In Vitro Propagation of Datura Quercifolia Plant - IJGHC

E-ISSN: 2278-3229IJGHC; March 2013 – May 2013; Vol.2, No.2, 301-305.E-ISSN: 2278-3229Research ArticleInternational Journal of Green and Herbal ChemistryCODEN (USA): IJGHAYAn International Peer Review E-3 Journal of SciencesAvailable online at www.ijghc.comGreen ChemistryIn Vitro Propagation of Datura QuercifoliaPlant1 Eefa Manzoor and 2 Bilal Ahmad Bhat*Department of Biotechnology, Sai Institute of Paramedical and Allied Sciences Dehradun, UttrakhandDivision of Social Science, Faculty of Fisheries, Rangil Ganderbal, Shuhama Campus, S.K.University of Agricultural Sciences and Technology Kashmir, Srinagar (J&K)Received: 8 April 2013; Revised: 8 May 2013; Accepted: 13 May 2013Abstract: In this paper, an attempt has been made to explore the possibility ofin vitro propagation of Datura plant. The preliminary tissue culture studycarried out showed that this plant has a potential for in vitro propagation andplantlet regeneration. Individual treatments and treatment combinationsvariedly influenced the germination of seeds in Datura quercifolia. Seed coatremoval was an effective treatment for inducing germination. Seedscarification followed by a GA 3 (200ppm) treatment for 48 hours seems to bevery effective wherein 87% germination was observed. Further, a good shootresponse was observed on various BAP + 2,4-D combinations. Multiple shootregeneration from shoot apices was recorded on BAP (2.5 µM) and BAP (10µM) + 2,4D (5 µM).Keywords : Datura quercifolia, tissue culture, inoculation, in vitropropagation, seed treatmentINTRODUCTIONThe word datura is derived from a Hindi word dhatura (Thorn apple). Datura is a genus of woodystalked leafy annuals and short lived perennials which can reach upto 2m in height. The plantsproduce spiny seed pods and large white or trumpet shaped flowers. Most parts of the plant containthe alkaloids Atropine, Scopolamine and Hyoscyamine. Datura quercifolia, commonly known as theoak-leaf thorn-apple or the Chinese thorn-apple, is a small shrub in the genus Datura. It can grow301 IJGHC; March2013 -May 2013, Vol.2, No.2, 301-305.

InVitro...Eefa Manzoor and Bilal Ahmad Bhatwithin dry or moist soils and requires large amount of sunlight to grow. The plant produce greencolored fruit with long sharp spikes or spines. The flowers are cream white to purple in color. Theleaves resmeble oak leaves, hence the etymology of the species epithet, "quercifolia". It is used as adecorative ormental plant. Datura quercifolia is a member of the nightshade family and is also calledjimsonweed. Relationship between alkaloid production and growth measured as bio mass increaseand cellular division frequency in Datura Stramonium in vitro root cultures was observed by 1 Baize etal. (1998). Improvement in seed germination percentage and seedling growth in Datura innoxia bypresowing seed treatment with vitamin B1, B6, B complex and C was recorded by Rajagopal et al 2 .(2001). Vitamins improved seed germination percentage and among concentrations 500 ppm provedoptimum for most parameters. An efficient protocol for in vitro production of Datura metel plantletfrom stem and leaf explants excised from in vivo source and cultured 3 on MS 1962 mediumsupplemented with NAA, IBA, 2.4-D, BAP and GA3 either singly or in combination was establishedby Sood and Guilleti 4 . Effect of light had a cardinal role that influenced growth and multiplication ofplantlets in vitro. Continuous illumination of the cultures promoted better and efficient growth. Thepresent study was carried out with following objectives in view (i) Breaking down the dormancy ofseeds of Datura quercifolia: various methods and treatments involved (ii) In vitro propagation ofDatura quercifolia.MATERIALS AND METHODSeeds of Datura quercifolia were collected from field gene bank of IIIM, Srinagar. The seeds weredivided into groups of 50 seeds each. The seeds were washed with 0.1% mercuric chloride for 5-7minutes and then with 70% alcohol for 1 minute. Then seeds were thoroughly rinsed with doubledistilled water. The seeds were then subjected to various treatments and placed on a moist silter paperin a petri-plate of 4 inches diameter. The various treatments given were:a. Chilling: Seeds were subjected to chilling (at 4 0 C) for 48 h and 72 using L.G refrigerator.b. Gibberellic acid: GA 3 treatment of 200 ppm prepared by dissolving 200 mg of GA 3 in 1000ml of distilled water. The seeds were kept in each of the solutions for about 24 hours followedby a wash in autoclaved double distilled water.c. Sulphuric acid (H 2 SO 4 ) treatment: Seeds were kept in a muslin cloth and then dipped inconcentrated H 2 SO 4 for 3 minutes followed by a wash in autoclaved double distilled water.Sulphuric acid Merck used was 99.9% pure.d. Sand paper scarification: Sand paper of “size 100” was used. The seeds were kept betweentwo sheets of sand paper and rubbed 8 times.e. For each treatment two replicates were used. A set of control was kept to comparegermination efficiency. MS medium was used for plant tissue cultures for morphogenesis andplant regeneration. The sterilized explants were inoculated in sterile MS medium containingdifferent concentrations of harrmones under aseptic conditions in a Laminar Flower andincubated under 16 h light period at 25 0 C.RESULTS AND DISCUSSIONSeeds for each treatment were kept in the lab conditions (average temperature 15-20 0 C). Replicateswere monitored in ordinary light and complete darkness. The germination of seeds was monitored andthe details recorded. Datura seed can show high or low germination rates and seedling emergence canbe quite irregular. Species such as D. stramonium (jimsonweed) tend to sprout readily, while otherslike Datura quercifolia may not germinate for 9 months to a year. Various treatments were given to aset of unchilled seeds. Un-chilled seeds kept in ordinary light without any treatment didn’t show anygermination. Seeds were scarified and also given GA 3 treatment (Table 1).302 IJGHC; March2013 -May 2013, Vol.2, No.2, 301-305.

InVitro...Eefa Manzoor and Bilal Ahmad BhatTable 1: In-vitro seed germination studies in Datura quercifoliaTreatmentNo. of daystaken for firstseed togerminateNo. of daystaken for lastseed togerminateGermination %L D L D L DControl 0 0 0 0 0 0Sand paperScarificationAcid Wash(3 min)GA 3 200ppm2 23 30 23 25 62 2 8 11 50 332 2 20 10 87 50L: Light, D: DarknessMaximum germination (87%) was recorded in seeds treated with GA 3 (200ppm) for 48 h andinoculated on simple distilled water kept under light. Seeds whose seed coat was scarified withsand paper and kept in complete darkness showed least germination of 6%. A set of seeds werescarified and chilled for (48-72h). Maximum germination in chilled seeds was observed in 48 hchilling (60%) kept in light (Table 1).Shoot tips excised from in vitro raised seedlings were used as primary explants and inoculated onMS medium augmented with different growth regulators. Various types of morphogeneticresponses recorded are depicted in (Table 2).Table- 2: Morphogenetic response of shoot tips of Datura quercifolia to variousPhytohormones.Medium* Nature of Response** Response %MS basal medium No Response 0%MS (x1/2) + BAP(2.5 µM) Initiation of shoots 30%MS + BAP (5 µM) No Response 0%MS +BAP (5 µM) + NAA (5 µM)Initiation of shoot multiplicationfollowed by stunted growth40%MS + 2,4-D (5 µM) + BAP (10 µM) Shoot multiplication and elongation 70%*10 replicates per treatment, **Data scored after 5 weeks of culture period303 IJGHC; March2013 -May 2013, Vol.2, No.2, 301-305.

InVitro...Eefa Manzoor and Bilal Ahmad BhatShoot tips cultured on MS (1/2x) + BAP (2.5 µM) showed initiation of shoots followed by stuntedgrowth pattern. Initiation of multiple shoots also took place on MS (1/2x) + BAP (5 µM) +NAA (5µM) which also was followed by stunted growth pattern. However a combination of 2,4-D (5 µM) +BAP (10 µM) resulted in multiple shoot formation and its subsequent elongation.The relevance and significance of different seed treatments for breaking dormancy and improvingseed germination is well known 5 . Treatments such as soaking before sowing, seed coat puncturing,scarification, acid wash, chilling, GA 7 .During the present investigation of Datura quercifolia it is observed that unchilled seeds subjected toscarification showed 50% germination and survival in light. In complete darkness 33% seeds recordedgermination. Beigh et al. 6 obtained 50% germination in scarified seeds of Podophyllum hexandrumnot subjected to any chilling. Similar results were also obtained by Andrabi 7 in scarified seeds ofAtropa acuminata kept in ordinary light. Further, unchilled seeds of Datura quercifolia subjected toacid wash showed 50% germination. In similar studies carried out by Bisht and Kediyal 8 on Atropabelladona seeds treated with sulphuric acid (75%) followed by sodium hydroxide (30%) recordedseed germination.Unchilled seeds of Datura quercifolia were scarified followed by 48 h dip in GA 3 (200 ppm). Bestresults (87%) were recorded in seeds treated with GA 3 (200ppm) in ordinary light .50% germinationwas recorded in seeds kept in complete darkness.Similar results were obtained in Atropa belladonna seed germination studies carried out byAndrabi 7 . The effect of pre-chilling followed by seed coat scarification showed best results (60%germination) in 48h chilled seeds in light. This was followed by 20% germination in seeds chilled for48h in complete darkness. 72h pre-chilling followed by seed coat scarification resulted in 16% seedgermination in light and 13% in complete darkness. It is observed that scarification together withother treatments do selectively influence seed germination. In present study maximum shoot numberwas obtained on MS enriched with 2,4-D (5 µM) + BAP (10 µM) using shoot tips as explants. Theseresults reveal that the behaviour of regeneration phenomenon shown by the plant is more or lesssimilar to other medicinal plants like Artemisia pallens 9 and Dioscorea floribunda 10 .CONCLUSIONOur study, conducted to explore the possibility of in vitro propagation of Datura quercifolia, amedicinally important plant. The preliminary tissue culture study carried out showed that this planthas a potential for in vitro propagation and plantlet regeneration. Individual treatments and treatmentcombinations variedly influenced the germination of seeds in Datura quercifolia. Seed coat removalwas an effective treatment for inducing germination. Seed scarification followed by a GA 3 (200ppm)treatment for 48 hours seems to be very effective where in rapid 87% germination was observed. Inour study good shoot response was observed on various BAP + 2, 4-D combinations. Multiple shootregeneration from shoot apices was recorded on BAP (2.5 µM) and BAP (10 µM) + 2,4D (5 µM).Similar reports of multiple shoot regeneration in shoot tip cultures of Lavandula officinalis on variousBAP concentrations and combinations has been recorded by Tyub et al (2004). In conclusion presentinvestigation forms an important preliminary step for breaking seed dormancy and inducing rapidseed germination in Datura quercifolia and its subsequent micro propagation. Extension of thesestudies will prove highly beneficial for future strategies in tissue culture studies for propagation of thisplant.304 IJGHC; March2013 -May 2013, Vol.2, No.2, 301-305.

InVitro...Eefa Manzoor and Bilal Ahmad BhatACKNOWLEDGMENTSThe authors are highly thankful to the referee(s) and the editor of the Journal for their valuablesuggestions, which helped in improving the quality of our research paper.REFERENCES1. A.M.Baize,A. Quiroz,J.A. Malnado-Mendoza and Loyola-Vargas, Growth patternsand alkaloid accumulation in hairy root and untransformed root cultures of Daturastramonium. Plant cell, Tissue and Organ Culture, 1998, 54(2): 123-130.2. R.Rajagopal, S.H. Afaq, Afridi and R.H.Mohammed, Germination and Growth ofDatura innoxia Mill, (II): Effect of pre-sowing seed treatment with 4 vitamins ongermination and seedling growth in pot culture. Hamdard Medicus, 2001, 44 (1): 99-108.3. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassays withtobacco tissuecultures. Physiol. Plantarum, 1962,15 : 473–497.4. Sood N. P and Guilleti, Efficient protocol for in vitro differentiation and plantletformation from callus culture of Datura metel. J Sci Pharmacy, 2002, 3 (2): 66-67.5. Thomas, Seed treatments and technique to improve germination. Scientia hortic1981, 32:47-516. S.Y.Beigh, M. Iqbal and I.A. Nawchoo, In vitro seed germination studies on Himalayas Podophyllum hexandrum Royle, an endangered medicinal plant of Northwest,2002, 22(3):132-134.7. F.Andrabi, Studies on seed biology for developing conservation and strategies forAtropa acuminata, a threatened medicinal plant of Kashmir Himalayas,2002,11(2):2-3.8. Bisht and Kediyal, Effect of chemical treatments on germination and survival of theseedlings in Atropa belladomna Linn. Indian J. Forestry, 1995, 11(3): 208-210.9. B.D.Benjamin,A.T. Sipahimalni, and M.R.Heble, Tissue culture of Artemesiapallens. Plant cell tissue and organ culture,1990, 21:159-164.10. J.Sengupta,G.C. Mitra, Organogenesis and tuberization in cultures of Dioscoreafloribinda. plant cell tissue and organ culture, 1984,3(4):325-332.Corresponding author: Bilal Ahmad Bhat; Division of Social Science, Faculty of Fisheries, Rangil Ganderbal,Shuhama Campus, S.K. University of Agricultural Sciences and Technology Kashmir, Srinagar (J&K)305 IJGHC; March2013 -May 2013, Vol.2, No.2, 301-305.