Tgp chi Cong ngh4 Sinh hgc 12(1); 73-84, 2014
N G H I E N C t l t l M Q T S O Y E U T O A N H H l T d N G D E N Q U A T R I N H P H A T S I N H P H O I V O T I N H S A M N G Q C L I N H {PANAX VIETNAMENSIS H A ET G R U S H V . ) IN VITRO Biii v a n The Vinh', VQ T h | Thuy', T h i i Thuong Hifin', VO Qu6c Luan', Nguyin B i Nam', NguySn PhAc Huy', Tr|nh Thj H u v n g \ Vii Thi Hien', Le Kim Cuong', Hd Thanh T i m ' , Do Manh Cuong', NguySn Vift Cufmg', Do Kh^c Thinh^, Duong Tan Nhut'
' Vi^n Nghien cuu Khoa hgc Tdy Nguyen, Vien Hdn ldm Khoa hoc vd Cong nghe Viet Nam
^Vi4n Khoa hgc Ky thudt Nong nghiep Miin Nam, Vien Khoa hgc Nong nghiep Viet Nam Ngiy nh^n bai; 11.02.2013
Ngiy nhandang: 20.3,2014 T 6 M TAT
Sam Nggc Linh (Panax vietnamensis Ha el Grushv.) la mot cay thuoc quy ciia Viet Nam. Trong nhimg nim gan day, vi?c nghien ciiru kha nang nhan giong va hao ton loai cay dugc lieu quy nay dang trd thanh van d€ rat cap thiet, Trong do, viec nghien ciiu qui trinh phat sinh phoi vo tinh sam Ngoc Linh dong mgt vai tro het sire quan trgng, Trong nghien ciiu nay, chiing toi lien hanh khao sat mgt so yeu to anh huong den qua trinh phat sinh phoi vo tinh sam Nggc Linh nhu nguon mau cay, thanh phan khoang trong moi truang nuoi cay, chat dilu hoa sinh tmdng thyc vat, spermidme, proline va ngu6n carbohydmte, Trong 3 nguon mlu ban diu gSm la, cuong li, cii ciia cay sam Nggc Linh in vitro 3 thing tuoi thi mau cay la voi kich thircrc 0,5 x 0,5 cm thich hgp ik nu6i cSy tao phoi Uen moi truong MS bo sung 1,0 mg/l 2,4-D kit hap voi 0,5 mg/l NAA va 0,2 mg/l kinetin. Anh huong ciia spermidine va proline len kha nang kich thich gia tang tan suat phat sinh phoi cung duyc nghien ciru, Ket qua cho thiy spermidine (0,1 mM) va proline (300 mg/l) co tac dyng gia tang tin suat phit sinh phoi (tuong img 93,3% vi 86,7%), Nhim toi uu h6a moi tnrong cam img tao phoi vo tinh sam Ngpc Linh, 3 ngu6n carbohydrate khac nhau (sucrose, glucose va fructose) d cac nong do 10 - 60 g/l da dirge thii nghidm. Trong d6, sucrose 6 nong do 50 g/i co hieu qua trong vi?c cam iing lao phoi vdi ti le dat 86,7% va so lugng phoi dat 167 phoi/miu Glucose va finctose to ra khong phii hgp de sii dung lam nguon carbohydrate bo sung vio moi tnrdng cam iing tao phoi doi vdi sam Nggc Lmh,
Tir khda: Carbohydrate. Panax vietnamensis, phoi vo tinh. proline, spermidine
DAT VAN DE
Sim Ngpc Linh (Panax vietnamensis Ha et Grushv,) thuOc hp Nhan sim (Aralilaceae), l i cay duQc li^u c6 thinh phan ginsenoside duac danh gia vio logi nhi^u nhit so vo'i cac loii khac ciia chi Panax tren the gidi Qua nghien ciiu c i c nha khoa hpc d i nh|n thay sam Ngoc Linh khong chi co cac tic dyng du^c ly d^c Irung cua chi Nhan sam m i con CO nhthig lac dung dien hinh nhu chong stress, chong ttim cam, tic dyng len su chong oxy hoa in vitro v i in vivo, .. Nhom chit co vai tto quyet dinh nhat den lac dyng dupc ly ciia loai s i m niy la cac saponin triterpenoic mi d^ii dien chinh la MR^, G-Rb| va G- Rgi (Tran Cong Luan, 2003). Sam Ngoc Linh li mot trong nhiing loai sam co ham lugng saponin khung dammaran cao nhit (khoang 12 - 15%) va lugmg saponin nhieu nhll so vdi cac loai khic cua chi Panax ttfin Ihi gidi (Nguyen Thugmg Dong el al, 2007).
Chinh nhd nhung gia trj duoc ly to ldn nhu viy ma nhu ciu doi vdi sim Ngpc Linh ngay cang gia ting. Tuy nhien, viec ttong dai Ira loai cay trong c6 gia tti niy dang gap phai rat nhieu kho khin nhu gidi han ve pham vi phan bo, thcri gian trdng keo dai, nhieu sau benh,.,, Vi vay, ttong vong vai n i m ttd lai day, nhieu nghien ciru da duoc lien hanh nhim nhan gidng vo tinh sam Ngoc Linh (Nguyin Ngpc Dung, 1995; Duong T i n Nhut et al, 2010), Tuy nhien, phuong phap nhin giong Iruyen thdng khdng dap ling duac nhu ciu ve cay gidng sam Ngpc Linh ngay cang ting tten thi trudng do Ii le son^ sot cua cay con in vitro khi chuyen ra vudn uam thip.
Nhan gidng vd tinh thdng qua con dudng phat sinh phoi da mang lai nhieu irng dung thyc tien va cd tinh thirang mai cao Viec irng dung phuang phip phit sinh phoi vd tinh d cay sam Ngpc Linh hira hen se mang lai nhieu uu diem vi cd the tao ra mpt so luting ldn cay con cd chat lupng lot trong mpt thdi
73
Biii van Thi Vinh eM/.
gian ngan, ti le sdng sdt ciia cay con ngoai vudn uam cao do cay con phat trien td phoi vd tinh theo con dudng tuong tir nhu phdi hiru tinh. Trong nd luc nghien ciiu irng dung nudi cay md de san xuat sd lucmg ldn cay con gidng sana-NgOG-iinh ddng-flMt- ve mat di truyen vi cd kha nang sdng sdt cao, chting tdi riin hanh nghien cutu mdt so yeu td anh hudng den qua trinh phat sinh phdi vo tinh sam Ngpc Linh nhu ngudn miu ciy, ham lupng khoang, chit dieu hda sinh trudng thuc vat ttong mdi trudng nudi cay, ngudn carbohydrate, spermidine va proline.
VAT LIEU VA PHU'ONG PHAP NguSn mlu in vitro
Ngudn mau sir dung ttong nghifin ciiu niy Ii cac CO quan nhu la, cudng Ii va cii duoc cat ra tOt cay sam Ngoc Linh in vitro 3 thing tudi cd chieu cao khoing 1,5 cm, ttpng lupng tuoi khoang 0,2 g da dn dinh qua 4 den 5 lan cay chuyen, hifin cd t^i Phdng Sinh hpc Phan tir va Chpn tao Giong cay ttdng, Vien Nghifin ciiu Khoa hpc Tiy Nguyen.
Moi trirdng nuoi cay
Mdl trudng MS (Murashige, Skoog, 1962), VMS (Mdi tnrdng MS cd thanh phin da lupng giim mpt niia), MS'/4 (Mdi trudng MS cd thatih phan da lupng va vi lupng giim mdt nua), SH (Schenk, Hidebrandt, 1972), V^SH (Mdi trudng SH cd thanh phan da luang giam mdt nira) va SWA (Mdi trudng SH cd thanh phin da lupng vi vi luong giam mpt nira) cd bd sung them 30 g/l dudng sucrose (ngoai tni thi nghifim vfi cac ngudn cacbonhydrat khac nhau), 8 g/l agar, cac chat dieu hda sinh trudng thudc nhom auxin (NAA, 2,4-D), cytokinin (kinetin), tiiy thudc vio ttmg thi nghiem se bd sung d cic ndng do khac nhau. Sau dd difiu chinh dp pH ciia mdi trudng tii 5,7 dfin 5,8.
Dieu kien nudi c^y
Trong phdng thi nghifim vdi nhiet dd 25 ± 2°C, cudng dp chieu sang khoing 35 - 40 jiraol. m'^.s'', thdi gian chieu sang 16 gid/ngiy.
Phuong phap
Khdo sdt dnh hie&ng cua nguon mdu ciy len khd nang phdt sinh phoi vo tinh sdm Nggc Linh
Cac mlu ciy la (0,5 x 0,5 cm), cudng la (dai 1,0 cm), cti (0,5 X 0,5 x 0,1 cm) dupc c5t khdi cay sam Ngpc Linh in vitro 3 thing tudi va nudi ciy tren mdi trudng SH bd sung 1,0 mg/l 2,4-D va 1,0 rag/l NAA.
74
Sau 8 tuin nuoi cly, tifin hanh ghi nhan cic chi tieu tin suit phat sinh phdi, s6 lupng phoi/mlu va ttpng luong cua mau ciy (mg).
-Khdo sdt anh htedne ciia thanh phin khodng trong moi trtecmg nudi cay lin kha nang phdt sinh phoi vd tinh sam Nggc Linh
Cic mlu ciy la (0,5 x 0,5 cm) dupc nudi cly tren moi ttudng MS, '/2MS, MS'/z, SH, '/iSH va SU'A cd thanh phin khoang da lupng va vi lupng khac nhau dupc bd sung 1,0 mg/l 2,4-D va 1,0 mg/l NAA Thinh phin vitamin ciia MS dugc sit dyng cd dinh ttong tat ci cac mdi trudng thi nghiem. Sau 8 tuin nuoi ciy, tifin hanh ghi nh^n cac chi tieu ti le hinh thanh md seo (%), tin suit phat sinh phdi, sd lupng phoi/miu va ttpng luong cua mlu cly (mg).
Khdo sdt dnh hwdng cua chit diiu hoa sinh tnrdng thyc v^t ten khd ndng phdt sinh phoi vo tinh sam Nggc Linh
Md seo duoc tao ra tu cac thi nghidm tten dupc cit thanh nhiing manh nhd (0,5 x 0,5 cm) vi nuoi cly trfin mdi trudng MS bd sung 1,0 mg/l 2,4-D kei hpp vdi NAA vi/hoac kinetin d cic ndng dd khac nhau (0,1; 0,2; 0,5 va 1,0 mg'l). Sau 12 ttiin nuoi ciy, tifin hinh ghi nh$n cic chi tifiu tin suit phit sinh phdi, sd luong phdi/mau va ttpng lupng cua miu ciy (mg). Mdi trudng cam img tao phdi (EM) toi uu ttong Ihi nghifim niy dupc sii dyng lam mdi ttudng CO bin cho cac thi nghiem tiep theo.
Khao sdt dnh hieong spermidine, proline vd nguon carbohydrate len khd ndng gia tdng tin suit phit sinh phoi vo tinh s&m Nggc Linh
Cac mau mo seo sam Ngpc Linh dupc ciy vao mdi trudng cira dng tao phdi (EM) cd bd sung prolme (thudc nhdm acid amine, ndng dO 100, 200, 300 va 400 mg/l) hoic bo sung spermidine (thupc nhdm polyaralne, ndng dp 0,01; 0,05; 0,1; 0,2 mM)- Cac ngudn carbohydrate khac nhau (sucrose, glucose va fructose) ciing duac bd sung vao mdi trudng cam ting tao phdi (EM) d cac ndng dp khac nhau (10,20, 30, 40, 5(), 60 g/l) dfi danh gia kha ning kich thich gia tang tin suit phit sinh phdi vd tinh d sam Ngpc Linh. Cic chi tieu nhu ti lfi phit sinh phdi (%), so luong phdi/mau duac ghi nhan sau 12 tuin nudi cay.
KET QUA VA THAO LUAN
Anh hudng cOa nguon mau cly ISn khi ning phit
sinh phdi vo tinh sSm Ng9c Linh
Tgp chi Cong ngh? Sinh hgc 12(1): 73-84, 2014
Nguon mau va thanh phin dinh dudng ttong mdi trudng nudi cly cd vai ttd quan ttpng ttong su phit sinh hinh thii cua man cay (Fleming, 2002).
Trong thi ngbipm nay, cac miu ciy la, cudng li vi cii dugc clt ra tit ciy sam Nggc Linh in vilro 3 thing tu6i bit dau phin iing hinh thinh md seo sau 2 tuan nudi cly trfin mdi trudng SH bd sung 1,0 mg/l 2,4-D va 1,0 mg/l NAA. Md seo phit sinh diu lien tren mlu cay cu, cd dang xdp, hinh thanh xung quanh vien cii. M6 s?o xdp, ttong, mpng nudc cung dugc tao thinh tir cac mau ciy Ii vi cudng li. Ddi vdi mlu ciy li, md s?o hinh thinh diu tifin d phln m$t cit tifip xiic vdi moi trudng sau dd phat trien d ci 4 phia ciia mit cat. Trong khi dd, ddi vdi mau ciy cudng li, md spo chi hinh thinh d hai diu, noi cd vet cit (Hinh 9ai, ai, as).
Sau 8 tuan nudi ciy, md seo phat sinh manh nhit doi vdi mlu ciy li vi cii (Hinh 93,, aj), dat ti
le 100% (Hinh I). Tren be mat khdi md seo xdp bat dau xuat hien nhiing cham nhd miu tim, phan Idn nhiing cau tnic nay sau dd phat trien thanh re bit dinh vdi cac dang hinh thii khic nhau (dii lieu khdng dupc trinh bay). Ben canh dd, cd sy xuat hifin cua nhung cau tnic phdi hinh cau mau trang tren be mat khdi md seo cd ngudn gdc tir mau cay li va cu vdi ti Ie 8% (Hinh 1). Mac du sd phdi hinh thinh tu khdi md seo co ngudn gdc tir man cay cu (7 phoi/miu cay) nhifiu han so vdi miu cay li (3 phoi/mlu ciy) nhung do sd lupng mau cay li/cay nhieu han rat nhieu so vdi miu ciy cii/cay nen tdng sd lupng phdi thu nhan duoc tir miu cay la nhifiu ban so vdi cu. Do do, trong cic thi nghifim tifip theo, chiing tdi su dung la lam ngudn v|t lifiu ban diu de nghifin cihi inh hudng cua cic yfiu td khac len qui trinh phit sinh phdi vd tinh d cay sam Ngpc Linh.
100 90 BO 70 60 50 40 30 20 10
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• Til? lao phoi (?o)
• So phoi
Hinh 1. Anh hudng cua ngu6n mlu cly len khi ning phit sinh ph6i v5 tinh sam Nggc Linh
Anh hirdng cua thinh phan khoing trong mdi trirjmg nuoi cay len khi ning phit sinh phdi vd tinh sim Ng9C Linh
Trong mpt sd nghien cuu vfi sam, mdi trudng MS thudng dugc sir dyng dfi nudi cay khdi tao md s?o (Huang et al, 2010), phit sinh co quan (Bonfill et al, 2003) hay tii sinh cay hoan chinh (Xiang et fl/.,2007; Duong Tin Nhut et al, 2010). Trong khj dd, mdi trudng SH l^i td ra hieu qui dfi nudi ciy rfi bit djnh vi I90 cu in vitro (Hoang Xuan Chien el al, 2011). Sy hip thu cic nguyen td khoang tir mdi trudng nudi cay trong qui trinh cam ung va sudt giai do(in diu phit sinh phdi vd tinh da dupc chimg minh (Dussert et al, 1995; Fisichella et al, 2000). Do dd, Uong thi nghifim niy, chung tdi sii dyng 2 mdi tnrdng khoing ca bin la MS vi SH, ddng thdi su
dung 4 mdl trudng cai bifin la !/;MS, MS'/:, '/2SH va SH'A cd thinh phan khoang da lupng va vi lupng khac nhau. Trong tit ca cac mdi trudng thi nghifim, chung tdi cd dinh thinh phin vitamin ciia MS, ddng thdi bd sung them 1,0 mg/l NAA va 1,0 mg/l 2,4-D.
Sau 8 man nudi cay, ket qua cho thay hau hfit
cac mdi trudng dfiu cim img su hinh thinh md s?o
vdi ti le cao. Moi trudng MS vi MS'/z cho ti le lao
phdi cao hem cic mdi trudng cdn lai (48%), tuy
nhien sd lupng phdi vd tinh hinh thinh nhifiu nhat d
miu la duoc nuoi cay trfin mdi Irudng cd thanh phin
khoing da lugng ciia MS (15 phdi/mau cay), Trfin
mdl trudng SWA vi 'ASH, cic miu cay deu phit
trifin Ihanh md seo mem, ket cau xdp vi dfi vd vyn
ma khdng cd su hifin difin ciia ciu triic gidng phdi
nio (Hinh 2).
Bui Van The Vinh etal Sy ket hgp cac t h i n h phan khoang da lugng
c i m ling sy phat sinh phdi vd tinh hieu qua nhat ddi vdi s i m Ngpc Linh la mdi tnrdng M S . Ket qua n i y cd thfi do ham lugng n i t a trong mdi trudng cao^ iLnhit l i g i p h a i lan so vdi nhung mdi tnrdng k h i c ; mdt nguyen nhan khac cd thfi la do sy can bang giira nitrate va ammonium ttong mdi trudng nudi cay. Moi trudng MS cd chiia 60 mM nita trong dd ham lugng NO3" chifim 39,4 mM va h i m lugng NH4* chiem 20,6 m M ttong khi tat ca cac mdi trudng k h i c , ham lugng n i t a khdng v u g t qua 30 mM. Ket qua tuong t u cung da d u g c bao c i o bdi Kim va ddng t i c g i i (2006) khi tifin h i n h nghien ciiu phat sinh phdi true tifip td mau cay la m i m c i y N h i n s i m . Sy hinh thanh cau tnic gidng phdi dat dugc tten mdi trudng M S ldn h a n so vdi trfin mdi trudng SH hay B5 vdi ti lfi N H / : N 0 3 " tdi uu la 21:39 (Kim et al, 2006). Khi nita hien difin d ndng dp cao, hinh thanh nfin nhifiu NH3, tdng hgp nen cac amino acid la nhiing hgp chat rat can cho sy tang trudng cua c i c ca quan sinh dudng (Marschner, 1983; Nguyfin Du Sanh, 1998).
Bfin cardi ham lugng nita tdng, cd thfi nh|in thiy rang sy k h i c bifit ve k h i nang hinh thanh md s?o phft sinh phdi cdn chiu anh hudng bdi ham lugng ion Kj v i Ca^*. Ion K* lifin quan dfin bam proton v i Uong sv van chuyen gian tiep cac chit hda tan (Briskin, Hanson, 1992) cd thfi la yfiu td gidi han sy phat sinh phdi vd tinh. Vi vay, ttong nudi cay huyfin phii Ca r6t, ham lugng potassium thap Uong mdi trudng nudi ciy lam han che sy hinh thinh phdi vd tinh ma khdng anh hudng dfin sy tang trudng tfi bao ttong khi ham lupng potassium cao lam tang manh sd lugng phdi (Brown el al, 1976). Sy phat sinh phdi vd tinh cimg cd the chiu anh hudng bdi calcium. Ca^^ ho£it ddng nhu la mdt tin hifiu thii cap ttong con dudng ttuyen tm hifiu kich thich ngoai bao bang each gan vdi calmodulin hay cac protein gan calcium khic dfi kifim soat nhieu dap img sinh ly tfi bao (Bush, 1995). CJ C i rdt, sy phSn cue hinh thai cua cac cym te bao sinh phdi ket hpp vdi Ca^* ndi bao ty do (Timmers, Schel, 1991). Hon nfla, sy gia tang ham lupng CaCl2 ttong mdi trudng cam ling hinh thanh sd lugng phoi nhieu hon (Silva, Ricardo, 1992).
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• Tl!?t9om6s¥o(%)
• Trpng lirpng (mg)
• Til? 190 phoi (%)
• S6 lu^g ptioi
Hinh 2. Anh huwng ciia thanh phan khoang trong mdi tnrdng nuai cay len kha ning phit smh phdi vo tfnh sSm Nggc Lmh Anh h u o n g cua chat dieu hda sinh t r u d n g t h u c
vat len k h i n a n g p h a t sinh phdi vo tinh sam Ngoc Linh
Cac miu md seo cd ngudn gdc tir la s i m Ngpc Linh in vilro dupc cat thanh timg manh cd kich thudc 0,5 X 0,5 cm v i cay vao mdi trudng MS cd bd sung 1,0 mg/l 2,4-D kfit hpp vdi N A A hoac kinetin d cac ndng dp 0,1; 0,2; 0,5 v i 1,0 mg/l (Hinh 9bi). Sau 12 tuin nudi ciy, kfit qua cho t h i y ti lfi m i u tao phoi va sd lugng phdi hinh thanh tten m l u khac nhau d cac nghiem thiic thi nghiem. Ddi vdi nghifim thiic ddi chiing (khong bd sung chit difiu hda sinh trudng), khdng xuat hifin phdi tten cac m l u md seo,
cac mau md seo d i n hda nau va chet. Cac nghiem thiic cdn lai bd sung 2,4-D kfit hgp vdi NAA ho|c kinetin d cac ndng dg k h i c nhau cho thay cd su xuat hien cac cau tnic phdi hinh cau. Sy kfit hgp gifla NAA va 2,4-D cho thay kha nang cam itng tao phoi tdt ban thdng qua cac chi tieu ti lfi phat sinh phdi vk sd lugng phoi/miu (Hinh 3).
Trong cac nghien ciiu vfi cam iing phat sinh phdi vd tinh d nhieu l o i i thyc vat Ihi auxin, d ^ biet l i 2,4-D t h u d n g d u g c sii dung nhifiu nh^t (Umehara, Kamada, 2005), Difiu nay d u g c chiing minh bdi nhiing thi nghifim d Ca rdt (Borkird et al, 1986), F. sellowiana (Cruz et al., 1990),
Tgp chi Cong ngh? Sinh hgc 12(1): 73-S4,20\4
Populus spp. (Michler, 1995), Cacao (Canhoto et al, 1999), Paspalum scrobilatum (Avci, Can, 2006). Nong dd auxin trong mdi tnrdng tao phdi dugc sir dyng khac idiau v i thay ddi tuy theo tirag loii cung nhu tirng loai kifiu gen ciia thyc v^t. Do dd, chiing tdi da su dung 1,0 mg/l 2,4-D cho tat c i c i c thi nghifim (day la ndng dp thich hgp trfin ddi t u g n g sam Nggc Linh d i d u g c nghien ciiu trudc day). Tuy nhifin, nfiu chi bd sung 2,4-D rifing rfi ma khdng cd sy hd trg cua cic chit dieu hda sinh trudng k h i c thugc nhdm auxin hay cytokinin thi khdng thu nhan d u g c md seo cd kha n i n g p h i t sinh phdi. Trong thi nghiem
nay, chiing tdi kfit hgp 2,4-D vdi mpt auxin k h i c l i NAA, d i y la chat cd k h i n i n g c i m ung p h i t sinh phdi vd tinh d mdt sd loii. N A A da dugc chiing minh l i rat hifiu qua trong vific tao phdi vd tinh d c i y D a u t u a n g va c i y C i rdt khi sii dyng vdi nong do 1,0 mg/l (Borkird et al, 1986).
Trong khi dd d thi nghiem ciia chung tdi, ti lfi phat sinh phdi cao n h i t (dat 60%) khi sii dung 0,5 mg/l N A A kfit hgp vdi 1,0 mg/l 2,4-D vdi sd phdi trung binh khoang 52 phdi/miu c i y (Hinh 3). Kfit qua nay phii hgp vdi nghien ciiu p h i t sinh phdi d Momordica charanlia L. (Ananya et al, 2009), Panax ginseng (Shohana et al, 2009).
J ffi
• Tl 1^ phat sinb phoi (%)
• S6 lupng phoi
BC EMl EM2 EM3 EM4 EMS EM6 EM7 EMS
Hinh 3. Anh hudng cua 2,4-D ket hgp vdi NAA ho^c kinetin l§n khi nang phit smh phoi v6 tinh sim Ngpc Linh BC d6i chiing (khOng b6 sung chit di^u hoa sinh trudng thyc v^t); EMI, EM2, EMS, EM4: 2,4-D (1,0 mg/l) k^t hpp v6i NAA (0.1;
0.2; 0,5 v i 1,0 mg/l); EMS, EM6, EM7, EMS 2,4-D (1.0 mg/l) kit hgp voi kinetin (0,1; 0.2; 0,5 v i 1,0 mg/l).
Cytokinin cd tic dyng kich thich sy hinh thinh phdi vd tinh tir nudi ciy md s?o khdng cd kha nang phit sinh phdi dupc bio cio Iren cic loii ciy dau nhu Phaseolus (Malik, Saxena, 1992), Trifolium (Maheshwaran, Williams, 1986), ciy Ba.u phyng (Gill, Saxena, 1992).
Cic lo^i cytokinin nhu kinetin dugc chiing minh l i cd tic dung t6t trong nuoi ciy ban diu dfi hmh thanh phdi vd tinh d rapt so loai than gd (Ehinslan el al, 1995). Tuy nhifin, nong dp cua kinetin dugc sir dung ciing khic nhau tiiy theo loii thyc vat. Kfit qui dugc chi ra trong thi nghifim niy cho thiy sy kfit hgp giiia 2,4-D v i kinetin khdng d^t hifiu qua cam img phal sinh phdi tdt nhu sy ket hpp giiia 2,4-D v i NAA. 0 nghifim thuc EM5 khdng diay cd diu hifiu phit sinh phdi, chi li khdi md seo ran chic, mau ving kem, mpt sd vi tri cd mau tim, O nghiem thiic EM6 bit diu cd sy xuat hifin phoi, my khdng nhieu nhung phdi hinh thinh cd mau tring sang, tuong ddi dong nhit v i phdi Idn ban so vdi d cic nghifim thiic ket hgp giOa 2,4-D v i NAA, Chinh vi viy, chimg ldi lien
hinh kfit hgp hai chat dieu hoa nay vio thi nghifim tifip theo dfi dinh gii khi nang cam img phit sinh phdi trfin ddi tupng sam Ngpc Linh
Khi kfit hgp 1,0 mg/l 2,4-D vdi 0,2 mg/l kinetin va NAA d cac none dd khac nhau cho t h i ^ kfit qua thu dupc tuang ddi tot va ddng dfiu d tit c i cac nghiem thiic, Cu the d nghifim thirc EM9, ti lfi m i u tao phdi la 33,3%, nghiem Ihirc EM 10 l i 53,3%, nghifim thiic EMI I dat cao nhit la 80%, den nghifim thuc EM 12 giam xudng cdn 6 0 % (Hinh 4). Sd phdi tao thanh Iren mau ciing tuong ddi cao so vdi thi nghiem chl ket hgp 2,4-D vdi NAA hoac kinetin va dat cao nhit d nghiem Ihirc E M I ! (117 phdi/miu). Phdi cd cic dang hinh cau, hinh lim, hinh thiiy ldi, hinh hai la mam (Hinh 9ci, C2, C3, C4). Phdi miu vang sang, to va tuang ddi ddng dfiu, khdng cd phdi nao bat thirdng, mpt so man co sy hinh thanh chdi.
77
Bui Van The Vmh et al
. M H _
.
• TiU phal sinh pb6i(%)
• 85 lupng phSi
Hinh 4. Anh hudng cCia 2,4-D ket hop v^i NAA va kinetin len kha nang phat sinh phoi v6 llnh sam Ngpc Linh. EM9, EM10, EM11, EM12: 2,4-D (1,0 mg/l) kit hop vo'i kinetin (0,2 mg/l) va NAA (0,1, 0,2; 0,5 va 1,0 mg/l)
Trong nudi cay md tfi bao thyc vat, sy phat sinh hinh thai bi anh hudng bdi cac thanh phan khac nhau ttong mdl trudng nudi cay, dac bifit l i ndng dp cac chit difiu hda smh trudng thyc vat nen viec danh gia anh hudng cua chiing Ifin sy tii sinh cay la rat quan ttong Sy phat sinh phdi vd titdi tir nudi cay md seo ttong mdi tnrdng cd chua cytokinin kfit hgp vdi auxin dugc bao cao d cac Ioai Ngd cdc (Bhaskaran, Smith, 1990), c i c loai thudc chi Trifolium bao gdm cd Ba la do, T. pralense L., va gan day bao cao ciia Haliloglu (2006) da cho thay kha nang phat sinh phdi vd tinh tir nudi cay m i u la Khoai mi ttong mdi trudng cd sy ket hgp cua auxin va cytokimn. Nhu viy, sy kfit hgp 2,4-D hay NAA vdi mdt loai cytokinin dugc cho la rat can thiet de kich thich phat sinh phdi vd tinh d mdt sd thyc vat. Mac dii phin ldn sy phat sinh phdi vd tinh dugc kich thich bdi viec sii dung auxin va cytokimn nfing lfi hoac kfit hgp ttong mdl trudng nhung khdng phai sy kfit hgp nao cua cytokinin va auxin deu dan den sy hinh thanh phdi vd tinh, Trong thi nghifim nay, chung tdi sir dung 2,4-D va NAA kfit hgp vdi kmetin cho hieu qua ti lfi phat smh phdi rit cao va cao nhlt d nghifim thiic E M U . Ket qua nay phu hpp vdi bao cao ttfin loai Pinus taeda (Vanildo et al, 2004) va ttfin cay Bdng vai (Hamidou etal, 2005).
Anh huong cua spermidine Ien kha n i n g gia tang tan s u i t p h i t sinh phoi vfi tinh s i m Ngpc Linh
Polyamine thyc chit la mpt dang cua acid amm va no duoc xem nhu l i radt chit difiu hoa sinh trudng. Do dd, chiing tdi da dua spermidine la mdt loai polyamine vao thi nghifim vdi myc dich khao sat kha nang phat sinh phdi tir md seo. Kfit qua thi nghiem cho thay khi bd sung spermidine vdi ndng do thap vao mdi trudng, rad seo dugc cam ting r l t
nhanh sau 4 tuan nudi cay. Tren be mat md seo da t h l y x u i t hifin l i m t i m nhiing phdi hinh ciu nho va sd lugng phdi tang d i n len. Sau 12 t u i n nudi ciy, s6 lupng phdi xuit hien nhifiu ttfin bfi mat, cd dii cac hinh dang nhu phdi hinh cau, hinh tim, hinh thuy I6i, hinh hai la m i m , . . . Phdi co raau ttang sang, khong t h l y xuit hifin phdi bien di. Khi tang d i n nong dp spermidine thi cac chi tifiu dugc ghi nhan ciing tang va d nghiem thiic bd sung spermidine vdi ndng do 0,1 mM cho hifiu qua cao n h i t vfi ti lfi phit sinh phoi (93,3%) va sd lugng phdi hiiih thanh tten mau (353 phdi) (Hinh 5, Hinh b2, h^, ha). Tifip tuc tang ndng do spermidine Ien 0,2 mM thi ti lfi m i u hinh thanh ph6i va so phdi hinh thanh tten mau giam xudng. Ket qua nay tuong tu vdi ghi nhan cua Kevers va ddng tac gia (2000) ttfin ddi tugng Panax ginseng. Nhung trfin cay C i phfi thi ndng dp thich hgp la 0,05 mM, tten cay Momordica charantia L. thi ndng do thich hgp la 0,001 mM (Ananya etal, 2009).
Polyamine dong vai ttd quan ttpng ttong si; bi$t hoa tfi bao dfi hinh thanh phdi (Rajam, 1997). Tuy nhifin, ca che chinh xac ciia nd thi chua dupc hieu mpt cich rd rang va den bay gid v i n cdn dugc tifip tuc nghifin cdn. O ndng dp cao, polyamine co tac dyng giiip cho tfi bao ldn len va phan chia. Trong giai doan cam iing phdi, polyamine se giiip cho tS bao vung md phan smh p h i n chia nhanh chdng vi giiip cho phdi dugc hinh thanh, Ndng dp polyamine giam cd thfi gia tang c i c tfi bao md seo nhung se lam giara sy hinh thanh phdi. Do do, viec sir d\ing polyamine kfit hpp thfim vdi c i c chat dieu hda sinh trudng khac ttong mdi trudng nudi c i y mang lai h i ^ qua v i cd y nghia ttong giai doan phit sinh phfli Cling nhu cic giai doan hinh thanh cay hoin chinb ft thyc v i t (Kevers et al, 2000).
78
Tgp chi Cong nghf Sinh hQC 12(1): 73-84,2014
400 350 300 250 200 IKI
50 0 •
353
OOlmM 5 3 . | H
O.lSmM
r,., H
^|p^
O l m M
-^
0 2mM
• Ti 1$ phat sinh pboi (%}
• S6ph6i Trong lugng (mg)
Hinh 5. Anh hufi-ng ciia spermidine Ign k h i nang gia tang tang tan suat phat smh ph6i va tinh sSm Ngpc Lmh
Spermidine la mdt polyamine mang tinh dac hieu dugc dung cho su phat sinh phdi vd tinh tit md te bao Ca rdt (Feirer et al, 1985) v i ttong su6t cic giai doan cam iing ciia m i n h ciy cd Dinh lang (Cvikrovi et al, 1999). Monteiro v i ddng t i c gii (2002) ciing d i khang dinh dugc vai ttd cua polyamine, dac biet la spermidine trong sy phat sinh phoi vd tinh Panax ginseng. Nhiing nghifin ciiu tten rad s?o cd k h i ning phit sinh phdi ciia Panax ginseng khi sii dyng spermidine cho hifiu q u i tuong doi tot (Kevers et al, 2000). Viec sd dung polyamine ket hgp vdi nhom auxm hay cytokinin giup cho rad nudi cay phat sinh phdi va nang cao tan suat tao phot. Trong nghifin cdu niy, chung tdi da ghi nhan dugc vai ttd cua spermidine trong vific ning cao tan suit phit sinh phdi vd tinh d cay sam Nggc Lmh khi sii dirng d ndng dd 0,1 mM kfit hgp vdi 1,0 mg/I 2,4- D, 0,5 mg/I NAA v i 0,2 mg/l kmetin.
Anh hudng ciia proline ifin kha nang gia tang tan s u i t p h i t sinh phdi vd tinh s i m Ngoc Linh
Vific bd sung cic acid amin ngoai smh vao mdi Irudng nudi cay dugc bao cao l i cd tic dyng thuc day sy phat sinh phdi vd tinh d mdt sd Ioai thyc vat (Wang et al, 2002). Amino acid la mgt ngudn nita hihi ca ( d ^ g khu) dugc chuyfin hda rat nhanh ttong t i bio thyc vat, kich thich Ifi bao sinh tnrdng va phat tri^n nhanh hon (Grimes, Hodges, 1990). Vi v i y viec b6 sung amino acid vao mdi trudng nudi cay cung cip cho tfi bao v i md mdt ngudn nito hiru ca phii hgp d mpt miic dd nhit dinh. 6 cac loii thyc vat khic nhau thi nhu ciu ve cic chat bd sung nhu amino acid v i Ihanh phin ciia cac chit dieu hda sinh trudng ciing khic nhau.
Ket qua Ihi nghiem cho thiy vific bd sung proline cd anh hudng tuang ddi tdt Ifin sfi lupng phdi vd tinh dugc hinh thinh sau 12 m i n nudi ciy, phdi dugc tao thanh tuong ddi ddng nhat. Khi tang din ndng dd proline td 100 mg/l Ifin 300 mg/l thi ti lfi mau t^o phdi, sd phdi hinh thinh tren miu va trpng lugng ciia mau cung tang dan. O nghiem thiic bd sung 300 mg/l prolme vao mdi trudng nudi cay, ti le m i u tao phdi cao nhit dat 86,7%, sd phdi hinh Ihinh tten mau khoing 167 phdi, ttpng lugng tuoi ciia mau la 40,55 mg. Khi ting ndng dp prolme Ifin 400 mg/l thi cic chi tifiu giam xudng ro ret, ti lfi miu tao phdi chi cdn 66,7%, sd phdi hinh thanh trfin mau khoang 83 phdi, trong luong tuoi cua miu l i 30,90 g (Hinh 6). Khi ndng dp proline qua cao se cd tic dung lic che su hinh thanh phdi vd tinh tu cic miu md seo Trong thi nghiera nay, moi truimg MS bd sung 1,0 mg/l 2,4-D, 0,2 mg/l kinetin, 0,5 rag/1 NAA v i 300 mg/l proline la nong do tdi uu anh hudng den kha nang c i m img phal sinh phdi vo Iinh va nang cao tan suit phit sinh phdi vd tinh thdng qua nudi cay md seo sam Ngpc Linh.
Proline rifing le hay kfit hpp vdi cac amino acid khac cd tac dimg kich Ihich phal smh phdi d cac loai thyc vat khac nhau nhir ttong trudng hgp cay Dau ninh v i Diu xanh (Shetty, McKersie, 1993), Proline dugc xera nhu li ngudn nita khu quan ttpng dugc chuyfin hoa va hap thu rat nhanh ttong te bao thuc vat, ngoai ra proline dugc xera nhu l i mdt ngudn nang luong san cd cho tfi bio Ben canh do, proline cdn cd chiic nang quan ttpng khac nhu l i bao ve cac enzyme chdng lai su suy thoii, difiu khifin ndng dp acid ttong cylosol. Ndng dp cua proline cd lac dung nhu li mpl chat dem ben ttong Ie bao chat, chdng lai sy gia Iang ndng dp cac ion ttong
79
Biii Van The Vinh et aL khdng bao, cd chuc nang nhu mpt chat difiu khifin cau
tnic cua nudc, lam tang tinh tan ciia protein vi cic phan lu sinh hpc khic dudi difiu kifin thfi nuac giam (Schobert, 1977).
Trong thl nghiem niy, chung tdi nhan thay vific sd dyng proline khdng lac dyng manh dfin hieu qui phat sinh phdi vdi tin suit cao nhu spermidine. Trong 4 ndng dp dugc thir nghifim thi d nghifim thiic bd sung
300 mg/l proline cd anh hudng dang kfi nhat dfin ti le phit sinh phdi va sd lugng phdi tao thanh. Ket qui nay khac vdi ghi nhan cua Manosh vi ddng tic gia (2007) tren ddi Uigng Dau tay (ndng do tdi uu la 500 mg/lX trfin ddi tugng-cay Dau phung (ndng dp thfch hgp la 100 mg/l), Victoria va ddng tic gia (1984) trfin dii Uigng cay Ca rdt (ndng dp thich hpp la 100 mg/l).
Difiu nay cho thly d mdi loii thyc v^t khac nhau thi prohne cd tic dyng d cac ndng dp khic nhau.
180 160 140 120
80 EO 40 20
''
,.-
arJr mZm^
m\MV
1 0 0 m g / l 2 0 0 m g / l 1671 1 1 1
mdt • T B T -
llJa
300 mg^ 400 mg^
• Tl Is phat sinb phoi (%)
Trflng hnmg (mg)
Hinh 6. Anh hudng cua proline Ign kha nang gia Iang tang tuan suit phit smh phoi vo tinh sim Ngoc Lmh
Anh hudng cua nguon carbohydrate Ifin khi ning gia tang tan suit phit sinh phdi vo tinh sim Ngpc Linh
Tfi bio, md hay co quan nudi cay thyc vit thdng thudng ddi hdi cung cip mdt lugng carbohydrate dfi thda man nhu cau vfi nang lugng cin thifit. Trong nudi cay rao, carbohydrate hoat ddng nhu la nhung tic nhin thim thau va hd trg cho sy phit trien tfi bao, Ngudn carbon chu yfiu dugc bd sung ttong radi trudng nudi cay la dudng, cac tfi bao thuc vit se su dyng ngudn dudng niy de tao nen bd khung carbon phuc vu cho vific tang trudng cung nhu phat sinh co quan, Dudng ciing la mdt yeu td quyfit dinh kha nang khdi phat cac co quan cua md cay.
Thinh phan carbohydrate ttong mdi ttudng nudi cay da dugc chiing minh li cd tic dpng den sy phat sinh phdi vfi tinh d nhieu loai thyc vit (Lou, Kako, 1995). Ket qua trong Ihi nghiera nay cho thay ca 3 loai dudng sir dung dfiu cd kha nang cam img phat sinh phdi, Tuy nhifin, ti le phat sinh phdi khi su dyng dudng sucrose la cao nhat, dat dfin 86,7%, ttong khi dd dudng glucose chi dat 56,7%, dudng fructose cho ket qui Ihap nhat, chi dat 40% (Hinh 7). Rieng chi tifiu sd phdi tao thinh tren mau, dudng sucrose cung cho kfit qua cao nhil (dat 167 phdi/mau), dudng glucose chi dat 75 phdi/mau, dudng fructose thip
nhlt (dat 35 phdi/miu) (Hinh 8). Ti lfi phit sinh phoi, sd phdi tao thinh ttfin miu deu tang dan cung vdi s\r gia tang ndng dp dudng vi dat kfit qui cao nhat la 6 ndng dp 50 g/l, sau dd giim din khi tifip tuc ting ndng dp.
Trong nghien ciiu nay, sy gia tang ndng dp dudng
thuc day sy gia tang cic cym tifin phdi va phdi vo Iinh,
difiu niy gidng vdi nhung nghifin ciiu da dugc bao cio
ttfin nhifiu ddi tugng khic (Luo et al, 1996). Mo s?o
hay cic ca quan nudi cay cua thyc vat ddi hdi phai co
su hgp nhlt cua ngudn carbon ttong moi trudng nuoi
ciy vi sucrose dugc sii dyng nhu la ngudn carbon
chinh ttong nudi cay md (Fuentes et al, 2000). MSc
du sucrose dugc diing nhu li ngudn carbon chinh
ttong qui trinh phit sinh phdi vd tinh nhung cd mpl
vai nghien ciin da chung minh dugc sg can thiet co
mdt ndng do carbohydrate cao, nd khdng chi ddng vai
ttd li ngudn dinh duang mi cdn thfi hifin tinh chat U
difiu hda thim thiu. Do do, ndng dg dudng cao khfing
nhiing cd vai ttd ttong sy phit sinh phdi vd tinh oi
cdn cd thfi tic ddng dfin sy tham thau tfi bao. Nong dp
dudng sucrose cao lie ddng dfin khi nang tham ihfiu
ciia te bio ttong qui trinh phit sinh phoi vd tinh cflng
dugc dfi cap bdi rapt vii nhdm nghien ciiu {May,
Trigiano, 1991). Do dd, vai ttd ciia dudng sucrose
ttong nghifin ciiu niy lim sing ha hai chiic ning ciia
Tgp chi Cong ngh4 Sinh hgc 12(1): 73-84,2014
carbohydrate, dd l i chuc nang dinh duong va chiic vdi mpt sd kfit q u i dugc thyc hien tren cay Asparagus ning difiu hda thim thiu. Kfit qua nay ciing phu hpp officinalis L. (Kanji, Yuji, 2000),
TII#phatainhptioi(%)
— Glucose
— Fnitose
10 mg/l 20 mg/l 30 mg/l 40 mg/l 50 mg/l 60 mg/l Hinh 7. Anh h u d n g cCia ngu6n carbohydrate len ti I? phat smh phoi vo tinh sam Ngoc Lmh (%)
S6 luang phoi
10 g/l 20 g/l 30 g/l 40 g/l 50 g/l 60
Hinh 8. Anh hudng cua nguon carbohydrate len so lu'gng phoi vo tinh sam Ngoc Lmh hinh thanh
KET LUAN
Mdi trudng thich hgp nhit dfi cam irng tao phdi vd tinh tli miu ciy la ciy sam Ngoc Linh in vilro la mfli Imdng MS cd bd sung 1,0 mg/l 2,4-D kfit hpp vdi 0,5 mg/l NAA va 0,2 rag/l kmetin trong difiu kifin chifiu sing 16 gid/ngay. Viec bo sung spermidine (ndng dp 0,1 mM) thupc nhdm polyamine hay proline (ndng dp 300 rag/l) thupc nhdm acid amine cd tic dung kich thich gia tang tin suat phit sinh phoi ciing nhu so luong phdi vd tinh hinh thinh, Vific sir dyng cic ngudn carbohydrate khic nhau nhu sucrose, glucose hay fructose dfiu cd khi ning cara iing sy phit sinh phdi vd tinh d sam
Ngoc Linh, tuy nhien, dudng sucrose d ndng dp 50 g/i cho hifiu qua c i m img sy hinh Ihanh va phat trifin phdi Idt nhat Cay con cd nguon gdc ni phdi vd tinh khi dugc chuyen ra vudn uam dat ti le sdng sdt cao (Hinh9di,d2, ds).
Lin cam on: Cdc ldc gid xin chdn thdnh cdm ffn de tdi:U'ng dung he thong chieu sdng dan sdc (LED) trong nghien cicu nhdn nhanh cdy sdm Ngoc Linh (Panax vietnamensis Ha et Grushv.) ven so lugng ldn phuc vu nhu cdu nhdn giong ciia tinh Qudng Nam (Vien Hdn ldm Khoa hgc vd Cong ngh$ Viet Nam) da ho tra kinh phi cho de ldi nghien ciru ndy.
Bui V i n The Vinh el al
Hinh 9. Qua trinh phat sinh phoi vo tinh sam Ngoc Lmh {Panax vietnamensis Ha et G m s h v ) . a,, aa, a j Mo s?o dugc hinh thanh t i i m l u cSy la, cuong i i va cu cua cay sam Nggc Lmh in vitro sau 8 t u i n nuoi c^y, b i , b?, b j , bj: Phoi v6 tinh flu^c hinh thanh ti> mo seo d cac thCTi d l l m 0 , 4 , 8 v a 12 t u i n nuoi cay tren moi t n r a n g MS bo sung 1.0 mg/l 2,4-D, 0.5 mg/I NAA, 0,2 mg/l kinetin va 0,1 mM spermidme, Ci, C;, Cj, C4 Phoi vo tinh sam Ngoc Lmh a cac giai i?oan hinh cau, hinh tim, hinh thuy [61 va la m i m , d i , d j , d j cay con sam Ngoc Lmh c6 ngu6n goctCi' phoi v o t i n h trong ong nghiem, chuan b| dua ra vi/tm
\yom va sau 3 t h i n g trong thLP nghiem.
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F A C T O R S E F F E C T I N G S O M A T I C E M B R Y O G E N E S I S O F N G O C L I N H GINSENG {PANAX VIETNAMENSIS H A ET G R U S H V . )
Bui Van The V i n h \ Vu Thi Thuy', Thai Thuong H i e n \ Vu Quoc Luan', Nguyen Ba Nam', Nguyen Phuc H u y \ Trinh Thi Huong', Vu Thi Hien', Le Kim Cuong', Ho Thanh Tam', Do Manh Cuong', Nguyen Viet Cuong', Do Khac Thinh^ Duong Tan Nhut"
'Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology
^Institute of Agricultural Science for Southern Vietnam, Vietnam Academy of Agricultural Sciences
SUMMARY
Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) is a precious and economic medical herbal of Vietnam, In recent years, with micro-propagation and conservation of this herb tendency, plant regenerarion is a necessary problem, in this, research about somatic embryogenesis play an important role. Therefore, this study was conducted for estimating affection of explant sources, mineral component of culture media, type, plant growth regulators, spermidine (polyamme groups), proline (amino acid groups) and carbohydrate sources on somatic embryogenesis of Ngoc Linh ginseng With three explant sources such as leaf, petiole and main root, leaf explants having 0,5 x 0,5 cm size were cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg.f' 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg.l'' kinetin and 0.5 mg,!'' naphthaleneacetic acid (NAA) to establish embryogenic culture. The effects of exogenous spermidine and proline on enhancement of somatic embryogenesis were investigated. The results showed that spermidme (0.1 mM) and proline (300 mg.l'') resulted in a high frequency of somatic embryogenesis (93.3% and 86.7%, respectively). To fiirther optimize a culture medium for induction of embryo formation of P. vietnamensis, three carbohydrate sources (sucrose, glucose and fructose) from 10 to 60 g.f' were tested. Among them, glucose and fructose were not suitable for somatic embryogenesis in this species while sucrose at 50 g.l"' produced ttie highest embryogenic frequency (86 7%) and number of embryos per responding explant (167).
This study confirmed the importance of spermidine, proline and osmotic potential provided by sucrose in enhancement of somaUc embryogenesis.
Keywords: carbohydrate.Panax vietnamensis, proline, somatic embryos, spermidin
'Authorfor correspondence: Tel: +84-63-3831056; Fax: +84-63-3831028; E-mail: duonstannhuKi
