High Frequency Axillary Bud Multiplication of Caralluma stalagmifera C.E.C. Fischer- A Medicinal Plant

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1 Original Artile High Frequeny Axillary Bu Multipliation of Caralluma stalagmifera C.E.C. Fisher- A Meiinal Plant V.Raja Sreelatha an T.Pullaiah* Department of Botany, Sri Krishnaevaraya University, Anantapur , A.P., Inia ABSTRACT Aress for Corresponene Department of Botany, Sri Krishnaevaraya University, Anantapur , A.P. Ojetive: The ojetive of the present stuy was to evelop a protool for miropropagation of the meiinal plant Caralluma stalagmifera (Aslepiaaeae) from noal explants. Methos: Noal explants of fiel grown plants were ulture on MS meium supplemente with BAP, 2-iP, GA 3 an auxins NAA, IAA an IBA. The regenerate shoots were roote on half strength MS meium supplemente with IAA, IBA an NAA. They were alimatise in plasti ups with polythene overs an then transferre to fiel. Results an Conlusion: Axillary u proliferation with a mean of 8.47 shoots/noe was ahieve when the explants were grown in an optimize Murashige an Skoog (MS) meium whih were supplemente with the phytohormones BAP (2.0 mg/l) + 2-iP (2.0 mg/l) + NAA (0.5 mg/l). When the noe segments grown in vitro were exise an ulture in a meium supplemente with the same onentration of the plant growth hormones that were use for the growth of the Axillary us, more than 15 shoots evelope in 30 ays. Conseutive exision an ulture of noes grown in vitro greatly augmente the numer of shoots whih emerge as well as shoot elongation when the onentration of the growth hormones use in the MS meium was BAP (2.0 mg/l) + GA 3 (1.0 mg/l). Highest numer of roots was oserve when half strength MS meium enrihe with NAA (0.5 mg/l) was use. Harening proess of the roote plants was ahieve in polyups having equal proportions of sterile peat mass, farmyar manure an soil, whih resulte in a survival rate of 70%. Keywors: Aslepiaaeae, Meiinal plant, Caralluma stalagmifera, Noal explants, Shoot multipliation, Plant tissue ulture, Miropropagation. Amerian Journal of Phytomeiine an Clinial Therapeutis

2 INTRODUCTION The ry an roky regions of South Inia are home to Caralluma stalagmifera (Aslepiaaeae), a suulent an perennial her having a sour taste. This plant as a whole has een use to extrat various novel steroial glyosies suh as Iniosies I-II an Stalagmosies I-V along with the wellknown steroial glyosies suh as Lasianthosies A-B an Carumellosie III 1. Butanol an aqueous extrats of C. stalagmifera have emonstrate antiarthriti an anti-inflammatory ativities in rats having kaolin inue arthritis an arrageenin inue paw oeema 2. The genus Caralluma has ertain plants whih are prolifi with esterifie polyhyroxy pregnane glyosies having anti-tumour properties, while there are other plants in this genus whih possess Carenolies 3,4 an flavone glyosies 5,6. Aoring to the Inian Folkore reports an the praties of trials an hunters, the extrats of Caralluma are known to stimulate weight loss y suppressing the appetite an this knowlege has een translate an markete y the inustry as GENASLIM, whih is a famous pill for the oese people 7. To meet the emans for meiines of heral origin, there is a strong nee to propagate elite plants suh as Caralluma. Conventional means of ultivation of these plants provie very low yiel ue to lower rate of survival. Hene, aoption of alternate plant propagation methos suh as in vitro propagation will ensure that the emans of the heral meiinal inustry are aequately met with. In vitro propagation assures the growth of true-to-type plants with limiting onitions of spae an time. The present ommuniation is the first report on in vitro propagation of Caralluma stalagmifera using noes erive from mature plants. MATERIALS AND METHODS Colletion of plant material an surfae sterilisation Caralluma stalagmifera was ollete from Gooty hills, Anantapur istit, Anhra Praesh, Inia an were potte in pots an maintaine at Sri Krishnaevaraya University, Anantapur. The soure of explants use for the stuy were young shoots that were growing atively. These explants were surfae sterilize y first washing them uner running tap water an then using 1% tween- 20 (etergent) for 10 min. This was followe y a susequent wash uner running tap water. Then, the explants were transferre to the Laminar Air flow hamer for further steps of surfae sterilization. The explants were washe with sterile oule istille water followe y a wash with 0.1% Meruri hlorie for 5 min an then susequent wash steps with sterile oule istille water. Explants were then sujete to 70% ethanol treatment for 1 min. an 3-4 rinses in sterile oule istille water. Meia an Culture onitions Stem segments, 1.0 m long, ontaining the axillary us were aseptially ut from the surfae sterilize shoot piees an inoulate onto the sterilize nutrient meium, one explant/tue. The amage ens were remove. The asal meium (MS) use was that Murashige an Skoog 8 supplemente with 3% (w/v) surose an 0.8% agar. Depening on the experiment the asal meium was further supplemente with ytikinins (BAP an 2-iP), Gierelli Ais (GA 3 ) an auxins (NAA, IAA an IBA) alone or as a omination mentione in Tales 1-2. Bateriologial agar was ae to the meium after it s ph was ajuste to 5.8. The gelle meium was ispense into 150 mm 25 mm rimless

3 ulture tues (20ml meium/ tue) an autolave at 1.06 kg/m-2 pressure an 121 o C for 15 min. Eah treatment onsiste of 15 ulture repliates an eah experiment was performe in tripliates. The temperature of the ulture room was maintaine at 25 o C with ool fluoresent white light (50µ mol m -2 S -1 ) for a photoperio of 16 hours. Shoot multipliation an rooting Regenerate shoots having more than two noes were exise from the primary ultures an suulture on meium supplemente with BAP 2.0 mg/l + 2-ip 2.0 mg/l + NAA 0.5 mg/l. Su ulture was repeate every 30 ays for shoot proution. Cultures were grown in 25 mm 150 mm ulture tues or in 300ml ulture ottles. To inue rooting, full an half strength MS meium supplemente with various onentration of growth hormones suh as IAA, IBA an NAA (0.1 mg/l to 3.0 mg/l) was use in whih exise regenerate shoots were inoulate. For inution of rooting regenerate shoots were exise an ulture on full strength an half strength MS meium with various onentrations of NAA, IAA an IBA (0.1 mg/l to 3.0 mg/l). Alimatization of plantlets The roote plantlets were transplante in plasti pots having san, peat mass an farmyar manure in the ratio of 1:1:1 after thoroughly washing them. These plantlets after transplanting were sujete to irrigation with ½ strength MS asal liqui meium evoi of surose, after whih they were overe with polythene overs. After 10 ays the polythene overs remove an well evelope plants were transforme to green house onitions an then they were transferre outoors after alimatization for a perio of two weeks. Statistial analysis Analysis of variane (ANOVA) was use to analyze the ata statistially an the means otaine were ompare using the Tukeys test at a proaility level of signifiane of 0.005%. RESULTS AND DISCUSSION Explants ulture on asal meium i not show any growth an eventually nerose after 15 ays as in Gymnema elegans 9, Tylophora inia 10 an Cynanhum allialatum 11. Externally applie hormones for u sprouting was neessary. MS meium ontaining BAP (2.0 mg/l) was more effetive than 2-iP (2.0 mg/l) for inuing proliferation of axillary us. In ontrast 2-iP was more effetive than BAP to inue shoot formation in Dealepis hamiltonii 12. The shoots otaine were longer in MS meium whih was supplemente with 2-iP (2.0 mg/l) when ompare to supplementation with the growth hormone BAP (Tale - 1). 2-iP was omine with BAP at ifferent onentrations, maximum response with mean numer of 2.88 shoots /explant an shoot length of 2.80 m was oserve when the omination of growth hormones use was BAP 2.0 mg/l + 2-ip 2.0 mg/l (Tale 1). Different omination of ytokinins in the ulture meia an inrease the numer of shoots forme as against using a single ytokinin (Prasa et al. 13 ) To unerstan the effet of ifferent growth hormones on the regeneration of shoots from noal explants, various experiments were performe. Assuming that omine effet of ytokinins, auxins an GA 3 oul improve further multipliation rate of shoots, a omine effet of ifferent onentrations an ominations were stuie (Tale 2). A maximum of 1.54 shoots / explant with 4.10 m of maximum shoot length was oserve when noal explants of Caralluma stalagmifera ulture on meium

4 supplemente with BAP 2.0 mg/l+ GA mg/l (Tale 2) (Fig 1A). This is the maximum length of shoots oserve out of all the onentrations of GA 3 with BAP 2.0 mg/l (Tale 2). Culture meium enrihe with BAP 2.0 mg/l + 2-iP 2.0 mg/l + NAA 0.5 mg/l emonstrate the est result with 88 % of response after 20 ays of ulture. On this omination explants proue 8.47 shoots/ explant with an average shoot length of 2.50m (Fig 1B). Derease or inrease onentration of NAA reue the shoot numer as well as length (Tale 2). Where as omination with IAA proue 6.22 shoots/explant an 2.30m of shoot length at 1.0 mg/l with 73 % of response (Tale 2). Use of IBA in plae of IAA was teste in Caralluma stalagmifera. BAP 2.0 mg/l + 2- ip 2.0 mg/l +IBA 0.5 mg/l proue maximum numer of 6.10 shoots/explant with 1.55 m of shoot length (Tale 2). Aition of GA 3 to ytokinins an auxins ominations reue shoot numer in noal explants of Caralluma stalagmifera (Tale 2). In our present stuy omination with ytokinins (BAP an 2-iP), NAA ontinue to give etter results than IAA an IBA (Tale 2). Holostemma annulare 14 showe similar results. Meiinal plants elonging to Aslepiaaeae suh as Gymnema elegans 9 Holostemma aa koien 15 an Hemiesmus inius 16 have een use in experiments to unerstan the synergisti effet of ytokinins in omination with auxins. Tieman an Hawkar 17 have reporte that uring the propagation of latex prouing plants, the presene of NAA resulte in suppression of shoot formation an inrease in allus formation. In our present stuy est results were otaine from two ytokinins (BAP + 2-iP) an auxins (NAA, IAA an IBA) ominations. Malathy an Pai 18 use a omination of two ytokinins an auxins in MS meium for enhane axillary u proliferation. Shoot forme in the meium ontaining BAP 2.0 mg/l + 2-iP 2.0 mg/l + IAA IBA in omination with ifferent onentrations of GA 3 were use as explants to suulture them onto the meium enrihe with BAP 2.0 mg/l + 2-iP 2.0 mg/l + NAA 1.0 mg/l. This resulte in signifiant multipliation of shoots though the onentration of plant growth hormones i not have any impat on the length of the shoots (Tale 2). When these shoots whih evelope were suulture in ulture meium having GA 3 an BAP in the onentration of 1 mg/l an 2 mg/l respetively, there was optimal shoot elongation with 40 ays (Fig 1C). Enhane shoot multipliation in susequent ulture is in aorane with pulishe literature on Aslepiaaeae meiinal plants like Hemiesmus inius 16, Holostemma aakoien 15, Aslepias erosa 19 an Ceropegia anelarum 20. Patnaik an Deata 21 have esrie that repeate suulture of Hemiesmus inius i not result in proliferation of shoots. In orer to enhane shoot multipliation ifferent auxins were omine with the optimize ytokinin onentration. Experiments involving Hemiesmus inius 21 an Aslepias erosa 19 have expoune the fat that auxins at low onentration oul moify positively the shoot inution response when omine with ytokinins. The aition of GA 3 to ulture meia resulte in a response whih was very muh omparale to that as seen with auxins, therey implying that GA 3 an e a replaement for auxins for shoot inution 22. Hene, shoot propagation woul e ieally aomplishe with an optimize ratio of gierelins an ytokinins. But in the present stuy the omination of GA 3 an ytokinin was generally less satisfatory for the inution of shoots than that of

5 auxins + ytokinins. However, GA 3 was preventive for shoot regeneration in sugar eet floral axillary us 23. Suulture of the shoot explants on to fresh meium was performe one in 4 weeks to provie an uninterrupte supply of shoots with no erease in their morphogeneti potential for a longer perio. When shoot explants of 5.0 m length were suulture onto the rooting meium whih ontaine half strength MS meium enrihe with ifferent onentrations of the 3 auxins: IBA, NAA an IAA, only NAA prove to show signifiant root inution potential ompare to the other auxins (Tale 3). Extensive allusing at the ase with out root formation was notie respetively when high onentration of NAA, IAA an IBA supplemente to the meia in Caralluma stalagmifera. Root inution was possile with all auxins. However, strong root systems were otaine on NAA supplemente meium, with 0.5 mg/l onentrations exhiiting the est response (Tale 3) (Fig 1D). At this onentration 73 % shoots evelope roots with an average 8.42 roots/shoot. Though auxins are known to e the ieal plant hormones responsile for root inution, the extent of rooting inue onsieraly varies from one plant to the other 24. The positive response of rooting in the present stuy is similar to oservation of other memers of Aslepias Dealepis arayalpathera 25 an Ceropegia anelarum 20. Harening proess of the plantlets that were regenerate in vitro was initiate y first washing the plantlets with water a ouple of times to lear all ulture meium that was attahe to the plantlets, followe y whih the plantlets were potte in 10 m pots having equal quantities of peat mass, san an farmyar manure. These plantlets were inuate in the ulture room for sometime. The potte plants were overe with polythene over to ensure high humiity an irrigate every two ays with ½ strength MS maro salts free of surose for 2-3 weeks. The plantlets were expose to outsie onitions graually from the polythene overing, thus maintaining an optimal alane of relative humiity an therey enhaning their rate of survival. After 15 ays the harene plantlets were transferre to the green house, where 75% of the plantlets survive (Fig 1E). There were asolutely no aerrations in the phenotype of the plants in the green house that were regenerate in vitro. CONCLUSION Thus, we an infer that the protool whih we have esrie in this researh work an e use to miropropogate Caralluma stalagmifera suessfully using the tehniques of axillary u multipliation that ensures the aggranization of the enemi meiinal plant. ACKNOWLEDGEMENTS Authors are thankful to the Counil of Sientifi an Inustrial Researh (CSIR) Government of Inia, New Delhi for its finanial assistane. Conflit of Interest We elare no onflit of interest. REFERENCES 1. Olaf Kunert, Belvotagi Venkatrao Aavi Rao, Gummai Srihar Bau, Meooyina Pamavathi, Boala Ravi Kumar, Roert Mihael Alex, Wolfgang Shuhly, Neojsa Simi, Doris Kuhnelt an Ahanta Venkata Narasimha Appa Rao: Novel Sterioal Glyosies from two Inian Caralluma speies, C. stalagmifera an C. inia. Helvetia Chemia Ata (2)

6 2. Rey, B.M., Byahatii, V., Appa Rao, A.V.N. an Ramesh, M. Anti inflammatory ativity of Stepelia noilis an Caralluma stalagmifera. Fitoterapia Deepak, D., Khare, A. an Khare, M.P. Plant pregnanes. Phytohemistry Deepak, D., Srivastav, S. an Khare, A. Pregnane glyosies. Progress in the hemistry of organi Natural Prouts Ramesh, M., Rama Kumar, M., Krishna Mohan, G., Ravi kumar, B., Appa Rao, A.V.N., Raha Kishan, M. an Mahava Rey, B. Flavone glyosie from three Caralluma speies. Biohemial Systematis & Eol Rizwani, G.H., Usmanghani, K., Ahme, M. an Ahma, V.U. Flavone glyosies of Caralluma tuerulata N.E. Brown. J. Pharm. Si., Lawrene, R.M. an Chouary, S. Caralluma fimriata in the treatment of oesity; 12 th Annual worl ongress on antiaging meiine Deemer, 2-5, U.S.A. 8. Murashige, T. an Skoog, F. A revise meium from rapi growth an ioassays with toao tissue ultures. Physiol Plant Komalavalli, N. an Rao, M.V. In vitro miropropagation of Gymnema elegans Wt. & Arn. a rare meiinal plant. Inian J. Exp. Biol Sharma, N. an Chanel, K.P.S. Effet of asori ai on axillary shoot inution in Tylophora inia (Burm.f.) Merrill. Plant Cell Tiss. Org. Cult Raghuramulu, D., Sri Rama Murthy, K. an Pullaiah, T. In vitro propagation of Cynanhum allialatum. J. Trop. Me. Plants Girihar, P., Vinokumar, D., Oul Rey, B. an Rajasekharan, T. Somati emryogenesis organogenesis an regeneration from leaf allus ullture of Dealepis hamiltonii Wight & Arn. an enangere shru. In vitro Cell. Dev. Biol. Plant Prasa, P.J.N., Chakrahar, T. an Pullaiah, T. Miropropagation of Cryptolepis uhanani Roem. & Shult. Taiwania Suha, C.G., Krishnan, P.N., Pushpangaan, P. In vitro propagation of Holostemma annulare (Rox.) K. Shum., a rare meiinal plant. In vitro Cell. Dev. Biol. Plant Martin, K.P. Rapi propagation of Holostemma aa-koien Shult. a rare meiinal plant, through axillary u multipliation an iniret organogenesis. Plant Cell Rep Sree kumar, S., Seeni, S. an Pushpangaan, P. Miropropagation of Hemiesmus inius for ultivation an proution of 2-hyroxy 4-methoxy enzalehye. Plant Cell Tiss. Org. Cult Tieman, J. an Hawker, J.S. In vitro propagation of latex prouing plants. Ann. Bot Malathy, S. an Pai, J.S. In vitro propagation of Hemiesmus inius. Fitoterapia Chi Won, L. an John, C.T. Propagation of esert milkwee y shoot tip ulture. Hort. Si Beena, M.R., Martin, K.P., Kirti, P.B. an Hariharan, M. Rapi in vitro propagation of meiinally important Ceropegia anelarum. Plant Cell Tiss. Org. Cult Patnaik, J. an Deata, B.K. Miropropagation of Hemiesmus inius (L.) R. Br. through axillary u ulture. Plant Cell Rep Sekioka, T.J. an Tanaka, J.S. Differentiation in allus ultures of uumer (Cuumis sativus L.). Hort. Si., Coumans-Gilles, M.S., Kevers, C., Coumans, M., Ceulemans, E. an Gastar, T.H. Vegetative multipliation of sugar eet through in vitro ulture of infloresene piees. Plant Cell Tiss. Org. Cult., Swamy. S.L., Puri, S., Singh, A.K. Effet of auxins (IBA an NAA) an seasons on rooting of juvenile an mature harwoo

7 utting of Roonia pseuiaaia an Grewia optiva. New Forest Suha, C.G., Krishnan, P.N., Pushpangaan, P. an Seeni, S. In vitro propagation of Dealepis arayalpathra, a ritially ethnomeial plant. In vitro Cell Dev. Biol. Plant Tale 1: Effet of various onentrations of BAP an 2-iP on multiple shoot inution from mature noal explants of Caralluma stalagmifera ulture on MS meium. Plant growth regulators (mg/l) Shoot sprouting frequeny (%) Shoot No. per explant Mean SE Shoot length (m) Mean SE BAP iP BAP + 2-iP a i e a e f h f e f g f a h g e f e a f f h e a Values represent mean + stanar error of 15 repliates per treatment in three repeate experiments. Means followe y the same letter not signifiantly ifferent y the Tukey test at 0.05% proaility level.

8 Tale 2: Effet of ifferent ominations of BAP, 2-iP, NAA, IAA, IBA an GA 3 on shoot regeneration of mature noal explants Caralluma stalagmifera Plant growth regulators (mg/l) BAP 2-iP NAA IAA IBA GA 3 Shoot sprouting frequeny (%) No. of shoots/ explant Mean + SE e Shoot length (m) Mean + SE a e a a a a Values represent Mean ± SE, of 15 repliates per treatment in three repeate experiments. Mean followe y the same letter are not signifiantly ifferent y the Tukey test at 0.05% proaility level.

9 Tale 3: Effet of various auxins on rooting response from in vitro regenerate shoots of Caralluma stalagmifera ulture on MS half strength after 30 ays Conentration of Auxin mg/l NAA IAA IBA % of response Numer of Roots/shoot Mean ± SE ± 0.05 Length of roots (m) Mean ± SE 2.13 ± ± ± 0.01 a 3.59 ± ± CP CP CP CP ± 0.02 e 1.80 ± 0.02 a ± ± ± ± 0.02 a ± 0.02 e 1.50 ± CP CP ± 0.03 e 1.45 ± ± 0.02 e 1.70 ± ± ± ± ± CP CP Values represent mean + stanar error of 15 repliates per treatment in three repeate experiments. Means followe y the same letter not signifiantly ifferent y the Tukey test at 0.05% proaility level. CP Callus Proution a

10 Figure 1: A E. High Frequeny axillary u multipliation of Caralluma stalagmifera a. Maximum shoots elongation oserve on MS + BAP 2.0 mg/l + GA mg/l.. In vitro regeneration of noal explants with multiple shoot evelopment on MS meium supplemente with BAP 2.0 mg/l + 2-ip 2.0 mg/l + NAA 0.5 mg/l.. Enhane shoot length oserve after su ulture on the MS meium ontaining BAP 2.0 mg/l + GA mg/l.. In vitro rooting if in vitro raise shoots on ½ strength MS meium fortifie with NAA 0.5 mg/l after 30 ays. e. Alimatization of in vitro grown plants of Caralluma stalagmifera.