Tgp chi Cong nghi Sinh hgc 12(2)- 355-364, 2014
M Q T S O K E T Q U A N G H I E N C U t J T A O V A N U O I R E B A T D 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 . )
Mai Trirdng', Tr^n Thi Ngoc H i ' , Phan Tirong Loc', Le TSn Dirc\ T r ^ n T r o n g T u ^ n ' , Bh Dang G i a p ' , Biii Dinh Thach', Nguyen Thj Thanh', Pham Dire Trf', Nguyen Dire Minh H i i n g \ Nguyen Van Ket^
Tran Cfing Luin^, Nguyin Hfru H 6 '
' Viin Sinh hgc nhiil ddi. Viin Hdn ldm Khoa hoc vd Cong nghe Viit Nam
^Trud'ng Dgi hgc Dd Lgt, Bg Gidg due vg Ddo tgo
^Trung tdm Sdm vd Dugc liiu Tp. Ho Chi Minh Ngay nhin bai: 10.5.2014
Ngay nhan dang: 19.6,2014 TOM TAT
N^i dung bii niy trinh biy mot so ket qua nghien cuu ve tao va nuoi re bat dmh sam Ngpc Linh tren moi trudng thach vi trong moi truong long. Muc dich nghien cuu la tao ket qua tien de cho nghien ciru nhan sinh khoi quy mo ldn loai mo biet hoa nay nham thu hgp chat thir cap. Ba loai mo diing tao re bat dmh bao gom m6 seo li chet, phfli/cvm phoi truong thanh mang choi va mo seo ciing. Re bat dinh hinh thanh ngay trong giai do^n nuoi cay minh la (nu6i ciy m$t giai do?n, khong qua cay chuyen) tren moi trudng MS bo sung 2 mg/l 2,4-D va 10% nudc dira; re bat dinh ciing hinh thanh tir mo seo cay chuySn (nucii cay hai giai doan) khi duoc nuoi tren moi trir&ng MS c6 3 mg/l NAA, MS c6 3 mg/l NAA, 2 mg/l 2,4-D, 5% (v/v) nudc dira va moi tnrdng B5 CO 5 mg/l IBA. Ngoai ra, su hinh thinh rl bat dinh tir phoi/cum phoi (da loai bo hoan toan re try) ciing da dup'c ghi nhan qua nuoi cay tren moi trudng 1/2MS bo sung 2 - 3 mg/l NAA. Re bat dinh tir m6 seo cdng (tir moi trudng B5 co 5 mg/l IBA) da dugc nghien cihi nuoi cay tiep tuc tren moi truong thach/long White co 5 mg/l NAA, B5 c6 1 - 5 mg/l IBA/NAA nham tim hieu kha nang tang sinh khoi qua qua trinh tao re thii cap, Nghien ciiu kha nang hinh thanh cac so khoi re va phan nhanh re tren cac moi trudng White/B5 co 1 - 5 mg/l IBA/NAA tu khue cSt re bat dinh ciing da dugc thuc hien.
Tir kh6a: Khiic cdi ri el Grushv)
,.phdi! 3 truang thdnh, re bdi dinh, sdm Nggc Linh (Panax vietnamensis Ha
MO DAU
Nhu chiing ta da biet, nuoi cay re bat dinh (adventitious root) in vitro l i mpt trong cie huong nghien ciiu rat quan trpng nham thu nhan cie hap chat thir cap (HCTC). He thong nuoi cay nay khac ph^ic cac nhupc diem cua he thong nuoi cay te bao trong mpt so truang hgp, do c i e uu diem sau: 1/ Re li mpl lap hop co he thong c i e te bao biet hoa va nhu vay d i mang mpt nhiem vu nhat dinh doi voi boat dpng song ciia ciy, do viy cilng lien ket vcri nang lire sinh tong hap HCTC; 2/ Dfii vai mot so cay duac li^u, re la nai sinh tong hap/tich tu c i e HCTC; 3/ Co the nhan nhanh sinh khoi re trong moi truong long ma khong bj han che vi hien tuang hoa kinh/hoa trong (vitrification) nhu trucmg hgp nuoi the choi; 4/
Tuang tu nhu a mpt so he thong nuoi cay khac, co the nu6i nhin quy mo lan re trong dieu kien khong ch6, khong phy thugc vao dieu kien moi trudng ben ngoai.
Nhu vay, nuoi cay loai mo a trang thii hoan chinh ve mat cau tnic va chuc nang, nhu re, la mgt tiep can co nhieu hua hen thu dugc nang suat cao HCTC v i thirc te da chung mmh dieu nay. R l bat dinh ciia nhieu loai thuc vat nhu Anlhemis nobilis (Omato et al, 1998), Cornus capilata (Tanaka et ai, 2001), Dubosia myoporoides x D. leichhardlii (Yoshimatsu et al, 2004), Echinacea purpurea.
Echinacea angustifolia (Wu et ai, 2006, 2007), Iris parviflora (Akashi el al, 2005), Rhus javanica (Taniguchi et al, 2000) da dugc nghien ciru nuoi nhan thanh cong va s i n xuat on dinh bang cac loai he thong bioreactor khac nhau vai quy mo tir 5 lit ddn nhieu nghin lit nham thu hoat chit.
() hg sam (Araliaceae), re bit dinh mgt so loii ham chua HCTC quan trpng nhu Panax ginseng da dugc nghien ciru nuoi cay (Son et al, 1999; Choi et al, 2000; Gu, 2003; Kim etal, 2005, 2006; Jeong et al, 2006), san xuat sinh khoi va thu hgp chit thir c i p 355
Mai Trudng et al.
quy mo bioreactor (Kim et al, 2002, 2005, 2007;
Paek, 2002) va san xuit quy mo Ion (Choi et al, 2000; Paek et al, 2005); Panax notoginseng (Gao et
al, 2005),.., Rieng d6i vai sim Nggc Linh - Ioaicimg hp Araliaceae, den nay trong nuoc da co mpt so cong trinh cong b6 k^t qui nghien cihi tao va nuoi ciy rl bit dinh (Duong Tan Nhut et ai, 2006;
Nguyin Trung Thanh et al, 2007; Nguyin Thinh Sum et al, 2009; Nguyin Thi Lilu et al., 2011;
Duong Tan Nhut et a., 2009, 2010; Duong Tan Nhut
et al, 2011; Nhut et ai, 2012) nhung chua ghi nhandupc cong bo lien quan den tao va nu6i ciy re bat dinh hinh thinh tii phoi soma/cum phoi trudng thinh mang choi, tir loai mo seo ciing (ngudn goc tir md seo CO khi nang sinh phoi) c6 kha nang duy tri kha nang tao re bai dinh trong thdi gian dai. Ket qua khue cit rl bit dinh ting sinh khoi trong dieu kien nudi cay long lac cung Ii diem mdi d nghien ciiii nay.
VAT LIEU VA PHLTONG PHAP Gi6ng sim
Gidng sam Nggc Linh {Panax vieinamensis Ha et Grushv.) (lay mau tii tinh Kontum).
Tao md s^o tit manh la va tao rS bat dinh tir mo seo la
La chet cay sam 6 thing tuoi (tu cu) d vudn uom tnidc het dugc rira sach bing nudc may trong 10 phiit, sau do dugc khu trimg lin lugt bang bang con 70% trong 1 phiit, nudc Javel thucmg mat 20%
(v/v, CO bd sung Tween 20) trong 10 phiit va dung djch HgCl2 0,1% (w/v) trong 5 phut, Li dupe cEt thanh cie minh kieh thudc 0,5 x 0,5 em va ciy vio mdi trudng tao md seo MS (Murashige, Skoog, 1962) CO 2 mg/l 2,4-b. Ghi nh|n kk qua tao mo seo sau 1,5 thing nuoi. Cit vi eiy chuyin (it nhat 2 lan, 20 ngay/lan) cie cum mo seo kich thudc 0,5 cm vao dTa petri 0 7 cm chira 20 ml moi trudng nhu tren de nhan mo seo. Sau do, mo seo (cilng vdi kich thudc nhu tren) duac ciy chuySn sang cie mdi trudng tao re bat dinh nhu: Moi trudng MS eo 2 mg/l 2,4-D + 10% nude dua, MS CO 2 mg/l 2,4-D + 3 mg/l NAA + 5% nudc dira, MS eo 3 mg/l NAA (30 g/l dudng saccharose) va B5 (Gamborg el al, 1968) + 1 - 5 mg/l IBA (50 g/l dudng saccharose).
Cay 10 cum mo seo/moi nghiem thuc, l^p l?i 3 lin. Ghi nhan k^t qua tao rl sau 2 thing nudi. Digu kien nuoi trong toi d nhiet do 24°C trong tii nuoi.
356
Tao phdi soma vk t^o rl bat djnh tir phdi soma trirdng th^nh mang chdi
Tao md s^o cd kha nang sinh phdi (KNSP):
Nudi cay md seo Ii (tir moi tnrdng MS co 2 mg/l 2,4-D) tren mdi tmong MS + 1 mg/l 2,4-D + 1 mg/I NAA + 0,2 rag/1 kinetm + 10% nudc dira (30 g/l dudng saccharose). Ghi nhan ket qua tao rao s ^ co KNSP sau 2 thing nuoi.
Tao ph6i tir md seo cd KNSP: Dimg moi trudng 1/2MS (1/2 khoang da lugng, gift nguyen vi lupng va vitamin) + 0,2 mg/I BA + 10% nudc dira (30 g/l dudng saccharose). Ghi nhin ket qua tao phdi sau 2 thing nudi.
Tao phoi trucmg thinh ddng thdi kich thich choi phit trien: Diing mfli trudng 1/2MS + 0,5 mg/l BA + 1 mg/l GAs (20 g/l dudng saccharose). Ghi nhin ket qui tao phoi trudng thinh (co diy dii hai la mim) sau 2 thang nuoi,
Phoi trudng thanh d giai do^n ed chfii (conversion into plant), sau khi eSt ngang d vi tri than sat goc ch6i, loai bo hoan toan phan re, dupc nuoi cay tren moi tnidng 1/2MS ed bo sung 2 mg/I va 3 mg/I NAA dS tao re bat dinh. Thu nghiam tao re bat dinh tir cum da phdi vi phdi chua cd ch6i cung dugc thuc hifin nhu tren. Ghi nhan ket qui t^o re sau 1,5 thang nuoi. Dieu kien nudi ngoai sing «= 3.000 lux d nhiet dp phong 25-28''C.
Thvt nghiem so sdnh hinh dang, tim hi^u s\r phin
nhanh cua re bat dinh hinh thanh tir md s^o vi phdi soma
Re bat dinh (dii 4 cm), hinh thinh tii m6 s?o nudi tren mdi trudng MS co 3 mg/I NAA, tir phoi soma nudi tren mdi trudng I/2MS cd 3 mg/I NAA, tii md seo cumg nudi tren B5 co 5 mg/l IBA, dupc nuoi cay tren mdi ttudng White cd 5 mg/l NAA va moi trudng B5 c6 5 mg/l IBA dl khao sit su phan nhanh tao rfi thii cap. Ket qua tao re thii cap dugc ghi nhan sau 1 thing nu6i ciy. Tuang tu, rl bit dinh (dai 4 cm) tu mo seo ciing cting dugc nuoi cay tren m6i trudng White co 3 mg/l NAA dl tim hiiu su phin nhanh tao rl thii cip cae cip (cip 1, 2 va 3). Kit qua tao re thii cap cie cap dupc ghi nhin sau 1,5 - 3,5 thing nudi cay. Dieu kien nuoi trong toi d nhi$t dg 24°C trong tii nuoi.
D I tim hieu sau hem sir phan nhanh thong qua si;
hinh thanh eac so khdi re (root pnmordia), cac khiic
cit rl (KCR) bit djnh sa cip (2 cm), tir md seo ciing,
dugc nuoi eay trdn cac mdi trudng White cd 5 mg/l
NAA, B5 CO I - 5 mg/l IBA/NAA. Su hinh thinh
Tap chi Cong nghe Smh hoc 12(2): 355-364, 2014 cac so khdi re dugc ghi nhan sau 20 - 40 ngiy nuoi trong to! (d nhiet do 24^0 trong tii nuoi), Nuoi re bat djnh trong moi t r u o n g long lac
Hai loai rl bat djnh cd nguon goc tir phoi soma va tii mo seo cung dupc dimg lam thi nghiem, KCR so cap (kliong co re nhanh) va KCR mang rl nhanh (dai = 2 cm) tir hai loai re co nguon goc noi tren dupc nuoi cay trong moi trudng long. Ciy luong KCR re 0,5 g; 3 g; 5 g (I %, w/v) vao binh lam giic 250 ml, 500 ml, 1.000 ml chira 50 ml, 300 ml, 500 ml, theo thii tu, moi trudng long B5 + 5 mg/l IBA (50 g/l dudng saccharose). Ghi nhin ket q u i [hi nghiem sau 2 thing nuoi lac (90 vong/phiil; dieu kien loi, nhiet do phong 25-28''C).
KET QUA VA THAO LUAN
Tao mo seo tir m i n h la va tao re bat djnh tii' mo SCO la
Sau khoang 1,5 thing nuoi tren moi trudng MS CO 2 mg/l 2,4-D nhau liiay mo seo hinli Lliaiih a vj tri mep cit va tren khip b l mat minh l i (Hinh la) Mo seo dugc nhan sinh khoi qua cit cum, cay chuyen
sang moi trudng nhu tren diing lam vat lieu cho tlii nghiem tao re bat dinh (RBD) qua sir dung cac chit dieu hoa smh truong NAA, 2,4-D ket hop vdi NAA v i nuoc dira. Tren moi trudng MS co 2 mg/l 2,4-D + 10% nudc diia nhan thay d ngay giai doan nuoi cay dau tien (chua can cay chuyen) d i co hien tugng hinh thinh RBD nhung sd luong rl hinh thinh it (khoing 2 0 % so mau cay), re ngan va mgt pliin mo seo trd nen xop trang d cuoi giai doan nuoi ciy (Hinh lb), chiing toi ggi diy li nuoi cay lao RBD mpt giai doan (one-phase culture), khac vdi nuoi ciy hai giai doan (two-phase culture) d cac trudng hgp khac dugc trinh bay dudi day. Khong ghi nhan dugc su hinh thanh re tren mdi trudng MS chi co 2 mg/l 2,4-D, Qua nghien ciiu tao RBD dimg kit hpp 2,4-D, NAA va nudc dira (5%) nhin thiy RBD hinh llianh kha nhieu tren khap be mat cum mo seo nhung re cung ngan, con nhan tliay mo seo tra nen mem han (Hinh Id). Loai mo nay hien dugc nuoi cay duy tri qua thdi gian da han mgt nam van cliua mat kha nang phit sinh hinh thai rl, 0 hai trudng hgp tao rl vira neu, moi trudng deu co nudc diia 5 - 10% (v/v);
theo chiing toi, auxin tu nhien c6 trong nudc dira (Yong el al, 2009) da kich tliicli tao rl tir mo nuoi cay.
Hinh 1 . Tao mo s^o ILF manh Id chet va r§ b i t djnh tif mo seo la chet tren mot so moi tru'dng khac nhau a. M6 seo hinh thanh t u manh la sau 1.5 thang nudi c i y tren moi tru'O'ng MS c6 2 mg/l 2,4-D b. S i / hinh thanh r l bat dmh tCr mo s$o sau 1,5 thang nuoi cay tren moi trudng MS co 2 mg/l 2,4-D va 10% nudc difa. c. Re bat dmh hinh thanh tif mo seo tren moi truffng MS c6 3 mg/l NAA sau 1,5 thang nuoi cay d . Rh bat dmh hinh thanh tif mo seo tren moi trudng MS co 3 mg/l hiAA, 2 mg/l 2,4-D vh 5% n u d ^ difa, sau 1,5 thang nuoi c l y
Tren moi trudng MS co 3 mg/l NAA, RBD hinh thanh kha nhieu iren be mat cum mo seo (Hinh lc).
Tao md seo tir la v i d i p iing tao RBD ciia mo seo
tren moi trudng bo sung NAA nham tao nguon vat lieu cho cac nghien ciiu ticp tlieo da dupc nghien ciru (Duong Tin Nhut et ai, 2009; 2010; 2011) va kit 357
Mai Trudng el ai qua nay phii hgp vdi ket qua nghien ciiu ciia cac t i c
gia neu tren. Tao RBD dung NAA ciing da dupc nghien ciiu dung mo seo cii (Nguyin Thi Lilu et ai, 2011; Nguyin Trung Thanh el ai, 2007).'
Mo seo l i cung da dugc cay chuyen sang moi trudng MS co 1 mg/l 2,4-0, 1 mg/l NAA, 0,2 mg/l kinetin v i 10% nudc diia de tao mo seo co kha n3ng sinli phoi (KNSP) va cay chuyin sang moi trudng B5 CO 1 - 5 mg/l IBA de tao loai mo seo ciing co kha nang tii sinh re bat dmh
Tao phoi soma v i tao r l b i t dinh tir phSi soma t r u o n g thanh m a n g chdi
Mo seo CO KNSP dupc ti£p tuc nuoi cay lan lupt tren hai moi trudng 1/2MS co 0,2 mg/l BA va 10%
nudc dira v i 1/2MS eo 0,5 mg/l BA va 1 mg/l GA3 de tao phoi trudng thanh, co choi vdi la that kha phit trien va re tru (tap root) dien hinh. Phoi trudng thanh mang choi (sau day ggi tit la phoi), sau khi dugc cit ngang than phoi d vi tri sit goc choi (Hinh 2a), dugc nuoi cay tren moi trudng 1/2MS co 2 va 3 mg/l NAA
d l tao RBD. K i t q u i budc dau cho thay tren mfii trudng CO 2 mg/l NAA s6 lugng re hinh tlianh khoang 2 - 3 (Hinh 2e,d,e,f); khac hon, s6 lugng re nhilu hon tren moi trudng cd 3 mg/l NAA (hinh 2g,h). Da ghi nhan dugc truong hpp RBD phit sinh r l nhinh (lateral) (Hinh 3d). Thu nghiem tao rl tren phoi CO l i m i m v i choi chua phat trien (Hinh 2b) cung da dupc thuc hien nhung ket q u i cho thay phoi CO dip ung Idiong t6t, theo chiing toi, co the do kich thudc phoi nho dii da cd cue re kha khat trien nhung chua CO chSi do vay chua du tieu chuan dip iing vai nudi ciy nay, Nguac lai, cum hai phoi (Hinh 2i) qua thii nghiem tao re da cho ket q u i tot vdi nhieu rl hinh thanh (Hinh 2j). Nhin chung, 6 cac trudng hop tren, d vi tri vet cat ngang than phoi don/cum phoi deu CO sir hinh thanh it nhilu mo seo, cac RBD hinh thinh CO kieh thudc ngang nhau ve chieu dai va dudng kinh va RBD co dang cum/chiim (numerous), Cac re khi dat chieu dai khoang 2 cm dupc tich ra va nudi rieng dS tiep tuc tang smh theo chieu dai va de nliinh tao re thii c i p .
(-!;ih 2. Tgo r l bit iJmh tif phoi soma trivdng Ihanh mang choi a. Phoi trirdng thanh cd ch6i dupc cat ngang (vi trl vach ngang) than phoi, bo phin l i , dung lam vgt lieu tao r l bit dmh. b, Phoi giai dogn gin trudng thanh vai than phoi ro rgt
•"i-.L^ng la mim chua phat tnen va chua co choi, c. Si/ hinh thanh hai re bat djnh tip u| tri goc chli don tren moi tru&ng I/2MS bo sung NAA. d. Si/ phan nhanh ciJa r l bit djnh sau hinh thanh e,f,g,h. Sy hinh thanh nhilu r l bit dinh i Cum 1:1 lOi soma (hai phoi) dung tao re bSt dmh (loai bo phan re d vj tri thanh danh dau). j . Rl b^t djnh dgng cgm hinh thinh tir
Tap chi Cdng nghi'Sinh hgc 12(2): 355-364, 2014 Nhu da trinh bay, mo seo nhan dupc qua nudi cay tren moi trudng MS co 2 mg/l 2,4-D dupc nuoi ciy tren moi trudng MS c6 I mg/l 2,4-D, I mg/l NAA, 0,2 mg/l kinelin va 10%. nudc dira de tao mo seo CO KNSP (Hinh 3a); ti^p theo, mo seo co KNSP dugc nuoi cay tren moi trudng B5 co 1 - 5 mg/l IBA (50 g/l dudng). Ket q u i budc diu cho thiy mo seo trd nen ciing din va sau khoang 1,5 thing nuoi RBD hinh thanh it hay nhilu tuang quan thuan vdi su tang n6ng dg IBA Tren moi trudng co I mg/l IBA, re phit sinh it (Hinh 3b), nhieu hon d nong dg 3 mg/l vi 5 mg/l (Hinh 3c,d), Khi duy tri nudi cay trong thdi gian 2,5 - 3 thing, nhan thay r l tang sinh phii tren bl mat moi trudng (Hinh 3e,f). The mo mang rl
niy CO thi dugc nhan bang each cat nho cum (= 0,5 cm), ciy chuyin (Hinh 3g) va sau do mo tiep tuc tang sinh kh6i va tai sinh rl bat dinh mdi (Hinh 3h).
Qui trinh nhan mo seo da ghi nhan dugc su hinh thanh mgt s6 ph6i/ch6i (Hinh 3f,h), theo chimg toi, chung la ket q u i ciia q u i trinh nuoi cay mo seo iren moi trudng cd nong dp dudng cao ket hgp co auxin (IBA), kel q u i nay phii lipp vdi ket qua ngiiien ciiu ciia Asaka el ai, (1993, 1994) ve anh liuong tich cue cua nong do dudng cao (100 g/l) den kha nang dip liivj tao phoi soma ciia mo seo Panax ginseng, tham ch- vai tro ciia dudng nong do cao doi vdi su sinh phoi con the hien d moi trudng khong can bo sung chat dieu hoa sinh truang.
^ » ^ . j ^ r 1
mi- m UkmM
Hinh 3. Tao r l bit djnh lir md s^o cung tren cac moi trudng khac nhau va nhan re bat d|nh, a. M6 s^o c6 kha nang smh ph6i dting nuoi cay tao mo seo cung {vi tri mui ten). b,c,d. Sy hinh thanh re bat djnh lif md seo cung tren mdi trudng B5 co b6 sung 1, 3, va 5 mg/l IBA. theo thi> ty. e. Re bat dmh phat tnen sau 3 thang nuoi cay tren mdi trudng B5 cd 5 mg/l IBA f.
Cgn canh r l bit dinh tren tren moi truong B5 c6 5 mg/l IBA g Nhan mo seo cifng mang re qua cat cgm va cay chuyin h Sy hinh thanh r l bat dinh tren cgm md seo cung cudi giai dopn cay chuyin.
Mai Trudng el at.
T h u nghiem so sanh hinh dang, tim hieu su phan n h i n h cua re bat djnh hinh thanU tii- mo seo va phoi soma
Re bat dinh (so cap) tir ba ngudn vat lieu tren da dugc lich ra va nuoi ciy liep tuc tren moi trudng White CO 5 mg/l NAA v i B5 eo 5 mg/l IBA d l so sanh ve mat hinh thai. Kit q u i buoc diu cho thiy kha nang phan nhanh ciia cluing phu thuoc vio moi trudng khoing ca ban, nong dp auxin bo sung vi du tren mdi trudng White c6 5 mg/l IBA rl phan nhinh k e m - trung binh 5 re nhanh/re so cap, rl phin nhanh rai rac, re nhanh ngan tlliiih 4a) \ a moi tiuoiig B5 cd 5 mg/l IBA tao dap irng phan nhanh tot hon - trung
binh 6 r l nhanh/rl so cip, re phan nhanh tap trung, rl nhinh dai (Hinh 4b) v i giua ehiing khong co sir khic biei nhau v l mat hinh thii.
De tim hiiu c i e c i p ciia su phan nhinh. re bit dinh so c i p (chua co r l nhanh, tir md seo cirng) dupc nuoi ciy tren moi trudng ihach White co 3 mg/l NAA. Kit q u i cho thiy, r l so c i p tang sinh, co klia nang phan nhinh tao re nhanh cap 1, 3 va den cap 3 sau 1,5 - 3,5 thing nuoi (Hinh 5a.b.c). Ket qui nay phii hpp vdi nghien cuu cua Nguyen Thanh Sum el at, (2009) v l sii dung moi trucmg White bo sung NAA trong nghien ciiu nhan re bat dinh sain Ngoc Linh.
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Hinh 4. So sanh s u phan nhanh va hinh dgng khiic cat re bat djnh tif nhieu nguon goc treri cac moi trud'ng khac nhau. a.
Hinh dang khue c l l r l bat dmh nudi c l y tren mdi trudng White bo sung 5 mg/l NAA, cd ngudn gdc tir mo seo nudi c l y tren mdi trudng MS c6 NAA (1), tif phdi soma tren mdi trudng 1/2MS cd NAA (2) va tif mo seo cdng nudi cay tren moi trucmg B5 cd IBA (3) b. Hinh dang khue c i t r l b i t d|nh nuoi c l y tren mdi trudng B5 bo sung 5 mg/l IBA, cd nguon goc tir md seo (1), tif phdi soma (2) va tif md seo cifng (3) nudi c l y tren cac mdi trudng n h u t r u d n g hgp t r u d c .
Hinh 5. S y phan nhanh tgo r l t h u cap cCia r l bat djnh (tif mo seo cdng) tren moi Irifd-ng thgch. a. Sg- phan n h i n h ciia re bat djnh sp cap tao r l nhanh d p 1 sau khoang 1,5 thang nudi cay tren mdi trifd'ng White cd 3 mg/l. b,c. Sg- hinh thanh re nhanh cap 2 va cap 3 tif r l bat dmh so cap sau khoang 3,5 thang nudi cay tren mdi t r u d n g White n h u tren (m re chi'nh 1 2, 3: re nhsnh cap 1, 2 va 3).
Z-SQ
Tap chi Cong nghi Smh hgc 12{2): 355-364, 2014 Nham tim hieu khi nang hinh thanh so khdi r l , cac khiic cat re so cip tii' nio seo cung (dii khoing 2 cm, chua co so khdi rl) (Hinh 5a) dugc b d tn' nuoi ciy tren White co 5 mg/l NAA, moi trudng B5 co 5 mg/l NAA v i B5 co 3 mg/l v i 5 mg/l IBA. Kit q u i cho thay tren moi trudng White (cd 5 mg/l NAA) so sd khdi re hinh thanh it (so lupng khoang 2) (Hinh 6b): ngugc lai, iren moi trudng B5 co IBA 3 mg/l v i 5 mg/l (Hinh 6c, d) va NAA (Hinh 6e) dap ting tao sfl lugng so khdi rl cao hon, theo thii ty a 3 - 5 va 4.
Theo chiing toi ket qua tren la do thanh phan khoang moi trudng White ngheo hem so vdi m6i trudng B5 va khong co vitamin, Ngoii ra, nhin thay tren moi trudng B5 c6 NAA, hai diu va giira khiic cat co hai phii ra, c6 nhieu so khdi re nhung nhii khong cao so vdi tren moi trudng ed 5 mg/l IBA. Tiep theo de tim hieu siu hon tac dong ctia IBA den su phat inen cua re nhanh tir so khai re, khiic cat re dupc nuoi cay tren moi trudng B5 co I, 3 va 5 mg/l IBA. Ket qua clio thay nong dp IBA 3 mg/l tao su phan nhinh
manh so vdi nong dg I mg/l, dac biet nong do 5 mg/l tao su phan nhanh rat manh (Hinh 6f),
Hinh 6 con cho thiy loan cinh he rl nuoi cay tren mdi irudng White CO 5 mg/l NAA (Hinh 6g), tren B5 cd 5 mg/l NAA (Hinh 6h) v i B5 co 5 mg/l IBA (Hinh 6i), Cung nhan thay tren moi trudng B5 CO NAA, re cd dutmg kinh to hon re tren moi trudng CO IBA v i k h i nang phan nhanh cua rl tren moi trudng B5 CO IBA cao hon so vdi moi trudng chua NAA (phii hgp vdi thir nghiem doi vdi khiic cat n£u tren), Ket qua tuong tu cung dugc ghi nhan doi vdi nuoi cay khiic cat re b i t djnh co ngudn goc tir phoi soma (trinh bay d b i o cio khic), Su phan nhanh cao dudi tic dpng ciia IBA doi vdi re trong nghien ciiu niy phii hpp vdi ket q u i nghien euu eiia Nguyin Trung Thanh et al. (2007), Nguyen Thi Lilu et al.
(2011), Ket qua nghien ciiu d ngi dung nay tao tien de ve ngudn vat lieu re b i t djnh diing cho nuoi cay long lac.
Hinh 6. S u hinh thanh cSc so- khdi r l d khiic c i t r l b i t d(nh va s u phan nhanh tao quan the r l t h u c i p qua nudi c l y tren cac mdi trudng khac nhau. a. Cac khue c i t r l bat ^inh diing lam thi nghiem. b,c,d,e. S u hinh thanh mdt va n h i l u sa khdi tk tir khiJc c i t nudi c l y tren moi Irifdng White cd 5 mg/l NAA (b) va B5 cd 3 mg/l, 5 mg IBA (c, d) va 5 mgfl N/W (e) sau 20 ngSy nudi f. S u khac nhag ve s6 l u v n g r l nhanh hinh thanh tif khiic c i t r l nudi c l y tren mdi trudng B5 co 1, 3 va 5 mg/l IBA, Iheo thif t u . sau 40 ngay nudi g. Re phan nhanh tren mdi trudng White cd 5 mg/l NAA h. C i n canh r l phan nhanh tren mdi trudng B5 c6 5 mg/t NAA. i. Can canh r l phan nhanh tren mdi tn/d-ng B5 co 5 mg/l IBA,
Nuoi r^ bat dinh trong moi t r u o n g long lac Cac khiic cit vdi nhieu r l nhinh tir mo see Cling va tir phoi soma, qua nufli cay tren moi trudng thach 8 5 cd 5 mg/l IBA (Hinh 7a,b) dugc nuoi ciy
trong moi trudng long vdi cae thanh phan nhu tren.
Nhin thiy r l tang sinh kha sau 1 tuan nuoi (Hinh 7c) va tang nhanh sinh khoi sau khoang 2 thing nuoi nhung dudi anh hudng cua t i c dgng lac cac khiic re ting sinh cupn lai tao 'biii r e ' (Hinh 7e). Re 361
Mai Tnrdng el ai d3 dupc nuoi cay quy mo binh tam giic 500 ml (doi
vdi rl bat dinh cd nguon gfic tii phoi) v i 1.000 ml (doi vdi re bat djnh tu mo seo ciing) (Hinh 7d), Hien rl dang dugc nghien ciiu nuoi cay trong bioreactor sue khi the tich 3 lit den 10 lit va sd liSu se dugc trinh b i y d mpt b i o cao khic. Da co cong
b6 ket qua nghien cuu nuoi nhin re b i t djnh trong moi trudng long nhung re bat djnh nudi cay d dang gin liln vdi mo me - mo seo (Duong Tan Nhut et al, 2006), Thu nghiem nu6i kliiie cat re chua co rl nhanh da khflng dat k i t q u i tSng sinh re (so lieu e i nhan).
Hinh 7, Nudi khiJc cat re bat dinh trong mdt trud'ng long a. Cac khue cit re tif mo seo cung, sau 2 thing nudi cly tren mdi trudng thgch B5 cd 5 mg/l IBA. dting lam vat lieu nudi cly trong mdi trifdng Idng, b. C^n canh khiic cit ri vdi nhieu re nhinh dimg nudi cly trong mdi trudng long, c. Cac khiic cat r l tif md seo cirng giai doan sau 1 tuin nudi cly trong mdi trifdng long lie, d. Khiic d t r l bit dinh cd nguon goc tir phdi soma (1) va tCr md seo cung (2) tSng sinh khoi trong mdi trudng Idng lie B5 cd 5 mg/l IBA chira trong binh tam giac 500 ml ua 1.000 mi, sau 2 thang. e. Can canh mpt "biii r l " sau 2 thing nudi Idng l l a
KET LUAN
Tren doi tugng sam Nggc Linh, qua dng dung phuang phap nuoi eay mo in vitro tr6n vat lieu ban diu l i la ch6t cay ex vitro, da thuc hien co ket qua nghien ciiu t£io v i nuoi rl b i t dinh tren moi trudng thach va trong moi trudng long tir vat lieu mo seo va phoi trudng thanh mang choi. Co sd khoa hpc ciia su phan nhanh tao ting sinh re bat djnh bieu hien ve mit hinh thai nhu su hinh thinh cac sa khdi r l cung da duge nghien ciiu. Ket qua nghien ciiu trinh bay d bii nay budc diu tao tien de quan trpng cho nghifin ciiu nhan sinh kh6i r l bit dinh quy mo ldn phuc vu san xuat hgp chat thir cap.
Loi cam orn: Cdc ldc gid xin chdn thdnh cdm on Bo Khoa hoc vd Cong nghi, Sd Khoa hoc vd Cong nghi Tp. Hd Chi Minh dd tdi trg cho cdc ngi dung riing a nghien cuu ndy. De tdi duac thuc hiin Igi Phong thi 362
nghiim Irgng diem phia Nam ve cong nghi H bdo thuc vdt, Viin Sinh hgc nhiet ddi.
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Mai Truong', Tran Thi Ngoc Ha', Phan Tuong Loc', Le Tan Due', Tran Trong Tuan' Do Dang Giap', Bui Dinh Thach', Nguyen Thi Thanh', Pham Due Tri', Nguyen Due Minh Hung', Nguyen Van Ket^
Tran Cong Luan^, Nguyen Huu Ho''"
'institute of Tropical Biology, Vielnam Academy ofScience and Technology
^Da Lat University, Ministry of Education and Training
^Ho Chi Minh City Research Center of Ginseng and Medicinal Materials
SUMMARY
This paper presents some results on the induction and the culture of the adventitious roots of the Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv,) on the agar/phytagel or in the liquid media. The aim of this study is to lay out the foundation for large-scale propagation of this differentiated tissue for the secondary metabolites production. For inducing adventitious roots, three kinds of tissue, viz,, callus from the leaflet disc, the mature somatic embryo with shoot and the compact callus were used. In the one-phase culture, the adventitious roots were generated from the callus on the MS medium with 2 mg/l 2,4-D and 10% coconut water; the adventitious roots were also generated from the subcultured callus (two-phase culture) on the MS medium with 3 mg/l NAA, MS medium with 3 mg/l NAA, 2 mg/I 2,4-D plus 5 % coconut water and the B5 medium with 5 mg/l IBA. Besides, the adventitious roots were also obtained from the mature somatic embryos (after removing the tap root) through culture using the 1/2MS medium with 2 - 3 mg/! NAA The adventitious roots from the very compact callus (via culture on the B5 medium with 5 mg/l IBA) were cultured subsequently for evaluating the mass production capacity via secondary root proliferation The formation of root pnmordia from root segments on the White/B5 media was also carried out.
Keywords: Adventitious root, callus, mature somatic embryo, Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.), root segment
'Authorfor correspondence: Tel: +84-4907404827; E-mail' n^uvenhllullo2008(i^amaiicom 364
