Factors affecting in vitro plant regeneration from cotyledonary node explant of Senna sophera (L.) Roxb. A highly medicinal legume

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1 Vol. 13(3), pp , 15 Jnury, 214 DOI: /AJB ISSN Ademi Journls Afrin Journl of Biotehnology Full Length Reserh Pper Ftors ffeting in vitro plnt regenertion from otyledonry node explnt of Senn sopher (L.) Rox. A highly mediinl legume Shhin Prveen nd Anwr Shhzd* Plnt Biotehnology Lortory, Deprtment of Botny, Aligrh Muslim University, Aligrh (UP) -22 2, Indi. Aepted 8 Jnury, 214 An effiient in vitro regenertion protool hs een developed for mediinl legume Senn sopher (L.) Rox. using Cotyledonry Node (CN) explnts. The plnt exhiits high mediinl potentil nd is eing used in severl trditionl nd homeopthy system of mediine. The present study desried n in vitro regenertion protool, where different ftors were optimized for mximum multiplition nd propgtion. The ge of the explnt, plnt growth regultors, sl medium, ph of the medium nd surose onentrtions mrkedly influened in vitro propgtion of S. sopher. Among 14, 21 nd 28 dy-old CN explnts, 21 dy-old explnts were found to e the most responsive. A mximum of 19.5 ±.51 shoots/explnt were produed from 21 dy-old seedling explnts, hving n verge shoot length of 5.23 ±.14 m in 86. ± 2.8% ultures fter 6 weeks of inution on Murshige nd Skoog (MS) medium supplemented with enzyl denine (BA) (5. µm) + nphthlene eti id (NAA) (1. µm) nd ontining 3% surose with ph vlue djusted t 5.8. The highest rooting frequeny (96. ± 2.8%) with mximum of 7.63 ±.23 roots/shoot hving n verge root length of 4.86 ±.35 m ws otined on hlf-strength MS medium with 1. µm indole-3-utyri id (IBA) nd solidified with.25% phytgel. The plntlets were limtized in sterile soilrite under ontrolled onditions, hrdened nd suessfully trnsferred to soil in nturl onditions with 9% survivl rte. The regenerted plnts showed no morphologil vritions in terms of lef shpe, flower shpe, pod size, numer of seeds et., when ompred with the nturlly grown plnts in the field. Key words: Senn sopher, fee, otyledonry node, in vitro shoot regenertion, rooting, limtiztion. INTRODUCTION Senn sopher (L.) Rox. (Cssi sopher L., Fee) is ommonly known s ksondi in Hindi. It is diffuse, shruy plnt or under shru out.7 to 3. m in height. The plnt hs een shown to exhiit ntiner tivity nd is reported to e extensively used in homeopthy (Pullih, 26). The leves possess purgtive nd ntidiureti properties. It is used s n infusion or deotion or s n expetornt for ough, old, ronhitis, sthm, hiups nd jundie nd in su-ute stges of gonorrhoe. Internlly, it is reported to t s ferifuge in rheumti nd inflmmtory fevers nd in skin diseses (Anonymous, 1992). The hemil nlysis of the seeds reveled the presene of sori id, dihydrosori id nd β-sistosterol (Bill et l., 25).The powdered *Corresponding uthor. E-mil: shhzd.t@mu..in. Arevitions: BA, Benzyldenine; Kn, kinetin; 2iP, 2-isopentenyle denine; IAA, indole-3-eti id; IBA, indole-3-utyri id; NAA, α-nphthlene eti id; GA 3, gierelli id; MS, Murshige nd Skoog medium; PGR, plnt growth regultor.

2 414 Afr. J. Biotehnol. seeds mixed with honey re used in dietes. In ethnootnil literture, it is reported to e effetive in the tretment of pityrisis nd onvulsions of hildren (Bill et l., 25). The methnol extrt of the leves of C. sopher n e used s soure of nturl ntioxidnts with potentil pplition to redue oxidtive stress with helth enefits (Rhmn et l., 28). Conventionlly, it is propgted through seeds; however, hrd seed ot nd seed dormny prevent its germintion in nture. Therefore, lterntive tehniques need to e developed for mss propgtion nd onservtion of this vlule mediinl plnt speies. There re onsiderle eonomi enefits in the development of indigenous mediines nd in the use of mediinl plnts for the tretment of vrious diseses. Advnes in iotehnology, espeilly in vitro tehniques provide n importnt tool for multiplition, onservtion nd propgtion of severl mediinl plnts (Khllfll nd Dffll, 28; Shhzd et l., 211; Li et l., 212). Till dte there is single report on miropropgtion of S. sopher (Prveen nd Shhzd, 21). Thus, keeping in mind the use of in vitro pprohes for the onservtion of vlule mediinl plnts; the present study ws foused for in vitro propgtion of S. sopher otyledonry node explnts. MATERIALS AND METHODS In vitro seed germintion nd explnt olletion The ertified seeds of S. sopher otined from Prem Nursery nd Seed Store, Dehrdun (Indi), were used to rise septi seedlings. Seeds were wshed thoroughly under running tp wter for 3 min to remove dherent prtiles nd kept in 1% (w/v) Bvistin (Crnendzim Powder), rod spetrum fungiide, for 25 to 3 min. After tht wshed with 5% (v/v) Teepol, liquid detergent, y ontinuous shking for 15 min nd then rinsed with sterile Doule Distilled Wter (DDW) for 3 to 4 times under the lminr flow hood followed y short tretment of 7% (v/v) ethnol for 3 to 6 s nd then surfe sterilized with.1% (w/v) freshly prepred meruri hloride (HgCl 2 ) for 4 min. Finlly, the seeds were wshed (5 to 6 times) with sterile DDW to remove the tres of sterilnt. The sterilized seeds were inoulted in the ulture flsk (1 ml, Borosil) ontining MS (Murshige nd Skoog, 1962) sl medium or hlf-strength MS medium solidified with.8% (w/v) gr (Bteriologil grde, Hi-medi) nd ontining 3% (w/v) surose nd gierelli id (GA 3 ) t vrious onentrtions (.5, 1. nd 2.5 µm) or without GA 3. Ten replites were tken for eh tretment nd in eh flsk 1 seeds were inoulted. Culture medi nd onditions Murshige nd Skoog (MS) medium supplemented with 3% (w/v) surose nd.8% (w/v) gr (Bteriologil grde, Hi-medi) were used throughout the experiments. The ph of the medium ws djusted to 5.8 using 1N NOH or 1N HCl prior utolving t 121 C t 1.6 kg m -2 pressure for 2 min. All the ultures were mintined t 24 ± 2 C under 16 h photoperiod with photosyntheti photon flux density (PPFD) of 5 µmol m -2 s -1 provided y ool white fluoresent tues (4W, Phillips, Indi) nd with 5 to 6% reltive humidity. Shoot indution, multiplition nd mintenne The MS medium supplemented with different ytokinins (BA, Kn nd 2iP) t vrious onentrtions (1., 2.5, 5., 7.5 nd 1. µm) singly or in omintion with different uxins (IAA, IBA nd NAA) t vrious onentrtions (.5, 1. nd 2. µm) were used for multiple shoot indution through CN explnts exised from septi seedlings. For shoot multiplition nd long-term estlishment, the regenerting tissues were regulrly su ultured onto the fresh medium omprised of MS + BA (5. µm) + NAA (1. µm) every 6 weeks. The perentge of explnts produing shoots, numer of shoots/explnt nd shoot length were reorded fter 6 weeks of ulture. Effet of different medi, ph nd surose onentrtions Different ftors like nutrients omposition (B 5, L 2, MS nd WPM medium), ph of the ulture medium (5., , 6.2 nd 6.6) nd surose onentrtion (1, 2, 3, 4 nd 5%) hve n influene on the indution nd prolifertion of multiple shoots. Thus, in the present investigtion, ll these three ftors were stndrdized for the optimum regenertion of shoots vi CN explnt in S. sopher. In vitro rooting in miroshoots Regenerted shoots of out 4 to 5 m length were exised nd trnsferred to the rooting medi omprised of MS sl nd hlfstrength MS medium solidified with.25% phytgel nd supplemented with different uxins like IAA, IBA nd NAA (.5, 1. nd 2.5 µm). Dt were reorded for rooting perentge, men numer of roots/shoot nd root length fter 4 weeks of trnsfer onto the rooting medium. Hrdening nd limtiztion Plntlets with well-developed root nd shoot system were limtized s per the stndrd proedure dopted y Prveen nd Shhzd (21). Dt olletion nd sttistil nlysis The dt for perentge regenertion, numer of shoots per explnts nd shoot length were reorded fter 6 weeks nd for rooting experiment fter 4 weeks. All the experiments were onduted with minimum of ten replites per tretment nd repeted three times. The dt were nlyzed sttistilly using SPSS ver.16 (SPSS In., Chigo, USA). The signifine of differenes mong mens ws rried out using Dunn s multiple rnge test (DMRT) t P.5; the results re expressed s men ± SE of three repeted experiments. RESULTS AND DISCUSSION Aout 99.3% seeds germinted on hlf-strength MS medium ontining 1. µm GA 3 (dt not shown). Cotyledonry nodes (CN) exised from 14, 21 nd 28 dy-old septi seedlings were used for the seletion of the most responsive ge of the explnt nd estlishment of in vitro ultures.

3 Prveen nd Shhzd 415 Tle 1. Effet of ge of otyledonry nodes on diret shoot regenertion in S. sopher ultured on MS medium ontining different onentrtions of BA. BA (µm) Regenertion (%) 14 dy-old explnts 21 dy-old explnts 28 dy-old explnts Men numer of shoots per explnt Regenertion (%) Men numer of shoots per explnt Regenertion (%) Men numer of shoots per explnt -.. e.. e.. e.. d.. e.. e d d d d d d d -Dt reorded fter 6 weeks. -Vlues represent Men SE of three repeted experiments with 1 replites eh. Mens followed y the sme letter within olumns re not signifintly different (P.5) using Dunn s multiple rnge test (DMRT). Effet of explnt ge on multiple shoot regenertion The ge of the explnt plyed n importnt role on the onset of morphogenesis in in vitro ultures s the explnts exised from seedlings or younger plnts hve more regenertion pity thn explnts olleted from mture plnts (Distnnjong nd Geneve, 1997). Therefore, to evlute the effiieny of shoot regenertion, otyledonry node explnts exised from seedlings of different ge group (14, 21 nd 28 dy-old) were ultured on MS medium ontining vrious onentrtions of BA. MS medium without BA served s ontrol tretment. All the three different ged explnts filed to respond on hormone free MS medium nd died within 2 weeks of inoultion. Among different ged explnts, 21 dy-old seedling explnts produed mximum of ±.29 shoots/explnt on MS + BA (5. µm) fter 6 weeks of inoultion in ± 2.33% ultures. On this medium, indution of shoot uds strted fter 9 to 1 dys of inution. On the sme omposition of medium, the explnts isolted from 14 dy-old seedlings produed 9.6 ±.23 shoots/explnt with ± 1.45% response, while, the explnts otined from 28 dys old seedlings produed only 6.56 ±.23 shoots/explnt in ± 2.33% ultures fter 6 weeks of inoultion (Tle 1). Similrly, Jeykumr nd Jyln (22) reported etter regenertion effiieny of CN explnts olleted from 2 dys old septi seedlings of Psorle orylifoli. Explnts ontining xillry/pil uds hve quiesent or tive meristems depending upon the physiologil stge of the plnt. These uds hve the potentil to develop into omplete plntlets. But, in nture, these uds remin dormnt for speifi period of time depending on the growth pttern of the plnt (Rni nd Rn, 21). Likewise, there re mny reports ville, where the ge of the explnt ffeted the rte of shoot regenertion (Gitong et l., 21; Devi et l., 211; Moheodini et l., 211). Effet of ytokinins on multiple shoot regenertion The est responsive CN explnts exised from 21 dy- old seedlings were ultured on MS medium ontining three different ytokinins (BA, Kn nd 2iP) for multiple shoot regenertion through diret orgnogenesis. MS medium without ytokinins (ontrol) did not stimulte ny morphogeni response nd the explnts died within two weeks of ulture. However, supplementtion of vrious ytokinins t different onentrtions filitted indution of multiple shoots from CN explnts. Among ll the three ytokinins tested, BA ws proved to e the est nd the numer of shoots inresed with n inrese in the onentrtion up to 5. µm, eyond tht deline in perent response s well s numer of shoots ws reorded (Tle 2). Enlrgement nd swelling of the explnts ws oserved within 4 to 5 dys of inution followed y emergene of green protuernes from the swelled region fter 9 to 1 dys. At 5. µm BA, mximum of ±.29 shoots/explnt ttining n verge shoot length of 4.23 ±.26 m ws otined in mximum of ± 2.33% ultures fter 6 weeks of inoultion (Figure 1A). Higher onentrtions of BA (7.5 nd 1. µm) found to e inhiitory for shoot regenertion euse of huge sl llusing whih hmpered the growth nd development of new shoots. At 1. µm BA, derese in the perent response (64. ± 3.5%) ws notied nd the numer of shoots ws redued to 5.9 ±.2 shoots/ explnt fter 6 weeks of ulture euse llus formtion ts s mehnil rrier to nutrient nd wter uptke s suggested y De Klerk (22). These findings sustntite with the erlier reports of Oimum spp. (Pttnik nd Chnd, 1996), Gymnem sylvestre (Reddy et l., 1998); Entd phseoloids (Ro nd Vishnupriy, 22) nd Sussure ovllt (Joshi nd Dhr, 23). To hek the growth of sl llus, the ultures were frequently trnsferred onto the fresh medium of sme omposition fter removing the llus mss fter every 2 weeks. In ontrst to BA, Kn nd 2iP were effetive t higher onentrtion tht is, 7.5 µm where indution of shoot uds egn fter 14 dys of inoultion in oth Kn nd 2iP supplemented medi. Shoots were not s helthy s tht of BA supplemented medi nd were hving smll

4 416 Afr. J. Biotehnol. Tle 2. Effet of different ytokinins on diret shoot regenertion from otyledonry nodes of S. sopher. PGR ( M) BA Kn 2iP Regenertion (%) Men numer of shoots/explnt Men shoot length (m) ef f de d de d l.. j.. g ij g ef hi e de de d fg f d l.. j.. f k i f jk h def gh fg d k hi def -Dt reorded fter 6 weeks. -Vlues represent Men SE of three repeted experiments with 1 replites eh. Mens followed y the sme letter within olumns re not signifintly different (P.5) using Dunn s multiple rnge test (DMRT). leves with thin stem. The lower onentrtion (1. µm) of oth Kn nd 2iP filed to provide ny response. However, s the onentrtion of ytokinins ws further inresed liner inrese in the numer of shoots ws oserved up to n optiml level (7.5 µm). The MS medium ontining 7.5 µm of Kn produed n verge of 6.56 ±.34 shoots/explnt with regenertion potentil of 6.33 ± 1.45%. But, 2iP t the sme onentrtion yielded only 4.23 ±.14 shoots/explnt in ± 1.45% ultures fter 6 weeks of inoultion. Beyond the optiml onentrtion (7.5 µm), derese in the response s well s numer of shoots ws reorded due to profuse sl llusing (Tle 2). Similrly, the superiority of BA on ud rek nd multiple shoot prodution hs lso een reported erlier in severl mediinl nd romti plnts inluding Muun pruriense (Sthynryn et l., 28); Clitori ternte (Singh nd Tiwri, 21); Justii gendruss (Thoms nd Yoihiro, 21) nd Veroni ngllis-quti (Shhzd et l., 211). Effet of ytokinin-uxin omintions on multiple shoot regenertion In generl, higher onentrtion of ytokinin nd lower onentrtion of uxin re required in medium to promote multiplition nd prolifertion of shoots (Kohlenh, 1997; Been et l., 23). In the present study, the optiml onentrtions tht is, 5. µm of BA ws lso tested with three uxins to evlute the synergisti effet of ytokinin uxin omintions. Among vrious omintions, BA + NAA omintion tretments provided est response (Figure 1B). Indution of shoot uds took ple within 8 dys of inution with slight swelling of the explnt on MS + BA (5. µm) + NAA (1. µm) nd yielded mximum 19.5 ±.51 shoots/explnt in 86. ± 2.8% ultures with 5.23 ±.14 m shoot length fter 6 weeks of inoultion (Tle 3 nd Figure 1C). The synergisti effets of BA-NAA omintion hve lso een reported in other plnt speies suh s Ruvolfi serpentin (Bksh et l., 27), Bliospermum montnum (George et l., 28) nd Boerhvi diffus (Bisws et l., 29). The numer of shoots per explnt ws found to e more in this study ompred to our previous report (Prveen nd Shhzd, 21) on the sme plnt, where thidizuron (TDZ) ws used. Though, the ddition of higher onentrtion (2. µm) of NAA resulted in llus formtion nd therey deline in men numer of shoots/explnt (13.93 ±.23) s well s in perent response (8.33 ± 1.45%). In the other omintion tretments, BA + IBA omintion ws etter thn BA + IAA. The medium omprised of MS medium with optiml onentrtion of BA (5. µm) nd IBA (1. µm) exhiited 83. ± 1.15% response produing n verge of ±.37 shoots/explnt, while IAA t the sme onentrtion provided ±.23 shoots/explnt with ± 1.45% response fter 6 weeks of ulture (Tle 3). Effet of different medi Some speies give similr response in ll the medi while others show preferene for speifi medium for the est-

5 Prveen nd Shhzd 417 Figure 1. Multiple shoot regenertion in S. sopher through otyledonry node explnts on MS medium supplemented with vrious PGRs. A) Indution of multiple shoots on MS + BA (5. µm), r =.73 m. B) Multiple shoots development on MS + BA (5. µm) + NAA (.5 µm), r = 1. m. C) Multiplition nd prolifertion of shoots on MS + BA (5. µm) + NAA (1. µm), r =.92 m. Tle 3. Effet of optiml onentrtion of BA with different uxins on diret shoot regenertion from otyledonry nodes of S. sopher. PGR ( M) BA NAA IBA IAA Regenertion (%) Men numer of shoots/explnt Men shoot length (m) d d de d e d ef d d d f Dt reorded fter 6 weeks. -Vlues represent Men SE of three repeted experiments with 1 replites eh. Mens followed y the sme letter within olumns re not signifintly different (P.5) using Dunn s multiple rnge test (DMRT). lishment nd prolifertion of ultures (MCown nd Sellmer, 1987). Amongst ll the four medi tested, MS medium ws found to e the most suitle medium for multiple shoots regenertion from CN explnt nd yielded mximum 19.5 ±.51 shoots/explnt with highest shoot length of 5.23 ±.14 m on the optimized medium tht is, MS + BA (5. µm) + NAA (1. µm) fter 6 weeks of ulture (Figure 2). On MS medium shoot uds were indued within 8 dys of ulture. Multiple shoot uds were differentited on WPM, L 2 nd B 5 medi ut showed limited growth nd development even if they were mintined for longer period in ulture. MS medium hs lso een found more effetive thn other medi in vrious mediinl plnts (Wondyifrw nd Surwit, 24; Wng et l., 28). However, ontrry to our results, WPM ws found to e the est medium for shoot multiplition ompred to MS nd AN (Anderson s rhododendron medium) medium in Vinium rtostphylos y Cüe et l. (213). Effet of ph The effet of different ph tested in MS medium omprised of optimized onentrtions of BA (5. µm) nd NAA (1. µm) on shoot regenertion from CN explnts. The idel vlue of ph for the mximum regenertion proved to e 5.8 where mximum of 19.5 ±.51 shoots/cn explnt ws otined fter 6 weeks of ulture. ph vlues elow the optiml level resulted in the prodution of loose or wtery medium whih ws slight idi

6 418 Afr. J. Biotehnol. 25 Men numer of shoots/explnt Men shoot length (m) 6 Men numer of shoots/explnt d Men shoot length (m) MS WPM L2 B5 Different medi Figure 2. Effet of different ulture medi supplemented with BA (5. µm) nd NAA (1. µm) on shoot regenertion from otyledonry nodes of C. sopher fter 6 weeks of ulture. in nture nd delined the numer of shoots to 1.63 ±.29 nd 6.83 ±.27 shoots/explnt t ph 5.4 nd 5., respetively. High ph vlues produed lkline medium whih ffeted the regenertion potentil of the explnts nd thus, redued the numer of shoots to 8.43 ±.29 nd 5.5 ±.17 shoots/explnt t ph 6.2 nd 6.6, respetively (Figure 3). It is known tht the medium ph hs effets not only on the uptke of nutrients ut lso on hemil retions espeilly those tlyzed y enzymes (Thorpe et l., 1991). Our findings sustntite with erlier reports on other plnts where ph 5.8 ws found to e the optimum vlue (Nir nd Seeni, 23). Hydrogen ion onentrtion (ph) of the ulture medium is one of the importnt ftors of physio-hemil environment during development of plnt tissues under in vitro onditions (Willims et l., 199). The optiml ph of the medium vries ording to the different phses of morphogenesis nmely; estlishment of ultures, shoot prolifertion nd indution of roots (Sorio et l., 1997; Ostroluká et l., 24). Effet of surose onentrtions Plnt ell, tissue or orgn ulture normlly require the inorportion of ron or energy soure to the ulture medium (Krhu, 1997) nd surose hs een used s the mjor ron soure in plnt tissue ulture (Fuentes et l., 2). The est response ws exhiited y the explnts on medium omprising 3% surose, produing mximum of 19.5 ±.51 shoots/explnt ttining the highest shoot length of 5.23 ±.14 m on the optimized medium tht is, MS + BA (5. µm) + NAA (1. µm) fter 6 weeks of ulture. While, elow this optiml onentrtion of sugr the explnts showed less response nd onsequently the numer of shoots delined to 9.63 ±.34 nd 5.66 ±.29 shoots/explnt t 2 nd 1% surose, respetively. Higher onentrtions of surose lso redued the regenertion effiy of the explnt nd numer of shoots ws redued to ±.23 nd 7.63 ±.29 t 4 nd 5% of surose, respetively (Figure 4). Similr to our results, Anith nd Pullih (22) lso otined optiml regenertion t 3% surose in omprison to other onentrtions of surose. However in Euomis utumnlis, mximum regenertion ws otined t 4% surose nd the lower onentrtions deresed the regenertion rte (Tylor nd vn Stden, 21); similrly, in Amygdlus ommunis shoot multiplition ws otined only t 5 nd 6% surose (Gürel nd Gülsen, 1998) whih is ontrry to our results. On the other hnd, mjority of miropropgtion proto-

7 Prveen nd Shhzd 419 Men numer of shoots/explnt Men shoot length (m) 25 8 Men numer of shoots/explnt d e Men shoot length (m) Different ph of medium Figure 3. Effet of different ph vlues on shoot regenertion from otyledonry nodes of C. sopher on MS medium ontining optiml onentrtion of BA (5. µm) nd NAA (1. µm) fter 6 weeks of ulture. Men numer of shoots/explnt Men shoot length (m) 25 6 Men numer of shoots/explnt e d Men shoot length (m) Cnentrtion of surose (%) Figure 4. Effet of different onentrtions of surose on shoot regenertion from otyledonry nodes of C. sopher on optiml medium omprised of MS + BA (5. µm) + NAA (1. µm) fter 6 weeks of ulture.

8 42 Afr. J. Biotehnol. Tle 4. Effet of different uxins on in vitro root indution in miroshoots of S. sopher in phytgel solidified medium. Tretment ( M) Response (%) Men numer of roots/shoot Men root length (m) MS h e f ½ MS g de ef ½ MS + IBA (.5) d ½ MS + IBA (1.) ½ MS + IBA (2.5) ½ MS + NAA (.5) de de d ½ MS + NAA (1.) d ½ MS + NAA (2.5) fg de de ½ MS + IAA (.5) fg e de ½ MS + IAA (1.) ef ½ MS + IAA (2.5) g e ef -Dt reorded fter 4 weeks. -Vlues represent Men SE of three repeted experiments with 1 replites eh. Mens followed y the sme letter within olumns re not signifintly different (P.5) using Dunn s multiple rnge test (DMRT). Figure 5. In vitro rooting nd limtiztion of regenerted plntlets of S. sopher. A) In vitro root indution in phytgel solidified medium ontining ½ MS + IBA (1. µm), r = 1.78 m. B) An limtized plntlet in soilrite, r = 1.3 m. C) In vitro regenerted plnt of S. sopher in soil showing development of infloresene, r = 8.3 m. ols generlly utilize 3% surose s per reommendtion of Murshige nd Skoog (1962). Exogenous supply of sugr inreses strh nd surose reserves in miropropgted plnts nd ould fvour ex vitro limtiztion nd speed up physiologil dpttions (Pospíšilová et l., 1999). In vitro rooting The development of helthy root system is required for the suessful estlishment of regenerted shoots in the externl environment. Hene, pproprite sized (4 to 5 m) miroshoots of S. sopher were exised from the regenerting ultures nd trnsferred to different rooting medi for the indution of in vitro roots. Indution of roots from the ut ends of the shoots took ple within 1 to 12 dys nd thin, drk rown to lk roots were produed in ll the tretments. The est rooting (96. ± 2.8%) ws hieved on hlf-strength MS medium solidified with.25% phytgel nd ontining 1. µm IBA, wherein roots were indued fter 7 to 8 dys nd n verge of 7.63 ±.23 roots/shoot hving verge root length of 4.66 ±.35 m were produed fter 4 weeks of trnsfer (Tle 4 nd Figure 5A). Higher onentrtion (2.5 µm) of IBA re-

9 Prveen nd Shhzd 421 redued the rooting potentil nd resulted in the formtion of short roots. NAA nd IAA proved to e less effetive thn IBA for root indution. Our results re in ordne with vrious findings where IBA proved to e more effiient for rooting thn other uxins in numer of plnts suh s Cunil gloides (Frro nd Eheverrigry, 21). Hrdening nd limtiztion Hrdening nd limtiztion of plntlets is essentil for the survivl of regenerted plntlets in the externl environment. Regenerted plntlets with 4 to 5 fully expnded leves nd well-developed root system were hrdened off under septi onditions in sterile soilrite. Hrdening of plntlets ws done y the proedure desried in the mterils nd methods. Almost 9% plntlets survived in the field onditions fter suessful limtiztion nd exhiited norml growth nd development pttern with no detetle morphologil vritions when ompred with the in vivo grown plnts (Figure 5B nd C). Conlusion The omprehensive protool desried here is effiient, reproduile nd ould e used in future for mss propgtion nd iotehnologil progrms for the geneti trnsformtion or mnipultion of highly mediinl legume S. sopher. ACKNOWLEDGEMENTS The uthor Shhin Prveen knowledges the reserh ssistne provided y UGC under the sheme of Muln Azd Ntionl Fellowship for the wrd of Senior Reserh Fellowship (SRF). The uthor Anwr Shhzd lso grtefully knowledges the finnil support promoted y the Counil of Siene nd Tehnology (UP-CST, vide no. CST/D 3836) nd UGC (vide no /21 (SR), Government of Indi, New Delhi. REFERENCES Anith S, Pullih T (22). Shoot regenertion from hypootyl nd shoot tip explnts of Steruli foetid L. derived from seedlings. Tiwni 47: Anonymous (1992). Cssi. The Welth of Indi: A Ditionry of Indin Rw Mterils nd Industril Produts (Vol III), CSIR, New Delhi, pp Bksh R, Jhn MAA, Khtun R, Munshi JL (27). In vitro rpid lonl propgtion of Ruvolfi serpentin (Linn.) Benth. Bngldesh J. Ind. 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