CALLUS INDUCTION

CALLUS INDUCTION AND PLANTLET REGENERATION IN WITHANIA SOMNIFERA (L.) DUNALGITA RANIa, G. S. VIRKa, AVINASH NAGPALabaDepartment of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143 005, IndiabAuthor to whom correspondence should be addressed: gndu.botanical@vsnl.comAbstractCallus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l−1 (9.1 μM) 2,4-D and 0.2 mg l−1 (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l−1 2,4-D and 0.2 mg l−1 KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N6-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l−1 (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l−1 (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.Received: September 27, 2002; Accepted: March 18, 2003Keywords: Withania somnifera, Solanaceae, cotyledonary leaf, hypocotyl, root, callusIntroductionWithania somnifera (L.) Dunal (Solanaceae) is used in the traditional system of medicine and is known for its antistress, anti-inflammatory, antiarthritic, and antitumor properties (Malhotra et al., 1961; Uma Devi et al., 1992, 1993). One of the problems for commercial cultivation of this plant is the long gestation period (4–5 yr) between planting and harvesting. For uniform strains, and better growth and productivity, micropropagation techniques are of special use. Micropropagation of W. somnifera employing different explants, such as shoot tips (Sen and Sharma, 1991; Furmanowa et al., 2001; Ray and Jha, 2001), nodal segments (Tiwari and Singh, 1991), axillary meristems (Roja et al., 1991), leaves (Baburaj and Gunasekaran, 1995), and axillary leaves, axillary shoots, and hypocotyl and root segments (Rani and Grover, 1999) has been demonstrated. Abhyankar and Chinchanikar (1996) showed direct shoot regeneration from leaf discs grown on MS medium supplemented with indole-3-acetic acid (IAA), 6-benzyladenine (BA), and kinetin (KN) in various combinations. Kulkarni et al. (1996) determined direct shoot formation from leaf explants of in vitro-grown seedlings using MS (Murashige and Skoog, 1962) medium containing IAA and BA. No protocol has yet been standardized for micropropagation through callus using a variety of explants in W. somnifera. The present report deals with callus induction and plantlet regeneration of W. somnifera from hypocotyl, root, and cotyledonary leaf segment explants of in vitro-raised seedlings.Materials and MethodsThe seedlings were raised from seeds collected from plants growing in the glasshouse of the Botanical Garden, Guru Nanak Dev University, Amritsar, India, under aseptic conditions. The protocol of Sen and Sharma (1991) was followed for germination of seeds. The seeds were soaked in water for 24 h, washed with 5% (v/v) teepol solution (5 min), surface-sterilized with 0.1% (w/v) mercuric chloride (12–13 min), washed three to four times with sterile double-distilled water and placed in Petri dishes containing half-strength MS medium and 1% (w/v) sucrose without plant growth regulators (PGR). The seeds were incubated at 25°C in the dark for 1 wk and then under white fluorescent light (40 μmol m−2 s−1) with 16-h day-length.Callus initiationFrom 10–15-d-old seedlings, hypocotyls, roots, and cotyledonary leaves were excised and divided into segments of 0.5–1.0 cm and cultured on MS-based solidified medium supplemented with different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and KN for callus formation. The cultures were evaluated in terms of fresh and dry weights of calluses after 30 d.Plant regenerationThe callus obtained from various explants was used for regeneration assessment on MS medium supplemented with various concentrations and combinations of 2,4-D, KN, BA, and 2-iP [N6-(2-isopentenyl) adenosine]. The shoots obtained from calluses were divided into single shoots and transferred to MS medium containing different concentrations and combinations of BA and 2-iP. After 30 d, the cultures were evaluated in terms of number and height of shoots. The shoot multiplication rate was also investigated by transferring single shoots that were obtained from the first subculturing onto the medium containing BA or 2-iP alone or in combination. The cultures were evaluated after 30, 45, and 60 d.Root formationIn vitro-regenerated shoots (1.5–2.0 cm) were transferred to rooting medium containing different concentrations and combinations of IAA, indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA). The number of roots per shoot and root length were recorded after 30 d of culture.Each treatment consisted of 25 tubes that were incubated at 25 ± 1°C with 16-h photoperiod for callus formation (two explants per tube), shoot regeneration, first subculturing, and root formation. However, each treatment of the second subculturing consisted of 10 flasks (four shoots per flask). The experiments were executed in triplicate.Acclimatization of plantletsThe plantlets were removed from rooting medium and washed thoroughly to remove the adhering agar. The plantlets were then transferred to plastic pots containing a mixture of sterilized sand and garden soil (1:1). After 20–22 d, the plants were transferred to earthen pots containing only garden soil and then transplanted to the field.The data pertaining to fresh and dry weights of calluses, number of shoots or roots per culture, and shoot and root length were subjected to a one-way analysis of variance (ANOVA) test, and the differences among means were compared by high-range statistical domain (HSD) using Tukey's test (Meyers and Grossen, 1974).Results and DiscussionCallus inductionTable 1 depicts callus induction from hypocotyl, root, and cotyledonary leaf segments. Maximum callusing (76%) was noticed on hypocotyl segments grown on MS medium supplemented with 2 mg l−1 (9.1 μM) 2,4-D. The callus formation frequency increased to 91% when 2,4-D was used in combination with 0.2 mg l−1 (0.9 μM) KN. There was 100% callusing from root and cotyledonary leaf segments grown on MS medium containing a combination of 2 mg l−1 2,4-D and 0.2 mg l−1 (0.9 μM) KN, and also from cotyledonary leaf segments grown with 2 mg l−1 2,4-D alone (Fig. 1a). Fresh and dry weights of calluses derived from cotyledonary leaf segments were found to be maximum when explants were grown on MS medium containing 2 mg l−1 2,4-D and 0.2 mg l−1 KN, and differed significantly from those obtained with other PGR combinations. No callus induction was observed on MS medium without PGRs. These results are in conformity with some of the earlier findings in other plants of the same family, such as Solanum laciniatum (Chandler et al., 1982), Solanum sarrachoides (Banerjee et al., 1985), Nicotiana tabacum (Rathore and Goldsworthy, 1985), Solanum dulcamara (Emke and Eilert, 1986), Solanum melongena (Filippone and Lurquin, 1989), Coptis teeta (Tandon and Rathore, 1992), Hyoscyamus muticus (Basu and Chand, 1996), and Solanum nigrum (Shahzad et al., 1999).Plant regenerationThe calluses derived from root and cotyledonary leaf segments grown in the presence of various concentrations and combinations of BA, KN, 2-iP, and 2,3,5-triiodobenzoic acid (TIBA) (1–4 mg l−1) did not regenerate shoots. Callus regeneration did not occur on MS medium without PGRs. Hypocotyl calluses obtained on MS medium containing 2 mg l−1 2,4-D and 0.2 mg l−1 KN showed shoot regeneration in 44% of cultures. The number and height of shoots were 4.0 ± 0.1 and 2.3 ± 0.1 cm, respectively, on the same medium (Table 2; Fig. 1b). However, no shoot induction was noticed in hypocotyl calluses using BA either alone or in combination with KN. In line with the present study, 2,4-D and KN have also been reported to induce shoot regeneration from calluses of plants, viz., Aloe vera (Roy and Sarkar, 1991) and Stephania cephalantha (Suzuki et al., 1992). Table 3 shows the effect of BA and 2-iP on the first subculturing of shoots obtained from hypocotyl calluses. A maximum percentage of cultures (61.3%) showing shoot multiplication, number of shoots per culture, and height of shoots was observed in cultures grown on MS medium containing 2 mg l−1 BA (Fig. 1c). Interestingly, when BA at 1 mg l−1 (4.4 μM) was used in combination with varying concentrations of 2-iP, an increase in concentration of 2-iP resulted in a decreased rate of shoot multiplication. However, 2-iP resulted in fewer cultures showing shoot multiplication, fewer and shorter shoots as compared to those obtained with BA. The shoots obtained from the first subculture after 30 d were separated into single shoots and each shoot was transferred to a flask containing MS medium supplemented with different concentrations and combinations of BA and 2-iP. The number and height of shoots recorded after 30, 45, and 60 d of subculture are given in Table 4. A maximum of 20 shoots per flask was obtained after 60 d of subculture with 2 mg l−1 BA (8.9 μM) (Fig. 1d). The differences in number and height of shoots recorded with different concentrations of PGRs were found to be statistically significant at the 5% level of significance. BA at 2 mg l−1 was found to be most effective for shoot multiplication, which is in agreement with earlier results showing that BA is the most effective cytokinin for shoot multiplication in many other plants. These include Trifolium pratense (Campbell and Tomes, 1984), Prunus persica (Hammerschlag et al., 1987), Dipterocarpus intricatus (Linington and Kew, 1989), Morus alba (Sharma and Thorpe, 1990), Aegle marmelos (Varghese et al., 1993), Morus spp. (Pattnaik and Chand, 1997), Bacopa monnieri (Tiwari et al., 1998), and Holarrhena pubescens (Sumana et al., 1999). Of all the explants used, hypocotyl was the most efficient. Although callus formation could also be induced in root and cotyledonary leaf segments, they were unable to regenerate in the presence of all the PGRs tested. Similar results have also been reported in Capsicum frutescens, where callus initiation occurred most readily from root segments, but shoot regeneration was found to be low (Subhash and Christopher, 1988). Poor organogenesis from root segments has also been observed in other plant species (Mathews, 1987; Ozias-Akins and Perera, 1990; Zhao et al., 1993; Abbas et al., 1996; Gomes-da-Cunha and Fernandes-Ferreira, 1996). In contrast to the results of this study, the calluses derived from cotyledonary leaf and root segments of Carthamus tinctorius (Rani et al., 1996) and Allium sativum (Myers and Simon, 1998) were efficient for differentiation.Root inductionThe shoots formed roots after being transferred to rooting medium for 12–15 d. Maximum shoots (87%) rooted when cultured on MS medium containing 2 mg l−1 (9.9 μM) IBA. However, the frequency of root formation was comparatively lower with higher concentrations of IBA alone or in combination with IAA or NAA and with all concentrations of IAA alone. The highest average number of roots (32.2) and their length (3.2 cm) were obtained with 2 mg l−1 IBA (Table 5; Fig. 1e). Among PGRs tested, IBA at 2 mg l−1 was found to be best for the induction of root formation. Our results are in accordance with the similar findings of some other plant species such as Eucalyptus sideroxylon (Burger, 1987), Quercus suber (Romano et al., 1992), Elaeagnus angustifolia (Iriondo et al., 1995), Capparis decidua (Tyagi and Kothari, 1997), Melia azedarach (Thakur et al., 1998), and Eucalyptus tereticornis (Sharma and Ramamurthy, 2000).Rooted plantlets, when transferred to soil, showed 60% survival (Fig. 1f). Using the present protocol, we were able to obtain more than 120 plantlets from the callus of one culture tube in 6 mo. On average, by repeated subculturing, it is possible to produce about 9000 plants in 1 yr.AcknowledgmentsThe authors wish to thank Prof. A. K. Thukral, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar for critical suggestions and his help in statistical analysis of the data. This paper/work is dedicated to the fond memories of the late Prof. I. S. Grover, our teacher and former Head, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar.ReferencesAbbas, A. M. , J. K. Jones , and P. D S. Caligari . 1996. Clonal propagation by in vitro culture of Corchorus (jute). Plant Cell Tiss. Organ Cult. 47:231–238. CrossRefgenSfxLinks('i1054-5476-39-5-468-Abbas1', '%26aulast%3DAbbas%26aufirst%3DA.%2BM.%26date%3D1996%26atitle%3DClonal%2Bpropagation%2Bby%2Bin%2Bvitro%2Bculture%2Bof%2BCorchorus%2B%2528jute%2529.%26volume%3D47%26spage%3D231%26id%3Ddoi%3A10.1007%252FBF02318977');Abhyankar, G. A. and G. S. Chinchanikar . 1996. Response of Withania somnifera Dunal leaf explants in vitro. Phytomorphology 46:249–252.genSfxLinks('i1054-5476-39-5-468-Abhyankar1', '%26aulast%3DAbhyankar%26aufirst%3DG.%2BA.%26date%3D1996%26atitle%3DResponse%2Bof%2BWithania%2Bsomnifera%2BDunal%2Bleaf%2Bexplants%2Bin%2Bvitro.%26volume%3D46%26spage%3D249');Baburaj, S. and K. Gunasekaran . 1995. In vitro differentiation of shoots from leaf callus cultures of Withania somnifera (L.) Dunal. J. Ind. Bot. Soc. 74:323–324.genSfxLinks('i1054-5476-39-5-468-Baburaj1', '%26aulast%3DBaburaj%26aufirst%3DS.%26date%3D1995%26atitle%3DIn%2Bvitro%2Bdifferentiation%2Bof%2Bshoots%2Bfrom%2Bleaf%2Bcallus%2Bcultures%2Bof%2BWithania%2Bsomnifera%2B%2528L.%2529%2BDunal.%26volume%3D74%26spage%3D323');Banerjee, S. , A. M. Schwemmin , and D. J. Schwemmin . 1985. In vitro propagation of Solanum sarrachoides Sendt. J. Plant Physiol. 119:461–466.genSfxLinks('i1054-5476-39-5-468-Banerjee1', '%26aulast%3DBanerjee%26aufirst%3DS.%26date%3D1985%26atitle%3DIn%2Bvitro%2Bpropagation%2Bof%2BSolanum%2Bsarrachoides%2BSendt.%26volume%3D119%26spage%3D461');Basu, P. and S. Chand . 1996. Anthocyanin accumulation in Hyoscyamus muticus L. tissue cultures. J. Biotechnol. 52:151–159. CrossRef, CSAgenSfxLinks('i1054-5476-39-5-468-Basu1', '%26aulast%3DBasu%26aufirst%3DP.%26date%3D1996%26atitle%3DAnthocyanin%2Baccumulation%2Bin%2BHyoscyamus%2Bmuticus%2BL.%2Btissue%2Bcultures.%26volume%3D52%26spage%3D151%26id%3Ddoi%3A10.1016%252FS0168-1656%252896%252901626-4');Burger, D. W. 1987. In vitro micropropagation of Eucalyptus sideroxylon. HortScience 22:496–497.genSfxLinks('i1054-5476-39-5-468-Burger1', '%26aulast%3DBurger%26aufirst%3DD.%2BW.%26date%3D1987%26atitle%3DIn%2Bvitro%2Bmicropropagation%2Bof%2BEucalyptus%2Bsideroxylon.%26volume%3D22%26spage%3D496');Campbell, C. T. and D. T. Tomes . 1984. Establishment and multiplication of red clover plants by in vitro shoot tip culture. Plant Cell Tiss. Organ Cult. 3:49–57. CrossRefgenSfxLinks('i1054-5476-39-5-468-Campbell1', '%26aulast%3DCampbell%26aufirst%3DC.%2BT.%26date%3D1984%26atitle%3DEstablishment%2Band%2Bmultiplication%2Bof%2Bred%2Bclover%2Bplants%2Bby%2Bin%2Bvitro%2Bshoot%2Btip%2Bculture.%26volume%3D3%26spage%3D49%26id%3Ddoi%3A10.1007%252FBF00035920');Chandler, S. F.; Dodds, J. H.; Henshaw, G. G. Factors affecting adventitious shoot formation in Solanum laciniatum Ait. callus cultures. In: 5th Meeting on Plant Tissue Culture; 1982:133–134.Emke, A. and U. Eilert . 1986. Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara. Plant Cell Rep. 5:31–34. CrossRefgenSfxLinks('i1054-5476-39-5-468-Emke1', '%26aulast%3DEmke%26aufirst%3DA.%26date%3D1986%26atitle%3DSteroidal%2Balkaloids%2Bin%2Btissue%2Bcultures%2Band%2Bregenerated%2Bplants%2Bof%2BSolanum%2Bdulcamara.%26volume%3D5%26spage%3D31%26id%3Ddoi%3A10.1007%252FBF00269712');Filippone, E. and P. F. Lurquin . 1989. Stable transformation of egg plant (Solanum melongena L.) by cocultivation of tissues with Agrobacterium tumefaciens carrying a binary plasmid vector. Plant Cell Rep. 8:370–373. CrossRefgenSfxLinks('i1054-5476-39-5-468-Filippone1', '%26aulast%3DFilippone%26aufirst%3DE.%26date%3D1989%26atitle%3DStable%2Btransformation%2Bof%2Begg%2Bplant%2B%2528Solanum%2Bmelongena%2BL.%2529%2Bby%2Bcocultivation%2Bof%2Btissues%2Bwith%2BAgrobacterium%2Btumefaciens%2Bcarrying%2Ba%2Bbinary%2Bplasmid%2Bvector.%26volume%3D8%26spage%3D370%26id%3Ddoi%3A10.1007%252FBF00716677');Furmanowa, M. , D. Gajdzis-Kuls , J. Ruszkowska , Z. Czarnocki , G. Obidoska , A. Sadowska , R. Rani , and S. N. Upadhyay . 2001. In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity. Planta Med. 67:146–149. CrossRef, MedlinegenSfxLinks('i1054-5476-39-5-468-Furmanowa1', '%26aulast%3DFurmanowa%26aufirst%3DM.%26date%3D2001%26atitle%3DIn%2Bvitro%2Bpropagation%2Bof%2BWithania%2Bsomnifera%2Band%2Bisolation%2Bof%2Bwithanolides%2Bwith%2Bimmunosuppressive%2Bactivity.%26volume%3D67%26spage%3D146%26id%3Ddoi%3A10.1055%252Fs-2001-11494');Gomes-da-Cunha, A. C. and M. Fernandes-Ferreira . 1996. Somatic embryogenesis, organogenesis and callus growth kinetics of flax. Plant Cell Tiss. Organ Cult. 47:1–8. CrossRefgenSfxLinks('i1054-5476-39-5-468-Gomes-da-Cunha1', '%26aulast%3DGomes-da-Cunha%26aufirst%3DA.%2BC.%26date%3D1996%26atitle%3DSomatic%2Bembryogenesis%252C%2Borganogenesis%2Band%2Bcallus%2Bgrowth%2Bkinetics%2Bof%2Bflax.%26volume%3D47%26spage%3D1%26id%3Ddoi%3A10.1007%252FBF02318959');Hammerschlag, F. A. , G. R. Bauchan , and R. Scorza . 1987. Factors influencing in vitro multiplication and rooting of peach cultivars. Plant Cell Tiss. Organ Cult. 8:235–242. CrossRefgenSfxLinks('i1054-5476-39-5-468-Hammerschlag1', '%26aulast%3DHammerschlag%26aufirst%3DF.%2BA.%26date%3D1987%26atitle%3DFactors%2Binfluencing%2Bin%2Bvitro%2Bmultiplication%2Band%2Brooting%2Bof%2Bpeach%2Bcultivars.%26volume%3D8%26spage%3D235%26id%3Ddoi%3A10.1007%252FBF00040950');Iriondo, J. M. , M. de la Iglesia , and C. Perez . 1995. Micropropagation of Elaeagnus angustifolia from mature trees. Tree Physiol. 15:691–693. MedlinegenSfxLinks('i1054-5476-39-5-468-Iriondo1', '%26aulast%3DIriondo%26aufirst%3DJ.%2BM.%26date%3D1995%26atitle%3DMicropropagation%2Bof%2BElaeagnus%2Bangustifolia%2Bfrom%2Bmature%2Btrees.%26volume%3D15%26spage%3D691');Kulkarni, A. A. , S. R. Thengane , and K. V. Krishnamurthy . 1996. Direct in vitro regeneration of leaf explants of Withania somnifera (L.). Dunal. Plant Sci. 119:163–168. CrossRefgenSfxLinks('i1054-5476-39-5-468-Kulkarni1', '%26aulast%3DKulkarni%26aufirst%3DA.%2BA.%26date%3D1996%26atitle%3DDirect%2Bin%2Bvitro%2Bregeneration%2Bof%2Bleaf%2Bexplants%2Bof%2BWithania%2Bsomnifera%2B%2528L.%2529.%26volume%3D119%26spage%3D163%26id%3Ddoi%3A10.1016%252F0168-9452%252896%252904462-7');Linington, I. M.; Kew, R. B. G. Micropropagation of Dipterocarpus alatus and D. intricatus. In Vitro 25:Pt. 2, 63A; 1989.Malhotra, C. L. , P. K. Das , N. S. Dhalla , and K. Prasad . 1961. Studies on Withania ashwagandha Kaul. Part III. The effect of total alkaloids on the cardiovascular system and respiration. Ind. J. Med. Res. 49:448–460.genSfxLinks('i1054-5476-39-5-468-Malhotra1', '%26aulast%3DMalhotra%26aufirst%3DC.%2BL.%26date%3D1961%26atitle%3DStudies%2Bon%2BWithania%2Bashwagandha%2BKaul.%2BPart%2BIII.%2BThe%2Beffect%2Bof%2Btotal%2Balkaloids%2Bon%2Bthe%2Bcardiovascular%2Bsystem%2Band%2Brespiration.%26volume%3D49%26spage%3D448');Mathews, H. 1987. Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek). Plant Cell Tiss. Organ Cult. 11:233–240. CrossRefgenSfxLinks('i1054-5476-39-5-468-Mathews1', '%26aulast%3DMathews%26aufirst%3DH.%26date%3D1987%26atitle%3DMorphogenetic%2Bresponses%2Bfrom%2Bin%2Bvitro%2Bcultured%2Bseedling%2Bexplants%2Bof%2Bmung%2Bbean%2B%2528Vigna%2Bradiata%2BL.%2BWilczek%2529.%26volume%3D11%26spage%3D233%26id%3Ddoi%3A10.1007%252FBF00040429');Meyers, L. S. and N. E. Grossen . 1974. Analysis of independent group designs. Behavioral research, theory, procedure and design 237–252. San Francisco. W.H. Freeman and Co.genSfxLinks('i1054-5476-39-5-468-Meyers1', '%26aulast%3DMeyers%26aufirst%3DL.%2BS.%26date%3D1974%26atitle%3DAnalysis%2Bof%2Bindependent%2Bgroup%2Bdesigns.%26spage%3D237');Myers, J. M. and P. W. Simon . 1998. Continuous callus production and regeneration of garlic (Allium sativum L.) using root segments from shoot tip-derived plantlets. Plant Cell Rep. 17:726–730. CrossRef, CSAgenSfxLinks('i1054-5476-39-5-468-Myers1', '%26aulast%3DMyers%26aufirst%3DJ.%2BM.%26date%3D1998%26atitle%3DContinuous%2Bcallus%2Bproduction%2Band%2Bregeneration%2Bof%2Bgarlic%2B%2528Allium%2Bsativum%2BL.%2529%2Busing%2Broot%2Bsegments%2Bfrom%2Bshoot%2Btip-derived%2Bplantlets.%26volume%3D17%26spage%3D726%26id%3Ddoi%3A10.1007%252Fs002990050473');Murashige, T. and F. Skoog . 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497. CrossRefgenSfxLinks('i1054-5476-39-5-468-Murashige1', '%26aulast%3DMurashige%26aufirst%3DT.%26date%3D1962%26atitle%3DA%2Brevised%2Bmedium%2Bfor%2Brapid%2Bgrowth%2Band%2Bbioassays%2Bwith%2Btobacco%2Btissue%2Bcultures.%26volume%3D15%26spage%3D473%26id%3Ddoi%3A10.1111%252Fj.1399-3054.1962.tb08052.x');Ozias-Akins, P. and S. Perera . 1990. Organogenesis in cultured adventitious root segments and in protoplast derived callus of sweet potato. HortScience 25:121.genSfxLinks('i1054-5476-39-5-468-Ozias-Akins1', '%26aulast%3DOzias-Akins%26aufirst%3DP.%26date%3D1990%26atitle%3DOrganogenesis%2Bin%2Bcultured%2Badventitious%2Broot%2Bsegments%2Band%2Bin%2Bprotoplast%2Bderived%2Bcallus%2Bof%2Bsweet%2Bpotato.%26volume%3D25%26spage%3D121');Pattnaik, S. K. and P. K. Chand . 1997. Rapid clonal propagation of three mulberries, Morus cathayana Hemsl., M. ihou Koiz. and M. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees. Plant Cell Rep. 16:503–508. CSAgenSfxLinks('i1054-5476-39-5-468-Pattnaik1', '%26aulast%3DPattnaik%26aufirst%3DS.%2BK.%26date%3D1997%26atitle%3DRapid%2Bclonal%2Bpropagation%2Bof%2Bthree%2Bmulberries%252C%2BMorus%2Bcathayana%2BHemsl.%252C%2BM.%2Bihou%2BKoiz.%2Band%2BM.%2Bserrata%2BRoxb.%252C%2Bthrough%2Bin%2Bvitro%2Bculture%2Bof%2Bapical%2Bshoot%2Bbuds%2Band%2Bnodal%2Bexplants%2Bfrom%2Bmature%2Btrees.%26volume%3D16%26spage%3D503');Rani, G. and I. S. Grover . 1999. In vitro callus induction and regeneration studies in Withania somnifera. Plant Cell Tiss. Organ Cult. 57:23–27. CrossRefgenSfxLinks('i1054-5476-39-5-468-Rani1', '%26aulast%3DRani%26aufirst%3DG.%26date%3D1999%26atitle%3DIn%2Bvitro%2Bcallus%2Binduction%2Band%2Bregeneration%2Bstudies%2Bin%2BWithania%2Bsomnifera.%26volume%3D57%26spage%3D23%26id%3Ddoi%3A10.1023%252FA%253A1006329532561');Rani, K. J. , T. N. Rao , G. Raghunatham , and P. V. Rao . 1996. Studies on callus growth and differentiation in safflower. Ind. J. Genet. 56:458–461.genSfxLinks('i1054-5476-39-5-468-Rani2', '%26aulast%3DRani%26aufirst%3DK.%2BJ.%26date%3D1996%26atitle%3DStudies%2Bon%2Bcallus%2Bgrowth%2Band%2Bdifferentiation%2Bin%2Bsafflower.%26volume%3D56%26spage%3D458');Rathore, K. S. and A. Goldsworthy . 1985. Electrical control of shoot regeneration in plant tissue cultures. Bio/Technology 3:1107–1109. CrossRefgenSfxLinks('i1054-5476-39-5-468-Rathore1', '%26aulast%3DRathore%26aufirst%3DK.%2BS.%26date%3D1985%26atitle%3DElectrical%2Bcontrol%2Bof%2Bshoot%2Bregeneration%2Bin%2Bplant%2Btissue%2Bcultures.%26volume%3D3%26spage%3D1107%26id%3Ddoi%3A10.1038%252Fnbt1285-1107');Ray, S. and S. Jha . 2001. Production of withaferin A in shoot cultures of Withania somnifera. Planta Med. 67:432–436. CrossRef, MedlinegenSfxLinks('i1054-5476-39-5-468-Ray1', '%26aulast%3DRay%26aufirst%3DS.%26date%3D2001%26atitle%3DProduction%2Bof%2Bwithaferin%2BA%2Bin%2Bshoot%2Bcultures%2Bof%2BWithania%2Bsomnifera.%26volume%3D67%26spage%3D432%26id%3Ddoi%3A10.1055%252Fs-2001-15811');Roja, G. , M. R. Heble , and A. T. Sipahimalani . 1991. Tissue cultures of Withania somnifera: morphogenesis and withanolide synthesis. Phytother. Res. 5:185–187. CrossRefgenSfxLinks('i1054-5476-39-5-468-Roja1', '%26aulast%3DRoja%26aufirst%3DG.%26date%3D1991%26atitle%3DTissue%2Bcultures%2Bof%2BWithania%2Bsomnifera%253A%2Bmorphogenesis%2Band%2Bwithanolide%2Bsynthesis.%26volume%3D5%26spage%3D185%26id%3Ddoi%3A10.1002%252Fptr.2650050411');Romano, L. A.; Noronha, C.; Martins-Loucao, M. A. Factors affecting in vitro rooting of Quercus suber. In Vitro 28:Pt. 2, 116A; 1992.Roy, S. C. and A. Sarkar . 1991. In vitro regeneration and micropropagation of Aloe vera L. Sci. Hort. 47:107–113. CrossRefgenSfxLinks('i1054-5476-39-5-468-Roy1', '%26aulast%3DRoy%26aufirst%3DS.%2BC.%26date%3D1991%26atitle%3DIn%2Bvitro%2Bregeneration%2Band%2Bmicropropagation%2Bof%2BAloe%2Bvera%2BL.%26volume%3D47%26spage%3D107%26id%3Ddoi%3A10.1016%252F0304-4238%252891%252990032-T');Sen, J. and A. K. Sharma . 1991. Micropropagation of Withania somnifera from germinating seeds and shoot tips. Plant Cell Tiss. Organ Cult. 26:71–73. CrossRefgenSfxLinks('i1054-5476-39-5-468-Sen1', '%26aulast%3DSen%26aufirst%3DJ.%26date%3D1991%26atitle%3DMicropropagation%2Bof%2BWithania%2Bsomnifera%2Bfrom%2Bgerminating%2Bseeds%2Band%2Bshoot%2Btips.%26volume%3D26%26spage%3D71%26id%3Ddoi%3A10.1007%252FBF00036108');Shahzad, A. , H. Hasan , and S. A. Siddiqui . 1999. Callus induction and regeneration in Solanum nigrum L. in vitro. Phytomorphology 49:215–220.genSfxLinks('i1054-5476-39-5-468-Shahzad1', '%26aulast%3DShahzad%26aufirst%3DA.%26date%3D1999%26atitle%3DCallus%2Binduction%2Band%2Bregeneration%2Bin%2BSolanum%2Bnigrum%2BL.%2Bin%2Bvitro.%26volume%3D49%26spage%3D215');Sharma, K. K. and T. A. Thorpe . 1990. In vitro propagation of mulberry (Morus alba L.) through nodal segments. Sci. Hort. 42:307–320. CrossRefgenSfxLinks('i1054-5476-39-5-468-Sharma1', '%26aulast%3DSharma%26aufirst%3DK.%2BK.%26date%3D1990%26atitle%3DIn%2Bvitro%2Bpropagation%2Bof%2Bmulberry%2B%2528Morus%2Balba%2BL.%2529%2Bthrough%2Bnodal%2Bsegments.%26volume%3D42%26spage%3D307%26id%3Ddoi%3A10.1016%252F0304-4238%252890%252990054-I');Sharma, S. K. and V. Ramamurthy . 2000. Micropropagation of 4-year-old elite Eucalyptus tereticornis trees. Plant Cell Rep. 19:511–518. CrossRefgenSfxLinks('i1054-5476-39-5-468-Sharma2', '%26aulast%3DSharma%26aufirst%3DS.%2BK.%26date%3D2000%26atitle%3DMicropropagation%2Bof%2B4-year-old%2Belite%2BEucalyptus%2Btereticornis%2Btrees.%26volume%3D19%26spage%3D511%26id%3Ddoi%3A10.1007%252Fs002990050765');Subhash, K. and T. Christopher . 1988. Direct plantlet formation in cotyledon cultures of Capsicum frutescens. Curr. Sci. 57:99–100.genSfxLinks('i1054-5476-39-5-468-Subhash1', '%26aulast%3DSubhash%26aufirst%3DK.%26date%3D1988%26atitle%3DDirect%2Bplantlet%2Bformation%2Bin%2Bcotyledon%2Bcultures%2Bof%2BCapsicum%2Bfrutescens.%26volume%3D57%26spage%3D99');Sumana, K. R. , K. M. Kaveriappa , and H. S. Bhat . 1999. In vitro micropropagation of Holarrhena pubescens. J. Med. Arom. Plant Sci. 21:299–303.genSfxLinks('i1054-5476-39-5-468-Sumana1', '%26aulast%3DSumana%26aufirst%3DK.%2BR.%26date%3D1999%26atitle%3DIn%2Bvitro%2Bmicropropagation%2Bof%2BHolarrhena%2Bpubescens.%26volume%3D21%26spage%3D299');Suzuki, S. , H. Fujino , Y. Tatsuo , N. Yamazaki , and M. Yoshizaki . 1992. Rapid propagation of Stephania cephalantha Hayata by tissue culture. Jap. J. Breed. 42:769–777.genSfxLinks('i1054-5476-39-5-468-Suzuki1', '%26aulast%3DSuzuki%26aufirst%3DS.%26date%3D1992%26atitle%3DRapid%2Bpropagation%2Bof%2BStephania%2Bcephalantha%2BHayata%2Bby%2Btissue%2Bculture.%26volume%3D42%26spage%3D769');Tandon, P. and T. S. Rathore . 1992. Regeneration of plantlets from hypocotyl-derived callus of Coptis teeta. Plant Cell Tiss. Organ Cult. 28:115–117. CrossRefgenSfxLinks('i1054-5476-39-5-468-Tandon1', '%26aulast%3DTandon%26aufirst%3DP.%26date%3D1992%26atitle%3DRegeneration%2Bof%2Bplantlets%2Bfrom%2Bhypocotyl-derived%2Bcallus%2Bof%2BCoptis%2Bteeta.%26volume%3D28%26spage%3D115%26id%3Ddoi%3A10.1007%252FBF00039923');Thakur, R. , P. S. Rao , and V. A. Bapat . 1998. In vitro plant regeneration in Melia azedarach. Plant Cell Rep. 18:127–131. CrossRefgenSfxLinks('i1054-5476-39-5-468-Thakur1', '%26aulast%3DThakur%26aufirst%3DR.%26date%3D1998%26atitle%3DIn%2Bvitro%2Bplant%2Bregeneration%2Bin%2BMelia%2Bazedarach.%26volume%3D18%26spage%3D127%26id%3Ddoi%3A10.1007%252Fs002990050544');Tiwari, S. K. and S. P. Singh . 1991. Micropropagation of Withania somnifera by tissue culture. Van. Sandesh 15:19–20.genSfxLinks('i1054-5476-39-5-468-Tiwari1', '%26aulast%3DTiwari%26aufirst%3DS.%2BK.%26date%3D1991%26atitle%3DMicropropagation%2Bof%2BWithania%2Bsomnifera%2Bby%2Btissue%2Bculture.%26volume%3D15%26spage%3D19');Tiwari, V. , B. D. Singh , and K. N. Tiwari . 1998. Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monnieri (L.) Wettst.]. Plant Cell Rep. 538–543. CrossRefgenSfxLinks('i1054-5476-39-5-468-Tiwari2', '%26aulast%3DTiwari%26aufirst%3DV.%26date%3D1998%26atitle%3DShoot%2Bregeneration%2Band%2Bsomatic%2Bembryogenesis%2Bfrom%2Bdifferent%2Bexplants%2Bof%2BBrahmi%2B%255BBacopa%2Bmonnieri%2B%2528L.%2529%2BWettst.%255D.%26spage%3D538%26id%3Ddoi%3A10.1007%252Fs002990050438');Tyagi, P. and S. L. Kothari . 1997. Micropropagation of Capparis deciduain vitro shoot proliferation on nodal explants of mature tree and seedling explants. J. Plant Biochem. Biotechnol. 6:19–23.genSfxLinks('i1054-5476-39-5-468-Tyagi1', '%26aulast%3DTyagi%26aufirst%3DP.%26date%3D1997%26atitle%3DMicropropagation%2Bof%2BCapparis%2Bdeciduain%2Bvitro%2Bshoot%2Bproliferation%2Bon%2Bnodal%2Bexplants%2Bof%2Bmature%2Btree%2Band%2Bseedling%2Bexplants.%26volume%3D6%26spage%3D19');Uma Devi, P. , A. C. Sharada , and F. E. Solomon . 1993. Antitumor and radiosensitizing effects of Withania somnifera (Ashwagandha) on a transplantable mouse tumor sarcoma 180. Ind. J. Exp. Biol. 31:607–611. Medline, CSAgenSfxLinks('i1054-5476-39-5-468-Uma1', '%26aulast%3DUma%2BDevi%26aufirst%3DP.%26date%3D1993%26atitle%3DAntitumor%2Band%2Bradiosensitizing%2Beffects%2Bof%2BWithania%2Bsomnifera%2B%2528Ashwagandha%2529%2Bon%2Ba%2Btransplantable%2Bmouse%2Btumor%2Bsarcoma%2B180.%26volume%3D31%26spage%3D607');Uma Devi, P. , A. C. Sharada , F. E. Solomon , and M. S. Kamath . 1992. In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor sarcoma 180. Ind. J. Exp. Biol. 30:169–172. MedlinegenSfxLinks('i1054-5476-39-5-468-Uma2', '%26aulast%3DUma%2BDevi%26aufirst%3DP.%26date%3D1992%26atitle%3DIn%2Bvivo%2Bgrowth%2Binhibitory%2Beffect%2Bof%2BWithania%2Bsomnifera%2B%2528Ashwagandha%2529%2Bon%2Ba%2Btransplantable%2Bmouse%2Btumor%2Bsarcoma%2B180.%26volume%3D30%26spage%3D169');Varghese, S. K. , J. A. Inamdar , K. Kalia , R. B. Subramanian , and M. Nataraj . 1993. Micropropagation of Aegle marmelos (L.). Cor. Phytomorphology 43:87–92.genSfxLinks('i1054-5476-39-5-468-Varghese1', '%26aulast%3DVarghese%26aufirst%3DS.%2BK.%26date%3D1993%26atitle%3DMicropropagation%2Bof%2BAegle%2Bmarmelos%2B%2528L.%2529.%26volume%3D43%26spage%3D87');Zhao, Y. X. , D. Y. Yao , and P. J C. Harris . 1993. Plant regeneration from callus and explants of Sesbania spp. Plant Cell Tiss. Organ Cult. 34:253–260. CrossRefgenSfxLinks('i1054-5476-39-5-468-Zhao1', '%26aulast%3DZhao%26aufirst%3DY.%2BX.%26date%3D1993%26atitle%3DPlant%2Bregeneration%2Bfrom%2Bcallus%2Band%2Bexplants%2Bof%2BSesbania%2Bspp.%26volume%3D34%26spage%3D253%26id%3Ddoi%3A10.1007%252FBF00029714');enlarge figureFig 1. Callus induction and plantlet regeneration in Withania somnifera. a, Callus induction from root segments on MS medium containing 2 mg l−1 (9.1 μM) 2,4-D and 0.2 mg l−1 (0.9 μM) KN. b, Shoot induction from hypocotyl callus with 2 mg l−1 2,4-D and 0.2 mg l−1 KN. c, Shoot multiplication of hypocotyl callus-derived shoots with 2 mg l−1 (8.9 μM) BA. d, Multiple shoots obtained after 60 d of the second subculture with 2 mg l−1 BA. e, Root formation with 2 mg l−1 (9.9 μM) IBA. f, Plantlet transplanted to a plastic pot.
Posted by myblogbd at 10:51 AM 0 comments
Labels: SCOOTERS