Tgp chi Cong nghe Sinh hpc 9(3): 317-331,20li
N G H I E N C m j M Q T S O Y E U T 6 T ^ O C U S A M N G Q C L I N H (PANAX VIETNAMENSIS H A E T G R U S H V . ) IN VITRO V A X A C D I N H H A M LU*ONG S A P O N I N T R O N G C A Y T A O TU" C U T R O N G T H t T N G H I E M 6 N U I N G Q C L I N H
H o i n g XuSn Chi^n', Ngd T h a n h Tiii', Nguyen Bd Tr^rc', T r i n Xufln r i n h ' , L i m Bich T h d o ^ T r a n Cong Lu§ii% Dirong T i n Nh^rt'
' Vien Sinh hpc Toy Nguyen
'Trung tdm Sdm vd Dupc lieu thdnh p/id H6 Chi Minh
Sam Nggc Linh Id mdi cdy thu6c quy ciia Viet Nam, vdi no lye bdo ton vd phdt t r i k lodi dugc lieu qu^
nay thi ung dyng ky thu^t nuoi ciy mo dugc xem nhu Id mgl gidi phdp hieu qud. Viec tao cu in vitro gdp phan lam tang siirc song cua cdy. nSng cao ty le sdng sdt khi dira cSy ra ngoai vudn uom. Trong nghien cihi ndy, quy trinh tgo cii in vitro vd xdc dinh h^ni lugng saponin o cdc cdy tring ihu nghiem 17 thdng tu6i da dugc trinh bd^. Cdc h? phiic hgp ciia GA/ABA vd auxin/cytokinin trong qud trinh hinh thdnh cii cung da dugc khao sdt.
Nong dp ABA thich hgp nhdl cho qud trinh t^o cii la 3,0 mg/l. GA3 lic chi kha ndng tao cu in vitro cay sdm Nggc Linh. Cdc chdi cay sdm Nggc Linh in vitro dugc cam img da lao cii thdnh cong trSn moi trudng SH co bi sung 1,0 mg/I NAA vd 2,0 mg/l BA trong dlfiu kien chieu sang 16 gid/ngay. N6ng do dudng sucrose tit nhit cho qua trinh t?o cu sam Id 50 g/I. Ham lugng saponin trong cii ciia nhihig cay 17 thang tudi nuoi tring ngoai tu nhien cung dd dugc xdc djnh la G-Rb, (0,21%), G-Rg, (0,17%), MRj (0,77%).
Tir khoa: ABA, cu. GA. G-Rb/, G-Rgi, MR2. Panax vietnamensis. saponin, sdm Ngoc Linh DAT VAN BE
Sam Nggc Linh la mgt loai sam ddc hihi cua Viet Nam vdi ten khoa hgc la Panax vietnamensis Ha et Gmshv. Dii chi mdi dugc biit din tir nam 1973, nhung qua nghifin curu cac nha khoa hgc da nhan thay sdm Nggc Linh khdng chi cd cac tac dung dugc ly dac trung ciia chi Nhan sam md cdn cd nhOng tac dung dien hinh nhu chdng stress, chdng trim cam, tac dyng len sy chdng oxy hda in vitro vd in vivo... Nhdm chat cd vai trd quyet dmh nhdt den tac dyng dugc ly ciia lodi sam nay la cac saponin triterpenoic ma dai dign chinh la MR2, G-Rbj va G- Rgi (Tran Cdng Lu^n, 2003). Sdm Nggc Linh la mgt trong nhjjng loai sam cd ham lugng saponin khung dammaran cao nhat (khoang 12 - 15%) vd lugng saponin nhifiu nhit so vdi cdc loai khac cua chi Panax tren the gidi (Nguyen Thugng Dong et al, 2007). Vdi nh&ng d^c diem dd, sam Nggc Linh khong chi la loai sam quy cua Vi?t Nam ma cdn cua cd the gidi.
Nhan gidng sam Nggc Linh g^p nhieu khd khan do chi trdng dugc d khu vyc quanh dinh nui Nggc Linh, thdi gian nudi trong keo dai tii 6 dfin 7 ndm cu mdi tich triJ du hoat chit d i thu hoach. Nhan gidng h&u tinh theo each thdng thudng (gieo h^it) khdng cho k i t qua cao vi nhiiu 1^ do: khd thu nhan hat, hat
khi gieo nam trong dat sau 1 thdi gian dai mdi nay mam, vi vay hat bi cac loai ddng vat, cdn tning gam nham dn... ngodi ra ty le nay m i m td hat chi dat khoang 30 - 40%. Theo sd lieu diiu tra mdi nhat, sam Nggc Linh da bi khai thac qua mdc, dudng nhu khdng cdn thay trong tu nhien va chiing dang nam trong danh myc 250 loai quy hiim cin dugc bdo ve.
Do do, yeu cau cap thiet la tim dugc ky thudt mdi giup nhan gidng nhanh va dem lai ngudn sinh khdi cd hieu qua lay tir loai cay dugc lieu nay. Trudc day da cd mgt sd nghien cuu trong nude thuc hien nham nhan gidng in vilro loai dugc lieu nay, tuy nhien hifiu qud nlian giong va kha nang sdng sdt ciia cay con khi ra ngodi tu nhien la khdng cd vi cay yeu va khdng tao dugc cu con d phdn goc, khi dp dung trong thir nghiem thi 100% cdy deu chet (Nguyin Nggc Dung, 1995). Tir nam 2006 din 2010, Duong Tdn Nhyt va ddng tac gia da budc dau nghien ciiu nhan nhanh re thir cap ciia sam Nggc Linh; su dung ky thuat HPLC (High-performance liquid chromatography) nham xay dung phuong phap dinh lugng ginsenoside-Rgi, Rb| va MR2 trong dich chiit sinh khdi sam Nggc Linh; tim ra dugc mgt sd yeu td dnh hudng den sinh khdi cua cay sam Nggc Linh nudi cay in vitro va budc dau phan tfch ham lugng saponin, nhan gidng vd tinh thanh cdng cay sam Nggc Linh, xac dinh dugc ham lugng saponin va du lugng mdt sd chat
jloaiig AiKin I. liicnc, dieu hda sinh trudng trong md s^o, chdi, rl sdm
Nggc Linh nudi cay in vitro vd budc diu dd Ihu dugc mgt sd ket qui khd quan.
Tir kil qud cila nh&n§ nghiSn ciiu IrSn v& qua cac khdo sdi dd cho thiy ring, m^t trong nh&ng d^c diem dan din sy khdc bi^t gi&a cdy s6ng sdt khi trdng ngodi ty nhien vdi cSy bj chet chinh Id cdy phdi cd cii phia dudi g6c. Cii ndy vira ddm nhi§m vai trd la CO quan dy IrO nguin dinh dudng cung cip cho cay khi ra ngodi ty nhien. vua Id c o quan sinh sdn chua cdc choi non giiip cay ndy mam khi ryi Id. Theo nhDng khdo nghiam ban dau ciia Nhyt vd d^ng tdc gid (2010), nh&ng cay sdm Nggc Llnh khi dua ngodi vudn uom neu cd cu phia dudi goc thi khd nfing song sdt rat cao tren 90%.
Chinh vi v§y, bdng k j thu$l nudi c i y in vitro, chiing ldi da nghien cuu quy irlnh tao cu cdy sam Nggc Linh giiip hodn thign quy trinh nhan giong cay sam in vitro giiip cdy con khi dua ra ngodi vudn uam cd siic sdng cao. re vd cii phdt triin tot, thich nghi tot vdi mdi tmdng ben ngoai, gdp phin cdi thign vd tdi tao dugc mgt lugng cay dd mat di trong ty nhien.
Vdi uu the nhimg cay cd cii nay chiing ta cung cd the cd hy vgng trong vi?c di thuc cay sdm den cdc khu vuc cd dieu kien ty nhien tuong tu d i trdng va phdt triin. Vdi ky thuat HPLC chiing tdi cung dd xdc djnh dugc ham lugng saponin cd trong cdc cay cd cu, dam bao rang cay nudi cay md van cd du hoat tmh khi trdng ngoai tu nhien.
ii'iiiy dg khac nhau Sau iiifirng lir 5.7 den 5.S
i ,(• ptionii lln ii,jliK'ni \<u nliicl do 25 ± 2"
ui<.[i!J .In ihiL-ii s;iii.u klin.int; 2SIHI - 3(100 lu^. ih
I'liiion^plDip
hhuri '•lit rd, mai irinnn: Ahount; t/.V. '.• W.V, .V) ' SII ill ho sun-: Ih.-iii 0.^ niii/l Bl. 11.5 nifi/l .\,l I 1-/I AC. ill 1^.1 sinrou'
Mni ii,„rii,. MS, VMS. '/SII v;i SH a> thar ph.iii Ml.iniiii. klioang da lugng vj \i luang khj iih.iii (laiiu kc Dl) dd. nuic ticu cua ihi nghiem nay I s(i sanh anil luronj; iiud mdi trudng MS va m{
iMiirti;.' SII IIL-11 kha iianii lao cu cay sam Ngoc Lin /;; I iiin I.nil i n ^u t h e cat nghien cim cac chat die lina iK']i Ihio ( at till licii nhif Iv Ic tjo CU. trpn lifi'iiL: linn, tlucii L,IU tiia chdi dirm; thu nhan sa
Uikinin tli'n si hinh ihiniL
(.at J i (ImiL ta> \:iii moi imtmg SH ci bn suni^ ^n y I AVSO\K ^ut^^^c. I g I AC \a bo sun|
noni; do N \ \ m o ii,.\ I ,(i, 2.11 mg l u a BA|0.0 11,^. 1,(1, :.ll mg ll iTLjt. iighii-m thuc khac nhau, Cai till lieu iilur t> Ic lao cu irong Immg tirm. chieuca(
tua thoi ilirgu Ihii iiliaii -^aii S man nudi ca\
VAT Li$U VA PHirONG PHAP Nguon m i u in vilro
Ngudn mau sir dyng la chdi cay sam Nggc Linh in vilro tuong ddi ddng nhit vd cd chiiu cao khoang 1,5 cm, trgng lugng tuoi khodng 0,2 g dd dn djnh qua 4 den 5 lan c i y chuyin, hign cd l^i Phdng Sinh hgc Phan tir va Chgn Uio Gidng cay trdng, Vign Sinh hgc Tay Nguyen.
Mdi trudng nudi c i y
Mdi tmdng MS (Murashige. Skoog, 1962), '/iMS (Mdi tmdng MS cd thanh phin da lugng gidm mgt niia), SH (Schenk, Hidebrqndt. 1972) vd /2SH (Mdi tmdrjg SH cd thdnh ph/i.n da lugng gidm mgt niia) cd bd sung them 30 g/I dudng sucrose, g g/1 agar, 1 g/1 than ho^t tinh (AC), cac chit dieu hda sinh tmdng thugc nhdm auxin (NAA), cytokinin (BA), gibberellin (GAj) tiiy thugC vao tihig thi
Khao sal ndng dp cua IB t len \ir hinh thdnh. s/n/
truoni- vd phdt trien t ua cu sdm \gpi Llnh in vitro ( at LIUH s.iiii Ngoi. l.inh dugc cay vao moi Huong s | | t o hi> •,iiiig 'll g I dutmg sucrose, 1 g'l
\ l , \.i \ | l \ o cat nong do ur 0 den 4 mg/l Cac chi iicii nhir l\ Ic lao cii. irong lutmg tuai. chieu cao ciia ilioi tinot ihii nh.in sau S luan nudi cay hhuii \ai linh hirim!- cua (7-tj len sir hinh thanh.
.-.inh triiiiiii; ui phdi irien cua cu sdm ^goc Linh (.at- choi s;im Ngoc Linh dugc cay tren nioi 11 none s i l t o bd -.niig M) g 1 dudng sucrose, 1 g'l AC va (. \ , o cac ndng do lir 0 den .1 mg'l. Cac chi tieu nhu l\ Ic tao cu. trong lin.mg luoi, cliicu cao cua choi ihrgc ihu nhan sau S lii.in nudi t.H
Khao sdt ndnfi dp sucrose len su hinh thdnh. sinh trinrng vd phdt trien cua vu sdm .\gpc Linh in vitro Cac chdi sam Ngoc Linh diroc ciy tren moi
Tpp chi Cong nghe Sinh hpc 9(3): 311-331,2011
tmdng SH cd bd sung dudng sucrose d cdc ndng dg tii 15 den 70 g/1. Cac chi tiSu nhu ty 1? tgo cii, trgng lugng tuai, chiiu cao cua chdi dugc thu nhan sau 8 tuan nudi cay.
Phdn tich hdm lirpng saponin Irong cSy sam Ngpc Linh sau 17 thdng trdng ngodi vw&n uam
Cdc cay sdm Nggc Linh in vilro cd cu dugc trong tai khu vyc xa Te Xdng, huy^n Tu Mo Rdng, tinh Kon Turn sau 17 thdng sinh tmdng va phat triin dugc thu nhan va sir dyng cho thi nghi?m nay.
Bdng 1. Chuo'ng trinh h$ pha d^ng nira gidi.
T h v i gian 0 - 1 0 phut 1 0 - 2 0 phut 20 - 40 phiit 40 - 60 phiit 60 - 1 0 0 phOt
A C N t N i n y c 1 5 . 8 5 - 2 5 : 7 5 25.75 - 30:70 30:70 - 60 40 6 0 4 0 - 80.20 80:20
C h i K m g t r i n h Ti$m ti4n T($m ti4n Ti$m tl^n Ti$m t i l n D i n g mdi
Diiu kien cho he thong HPLC (High-performance liquid chromatography)
Thi tich tiem mau: 20 jtl. Tdc do ddng: 0,5 ml/phiit. Cot RP C18 Supelco (250 mm x 4,6 mm - 5 nm). Nhiet do cot; 25°C. Detector; PDA d budc sdng phat hien MR,. 190 nm; G-Rb, va G-Rg,: 203 nm. Pha giai mdu cdc chuin trong dung mdi ACN:Nudc (70:30) cd ndng dg nhu bang 2.
Tiem cac dung dich mlu chuin vao mdy HPLC vdi chuang trinh rira gidi, ghi nhan dien tich peak va dyng dudng bieu dien mdi tuong quan giua ndng do va dien tich peak.
Bang 2. Nong do giai mlu cdc chit chuin.
srr
1 2 3 4 5
MR2 72 144 216 288 360
((ig/ml) G-Rg, ((ig/ml) 19,4 38.8 58,2 77.6 97
G-Rbi (jig/ml) 24 48 72 96 120
Chuan bi mlu thir
Can khodng 0,5 g sam, chiet sieu am vdi methanol cho din khi ki§t hoat chat, Igc. Tap tmng djch chiet cd quay dp suat gidm den cdn. Hda can vdi 20 ml nude cat, sieu dm cho tan hit can. Dich nude
dugc ldc vdi ether ethylic (10 ml x 3 idn), logi djch ether ethylic. Djch nude dugc ldc tiip vdi n-butanol bdo hda nude cho den khi ki?t hoat chit. T|ip tmng djch n-butanol bdo hda nude cd den cdn. Cdn ndy dugc hda l^i trong hon hgp acetonitril vd nude ty 1^
(70:30), djnh miic chinh xac 5 ml Igc qua mdng 0,45 nm. Djch Igc dugc tiem vao mdy HPLC ghi nhan di^n tich peak
Cdng thdc xdc djnh ham lugng:
;r(%) = cx5x-jcio"'
p
Trong dd: C; Ndng dg mdu thu thu dugc dya trfin dudng chuin (ng/ml). 5: Dg pha lodng mau, b Khoi lugng 100 g nguyen li^u (g). p: Khii lugng nguyen ligu dem thii da trir im (g).
Xu 1^ so li^u
Mdi thi nghiem dugc lap lai 3 Idn, sd li?u dugc xir ly bing phin mem Microsoft Excel va phuang phdp Duncan's test (Duncan, 1995) vdi P = 0,05.
KET QUA VA THAO LUAN
Khao sdt cdc mdi trudng khoang MS, '/iMS, SH,
•/iSH c6 bo sung them 0,5 mg/l BA, 0,5 mg/I NAA, 1 g/I AC, 30 g/1 sucrose.
Sau 3 thdng nudi cay, cimg vdi sy phat triin cua cdy, tir goc cua cdc mau cay bit diu xuit hi^n cac cii d hau het cdc mdi trudng. Sdng chii y la d mdi tmdng SH ty le tao cii la rdt cao (90%) gdp ddi so vdi mdi tmdng MS. Cac chi tieu khac nhu chiiu cao va trgng lugng tuoi ciing cao hon so vdi cdc mdi tmdng khdc (Bdngp). Cii hinh thdnh tren mdi tmdng nay cung to hoti han so vdi tren mdi tmdng MS va /2MS, cii CO it chdi, chdi cd Id xanh tdt (Hinh 1),
Trong_ mdi tmdng SH ham lugng cac vitamin
cao hon rat nhieu so vdi mdi tmdng MS, ddc bi?t Id
thiamine (Bl) cao gdp 50 lin va myo-inositol gdp 10
lan. Hau het cdc md nudi cay deu cd kha ndng tdng
hgp tat cd cdc loai vitamin can thiet nhung khdng
day du ve sd luong. Vitamin cd vai trd xiic tac cdc
qua trinh trao ddi chat diln ra trong te bdo, cho nen
mudn d^t dugc sy sinh trudng tdi uu cua md nudi
cay thi cac nhd nghien ciiu thutmg dua them vao mdi
tmtmg mgt sd vitamin nhu: thiamin (vitamin Bl),
nicotinic acid (vitamin B3), vitamin B5, pyridoxine
(vitamin B6). Trong sd dd vitamin Bl dugc coi la
vitamin thiet yeu ddi vdi su smh trudng va bien
319
dudng cua te bdo thyc v^l. Sy phosphoryl hda biin thiamin thanh thiamin-pyrophosphate, chit ndy Id cocarboxilase tucmg dng vdi sy khir carboxyl cua cdc cetonic acid. Vitamin Bl dugc long hop irong 1^
hay cdc chdi non (dnh sdng Id y i u t i thiit yeu), Myo- inositol (hay meso-inositol) Id mOt d i n g phin ciia inositol, cd vai trd quan irgng trong sy truyin tin hi?u hormon vti lien kit vdi auxin (Bin Trang Vi?L 2002), Trong mgt so nghien cdu ve sam. dflc bi?t Id
Bdng 3. Anh hudng ciia cdc mfll Iru'&ng khodng MS, l ^ S , SH, %3H din khd ndng hinh thdnh cO sdm Ngpc Linh- Hoang Xuan ChiSn elai nghifin ciru cua Pack vd ddng tdc gid (2000) thi ni6 trudng SH lai td ra rit hi^u qud de nudi ciy rl bi djnh cdy Nhdn sSm trong h0 thong bioreator. Pdtkvi ddng tdc gid (2000) cung dd bao cdo trong so c^
mdi trudng thii nghigm thi mdi trudng SH cho s6 re trung binh cao nhit vd sy tdng trudng ciia re thi ehjij dnh hudng m^nh bdi cdc chat dieu hda tdng tnrdng thyc v$t cung nhu c i c muoi c o ban cua mdi tmdng thir nghiam.
M6t tnj>d>ng MS VMS
SH ' . S H
Ty 1$ t f o cu 43,33%
65,72%
90%
61,50%
(%)
Chi&u cao 2,89cd*3,51b
4,083 3,01c
(cm) Trgng l u ^ g tvcr\ (g) 0,20d
0,4Bb
0,53a 0,40c
Hinh thdi cu
CO nhd, ch6i nhd, c6 nhi^u r l ta Cu r i t nhd. cd nhilu chdi, khOng ' thiy r l xuit hi$n
CO to, chAc. il choi, chAi to, r l it Cu lo. jl r l
Chu thich "Nhiing chir cai khac nhau (a, Irong Duncan's lesl
u trong cac col b i l u di^n s y khac nhau cd nghia vdii P =
^mw^^^
T" W]
ll Irun cac niui i r u o n g klioang lan l u o l lu' Irai gua MS, '/;MS. SH, VJSH
Ihirm \-jo dd, IT mdi Inrdng SII khung cu mil Nli.,NOi Cling Id mgt yeu td dan dcii sy lan cu ciia (.•ay. Trong su phat trien va lang Irirang cuu lliyc vat.
niirogcn cd vai tro chung I:i iigincn lieu dc idng hgp nen cac amino acid, nucloulidc. honmmc va ciK-n/yme, Tuy nhicn, Irong sy hinh thanh va phai
20
Incn cu. nitrogen lai cd tac dgng nhu mdi chat lic che qua trinh tao cu va tang trudng cu. 0 giai doan \p cii. niu nitrogen hien dien d ham lugng cao se lani lang ndng do GA ndi sinh trong thuc vai (GA lie che qua trinh tao cu). Han nira khi nitrogen hien dieno ndng do cao, hinh thanh nen nhiiu NH,, trong la.
Tpp chi Cong ngh? Sinh hpc 9(3): 317-331, 2011 tdng hgp nen cac amino acid la nhiJng hgp chit rit can cho sy tang tmdng cua cac co quan sinh dudng hon la tao cu. (Nguyin Du Sanh, 1998). Nitrogen anh hudng len sy tao cii vd tang tmdng cii, do nd tdc dgng len su phat triin than. Khi cung c i p nhiiu nitrogen khoang, cd uu tien phdt triin chdi, than. Id, khdng thich hgp cho su tao cii (Marschner, 1983). Sy gidm hdm lugng nitrogen thdng qua vi^c giam hdm lugng mudi nitrat trong thdnh phan mdi tmdng MS giup cho su hinh thdnh cu (Zarrabeitia el al, 1997).
Khi ham lugng nitrogen thip, cd tdc dyng Idm gidm hay ngirng qud trinh sinh sdn sinh dudng, giup kha nang tao cu. Mgt sd tdc gid dd chiing minh lifin quan giiira lugng nitrogen vdi hormon thyc vat (Palmer, Smith, 1969; Wang, Hu, 1982). Nitrogen cao se 1dm tdng lugng cytokinin giup chdi tdng tmdng, lam anh hudng din sy hinh thanh cu. Ngugc
lai, thieu nitrogen, tugng ABA tdng cao giiip cho sy K^o cii (Krauss, Marchner, 1982; Haeder, Beringer 1983).
Nhu v | y , d mdi tmdng SH nong dg vitamin cao va sy suy gidm nitrogen trong mdi trudng dd tdc dOng den sy t?o cu thdng qua vi§c 1dm gidm hay ngung tr? sinh sdn sinh dudng vd giiip cho qua trinh tich luy chit dy tr& hinh thdnh cii sam Nggc Linh.
Khdo sdt c i c ning dg auxin vd cytokinin din sy- hlnh thdnh cii
Trong t^o cii in vilro, cd nhieu sy can bdng quan trgng cdn thiet, nhat Id can bang auxin/cytokinin vd can bdng GA/ABA. Trong thi nghi?m nay, can bing auxin/cytokinin dugc khao sdt tren sy tao cii sam Nggc Linh in vitro thdng qua can bdng cua NAA va BA.
Bang 4. Anh hudng cua cdc nfing dO NAA vd BA din sg hinh thdnh cu sSm Ngpc Linh.
Nghiem t h i j c ' F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16
NAA 0 0,5 1 2 0 0,5 1 2 0 0.5 1 2 0 0,5 1 2 Chii t h i c h : 'NhOng trong Duncan's test B A 0 0 0 0 0,5 0,5 0,5 0.5 1 1 1 1 2 2 2 2 1 chO' cai
T r g n g t u ^ n g t i P o i ( g ) 0 . 3 3 r 0,47e 0,30fg 0,23 0.55cd 0,91a 0,74b 0.26g 0,52de 0.33f 0,53d 0.32f 0,44e 0,56c 0,45e 0,26g khac nhau (a, b.
Chieu cao cSy (cm) 5.50e 5,53de 4,70g 2,40k 3.63i 4.17h 4,80g 4.27h 5.93d 4.20hi 5,17f 4,87g 6.37c 8,80a 7.20b 3,05j
T y le tao
(%)' '
41.40%
0%
35,18%
30,33%
0 % 0%
0%
43,05%
73.70%
65.30%
0%
0%
79,16%
83.48%
89.75%
87,48%
c. .) d u g c ndu trong cdc cOt
oil Hinh thai c u Cu rat nho, c6 n h i l u chdi ben Khflng c6 cO, x u i t hi#n n h i l u mo seo, r l Cu rat nho, r l nhilu, dinh kem mo seo Cu nho. It r l . dinh \vbm md s^o Khdng co cii. co x u i t hien mo seo, c ^ l i Khdng co cu. chdi n h i l u Khflng c6 cd, m6 seo n h i l u CO r i t nhd, cd re CO CO nhieu chdi, it r l CO to, chSc, cd n h i l u ch6i, cd re Khdng cd cd, n h i l u md s?o Khdng cd cu, cd r l , it mfl seo CO cd n h i l u c h i i nhd, cd r l tcr Cu hinh chop cd it c h i i . il r l CCi hinh chdp to. chic. It ch6i, choi to, r§ it CCi hinh chdp to, ft r l , chdi nhd b i l u d i l n s g khac nhau cd nghTa vai P = 0,05
CI thi nghiem tii Fl d i n F4 trong mdi tmdng chi bd sung NAA, khi khdng cd sy hien di§n cua BA, ty 1^ tao cii r i t thip va cu hinh thanh d cac mdi tmdng
nay tuong doi nhd, d mdi tmdng F2 cd bd sung 0,5 mg/l NAA hiu nhu khdng tao cii (Bang 4). Nhin chung cd su tao md seo tu mau cay trfin mdi trudng
n u a n g >vuan v^nien el cd bd sung NAA. Nguy€n nhdn Id do sy phdn chia
qua miic ciia Idp te bdo biiu bl vd nhu md vd do tdc d$ng kfch thfch phdn chia t i bdo ciia NAA (Bdng 4).
Dudng nhu sy phdn chia ndy khdng xdy ra d6ng thdi vdi sy tich liiy tinh b$t nen d^ng mo s?o ndy cd mdu trdng ngd, d l hda ndu, hai dye. Khii md si;o ndy id mgt ldp te bao khdng cd hinh d^ng c i djnh do Iiin kit Idng I6o giita cdc t i bdo, chiia nhiiu nude.
NAA cd bdn chit Id mgt auxin. Theo Biii Trang Vigt (2000), auxin cd vai trd Id chit kich thfch viing tugng ting phdn chia, dnh hudng \&n ho^l dOng ciia tugng ting sinh md, kfch thich 190 md s^o vd kfch thfch hinh thdnh rl sa khdi nhung l^i ire c h i sy tdng tmdng cda re khi cd m$t d n^ng dd cao.
Trong qud trinh 190 cii, sau giai do^n cdm ting t^o cii, d CO quan tiin 190 cu Ihyc vat bit diu phdn chia va gia tdng kfch thudc te bdo dong thdi tfch trii chit dy trii. Liic ndy auxin ddng vai trd rit quan trgng, d^c bi^t Id kfch thfch sy phdn chia vd tdng rgng cua cac te bao viing tugng ting, tao khdng gian tich trit chit dy tri? (Nguyin Du Sanh, 1998). Ngoai ra, auxin cdn giiip cho t i bao t | p tmng chit dinh dudng, thu hut chat dinh dudng va gia tdng dp suit tham thau trong te bao. Trong thi nghigm t^o cii, khi phoi ra dudi cudng dg dnh sang cao cii se cd d^ng biit chi. Dieu ndy la do dudi tdc dgng ciia anh sdng, auxin bj phdn hiiy ddng thdi t i bdo mau hda mgc (do mat nude) nen sy tang tmdng khdng xdy ra (Nguyen Du Sanh, 1998).
Ngugc lai vdi mdi tmdng chi bd sung NAA, d cac mdi tmdng F5, F9 vd F i 3 chi bd sung BA tuang duong d cdc ndng dg 0,5; 1 va 2 mg/l, chiing tdi nhan thiy d ndng dg BA thip (0,5 mg/I) khdng cd sy tao cu, tuy nhien, khi gia tdng nong dg BA I6n cao trong mdi tmdng dd cd sy hinh thdnh cii, ty I? hinh thanh cu cung rit cao d F9 (73,70%), F13 (79,16%), cii dugc t^o thanh cd mang theo nhiiu choi (Bdng 4).
Theo ket qud cdc nghien ciiu chung ve sy t^o cii trudc day, vai trd ciia cytokinin rit quan trpng. Nhiiu thi nghi?m cho thiy cytokinin cd ho^t tfnh kich thich tao cu m^nh hon auxin vd cd vai trd quan trgng trong ca hai giai doan I90 cii. Hdm lugng vd hop! tfnh cua cytokinin tdng cao trong suit qud trinh t^o cii d Khoai ta^. Trong giai dopn dau, cytokinin t^p trung nhiiu d dau miit than bd (stolon) Khoai tay vd giup te bdo ndy phan chia sau mgl thdi gian va bdt diu tich luy tinh bpL Trong nghien cim ve sy t^o cii ciia cd dng, Nguyen Du Sanh (1998) cung ghi nh§n thiy ning dg cytoknin cao trong suot qud trinh tao cu ca giai doan cdm ung iSn giai doan tdng tmdng cu Cd dng. Cytokinin tac d$ng
bing c ^ h kfch Ihfch ting hgp enzyme tong b q ) d b$t strarch synthase hay LIDPG-udophosphoglyc«
ding thdi lie che ho^it tfnh ciia enzyme phdn giiia b^t (Nguyin Du Sanh, 1998; Biii Trang Vi|t, 200!
Gabryszewska vd Hempel (1985), Pierik vd ddngj gid (1988) da chimg minh ring vi^c b i sung BA kf thfch phdt triin thdn rl p h ^ nh^nh. (!f m$t ^ lodi c khdc nhu Liiy th) ddi hdi nong d$ BA t h ^ trong qi trinh t^o cii. N i n g dd BA cao dugc sir dyng cho t l img t90 cii Ngh^ (Shirgurkar et al, 2001; Sanghami
Nayak, 2000). 1 Nhin chung cytokinin cd vai trd rdt Idn trong s
hinh thdnh cu chung d c^c lodi thyc v$t cd cii. Tht kit qud cua thi nghiam ndy trSn ddi tugng cdy idi Nggc Linh, cd t h i do tugng cytokinin ngi sinh tron md ciy thip nen lugng BA ngo^i sinh cd ho^l tin thiic d i y qud trinh tao cu.
NAA vd BA khi ket hgp vdi nhau d in$t ndngit thich hgp t^o nen m0t sy cSn bdng giiJa auxinJ cytokinin trong md c i y vd quyit dinh t90 chii. tufr hay hinh thanh cii. d nghifm thiic F I 4 khi bd sisij NAA (0,5 mg/l) kit hgp vdi BA (2 mg/l) da cho thi;
cdc chi s i v i trgng lugng tuoi (0,56 g) vd chieu cat (8,80 cm) la t i t nhit, ty 1$ cu hinh thanh ca<
(83,48%), tuy nhien d n ^ i g m thuc F15 cd b i siinj NAA (1 mg/l) kit hgp vdi BA (2 mg/l) cdc chi sivi trgng lugng tuoi (0,45 g) vd chiiu cao (7,20 cm;
khdng cao bdng nhung hinh thai cay vd cu lai thi hi?n tdt hon, ty Ig hinh thanh cii (89,75 %) cao bot (Bang 4). C h i i cdy hinh thdnh tn nghiam thiic^l5 tc khde hon so vdi chdi cSy dm yiu va cao ciiaiighi^
thurc F14. Cu sdm hinh thdnh c6 hinh chdp vdi cm chinh vd cdc mdt ngii ndm xung auanh cac dot tuone ty nhu cay ngodi ty nhifin. Sy ket hgp ciia NAA vi BA cho thiy cdc chi tieu v i ty l§ t^o cii, hinh thai ci^
trgng lugng tuai vd chiiu cao cay Id t i t hon khi bfi sung ri€ng le.
Sy 190 md s§o, 190 cii hay cac d^ng bit thuffiig ciia cu cd the do sy ho9t dgng qud miic ciia NAA.l^
BA. NAA giiip cho sy phdt sinh mo s?o tir mau cay cdn BA gifip cho sy phdn chia t i bdo. Kit hgp hai dut diiu hda nay theo mdt ty 1^ thfch hgp sS kfch thich cu cdc qud trinh phdt sinh hinh thdi khac nhau. ZhengP d i n g tac gia (2008) cho ring cdc chdt diiu hoa m trudng thyc V9t ddng mgt vai trd rat quan trong tioAg sy hinh thdnh cdc ca quan, va d ^ biet nh$n thay c6 sir anh hudng qua I9J giOa auxin vd cytokinin trong qua trinh t9o cu Giing d ndng dd dudng cao. Raghu (1997) k i t hgp giaa NAA va BA d n i n g dgng dirdng||
cho sy hinh thdnh cii Nghg. Su k i t hgp giiia aui cytokinin cung cdm ling su hinh thdiih vd phat tt
Tap chi Cong nghi Sinh hpc 9(3): 317-331,2011 ciia cau tnic luu trQ' nhu cu vd re cu, thi hien d mgt sd loai cdy trdng nhu Khoai md, Khoai tdy vd Lily (Ja'sik, Mantell, 2000; Ja'sik, Klerk, 2006; Kim et al, 2005; Romanov et al, 2000).
Nhu V9y, tren ddi tugng cdy sam Nggc Linh khi tao cu cin cd sy kit hgp NAA (I mg/l) vd BA (2 mg/l), mite can bdng ciia auxin vd cytokinin ndy cho ty le hinh thanh cii va hinh thai cd Id tdt nhit.
Khdo s i t nong d$ cua ABA len sy: hinh thdnh, sinh trirdng vd phiit t r i i n ciia cd sdm Nggc Linh in vitro
Sy t9o cu dugc ghi nhan sau 2 thang d tit ca cdc ndng dg cua ABA. Tan so 190 cu tdng din khi gia tan§ ndng dg ABA din 3 mg/l, va bdt diu suy gidm d ndng dg cao (4 mg/l) 0 nghiam thiic cd b i sung 3 mg/l ABA tan so 190 cu ciing nhu trgng lugng tuai Id cao hon so vdi cdc nghiem thiic cdn 19! (Bdng 5). 6 cac ndng dg thip, ABA kich thfch nhg qud trinh 190 cu, hinh thdi cu cd d9ng hinh cdu. Vi ABA Id chit lic che tdng trudng, ngdn can su tSng tmdng, keo dai sy ngu ciia chdi vd hat cho nen d nhiing cay cd cu to, hinh thdi cay khdng dugc khde m9nh, cd xu hudng di dang khi cd mat ABA d ndng dg cao.
Nhu vay, chung ta cd thi nhstn thiy ndng do ABA ngo9i sinh cd anh hudng ddng k i d i n sy hinh thanh cu sam Nggc Linh in vitro. ABA dugc hinh thanh trong la, kich thfch sy tao cu dac biet. Mdt sd nghifin cuu cho thdy co c h i tac dgng ciia ABA Ifin qua trinh tao cu la do tac dung kfch thich hip thu
sucrose trong cdc t i bdo, Idm tSng dp suit thim thiu (Nguyen Du Sanh, 1998). Nhieu thi nghiem cho thdy ABA khi tiin hdnh thit nghiam xir ly ABA tren cdy nguyen hay ddt thdn tach rdi diu cd sy xuit hi?n cua ABA d giai dogn mgt (giai dogn cam dng 190 cii) nhung cdc phin ndy se khdng tdng trudng niu tiip tyc xd I^ vdi ABA.
Cdc bdo cdo tren ihe gidi trudc day cung dd chi ra rdng, trong thdi gian dai d nhi?t dg thip lugng ABA ngi sinh tdng I6n d cdc cay cd cii, ngodi ra ABA cdn dugc kfch ho9t bdi mdt lo9t cdc gen lien kit vdi nhigt dd thip, h9n hdn, mlin, khd, protein luu tr&, ngii nghi, ndy m i m , ddng khi khdng (Gusta et al., 2005). Khi qua giai do9n ngii ddng, nhi?t dd i m trd l9i, lugng ABA suy giam vd din tdi giai do9n pha ngii hodn todn. M i i quan h? gi&a sy suy gidm ABA vd phd ngii ddng dS dugc cdng b i vdi kit qud cua nghien cihi ve Polianlhes tuberosa (Nagar, 1995) vd Allium wakegi (Yamazaki et al, 1995; 1999a; b;
2002). (3 cay sdm Nggc Linh, vdo cdc miia l9nh, cay cung cd ca chi ryi Id sinh 1^ vd roi vao trang thdi ngu ddng, dieu nay cd the da tac ddng din kha ndng sdn sinh ABA ngi sinh trong cay, khiin cu phat triin trong cac giai doan nay.
Ngoai ra, Krauss va Marschner (1982) cho ring ABA cd the uc che tdc ddng ciia GA. Trong khi GA kich thfch sy keo dai Idng vd ddt than (sy tdng tmdng theo chieu dgc) thi ABA kfch thich sy ddi hudng tang tmdng, 1dm cho t i bdo tang trudng theo chiSu ngang.
Bang 5. Anh hucmg cOa ABA den si
Ndng d$ (mg/l) 0 0,1 0,5 1
1,5 2 3 4
ir hinh thanh cu sam
T r g n g l u w i g t u w i (g) 0 . 0 7 r
0.17e 0,25d 0.40ab
0,30c 0,37b 0,42a 0.35bc
Ngoc Linh.
C h i l u cao (cm) 2.1e 2.gde 3.6c 4,8a
3,0d 3,8bc 3,5c 3,7bc
TV l« (%) 4 0 % 35%
46%
50%
52%
60%
66%
6 1 %
Hinh thdi cu
Cu hinh chdp nhd, khdng cd r l . it chdi Cu hinh cau, cd nhieu choi CO b l u . cd re cr cu6i cu CO hinh chdp nhd cd mdu xanh den, n h i l u r l
Cu cd n h i l u choi, it r l Cili nhd, cd n h i l u c h i i
CO cd hinh chdp, ddi. It r l , chi cd mdt choi CO hinh chdp phinh to, cdy dj dang. k6m phdt t r i l n
ChO thich: 'NhO'ng chO' cdi khac nhau (a, b, c ..) dugc neu trong cdc cdt bilu diln sg khdc nhau cd nghTa vdi P = 0,05 trong Duncan's test.
Hodng Xuan ChiSn et al Khdo sdt dnh huxVng cua GAj lin s\f hinh thiinh,
sinh t r u d n g vd phdt t r i i n cita cii sdm Ngpc Linh Sau 2 thdng nudi ciy, vdi sy cd mdt cua GAj trong mdi tmdng, khd ndng 190 cii ciia sfim Nggc Linh dd bj suy gidm. d cdc nghi?m thiic cd nong dO GAi thip, cdy vSn tgo cii nhung vdi ty 1$ khdng cao.
cu nhd, cdy k6m phdt triin. Khi gia tdng ning dd din 0,5 mg/l ty I? t90 cii ciia cSy chi cdn 5%, n i n g d$
trfin 0,5 mg/l cdy khdng cd khd ndng I90 cii. Tuy d nong dd cao cdy khdng I90 cii nhung cdc chi s i v i trgng lugng tuoi vd chiiu cao cdy Igi gia tdng, dieu ndy cd thi giai thfch Id do GAidd kich thfch sy phfin bao vd keo ddi t i bdo md phdn sinh dudi dinh khiin cay cao len (Bdng 6).
Vai trd ciia GA3 trong vi^c kiim sodt sy hinh thdnh cii cung dugc mgt so tdc gid ghi nh^n: GA?
dugc coi la chit cdn lugng cii (Matthyse, Scott, 1984; Wareing, 1984; Chailakhyan, 1985), gidm ho9t dgng GA3 din din sy 190 cii (Okazaw'a, 1988; Smith, Rappaport. 1969). thi nghiam niu cdt bd choi vd Id non, sy tugng cu khdng xdy ra, nhu vay sy tugng cu khdng dugc kich thfch bdi GA3 (Vince Pme, 1985), dnh hudng cua sy 190 cii co the han che nhd them chlor cholin chlorit (CCC) vdo mdi tmdng nudi ciy.
GAj ddng mgt vai trd quan trgng trong sy hinh thanh cu Gimg va nang cao trgng lugng tuai ciia cii in vitro
(Yongqiang Zheng el al, 2008).
Moi lifin h i giOa ty 1? ABA/GA trong qud trinh cdm irng 190 cii cQng dd dugc cdc nhd khoa hpc nghien ciru, GA/ABA dugc hinh thdnh trong la va dnh hudng tryc tiip d i n sy hinh thanh cu.
Trong d i i u ki^n ngdy ddi vd nhi^t dg cao, GA dugc t i n g hgp nhiiu 6 Id non trong khi ABA dugc t i n g hgp d Id thip. Khi dd cd GA, ABA di chuyin xumig dudi v i phia cO. Trong diiu ki?n ngdy ddi, J ca quan 190 cii, can bdng GA/ABA nghiSng vi phia GA, sy hinh thdnh cii bj ire chi. Tuong ty trong diiu ki^n ngdy ngin, nhigt dg thip, hdm lugng ABA tdng cao, can bdng sS nghieng v i phfa ABA, ty 1|
G/WABA thap. Kit qud 1dm can than ngam k ^ dai (d Khoai tdy) vd 1dm ddi hudng ifing tmdng, kfch thfch sy hinh thdnh cu, ty Ig auxin/cytokinin cung xuong thip din din sy phdn bdo mdnh li^t d cdc ca quan tiin tgo cii, kich thich sy hinh thdnh cu.
Ngodi ra nong dg GA3 vd ABA cung thay doi manh vdi khi cd sy gia tdng cua sucrose (Zheng (st.
al, 2004). «- Qua ket qua tren cd t h i thay ring GAj la mot chat dieu hda qud trinh hinh thdnh cu Id tdc nhan kich thfch keo ddi than, ngSn can sy tao cii va tang tmdng cu.
Bdng 6. Anh hirdng cua GAj din khd ndng hinh thdnh cu sdm Nggc Linh Ndng dg (mg/l)
0 0,05 0,1 0,5 1 1.5 2 3
Trgng lu-gng tuwi (g) 0,36de*
0,27e 0,28e 0.27e 0.37d 0,75a 0,52c 0.70b
Chiiu cao 3,90a 2,85e 2.57g 3.6gbc 3.71b 3.50c 3.30d 2.51f
(cm) Ty 1$ tfo 42%
30%
23%
5%
0%
0%
0%
0%
cii(%) Hinh thdi cu
CO hinh chdp, it r l ^ Cu nhd, it r l , It chii
Cu nhd hinh chdp, cd nhilu CO nhd, phlnh, nhilu chii. il r l
ChO thich: 'NhO'ng chO- cdl khdc nhau (a. b. c . ) dugc nfiu trong cdc cdl biiu diln sg khdc nhau cd nghTa vdi P = Irong Duncan's test.
Khdo sdt ning dg ciia sucrose l£n sv hinh thinh, sinh trudng vi phit triin cua cii sSm Ngfc Linh
in vitroDudng la mgt trong nhCTng nhdn td quan trgng trong nudi cdy md thyc V9t, ngoai cdc chat d i i u hda
tdng tmdng thyc vSt thi n i n g dg dudng co anh hudng ddng ke d i n chieu cao, trgng lirgng cdy va kha ndng t90 cd. Khi n i n g dd dudng sucrose iro^
mdi tmdng thay d i i nd cd anh hudng ddng k i ^ qud trinh 190 cii ciia cay. Trong thi n g h i i m ^ b chung tdi sir dyng mdi tmdng SH cd b i sung sucrose
Tpp chi Cong nghe Sinh hoc 9(3). 317-331,2011 tir 15 din 70 g/1 vd kit qua thu dugc cho thiy cd sy khac bi^t d trgng lugng, chiiu cao cay cung kha ndng tao cu (Bang 7).
Sau 2 thang nudi cijf, chimg tdi nh9n thiy d ndng dg sucrose 50 g/1 cho ket qud tit nhit cho qud trinh tao cii sam Nggc Linh. Cu hinh thdnh vd kfch thudc tdng khi ndng dg dudng tdng len. Mac dii ty Ig t90 cu khi d ndng dg dudng cao (70 g/1) len din 9 3 % nhung ddng thdi la ngin lai vd heo ua d nong dd ndy (Bang 7). Thyc vat nhan nang lugng tir qud trinh quang hm).
Tuy nhien, trong nudi ciy md thyc v^t, cSy con thieu kha ndng tu dudng do khiem khuyit chirc ndng cua lye l9p. Vi the ngudn carbon b£n ngodi rit quan trgng di sdn xuat du lugng carbohydrate nliim ddp img nhu ciu phdt triin ciia te bao.
Ngudn carbon sir dyng trong nuoi cay md thyc vat chii yeu Id dudng sucrose, fructose, glucose vd mdt ft la maltose, galactose, mannose, lactose. Tuy nhien, ngudn carbon tdt nhdt vd phd bien nhat trong nudi cay md Id sucrose d ndng do khoang tir 2 % - 5%. Khuri va Moorby (1995) da lap thf nghiam va nhan thay sucrose dugc su dyng nhanh ban va tap trung d rd nhieu han. Dieu dd cho thdy mdi tmdng cd chiia sucrose thfch hgp cho sy tao cii han cdc loai dudng khac.
Theo cac ket qua thu dugc gan day, sucrose cdn cd vai trd dieu chinh lugng GA (gibberellin) ngi sinh trong cay than cii (Xu era/., 1998).
Ndng do sucrose dnh hudng rat Idn den sy hinh thdnh cii va chat lugng ciia cu in vilro. Mdi tmdng cd ndng do sucrose cao giiip tao cu sam Nggc Linh vdl trgng lugng tuai cao (Bang 7).
Vai trd ciia dudng ddi vdi su hinh thanh cu da dugc nghifin ciru tren nhieu ddi tugng; dudng Id ngudn carbon c i n thiit trong mdi tmdng nudi cay vd ddng thdi la ngudn nguyen lieu quan trgng trong viec tich luy tinh bgt d i n d i n su tao cii va sy phinh to ciia
Bang 7. Anh hudng cua ning dd dudng sucrose din kha nSng hinh thdnh cu sdm Nggc Linh.
cu in vilro. Dantu vd Bhojwani (1987) ghi nhan ring ndng d§ sucrose cao thich hgp cho sy hinh thdnh cu cd kich thudc Idn trSn hoa Lay-an (dudng kinh tir 10 den 23 mm). (5 nong dd sucrose thap, kha ndng tfch liiy tinh bgt thip d§n den khd ndng t90 cu h9n che vd trgng lugng tuai ciia cii thip. Tuong ty trong thi nghiam nay chiing tdi nhan thiy d nong dg dudng cao (50 g/1) cho kich thudc ciia cu Idn nhit (Duong T i n Nhyt, 2007). Sucrose ddng mgt vai trd quan trgng trong hinh thdnh cii Girng in vitro. Sucrose tgo bd xuong carbon vd ndng lugng cho cam irng va hinh thdnh cii Z officinale (Yongqiang Zheng et al, 2008).
Trong chu trinh tdng trudng va phat triin ciia thuc vfit qud trinh quang hgp t9o cac san phim ddng hda. Mgt phan tham gia vao cSu triic thyc vfit phdt trien, mgt phin dugc tich liiy trong cac ca quan du trir nhu trai, hgt, than, cii, ban than cay, trong qud trinh phdt trien se ty tich luy chat ddng hda dudi d9ng cii (Nguyin Du Sanh, 1998).
Nhirng cau tnic cii dac biet nay cd mdt hodc hai chiic ndng sinh hgc. Tmdc het, chitng la co quan du trir cacbon va nitrogen d nhung dang cd the cung cap cho thyc vfit khi can thiet. Chiic nang thir hai ciia chung la co quan sinh san. Trong tmdng hgp nay, cu can tich trir ddy dii lugng sinh dudng can thiit cho nhu cdu sdng ddc lap cua cay sau nay. Cac lodi thuc vat t90 ra cac bd phfin dac biet nay thudng la cac loai cay luu nien Sau mgt thdi gian hay mdt miia thyc vat se sdng sdt dudi dang mdt co quan trong dfit va d trang thdi ngii. Co quan nay cd mang sdn chdi va se tao cay mdi vao vu sau. Cay sdm Nggc Linh trong tu nhien cung vfiy, chiing thudng cd mgt khoang thdi gian d trang thai ngii, trui la va tan di, sau dd mdi phat trien trd l9i.
Nhu vfiy, vdi viec bd sung lugng dudng sucrose vdi ndng dg 50 mg/l cd the giiip sam Nggc Linh tao cii to va hinh thai cay khde manh.
Sucrose (g/I) 15 30 45 SO 70
Trgng lirgng 0,34c 0.43b 0.46ab 0,51a 0,31c
tuwi (g) C h i l u 2,0e 2,5c 2,9b 3,1a 2.2d
cao cdy (cm) Ty Ig t^o cO 33%
4 2 % 65%
88%
93%
(%)
Hinh thdi cu CO nhd, cd chdi n h i l u CO nhd, cd r l CO lo, hcri phinh CO hinh chdp. lo CO hinh chdp nhd, c3y k ^ phdt trien Chu thich: *NhOng chu- cdi khdc nhau (a, b. c . ) dugc neu trang cdc cflt bilu diln sg khdc nhau cd nghTa v*i P = 0,05 In^ig Duncan's test.l l i K i n g X i k i n t h i c n ('/(,/
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c o n CO chira 11 NizuL' Linh
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I nil,I 1 ( , - K . , .
au k h o a i i j ; 25 phiil xuii i h i i l ,\ual hii-n d m h cua IlK'll 1
M K . v,i 16 phi'il Mi.il hKMi d m h cua ( i - R b , . { ) buiic s o n i j I'KI m i l cac d m h x i i a l hiOn r d n h u n g co hiyn u r g i i y l u u dirdng! n e n , t t hu<rc s d n g 2 0 3 n m khnnj;
th.iy i n d i i i l i cii.i M R : ( H i n h 2 . 3; 4 ) D o n g thni c h i m ^ I n i \ a c d i n h d i r a c phiremg t r i n h tuyen tinh. h^
so luaii,ij i | i i a n i u , i h,i l<i,ii ••apnnm M R ; . G - R g , . G- K l i | . u i n i j n h u \ , i i t i m h d i n r c g u n han d m h luong la u'ini l i , m | i h [ i i i i i c n c i M c h m i t !
I i
H i n h 2. Sac.ki do MR . G-Rb,, G-Rg iren n n Ngoc Linh 17 Ihang tuoi so vo'i sam | y nhien ua sinh khoi ch6i
N h u V Liua k c l qua d m h I
m d n g \ a ( h n h I i r o n g b a n g I I I ' L C , ilira I r c n sir t i d i c h i i u Vdl 1 chal c h u a n c d d m " . \ J so s<inli \ d i s a m N g g c L i n l i l u n h i c n . c o the i h . u cac c;iy 17 t h i i n g t u d i c d su hicndiC-n cua M R . , d - R b , \ a ( i - R g , H a m l u g n g M R ; c l i i O m l y Ic c a n l i i m han su \ u i C,-\ig, v.i
326
1 K'ing sac k\ l o p t i - R b , ( B a n g S) K c l c h u n g l u rang diroi t l i c u k i c n t u iihiL'ii n h u n g cay c d n g u o n giic in v(fro-
\ a n c u kha n a n g k i c h i h i c h cac le bao t r o n g qua trini s m h l o n g h a p n e n M R ; m d t l o a i s a p o n i n c h i i yeu ciia than re \ a re c u sam N g o c L i n h V e t h a n h phan cac s a p u m i i \ a m u c d g t u a n g d d n g cua s a p o n i n trong cac
Tpp chi Cong nghe Sinh hpc 9(3): 317-3 31, 2011 cay nudi ciy cd nguin gdc in vitro so vdi thdn re va ri cu sam Nggc Linh tg nhien tren sdc ky dd HPLC (Hinh 8), cho thdy ca hai sdc ky d i ciia mau sinh khdi chdi va sdm Nggc Linh cd sy tuang d i n g cao, cd the danh gia thanh p h i n cdc saponin ciia cay 17 thang tudi tuang ddng vdi saponin trong than r i va re
cu ciia cdy tg nhien.
Vdi nhSng kit qud nay chiing ta cd thi tin tudng rdng nhiJng cay cd ngudn g i c in vitro khi dem trdng ra ngodi tg nhifin vin cd diy dii cdc boat chit va boat tinh nhu cdy sam tg nhien. Xda bd di noi lo nggi v i cfiy nudi ciy md md khdng cd ho?it chat.
S003 40CH 30O|
2 0 0 ^ 1DCH
. ; U J p m . 4 n
J
m (1 OO)
G-Rg",
s
G-Rb,
i
Hinh 4. PeakG-Rgi, G-Rb, lr£n sac kidd HPLC cua saponin toan phdn sim nudi cly dugc trdng 17 thdng d budc sdng 203 nm.
Bdng 8. Ham lu^ng cac saponin trong ciy sdm Ngoc Linh 17 thdng tudi.
Khoi l u y n g nguygn li?u (mg)
S a p o n i n chuin
Di$n tich peak
Hdm lug-ng s a p o n i n t r o n g m i u
Ham l u ^ n g (%} saponin t r o n g m l u
Hdm lipgng (%) saponin t r o n g m l u t i n h theo dworc l i ^ u k h d k i f t
Ham lu>9>ng (%) s a p o n i n t r u n g b i n h 234242
234156 234317
0,8136 0,8133 0,8138
0,15 0.15 0,15
0,17 0,17 0.17 787585
787551 787498 826019
0,69 0.69
0,77 0,77 0,77 1,0074
1,0073 1,0075
0,18 0,18 0.18
0,21 0,21 0,21
Hodng Xudn Chiin etal
Hinh 5. Cu sdm Nggc Llnh in vitro, sg phdt Irlin cOa cdy slm cd cti t^l nui Ngpc Linh vd mlu sdm phfln tich. a. C i ) ^ Nggc Linh in vilro: b. Cly slm cd cu tnfdc khi dua ra ngodi vi^lm uom: Ci, Ci, Cj. Cdc d^ng cii sdm in vitro; d. Vutti trM sflm o) cu t3i khu vgc xS T l Xing, huy^n Tu Mff Rdng, tinh Kon Turn; ». Cdy sIm 17 thdng tudi d u ^ thu thdp tai virdn^t Cdy sdm cd cd 17 thing luii trvdc khi r ^ n lich: g. CO sfim siy khd dugc lo^l bd Id trvdc khi phdn lich.
KfeT LUAN
Mdi trudng thich hgp nhil di 190 cii sam Nggc Linh in viiro la SH cd bo sung 1 mg/t NAA, 2 mg/l BA Irong diiu kign chiiu sing 16 gid/ngay. Cic hp phiic hgp cua GA/ABA vi auxin/cytokinin trong qui trinh hinh thdnh cu cung da dugc khao sdt. Ning dg ABA thich hgp nhit cho qui trinh tgo cu li 3 mg/l.
GA3 ire chi khi ning t?o cu in vitro cdy sam Nggc Linh. Ndng dg dudng sucrose tit nhit cho qui trinh t9o cu sam Id 50 g/1. Qua hg thong HPLC chung toi
ciing di nh^n thiy trong cay sam Nggc Linh nuoi cay md trdng thii nghi?m 17 thing t^i niii Nggc Linli cd chiia cd 3 loai saponin quan trgng ciia Sam Ngoc Linh dd la MRj, Rg,, Rbj. Ham lirgng saponin trong ctj ciia nhiing cay nay ciing da dugc xdc dinh M MR2 (0.77%). G-Rg,(0,17%), G-Rb, (0,21%). '|
Ldi cim on: Xin chdn thdnh cdm cm Ban Khoa hpc vd Cong nghp dia phucmg - Bp Khoa hpc vo Cdng nghe dd ho trp kinh phi cho cho di tdi nghie^^'
Tpp chi Cong nghe Sinh hpc 9(3): 317-331, 2011 ndy. Di ldi thupc nhiem vy khoa hpc vd cong nghe cap thiet mai phdt sinh d dia phirong.
TAI LI$U THAM KHAO
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cinraiion on yrowih and cndohormone conceniraiion llon^ot iiibcmnyLT China \ciiZ 15-P F A C T O R S E F F E C T I N G IN VITRO M K K O U H I / O M i : F O R M A I I O N OF PAKiX VIETNAMENSIS H A E T G R U S H V . A N D QV \ M I I V I ION O F S A P O N I N C O N T E N T OF T H E R E F R O M G E N E R A T E D P L A N T L E T S ( i K O W N IN NCiOC LINH M O L N T A I N Hoang Xuan Chien', Ngo Thanh Tai', Ngu>i-n Ba
Tran Cong Luan^, Duong Tan Nhut'' * 'Tay Nguyen Institute of Biology
Research Center of Ginseng and Medicinal Mateimh IUH himinh Ciiv
rue', Iran Xuan linh , Lam Bich Thao".
SUMMARY
Ngoc Linh ginseng (Panax vietnamensis Ha tl driisln I is pharmaceutical compositions in which saponins arc ilic niosi i and development this precious medicinal species, the apphcaiKi effective solution. The growth and development of micri>rhi/_hoi
l-known Vietnamese ginseng for its rich
;iiii components In an effort to prescr\e i^MK- cullure techniques is considered an i\ cd planikts was enhanced ll would be
* Author for correspondence: Tel: 84-63-3831056. Fax 330
S4-63-3S3l()2<'i. F-mail. iiiionglaimhiilidgimiilcom
Tpp chi Cdng nghe Sinh hpc 9(3): 3\1-33\,20U
high survival rate when acclimatized in the field In this study, in vitro microrhizomc formation and determination of saponins in 17 month-old plant were presented. Microrhizomc formation was induced from the in vitro shoots of Panax vietnamensis on SH medium containing I mg/I NAA and 2 mg/I BA under 16h- photoperiod. The effect of GA/ABA or auxin/cytokinin microrhizomc formation was also investigated. The most suitable ABA concentration for the microrhizomc formation was 3 g/I. GAj inhibited microrhizome formation of Ngoc Linh ginseng. The most suitable sucrose concentration for the formation of microrhizome was 50 g/1. The rhizomes of 17 month-old plant growing in the wild were careiully selected in order to identify G-Rb I, G-Rg/, Affl^ concentrations which were 0.21,0.17 and 0.77, respectively.
Keywords. ABA. GA. G-Rb,. G-Rgi, microrhizome. MR2, Ngoc Linh Ginseng. Panax vietnamensis. rhizome, saponins
