Somatic Embryogenesis and Plant Regeneration from Petiole-Derived Callus of Spine Gourd (Momordica dioica Roxb. ex Willd)

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1 Funtionl Plnt Siene n Biotehnology 27 Glol Siene Books Somti Emryogenesis n Plnt Regenertion from Petiole-Derive Cllus of Spine Gour (Momori ioi Rox. ex Will) M. Thiruvengm * K. T. Rekh C. H. Yng Grute Institute of Biotehnology, Ntionl Chung-Hsing University, 25, Kuo-Kung ro, Tihung - 2, Tiwn, R.O.C. Corresponing uthor: * thiruv3@gmil.om ABSTRACT An effiient protool for plntlet regenertion from the ell suspension ultures of spine gour (Momori ioi Rox. ex Will.) through somti emryogenesis is reporte. Petiole-erive emryogeni lli were ulture on Murshige n Skoog meium ugmente with 4.5 M 2,4-ihlorophenoxyeti i (2,4-D) n 1% oonut milk (CM). The mximum frequeny of somti emryos (36.3%) ws oserve on MS meium supplemente with 2.2 M 2,4-D for three weeks of ulture. Ontogeni stuies of somti emryos revele tht the ells estine to eome somti emryos ivie into spheril proemryos n then progresse to gloulr, hert n further ifferentite properly into torpeo n otyleonry stges within 5 weeks. Emryo evelopment ws synhronous n strongly influene y the 2,4-D onentrtion. The MS liqui meium ugmente with 2.2 M 2,4-D n.5 M L-glutmine ws effetive to hieve high frequeny of somti emryo inution (44.5%). The otyleonry-stge somti emryos were trnsferre to MS liqui meium with no plnt growth regultors to hieve omplete mturtion within 7 ys. Lk of 2,4-D in suspensions inrese somti emryo mturtion with erese normlities. Surose ws foun to e the est ron soure for llus inution, emryo mturtion n emryo germintion. Reltively, only few numers of emryos evelope into root/shoot when trnsferre to 1/1 MS soli meium ontining.5 M sisi i (ABA), 2% (w/v) surose n.2% (w/v) Gelrite. Aout 11% of somti emryos germinte into morphologilly norml fertile plnts within 2 weeks. Regenerte plntlets were suessfully hrene, with survivl rte of pproximtely 6%, n estlishe in the fiel. This regenertion protool ssure suessful emryo inution, mturtion n plntlet onversion. Keywors: llus inution, germintion, hrening, mturtion, suspension ulture Arevitions: 2,4-D, 2,4-ihlorophenoxyeti i; ABA, (±)-is, trns-sisi i; BAP, N 6 -enzylminopurine; CM, oonut milk; IAA, inole-3-eti i; IBA, inole-3-utyri i; Kn, kinetin; NAA, -nphthleneeti i; PCV, pke ell volume INTRODUCTION Spine gour (Momori ioi Rox. ex Will.) is meiinlly n eonomilly importnt plnt, ultivte for its fruit, whih is use s vegetle. It originte from the Ino-Mlyn region (Rshi 1976; Singh 199) n hs een highly ultivte in Asi espeilly in Chin, Tiwn, Ini, Bnglesh, Pkistn n Sri Lnk. The fruit is rih soure of protein, rohyrtes n vitmin C (Rshi 1976; Bhuiy et l. 1977). In ition to its nutritious vlue, the plnt exhiits wie rnge of nti-ner properties (Li et l. 1999), ue to the presene of terpenes n sterois in roots (Luo et l. 1998) n nti-miroil tivity s it ontins lklois n glyosies (Bhosle n Prtkr 25). Besies, it is lso use in the tretment of leeing piles, urinry omplints, snkeite, sorpion stings n even s n ntisepti (Chopr et l. 1986). Improvement of this rop hs not een equtely ttempte, euse of its ioeious nture n its vegettive moe of propgtion. At present, its propgtion epens entirely on unergroun tuerous roots, whih oupy the ultivle ln for long perio, until the next plnting seson. Mintenne of tuer qulity uner fiel onitions n its storge re lso iffiult. Improvement through lssil reeing hs lso een met with limite suess ue to the limite geneti vriility in germplsm (Ni et l. 22). The estlishment of n effiient regenertion system through somti emryos will llow for rop improvement through progrmme tht inlues somti hyriiztion, in vitro seletion n geneti trnsformtion. Only few results on M. hrnti in vitro stuies suh s iret shoot regenertion of ifferent explnts hve een reporte (Islm et l. 1994; Wng et l. 21; Sultn n Mih 23). In M. ioi results were reporte only on iret orgnogenesis (Thiruvengm n Jyln 21; Ni et l. 22; Bhosle n Prtkr 25; Thiruvengm et l. 26). Although we hve reently reporte the evelopment of somti emryos in itter melon (Thiruvengm et l. 26), to our knowlege, no report on spine gour regenertion vi somti emryogenesis from petiole hs yet een esrie. This work esrie somti emryogenesis, ontogeny n histology of emryo evelopment n the effet of growth regultors n rohyrtes on high frequeny inution of somti emryogenesis in this nutritionlly n meiinlly importnt plnt. MATERIALS AND METHODS Plnt mteril Tuers of Momori ioi Rox. ex Will (one yer ol) were ollete from Tihung mountins n the plnts were rise in greenhouse t the Ntionl Chung-Hsing University, Tihung, Tiwn. Petiole explnts were ollete n wshe in running tp wter for 5 min n surfe sterilize in 7% (v/v) ethnol for 1 min. Further, explnts were trete in 1.% (v/v) soium hypohlorite solution for 1 min with osionl gittion. Finlly, the petioles were rinse with sterile istille wter for seven times n slie into explnts of pproximtely.3-.5 m 2. Reeive: 27 Novemer, 26. Aepte: 14 Deemer, 26. Originl Reserh Pper

2 Funtionl Plnt Siene n Biotehnology 1(1), Glol Siene Books Cllus inution Petiole explnts were ple in plsti Petri ish (9 15 mm; Alph Plus Sientifi Corp., Tiwn) with 25 ml meium, onsisting of MS slts (Murshige n Skoog 1962), 3.% (w/v) surose,.2% (w/v) Gelrite (Sigm, St. Louis, USA) with ifferent onentrtions (.-6.7 M) of 2,4-ihlorophenoxyeti i (2,4-D; Sigm, St. Louis, USA) lone n in omintion with 1% oonut milk (CM; Coonut milk ws prepre s esrie in Bhojwni n Rzn (1983) n e efore utolving) for llus formtion. The meium ph ws juste to 5.8 y 1N NOH or 1N HCl prior to steriliztion. The meium ws then soliifie with.2% Gelrite n utolve t 121 C n 1.5 kg/m -2 for 2 min. The inution onition for ulture, unless stte otherwise, ws mintine t 25 ± 2 C n 16/8-h (light/ rk) photoperio of ool white fluoresent light (25 μmol m -2 s -1 ). Suspension ulture For initition of ell suspension ultures, two-week-ol greenyellow, frile lli (>5 mg fresh weight eh) were septilly trnsferre to 125 ml Erlenmeyer flsks (Pyrex, USA) ontining 25 ml of liqui MS meium supplemente with vrious onentrtions ( M) of 2,4-D n gitte on gyrtory shker (95-11 rpm) in stnr growth room onitions. Suspensions were suulture every 7-1 ys n supplemente y fresh MS meium with 2,4-D. At the en of the seon suulture, the ells were filtere through 15 M stinless steel sieves to seprte possile emryogeni ells n smll lumps of ells. Cells from the suspensions were oserve uner mirosope uring the ulture perio. The growth rte of suspene ells ws monitore for the first 12 ys y etermining the pke ell volume (PCV) of smples from 1 replites. PCV ws mesure fter entrifuging the suspension t 2 rpm for 1 min in grute entrifuge tue (Kumr et l. 1988). MS sl meium lking 2,4-D serve s the ontrol. Emryos t ifferent stges of evelopment were seprte mnully n suulture in MS liqui meium with ifferent 2,4-D onentrtions. After two weeks of suulture, gloulr, hert, torpeo n otyleonry shpe emryos forme. Stuy of ontogeny n frequeny of somti emryos The ultures in liqui meium were exmine every y uring the experimentl perio to tre the ontogeny of somti emryos. Smples from 1 replite flsks per 2,4-D tretment were tken t rnom in orer to etermine the frequeny of ifferent stges of somti emryos. The results o not represent the ontinuous monitoring of ny prtiulr single ell, ut the oservtion of ifferrent ells t ifferent stges of growth (Epen n George 199). Soil trnsfer After germinte plnts were remove from the ulture tues n wshe in running tp wter, they were trnsferre to 6-m plsti pots ontining re soil, perlite n vermiulite mixture (3:1:1) mintine in humiity hmer (Conviron, Winnipeg, Cn) uner 16 h photoperio t 25 ± 2 C. After three weeks of hrening, the plnts were trnsferre to the fiel. Histology The somti emryos were fixe in solution of formlin, lohol n eti i (FAA) of 1 ml, whih ontins 5.4 ml formlin (37%), 65.6 ml ethnol (96%), 5 ml glil eti i n 24 ml Mturtion n germintion Mturtion of emryos i not progress in MS liqui meium with 2,4-D, hene otyleonry-shpe emryos were trnsferre into 15 ml Erlenmeyer flsks ontining 25 ml of hormone-free MS liqui meium for mturtion n further evelopment. Then the mture emryos were ulture on ifferent MS mei: MS + 3% surose, MS + 2% surose, 1/2 MS + 2% surose, 1/1 MS + 2% surose, MS + 3% surose +.5 M ABA, 1/1 MS + 2% surose +.1 M ABA, 1/1 MS + 2% surose +.5 M ABA, n 1/1 MS + 2% surose + 1. M sisi i (ABA; Sigm, St. Louis, USA), eh with.2% Gelrite for plntlet onversion. The ultures were mintine in stnr growth room onitions n plntlet onversion frequeny ws oserve. Effet of mei, growth regultors, rohyrtes n mino is Two-week-ol, green-yellow, frile emryogeni llus (75 mg fresh mss) erive from petiole explnts were ulture in liqui on ifferent sl mei, inluing MS meium, B5 meium (Gmorg et l. 1968), LS meium (Linsmier n Skoog 1965) n SH meium (Shenk n Hilernt 1972), were teste. Different onentrtions (.-4.5 M) of uxins (IAA, IBA, NAA n 2,4-D) iniviully or in omintion with ytokinins (.5 M BAP n Kn) were teste for somti emryo inution. The effets of ifferent rohyrtes suh s surose, frutose, gluose n mltose t vrious onentrtions rnging from -5% n mino is (L-lnine, L-proline, L-glutmine) t onentrtions rnging from.-.6 M were stuie on the ifferentition of somti emryos. The frequeny of emryo inution n stges of somti emryos were reore. Fig. 1 Somti emryogenesis from petiole explnts through suspension ultures of spine gour (Momori ioi). (A) Petiole erive emryogeni llus; (B) Spheril emryogeni ells; (C) Elongte ells; (D) Two ell stges; (E) Four ell stges; (F) Proemryos; (G) Gloulr emryo; (H) Erly hert-shpe emryo; (I) Hert-shpe emryo; (J) Torpeo shpe emryo; (K) Mture otyleonry shpe emryo with shoot primori n root primori; (L) Longituinl setions of hertshpe emryo; (M) Germintion of somti emryos; (N) Seril rrngement in germintion of somti emryos; (O) Hrene plnt. Br represents.4 mm (A-K);.5 mm (L);.5 mm (M-O). 21

3 Somti emryogenesis n plnt regenertion in spine gour. Thiruvengm et l. istille wter (Gerlh 1984). Smples were ut n emee in hyroxyethylmethrylte (Histo-Tehnique-Set Tehnovit 71; Kulzer, Wehrhein, Germny). At the eginning of this proess, the speimens were ehyrte in 2-h steps through gre series of ethnol (7%, 9%, 96% n 1%). The smples were then preinfiltrte overnight with mixture of equl prts of 1% ethnol n Tehnovit 71 se liqui. The somti emryos were then trnsferre into n infiltrtion solution of 1 ml Tehnovit 71 se liqui n 1 g hrener I for 1 y. A vum ws estlishe for 3 min t the strt of the lst two proesses. The ultures were emee in Teflon mouls with mixture of 15 prts of infiltrtion solution n one prt hrener II. The smples were polymerize for 1 h t room temperture n further for 6 h t 37 C. Speimens were mounte on lok-holers with Tehnovit 3. Slies (6 M) were ut t room temperture using Jung CM 18 mirotome equippe with type 818 isposle mirotome les (oth from Lei Instruments, Nussloh, Germny). Slies were strethe on th of istille wter n mounte on slies. They were then stine with.5% toluiine lue O (Sev, Heielerg, Germny) issolve in 1% soium tetrorte ehyrte uffer rinse in istille wter, rie n overe with Entelln (Merk, Drmstt, Germny) n overslip. This proeure stins the ytoplsm n unlignifie ell wlls lue (Gerlh 1984). Mirosopi nlysis ws performe using Leitz DMR mirosope (Lei, Wetzlr, Germny) with Wil MPS 48/52 mer (Lei, Heerrugg, Switzerln). Sttistil nlysis For llus inution, t lest 5 explnts were use n eh experiment ws repete three times. Smples of suspension ultures were tken rnomly t the en of eh suulture n the numer of emryos ws ounte uner mirosope. Counts were me from 1 ifferent inepenent smples n perentge of emryos ws lulte on the sis of the totl numer of proemryogeni ells present in the fiel. The nlysis of vrine (ANOVA), pproprite for the esign ws rrie out to etet the signifine of ifferenes mong the tretment mens. The tretment mens were ompre using Dunn s multiple rnge test t P.5, % level y SPSS softwre version (SPSS 14 for winows, SPSS, Chigo, IL, USA). RESULTS Cllus inution Tle 1 Effet of MS soli meium ugmente with 2,4-D n 1% oonut milk (CM) on the inution of emryogeni llus from petiole explnts of spine gour. Conentrtion of growth regultors ( M) Explnts prouing emryogeni llus Nture of llus Aventitious rooting 2,4-D ± 1. i GF ± 3. h GF ± 2.5 g GC ± 3. f BF ± 1. BF ++ 2,4-D ( M) + 1% CM ± 2. e GF ± 3. GWF ± 2.5 GYF ± 3. BF ± 2. BF ++ Eh vlue represents the men ± SE of three replites per tretment. The t were sttistilly nlyze using Dunn s Multiple Rnge Test (DMRT). In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. BF = rown, frile; GC = green, ompt; GF = green, frile; GYF = green-yellow, frile; GWF = green-white, frile., No ventitious rooting; +, less rooting; ++, profuse rooting. Petiole explnts from one yer ol greenhouse-grown plnts of M. ioi were ulture on MS meium with vrious levels (.-6.7 M) of 2,4-D lone n in omintion with 1% CM for inution of llus. After 2 weeks of ulture inution inite tht MS soli meium ontining 4.5 M 2,4-D n 1% CM proue green-yellow frile lli (Fig. 1A). The llus showe ense ytoplsm, smll vuoles n lrge nulei with eeply stine nuleoli when oserve uner ompoun mirosope. Suh potentilly emryogeni lli were suitle for suspension ultures. The mximum effiieny of llus n typil emryogeni ells ws note on MS meium ontining 4.5 M 2,4-D n 1% CM while t 2,4-D onentrtion higher thn 4.5 M, the llus turne rown with numerous ventitious roots (Tle 1). Suspension ulture, ontogeny n histology of somti emryos The green-yellow frile emryogeni lli were suspene in MS liqui meium ontining vrious onentrtions (.-4.5 M) of 2,4-D to estlishe the somti emryogenesis. Ative ivision n growth of ells were oserve in 2.2 M 2,4-D until the 7 th n 8 th y of ulture. Initilly, the lli exhiite highly vuolte ells. After 6 ys, the llus ells showe two morphologilly istint kins of ells, nmely, spheril (Fig. 1B) n elongte ells (Fig. 1C), oth with visile ytoplsm n nuleus. Eh spherel emryogeni ell trnsversely ivie into two ells, four ells n susequently into group of ells (Fig. 1D, 1E) tht ws onsiere to e the proemryo (Fig. 1F). The proemryo further ivie n forme gloulr emryos (Fig. 1G), hert shpe emryos (Fig. 1H, 1I) within perio of two weeks, whih lter evelope into torpeo n otyleonry emryos (Fig. 1J) within week. The optiml frequeny of somti emryo formtion ws hieve in 2.2 M 2,4-D t shking spee of 1 rpm were estlishe in ell suspension ulture (Fig. 2). On the sme MS meium with 2,4-D, the torpeo n otyleonry emryos i not evelop ut, some norml strutures ppere suh s stunte emryo polrity, poorly evelope otyleons, trumpet with lefy struture, whih lter turne to green lli (t not shown). Thus torpeo shpe emryos relluse on 2,4-D ontining meium. The histologil exmintions revele tht the gloulr stge somti emryos re Emryogeni llus n somti emryos (% formtion) E 6 2 PCV (ml pellet/ml ulture) on 7th y Proemryogeni mss/ells (1-) Gloulr stge emryos Hert-shpe stge emryos Torpeo-shpe stge emryos e Fig. 2 Influene of MS liqui meium ontining vrious onentrtions of 2,4-D on ifferentition of somti emryos in spine gour. The t were sttistilly nlyze using Dunn s multiple rnge test. In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. Eh vlue represents the men of 1 inepenent replites. The numer of pro-emryogeni ells ws ompre with the totl numer of llus ells present in the mirosopi fiel. The frequeny of ifferent stges of somti emryos were etermine y tking the smples from 1 replite flsks per 2,4-D tretment. Vertil rs represente S.D. 2,4-D ( M) e e 22

4 Funtionl Plnt Siene n Biotehnology 1(1), Glol Siene Books new iniviuls n evelop to ipolr strutures showing lerly well evelope shoot n root meristems, whih onsist of smll ells with ense ytoplsm (Fig. 1L). Mturtion n plntlet formtion of somti emryos Cotyleonry-shpe emryos, when trnsferre to MS meium without 2,4-D, gretly improve mturtion n further evelopment within 7 ys of ulture (Fig. 1K), wheres in the sme system, the presene of 2,4-D resulte in mlforme emryo strutures, whih lter evelope into frile llus. Bse on our experiment, fter mturtion, the emryos h the potentil pity to synthesize their own hormones for suessive funtion uring further evelopment n onversion. Consequently, the mture somti emryos proue shoots n roots from the poles, enhne extension of hypootyl n plntlet evelopment on soliifie 1/1 strength MS meium ontining 2% surose,.5 M ABA n.2% Gelrite within 14 ys (Fig. 1M, 1N). Full strength MS meium onsisting of 3% surose n.5 M ABA onsierly reue emryo germintion n rellusing (Tle 2). However, 1/1 MS meium with 2% of surose n.5 M ABA gretly enhne emryo germintion n effetively stimulte plntlet evelopment from somti emryos, in whih the verge frequeny of germinting emryos into tiny plntlets (11%) ws hieve (Tle 2). These plntlets, when trnsplnte into 6-m pots (Fig. 1O) ontining re soil, perlite n verminulite (3:1:1), susequently resulte in 6% survivl n grew to mturity in the greenhouse. A totl of 2 months ws require for the proution of plntlets tht were rey to e potte. Tle 2 Conversion of mture otyleonry-stge somti emryos into plntlets of spine gour. Mei omposition No. SE plte* Regenertion Remrks MS + 3% surose 25 Anorml rooting MS + 2% surose ±.2 e Browning n rellusing 1/2 MS + 2% surose ±.5 Rellusing 1/1 MS + 2% surose ±.8 Hypootyl extension oserve MS + 3% surose +.5 M ABA ±.5 Shoot n root pole formtion lter rellusing 1/1 MS + 2% surose +.1 M ABA 1/1 MS + 2% surose +.5 M ABA 1/1 MS + 2% surose +1. M ABA ±.6 Hypootyl extension with greening t shoot primori ±.4 Enhne extension of hypootyl n plntlet evelopment ±.8 Hypootyl extension with shoot primoril initition * Totl No. of otyleonry stge somti emryos (SE). Eh vlue represents the men ± SE of three replites per tretment. The t were sttistilly nlyze using Dunn s Multiple Rnge Test (DMRT). In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. Effet of growth regultors, mei, rohyrtes n mino is Among the uxins (IAA, NAA, IBA, 2,4-D) n ytokinins (BAP, Kn) teste, 2,4-D ws the most effetive for inuing somti emryogenesis. The highest frequeny of emryo inution ws oserve on mei with 2.2 M 2,4-D (Fig. 2), while the lowest ws on mei with 1.1 M. The other uxins (.-4.5 M of IAA, NAA n IBA) oul not inue emryogenesis (Tle 3). The llus eme green with the ition of ytokinins (BAP n Kn) n ompletely inhiite somti emryogenesis (Tle 3). The effet of rohyrtes (frutose, gluose, mltose, surose), sl meium (MS, B5, LS, SH) n mino is (L-lnine, L- glutmine, L-proline) with 2.2 M 2,4-D ws ssesse on Tle 3 Influene of ifferent growth hormones on ifferentition of somti emryos from petiole-erive llus of spine gour. Conentrtion Proemryogeni Different stges of somti emryos of growth regultors ( M) mss/ells (1 ) Gloulr stge Hert stge Torpeo stge IAA ±.6 k 1. ±.2 e ±.5 fg 2.5 ±.6 IAA + BAP ±.2 o ±.4 kl IAA + Kn ±.4 op ±.5 m IBA ±.4 kl 3. ± ±.8 fg 3.5 ±.6 1. ±.2 IBA + BAP ±.2 op ±.2 mn IBA + Kn ±.2 qr ±.4 op NAA ±.8 f 3. ±.5 1. ± ± 1. e 3.4 ± ±.8 NAA + BAP ±.2 o ±.4 m NAA + Kn ±.4 op ±.2 q 2,4-D + BAP ± ± 1. 2,4-D + Kn ± ±.5 Eh vlue represents the men ± SE of three replites per tretment. The t were sttistilly nlyze using Dunn s Multiple Rnge Test (DMRT). In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. ( = Nil response). the inution of somti emryogenesis. The supply of 3% surose promote effetive somti emryo inution (Fig. 3) ut the sme surose onentrtion inhiite emryo onversion (Tle 2). A low frequeny of somti emryo inution ws oserve on MS with 3% frutose, gluose n mltose (Tle 4). Somti emryos were not otine on B5, LS n SH meium (Fig. 4). The effetive enhnement of emryo inution, growth n evelopment ws hieve with.5 M glutmine (Fig. 5). Hene, the supply of proline n lnine ws less effiient in emryogenesis thn tht of glutmine (Tle 5). DISCUSSION The regenertion of plnts through emryogeni suspension ulture is n iel tool for the effiient in vitro seletion n proution of trnsgeni plnts (Christou 1997). Emryogeni suspension ultures hve een estlishe in very few uurits, inluing melon (Orite n Oosw 1986; Kgeym et l. 1991), uumer (Chee n Trioli 1988; Bergervoet et l. 1989) n itter melon Thiruvengm et l. (26). The present stuy of emryogeni suspension ultures for regenertion of spine gour hve een estlishe for the first time. The omplete proeure from inution of somti emryos to plntlet reovery oul e omplete within 6 ys. This proeure is novel from erlier reports on somti emryogenesis of melon (Orite n Oosw 1986), uumer (Chee n Trioli 1988), n itter melon (Thiruvengm et l. 26). The hoie of initil explnt is ritil ftor for emryogeni llus inution n initition. In the mjority of uurits seeling mterils hve een the most res- 23

5 Somti emryogenesis n plnt regenertion in spine gour. Thiruvengm et l. Somti emryos (% formtion) Torpeo stge Cotyleonry stge Surose (%, w/v) ponsive explnts for the inution of somti emryogenesis. Deeujon n Brnhr (1993) emonstrte tht most seeling mteril, suh s otyleon n hypootyl tissues were suessful for somti emryogenesis in uurits. There re severl reports tht petiole explnts re more suitle thn other plnt prts for in vitro plnt regenertion. Petiole explnts were use for plnt regenertion vi somti emryogenesis in Ros hyri (Mrhnt et Fig. 3 Influene of vrying onentrtion of surose on somti emryo formtion of petiole-erive llus in MS liqui meium with 2.2 μm 2,4-D. The t were sttistilly nlyze using Dunn s multiple rnge test. In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. Eh vlue represents the men of 1 inepenent replites. The numer of pro-emryogeni ells ws ompre with the totl numer of llus ells present in the mirosopi fiel. The frequeny of ifferent stges of somti emryos were etermine y tking the smples from 1 replite flsks per 2,4-D tretment. Vertil rs represente S.D. Stges of Somti emryos Gloulr stge Hert stge Torpeo stge Germintion of emryo MS B5 LS SH Different types of mei Fig. 4 Effet of ifferent sl mei with 2.2 μm 2,4-D on somti emryogenesis of petiole-erive llus in spine gour. The t were sttistilly nlyze using Dunn s multiple rnge test. In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. Eh vlue represents the men of 1 inepenent replites. The numer of pro-emryogeni ells ws ompre with the totl numer of llus ells present in the mirosopi fiel. The frequeny of ifferent stges of somti emryos were etermine y tking the smples from 1 replite flsks per 2,4-D tretment. Vertil rs represente S.D. Somti emryos (% formtion) Gloulr stge Hert stge Torpeo stge Cotyleonry stge L-glutmine M l. 1996), Begoni grilis (Cstillo n Smith 1997) n Herleum nins (Wkhlu n Shrm 1998). Eqully, in the present stuy, petiole explnts were foun to proue somti emryos in M. ioi. Auxins were solutely neessry for somti emryo inution ut were often omitte in the perio of mturtion n germintion (Choi et l. 1991; Mohme et l. 24; Thiruvengm et l. 26). The pretretment of 2,4-D for somti emryo inution in melon n sqush were oserve y Kintzios et l. (22). Similrly, the present investigtion lso emonstrte the effet of 2,4-D s vitl role for inuing somti emryogenesis. The inution of frile lli with emryogeni ompetene is prerequisite in somti emryogenesis for the regenertion of omplete plntlets. In our experiments, green-yellow frile lli were inue on fortifie MS meium ontining 4.5 M 2,4-D with CM (1%) for two weeks of ulture. Similr results y Binzel et l. (1996) note tht MS meium ontining 9 M 2,4-D with 1% CM improve the potentil of emryogenei llus in Cpsium nnum. The ition of BA or Kn to the ulture meium h either negtive or no effet on somti emryogenesis inution s emonstrte y Jelsk (1986) n Noel et l. (1992) in Cuurito pepo. In ontrst, 2,4-D with BAP or Kn ws responsile for inuing somti emryos in Cuumis melo (Orite n Oosw 1986), Cuumis stivus (Bergervoet et l. 1989) n Cuurito pepo (Kintzios et l. 22). An ttempt hs een me to evelop the ontogeni stges of somti emryo ifferentition from liqui ultures of uurits. Jelsk (198) n Jelsk et l. (1985) reporte tht the presene of 2,4-D in the ulture meium inhiite emryo evelopment of sqush eyon the gloulr stge. The omplete removl of 2,4-D t lter stges of ulture stimulte further emryo evelopment into the hert n torpeo stges in Momori hrnti (Thiruvengm et l. 26). This prolem ws voie in the present stuy, n inution n evelopment of emryos up to the torpeo n otyleonry stges were hieve on MS meium ontining 2,4-D. Similr results were oserve y Mlepszy et l. (1982) n Mlepszy n Nolsk- Orzyk (1983), in whih MS meium supplemente with Fig. 5 Effet of L-glutmine on evelopment of somti emryos from petiole erive llus on MS liqui meium ontining 2.2 μm 2,4-D. The t were sttistilly nlyze using Dunn s multiple rnge test. In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. Eh vlue represents the men of 1 inepenent replites. The numer of pro-emryogeni ells ws ompre with the totl numer of llus ells present in the mirosopi fiel. The frequeny of ifferent stges of somti emryos were etermine y tking the smples from 1 replite flsks per 2,4-D tretment. Vertil rs represente S.D. 24

6 Funtionl Plnt Siene n Biotehnology 1(1), Glol Siene Books Tle 4 Influene of rohyrte type n onentrtions on somti emryo formtion of petiole-erive llus in MS liqui meium with 2.2 M 2,4-D. Crohyrte Gluose Frutose Mltose Conentrtion Different stges of somti emryos Gloulr Hert Torpeo stge stge stge 1 1. ±.2 m 2 3. ±.5 i 1. ±.2 fg 1. ± ± ± ± ± ±.4 2. ± ± ± ±.8 g 1. ±.2 fg ±.4 e 2.6 ± ± ± ±.4 1. ± ±.5 ij 2.2 ±.6 e ±.5 kl ±.4 gh 1.2 ±.2 f ±.4 f 2.2 ±.2 e ±.5 k 4 5 Eh vlue represents the men ± SE of three replites per tretment. The t were sttistilly nlyze using Dunn s Multiple Rnge Test (DMRT). In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. ( = Nil response). Tle 5 Effet of mino is on somti emryogenesis of petioleerive llus in MS liqui meium with 2.2 M 2,4-D. Amino i Conentrtion Different stges of somti emryos Type ( M) Gloulr stge Hert stge Torpeo stge L-lnine 36.3 ± ± ± ± ±.4 1. ± ± 1. e 2.6 ±.2 ef ± 1. f L-proline 36.3 ± ± ± ± ± ± ± ± ± ± ±.5 e 1. ± Eh vlue represents the men ± SE of three replites per tretment. The t were sttistilly nlyze using Dunn s Multiple Rnge Test (DMRT). In the sme olumn, signifint ifferenes oring to the lest signifint ifferene (LSD) t the P.5 level re inite y ifferent letters. ( = Nil response). 2,4-D (.4 mg/l) ws effetive for the inution of somti emryogenesis from lef n stem explnts of C. stivus. Emryo mturtion egins fter the ompletion of histoifferentition in somti emryos. In C. stivus, hormone-free MS sl meium ws foun to e optiml for mturtion n further evelopment (Mlepszy n Nolsk-Orzyk 1983; Rjsekrn et l. 1983; Orezyk n Mlepszy 1985; Wyszogrozk n Shhin 1985). Similr oservtions were me in our stuy, where the omplete removl of 2,4-D from the ulture meium improve emryo mturtion. Our results ontrst to the reports of Tei et l. (1991) in whih the mturtion of somti emryos from otyleon, hypootyls, stem n petiole explnts of C. melo ourre on 1/2 MS meium fortifie with GA 4 (.2 mg/l). Lter the mture emryos were trnsferre to 1/1 MS meium ontining ABA.5 M for suessful germintion. This result ws oherent with tht of Kim n Soh (1996) in Allium sp., Nkgw et l. (21) in Cuumis melo, n Mohme et l. (24) in Mrotylom uniflorum for somti emryo germintion inue y ABA. Our histologil report lerly onfirme the formtion of ipolr strutures in spine gour. Gloulr emryos normlly onsist of smll ells with ense ytoplsm n lrge nulei n hve een oserve in regl gernium (Wilson et l. 1994). The results of this report re onfirme y the pre-sent stuy. The regenerte somti emryos evelope s new iniviuls tht onsiste in the gloulr stge of the typil smll ells with ense ytoplsm n evelope to ler ipolr strutures (Fig. 1L). In poppy (Kim et l. 23), the optimum onitions for mintenne of ell suspension ultures were the shking spee n pke ell volume (inoulums per 25 ml meium). Similr results were oserve in the present stuy where gittion of the ultures t 1 rpm ws foun to e optiml. The omponents of mei n vrious onentrtions of rohyrtes n mino is n exlusively ply vitl role in etermining the emryogeni ompetene of ell suspension n mturtion of somti emryo. This investigtion on meium omposition revele tht MS mei were highly signifint for the inution of somti emryogenesis, ue to high levels of nitrogen in the form of mmonium nitrte, whih is responsile for oth emryo initition n mturtion. Similr results were reporte in the leves of sqush n melon (Kintzios et l. 22), uumer (Clleut et l. 1987; Clusters n Bergervoet 199). The ourrene of vrious rohyrtes hs een foun to ffet the initition of somti emryos in spine gour, with 3% surose resulting in highest frequeny, followe y gluose n frutose with low frequeny of emryo initition. Mltose ws ompletely ineffetive for somti emryogenesis in spine gour. Similr results were lso otine in penut n soyen (Epen n George 1993; Smoylov et l. 1998). Guis et l. (1997) reporte tht gluose ws the most importnt sugr for somti emryogenesis in melon. However, our experiments onfirme surose plys n importnt role s n energy soure, n when e to the meium might e essentil for somti emryogenesis. Our results support similr oservtions me in uumer (Chee n Trioli 1988), melon (Nkgw et l. 21) n itter melon (Thiruvengm et l. 26). Glutmine ppers to e suitle nitrogen soure to support tive protein synthesis ssoite with somti emryo inution n mturtion. In our stuy, the ition of glutmine t.5 M resulte in the gret improvement (44.5%) of emryogeni frequeny n evelopment. The similr results were emonstrte y Skokut et l. (1985) n Sturt n Strikln (1984) in Meigo stiv, Khlifi n Tremly (1995) in Pie mrin n Vengtesn et l. (25) in Cuumis stivus. This emphsize tht the exogenous supply of these sustnes oul foster the physiologil mturity of emryos. CONCLUDING REMARKS The protool evelope oul estlish the potentil to proue Momori ioi plntlets from petiole explnts through somti emryogenesis. The entire proeure from inution of somti emryos to plntlet reovery oul e omplete within 6 ys. Suh novel protool n emphsize the gret potentil of iotehnologil pprohes suh s in vitro seletion, proution of syntheti see n evelopment of geneti trnsformtion stuies in spine gour. REFERENCES Bergervoet JHW, Mrk FV, Custers JBM (1989) Orgnogenesis versus emryogenesis from long term suspension ultures of uumer (Cuumis stivus L.). Plnt Cell Reports 8, Bhojwni SS, Rzn MK (1983) Plnt Tissue Culture, Elsevier, Amsterm, 52 pp Bhosle DS, Prtkr GT (25) Cllus ultures from Momori ioi (Rox.). 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