An efficient regeneration system, through somatic embryogenesis was developed for Curculigo orchioides Gaertn - an endangered medicinal herb. Somatic embryos weredeveloped on MS medium containing 8 - 15ÃŽÂ¼M BA from leaf explants. The highest, 69 % leafexplants responded in terms of embryogenic calli with average 8 embryos on MS mediumcontaining 8ÃŽÂ¼M BA. Regenerated plantlets were transferred to autoclaved mixture of soil:sand: compost (1:1:1; v/v/v) for hardening. Genetic fidelity of somatic embryogenesis derivedregenerant was assessed using random amplified polymorphic DNA (RAPD)

Adiyecha, Roshni

Jasrai, Yogesh T

Patel, Swati

English

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    Research in Plant Biology, 1(3): 48-52, 2011                                                                 ISSN : 2231-5101 www.resplantbiol.com    Regular Article Induction of somatic embryogenesis and genetic fidelity of endangered medicinal herb Curculigo orchioides Gaertn  Swati Patel2, Yogesh T Jasrai1* and Roshni Adiyecha1  1Department of Botany, University School of Science, Gujarat University, Ahmedabad-380009, Gujarat, India 2C.G.Bhakta Institute of biotechnology, Gopal Vidhyanagar, Bardoli, Surat-394350 *yjasrai@yahoo.com   An efficient regeneration system, through somatic embryogenesis was developed for Curculigo orchioides Gaertn - an endangered medicinal herb. Somatic embryos were developed on MS medium containing  8 - 15µM BA from leaf explants. The highest, 69 % leaf explants responded in terms of embryogenic calli with average 8 embryos on MS medium containing 8µM BA. Regenerated plantlets were transferred to autoclaved mixture of soil: sand: compost (1:1:1; v/v/v) for hardening. Genetic fidelity of somatic embryogenesis derived regenerant was assessed using random amplified polymorphic DNA (RAPD).  Key words: Somatic, embryogenesis, fidelity             Curculigo orchioides Gaertn (family-hypoxidaceae) commonly known as kali musli, is an endangered medicinal herb. In india it is distributed in sub-tropical Himalayas from Kumaon eastward  and in the western ghats from Gujarat-Maharastra southwards. It appears first with onset of monsoon and last to disappear on completion of monsoon (Francis et al. 2007). The leaves, roots and rhizome of Curculigo are medicinally useful (Bhamare, 1998). The plant possesses uterine stimulant (Dhar et al. 1968), hypoglycaemic, spasmolytic and anticancer, (Dhar et al. 1968; Aruna and Sivarama-krishnan, 1990), phagocytic (Kubo et al. 1983) immuno-adjuvant (Oru and Kogyo, 1983), antineoplastic, immuno-stimulant and hepatoprotective activities (Latha et al. 1999; Rajesh et al. 2000). Removal of plants for medicinal and edible, tuberous roots as a substitute for safed musli, coupled with extensive denudation of forests floor caused by cattle grazing (Jasrai and Wala, 2000), poor seed setting and germination are some of the major causes that contribute to the herb being categorized as a threatened plant (Augustine and D’sousa, 1997). Hence the methods for large scale in vitro propagation are needed to meet the commercial demand and to conserve this valuable plant.  Somatic embryogenesis has a tremendous potential for large scale production of plant material (Garcia and Martinez, 1995) and is considered as an effective aid in genetic transformation studies. It represents an alternative tool for massive clonal propagation. This appears to be a potential solution to the problem of field propagation, especially in area with frequent disease transmission and maintenance of cultivars that have been selected for important genetic characteristics (Amirato and Styer, 1985).     Swati Patel et al. / Research in Plant Biology, 1(3): 48-52, 2011   49  In this study an efficient, rapid and reproducible plant regeneration system was established for Curculigo through somatic embryogenesis. Direct regeneration of somatic embryos is important for the conservation of this endangered species, as rare somaclonal variants are likely to arise from indirect regeneration through callus.  Materials and method Young leaves of Curculigo orchioides shoots were used as an explant. Leaves were surface sterilized with 0.1% of HgCl2 (2 min). Treated leaves were thoroughly washed with sterilized distilled water (4 times) and inoculated aseptically on MS medium (Murashige and Skoog, 1962).   Culture media and growth condition: Leaf pieces (0.5 cm long) were inoculated on MS medium with 4.4µM , 8µM and 15µM BA with  3 % sucrose (w/v). The pH of media was adjusted to 5.8 and autoclaved at 121 ºC (20 min). For all media, 0.8 % agar-agar was added as gelling agent for semi-solid medium. The cultures were incubated at 25ºC under 16 h photo-period with 55 µ mol m-2s-1 photon flux density. Seventy replicates each were studied for somatic embryogenesis.  DNA extraction and RAPD analysis: Young leaves from regenerants were subjected to DNA extraction and purification (Doyle & Doyle, 1987). The modified PCR conditions were optimized (Akhare et al. 2008). The amplification products were revealed using agarose gel (1.2 %, w/v) electrophoresis system. All the reactions were repeated at least twice, and only the consistently reproducible bands were considered.   Results and Discussion  The present study was conducted for in vitro propagation of Curculigo orchioides through indirect somatic embryogenesis. The leaf-age of explants plays a major role in somatic embryogenesis (Thomas and Jacob, 2004). Low frequency of embryogenesis and embryogenic induction related to the age of explants suggest that the intrinsic physiological stage of explant plays decisive role in the induction of embryogenesis. Such observations were reported in somatic embryogenesis from zygotic embryo culture (Mathur, 2000; Gogate and Nadgauda, 2003). The middle lamina of the in vitro derived leaf explants (0.5 cm long) inoculated on MS media with 8µM and 15 µM BA showed callus formation. Callus formation was initiated within 2 weeks from the cut ends of the leaf explants. High frequency of callus formation was obtained in next 15 days after initiation (Fig -1 a). The callus was compact and pale-white in colour. Eventually, translucent patches of embryogenic cells were differentiated from the well developed callus. On the MS media with 8 µM BA, about 69 % cultures responded in terms of embryogenic calli, while about 40 % cultures developed embryogenic calli on MS media with 15 µM BA. Earlier, similar (89 %) response was obtained with 8 µM BA for direct embryogenesis (Thomas and Jacob, 2004). The translucent emebryogenic calli when subcultured to the fresh medium (Dodeman et al. 1997) transformed into green embryoids (Fig-1b). The growth of embryoids was slow and occasionally get detached from the other cells and continued their growth.    Average 8 embryos were formed on the MS medium with 8 µM BA while 4 embryos were developed on MS medium with 15 µM BA. It is very difficult to observe the different stages of emryogenesis in monocots (Thomas and Jacob, 2004). Moreover, the heart shaped stage is absent in monocots as they have only one cotyledon (Sivakumar et al. 2003). The histological analysis of the embryogenic calli clearly showed globular shaped embryo (Fig-      1c). Wheto MS meand suppof BA (0(Augustiplantletsthermocosand anhardeninirrigated         Fig 1: Sosomatic e Inembryogembryosto-type reliable evaluatin(Leroy eSn these  emdium withlemented w.44µM), devne et al. 2008. Plantlets l cups cond compog (Fig-1d) with ¼ matic embrymbryo, c- em plants reenesis, the determinesplants. Mothan pheng tissue cut al. 2000).wati Patel et bryoids wer ½ strength ith lower eloped sho), giving riswere thentaining mixst (1:1:1; . The plastrength M ogenesis inbryoids in segenerated  quality  the produlecular toootypic obslture induc The genetal. / Researche transferrenitrogen salconcentratioots and rooe to comple placed ture of sov/v/v) fntlets weS mediu Curculigo oction  d- Plavia somatof somatction of truls are moervation fed variationic fidelity  in Plant Bio 50 d ts n ts te in il, or re m (withoshockand Sweeksintermand Rplantlrhizomany survivrchioides Gantlets regeneic ic e-re or s of regenembryanalysampliacrossNo vregenaclogy, 1(3): 48ut sucrose) of changesen, 1985). Fo humidity wittent wateicardo, 19ets showede, roots anmorphologal. ertn., a- emrated  beingerated planogenesis wis. Of the fied produc all the micariation werants. -52, 2011  for one we in its new er an initial pas maintair mist (Jasra95). On est tremendd lush greenical variatbryogenic c  hardened. ts, derived as analyzed10 primersts that werropropagateas observek to recovenvironmenteriod of abned by sprai et al. 1999;ablishmentous growth shoots wition with  alli, b-translthrough som through R tested, OPe monomod plants (Fed amongb d r the  (Kar out 2 ying  Rolf , the  of hout 95% ucent atic APD E 18 rphic ig-2).  the          Fig 2: RACurculigoregenerathrough embryog       Ederived p(Jayanthinotoginsemariana (integrityregeneraoleracia wlack of vregenerainternal formationabnorma Teffective through orchioidesoptimumfound toformationpropagatregenera ReferencAkhare, DhumRAPDidentparen3:18-2SPD profile orchioidesnts derivedsomatic enesis. arlier uniforlants was n and Mng (ShoyamIsabel et al. among thents within as confirmariation in nts could bgenetic con, may be tl types (Lerohe presentprotocol osomatic em with genet concentrat be effectiv. This wion of C. otion.  es A.A., Sakhale, D.B.  profile sification ots. Internat4. wati Patel et  of   mity in somoticed in Tyandal, 2a et al.  1993). Simi somatic emthe cultivaed by ISSR somatic eme due to trols throughrough seley et al. 2000 study esf plantlet bryogenesisic fidelity evion of BAe on callusould help rchioides thrare, S.B., and Kharktudies in f hybridsional J. of Ial. / Researchatic embrylophora indi001), Pana1997), Piclarly, genetbryo-deriver of Brassianalysis. Thbryo derivethe stringehout embryction again). tablished aregeneratio in Curculigaluation. Th (8 µM) w and embrylarge scaough in vitKulwal, P.Lar, A., 200Sorghum f and thentegrat. Bio in Plant Bio 51 o ca x ea ic d ca e d nt o st n n o e as o le ro ., 8. or ir l., AmirafoemBiPrArunaPlag10Augst20froGaInAuguReCuDeBhamofNPhDhar,MScac24DoyleDNquBuDodem19so48Franc20CuenDeGarciaemcvPllogy, 1(3): 48to, P.V. andr large scalbryos inotechnologyess: New Yo, M. and Sivant produainst cancer11. ine, A.C., N08. Inductiom antherertn-An end. J. Biotech.,stine, A.C. generation rculigo orchv. 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Induction of somatic embryogenesis and genetic fidelity of endangered medicinal herb Curculigo orchioides Gaertn

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Induction of somatic embryogenesis and genetic fidelity of endangered medicinal herb Curculigo orchioides Gaertn

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