no work has been carry out in S bimaculata for in vitro propagation and conservation. This study primarily focuses on in vitro propagation and conservation of S bimaculata using shoot tip explant from in vitro grown seedling and simultaneous preparation of Chromosomal database from gamatic (n) somatic(2n) tissues. Introduction Chromosome analysis in any species is carried out at the somatic and gamatic level primarily to determine the stability of the plant at cellular level and also for genetic and biotechnological studies . No systematics effort has been made to formulate alternative method for its propagation and germplasm conservation.
- 5. Objectives The main objective of this study was for the preservation of medicinally important plant through in vitro propagation and to reduce its random harvesting stress in its natural habitat.
- 6. Material and methodology Collection of plant material Plant material collected as explant they may be any plant parts as shoot tip, leaf tip, pollen grain, floral part ,root part stem are use as explant they are totipotent cell.In the experiment shoot tip was used as explant which was derived from seed capsule germination. Washing and surface sterilization of explant First explant (shoot tip)was wash by tap water. Surface sterilization by 0.1% Hgcl2for 25 to 30 min. Then wash by distilled water 4 to 5 time to remove sterilant. Preparation of culture media and culture condition A standard medium was used for in vitro propagation there are different types of media for tissue culture according to the explant use for culture Mostly MS medium was prefer for most of cases .
- 7. Transfer of explant in culture media (inoculation) The explant was transfer to culture media in specific control chamber such area is free from bacteria and other microbes the transfer is done in laminar air flow chamber. Growth chamber (incubation) The culture are incubated in the growth chamber tissue culture room at 25 c having 50 to 60% relative humidity and 16 hours of photoperiod. Regeneration Plantlet regenerate after transferring the portion of callus in other medium and induction of root and shoot or directly from explant. Hardening of plant Plant with developed root was transfer to earthen pot containing soil sand and compost they are sensitive to external environment so humidity should be maintain between 80 to 90 % by covering with polythene bag up to 2 weeks.
- 8. Chromosomal analysis A healthy root tip was randomly taken from micro propagated plants growing on BMA root tip was treated with 2mM hydroquinolene for 3 and half hours at 12 to 14 c followed by over night Fixation in acetic alcohol. Hydrolysis of root tip was performed by 1N HCL for 12 to 15 min at room temp then stain in 2% aceto orcein for 3 hours and squashed in 45% acetic acid to obtained well scattered metaphase plate. Chromosome per cell were recorded and photograph were taken under Ziess photomicroscope.Meiosis was studied in flower bud collected from in vivo donor plant.
- 9. Statistical analysis All the experiment were repeated more then three times the experiment were set up in a Randomized design data were analyzed by analysis of variance (ANOVA) to detect significant between means. Means differing significantly were compared using Duncans multiple range test DMRT at 5% Probability level variability of data has also been expressed as the mean standard error.
- 11. Fig; In vitro propagation of swertia bimaculata different step with chromosomal analysis.
- 12. Result and discussion In vitro grown shoot tip or other parts are used as explant. shoot multiplication was achieved on BM with different level of BA with in 10 weeks of culture the number of shoot was further increase by addition of 10mm KNO3 it show similar finding with many plants. Regenerated shoot isolated from the culture were placed on BM with or without IAA ,IBA healthy root were induced in about 100% shoot on BM with 4-5 weeks of culture under A photoperiod of 16 hours light and 8 hours dark cycle then after 3-4 weeks the plant is transfer To earthen pot with maintaining humidity eighty to ninety percent.
- 13. Meiotic and mitotic chromosomal analysis were reveled to numerical stability of chromosomes between somatic cell and reproductive cell the stability of regenerated plants at the chromosomal level has direct relationship with the protocol system used thus the present protocol proved to be very efficient and repeatable and can be used for large scale conservation of germplasm. Result and discussion
- 14. conclusion In conclusion the present study describe s a simple efficient reproductable protocol for the Production of diploid plants the complete protocol of plant production required 20 weeks times. The chromosomal analysis prepared would be useful for any future study. Many medicinally and economically important plants can be regenerate through in vitro propagation and can be preserved for future need which directly reduce its pressure on natural habitat due to random harvesting of such important plants.
- 15. Acknowledgment I would like to thanks Prof Dr. Bijaya pant ,Dr. Sanjaya Kumar Jha and Dr.Chandra Prasad pokhrel for kindly support to provide full ideas, mathod and constant encouragement to prepare this research paper presentation.
- 16. REFERENCES Dafander,A. and Jha,T.B,In vitro propagation and conservation of Swertia bimaculata. Indian journal of Biotechnology , vol 11 july 2012 page 295- 299 Pant,S. Swertia in Nepalese Himalaya. Gention research Network (2006). Dubey,R.C.(2006).A Textbook of Biotechnology.S. Chand and Co .Ram Nagar, New Delhi.