KHOA HOC C 6 N G NGHE
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TRUONG TAN SANG Chu t!ch
Nu6c COng hda xS hpi chCi nghTa Vl^t Nam
IMGHIEIM C i i u iXlAIMG CAO K H A I\IA!\JG T A O iVIO SEO P H d l H O A V A T A I SIIMH CAY IIM VITRO T I J P H O I IMOIM IVIOT SO GldlMG L U A tIMDICA PHLJC V U BIEIXI (MAP GIEIXI
Nguyen Van Khi^m^ Nguyen Van CuuS Phung Thi Phirong NhungS
LATU Thi My DungS Pham H6ng QuanS Do Nang Vinh^
T6MTAT
Trong nghien cixu nay, he thong tai sinh cay in vitro cai tien tir mo seo phoi hoa co nguon goc tir phoi non cua cac giong liia Indica trong a nuac ta da duoc thiet lap. Mpt so yeu to khac nhau nhu moi truong va nong dp cac chat dieu hoa sinh truong anh huong den phan ting tao mo seo va tai sinh cay da diroc nghien ciiu.
Phoi non 12 ngay tuoi dupe nudi cay xap trong moi tarong ca ban N6 cho phan iing tao nio seo va tai sinh cay tot hon so voi moi truong co ban MS. He thong tai sinh cay tir pho- nan gom cac buoc: Buoc thii nhat, phoi non dupe nuoi cay tao mo seo trong moi truong N6 bo sung 2,4-D (2 mg/1), kinetin (0,1 mg/1), sucroza
(30 mg/1), cazein hydrolysat (300 mg/1), Lprolin (500 mg/1) duoi anh sang lien tuc 2000 lux a32"C trong 2 tuan. Buoc thir hai, mo seo phoi hoa dupe nuoi cay tao choi trong moi truong N6 bo sung BAP (2 mg/1), a- NAA (0,1 mg/1), cazein hydrolysat (2,0 g/1), L-prolin (500 mg/1), sucroza (30 g/1) o 26"C, chu ky 16 h chieu sang : 8 h toi trong 2 tuan; buoc thir 3: de keo dai choi va tao xh, mo seo co cac mam choi dai hon 2 mm dupe nuoi cay trong moi truong N6 khong co chat dieu hoa sinh truong trong 2 tuan. Cay tai sinh hoan chinh dupe cay chuyen trong trong nha kinh. Hp thdng tai sinh cay nay co the dupe sir dung de bien nap eac glen khac nhau vao phoi non cac giong liia.
Tir khoa; He thong tai sinh cay, tai sinh cay in vitro, mo see phoi hoa, phoi non, moitrudngNG.
sinh cay phuc vi.i bien nap gien vao cac giong liia trong loai phu Indica (Amin va cong su, 2004; Hoque
I. DAT VAN DE
a Ilia hi$u qua tai sinh cay phu thuoc dang ke vao giong, nhat la cac giong liia trong loai phu Indica.
Cho nen trong nhung nam gan day da c6 mot so nghien cuu tien hanh toi uu hoa moi truong nuoi cay tao mo seo va tai sinh cay de su dung he thong tai
va cong su, 2005; Kumar va cong su, 2005; Lin va cpng su, 2005; Visarada va Sarma, 2004).
Cho den nay da co mot so nghien ciiu chi ra rang mo seo co nguon goc tir phoi non co tiem nang tai sinh cay cao. Do vay, de cai thien hieu qua tai sinh va bien nap gien b cac giong liia Indica va mot so ' Vien Di truyen Nong nghipp
giong Ilia Japonica kho tai sinh, phoi non da duoc su dung de bien nap va da thu duoc hi6u qua bie^n nap cao (Hiei va cong sit. 1994; Saker va cong su, 2006;
Hiei va cong su, 2006; Hiei va Komaii, 2008).
Hiei va Komari (2008) da chi ra ning C-M- yru to quan trong de bien nap tlianh cong ph(M non liia (V cac giong liia Indica va Japonica la chat lucrng ph6i, tuoi phoi va xu ly \A\o\ vcVi nhiet va ap su;it truoc khi bien nap. Ho da nhan thfiy rAng chat luc.mg phoi non phu thuoc vao do klioc ciia cay va giai doan sinh truong, phat trien ciia cay.
Nghien cuu chuyen gien vao liia (V nuoc ta moi chi duoc bcit dau khoang 15 nSin gan dSy. Mac du phoi non la vat lieu tot cho bi^n nap va tai sinh liia nhung cho den nay co the a nuoc ta hi^n chua co nghien cuu nao v^ tao mo seo va tai sinh cay tir phoi non phiic vu cong tac bien nap gien liia. Trong cac nghien cuu ve bien nap phoi non liia mod chi co mot nghien ciru cua Tran Thj Cue Hoa va cong sir (2004) da bien nap gien cnlAc va r/ylAb vao phoi non giong Ilia IR64, K105 va Mot bui su dung Agrobacterium dat hieu suat chuyen gien tir 0,79- 5,83% phu thupc vao vector bien nap va giong liia.
Do do, nghien ciru tai sinh phoi non cac giong lua phuc vu cong tac bien nap a nuoc ta la rat can thiet. Trong pham vi de tai nay da nghien ciru anh huong cua tuoi phoi, giong liia, moi truong va chat dieu hoa sinh truong den tao mo seo va tai sinh cay.
Duoi day la mpt so ket qua nghien cuu cai thien de nang cao hieu qua tao mo seo phoi hoa va tai sinh cay tir phoi non ciia mpt so giong liia Indica dang dupe trong o nuoc ta. Quy trinh tai sinh cay tir phoi non se dupe ap dung de phuc vu cho cong tac bien nap cac gien vao phoi non liia trong thoi gian toi.
L VAT UEU VA PHUONG PHAP
1. vat li^u nghien ciiru
Vat li$u nghien cuu gom co 18 giong liia thuan Indica. Khang Dan 18, DT36, DT37, DT42, Huong Xuyen 5, Phieu Huong 1, Xi-23, QRl, IR64, Bac Thom
so 7, Bac Thom so 8, VSl, Liia Thom LTl, Lua Thom LT2, Liia Thom LTIO, Khang Dan dot bien, Tieu Huong 138 va IR56.
2. Chuan bi mau, khiJr trung hat vk nu6i c^y Mau dupe gieo trong 6 chau trong di^u kidn nha kinh tai Tram Thuc nghiem Van Giang, Hung Yen. cay liia dupe theo doi d^n khi tro bong. Bong liia sau khi thu phan vao cac giai doan 8, 12, 16, 20 ngay dupe thu hoach. Mau dupe bao quan a 4°C den khi su dung cho thi nghiem. Hat liia dupe hoc v6.
khur triing hat bang con 70" trong 30 giay va rua bang nu6c cat v6 triing de loai bo con. Sau do, hat dupe khiT triing bang HgClg 0,1% trong 7 phiit va nia bang nuoc cat v6 triing 5 lan d^ loai bo HgClg.
Phoi hat non dupe tach khoi hat liia bang dao va panh cay v6 trung trong bdc cay laminar va cay xap phoi trong dia cay petri duong kinh 6-9 cm chiia moi truong tao mo seo bo sung chat dieu hoa sinh truong 2,4-D va kinetin, o 32"C trong dieu kien anh sang lien tuc, 2000 lux. 1 y le phoi hat non tao mo seo va mo SCO phoi hoa diim* ghi nhan sau 2 tuan nuoi cay trong tu nuoi RUMEI) co dieu khien nhiet do, dp am, anh sang. Tai sinh cay dupe thuc hi^n bang cay chuyen mo SCO phoi hoa nu6i caiy trong moi truong tai sinh choi chua chfit di^u hoa sinh truong trong thoi gian 2 tuan. Mau nuoi cay dupe giur 6 26"C chu ky 16 gia chieu sang: 8 gio toi. Ty le mo seo tao choi va so choi/mo seo du()c ghi nhan sau 2 tuan nuoi cay. De tao cay hoan chinh, sau 2 tuan nuoi cay nhCrng mo SCO tao mam choi xanh dai tren 2 mm dupe cay chuyen sang moi truong khong co chat dieu hoa sinh truong, o 26"C chu ky 16 gio chieu sang: 8 gid toi.
cay tai sinh hoan chinh dupe cay chuyen trong chau dat o nha kinh.
Moi truong tao mo seo la moi truong MS (Murashige va Skoog, 1962) hoac N6 (Chu va cpng su 1975) + cazein hydrolysat (300 mg/1) + L-prolin (300 mg/1) + sucroza (30 g/1) + phytagel (4 g/1) + 2,4-D (0-3 mg/1) + kinetin (0-0,25 mg/1).
Moi truong tai sinh cay gom tai sinh choi (buoc I): MS hoac N6 + cazein hydrolysat (2,0 g/1) + L prolin (300 mg/1) + sucroza (30 g/1) + phytagel (4 g/1) + BAP (0-3) mg/1 -H a-NAA (0-0,5 mg/1).
Moi truong keo dai choi va tao rk (buoc ID: MS hoac N6 -t- cazein hydrolysat (2,0 g/1) -i- I^prolin (300 mg/1) + sucroza (15 g/1) + phytagel (4 g/1), khong bo sung chat dieu hoa sinh truong.
Ty le tao mo seo = so phoi non tao mo seo/tong so phoi non nuoi cay.
Ty le tao mo seo phoi hoa = so phoi non tao mo seo phoi hoa/tong so phoi non nuoi cay.
Ty le tai sinh cay= so mo seo phoi hoa tai sinh/tong so mo seo phoi hoa nuoi cay.
Ty 1^ tai sinh cay= so cay tai sinh/njo seo nuoi cay.
n.KFr QUA VA THAO LUilN
1. Anh huong ciia mdi trudng nu6i d(y d^n tao m6 seo vd t ^ sinh cSy
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2012
KHOA HOC C 6 N G NGHE
Hai moi trmVng co ban la MS va N6 duw su So lieu a hang 1 cho thay ty \v tao nio seo va mo dung de nghien cu\i anh huc'mg ciia HKM truinig nuoi seo plioi hoa iihu thuoc dang ke vao giong va moi cay den tao mo seo va tai sinh cay tir phoi non ciia 18 truong ar ban nuoi cay. So lieu a bang 1 la gia tri giong Ilia India khac nhau. Ca hai moi tmong sir trung binli ciia 3 lan nuoi cay lap lai voi tong cong dung la MS va N6 co thanh phan da luc.mg, vi lumig 100 i)h6i (mni l.m nuoi cfiy 30-35 ])h6i). Nhin chung, khac nhau nhung co ciing ndng dp cac vitamin. D.i moi trirdug N(i cho ly le tao nio seo va mo seo ])li(')i sir dung ca hai moi tnrimg nay bd sung dumig hoa cao lioii nuu tniinig MS tu .3-10%. Su khac nhau sucroza (30 g/1) c;izein hydrolysat (300 mg/1), 1.- nay co the do tli.inh i)lian va ham lii(,nig chat khoang prolin (300 mg/1) va chat dieu hoa sinh tnn'nig 2. l-l)
(2,5 mg/1), kinetin (0,25 nig/1) de nghien cini anh hudng den tao mo seo va ni6 seo i)h6i hoa. Doii.i; thoi da bd sung cazein hydrolysat (2 g/1) -i- L-prolin (300
da, vi liioiig khac nhan co trong moi tnroiig nuoi eay.
Nlur vay, thanh i)h.in, ham Itroiig eac nguyen to da lirpiig va VI luong co anh huVmg den tao nio seo va nio seo jjhoi hoa ciia phoi non. So lieu o bang 1 cung mg/1) + sucroza (30 g/1), P,.\P (L',() mg/1), a-^\.\ cho thay trong moi truong co chat dieu hoa sinh (0,1 mg/1) vao cac nioi liirinig nay de nghien cmi .inh trmVng gidng nhau ty le tao ni6 seo phoi hda d c;ic hudng den tai sinh cay, gidng cung kluic nliau dang ke, dao ddng tir 57,46 -
S() lieu \'e anh hux')ng ciia moi ti-utmg caIx'ui deii ta<. ^^•"^^'^ (^'"i ^'"•''"'''' ^•^^'"^^^ ^(5) va tu 52,0 -72,67% (ddi mo seo va mo s(X) jihoi hiia ciia 18 gidng liia dinv Uie ^'" "^*'' '•''ir<«ifl MS),
hien dbang 1 va anli hut'nig den tai sinh chdi <V Ixing 2.
Bang 1. Anh hudng cua moi truong co ban^d^'n Uio ni6 seo va mo seo phoi hoa TT
1
1 •) 1 w
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
(iidng lua
Khang Dan 18 Huong Xuyen 5 DT36
D137 DT42
Phieu Hmnig 1 QRl
Xi-23 1R64
Bac Thom sd 7 VSl
Bac Thom sd 8 Liia Thom LTl Liia Thom LT2 Liia Thom LTIO Tieu Huong 138 Khang Dan DB IR56
Moi tnnnig MS Ty le tao mo
seo (%) a B ± SE) 70,15 ± 2.16 81,00 ± 2,53 70.04 ±2,15 78,33 ± 2,45 66,00 ±0,18 89,54 ± 2,66 62.00 ± 1,82 67,33 ±1,67 72,00 ±1,80 75,63 ±2,14 78,05 ± 2.25 77,56 ±2,14 75,42 ± 2,38 78.44 ±1,88 72,45 ±2,11 70,46 ±1,92 68,54 ±2,15 64,25 ±1,68
ly le tao ni6 seo phoi hoa (%)
a B ± SL) 63,67 ±2,12 72.67 ± 2,33 56,54 ± 2,45 62,00 ±2,22 56,00 ±0,58 72,45 ±1.15 52,00 ±1,67 55,67 ±1,44 64.24 ±1,67 64,00 ± 2,21 67.33 ±2,15 65,58 ± 2,35 68,82 ±2,15 69,00 ± 2,24 67,52 ±1,67 68,00 ± 2,18 61,45 ±1,45 56,35 ±1,22
Moi tn 1 > le tao mi) seo
(%) (vn 1. SI-;) 71,()()± 2,2S h,'"),15±2.58 75.34 ± 2.25 .S1,45±2,S5
<i!),08±l,12 92,46 ± 2,65 68.52 ±1.75 71,18±1,54 78,33 ± 1,78
SI,33 ±2,55 cS2,78 ± 2,45 S.1.74±2,25 85,41 ±2.57 83.44 ±1.12 82.56 ±1.85 75,48 ± 2,05 71,50 ± 2.34 66.33 ± 1,45
nnig N(i
'1 y le tao mo seo I)h6i hoa (%)
(IE ± SE)
65,67 ±1,86 1 80,45 ±2,15
61,52 ±2.54 66,54 ± 2.12 57,46 ± 0,65 78,00 ±1.25 58,45 ±1.86 60.48 ±1,55 70,38 ± 1.75 74.00 ± 1,24 77.45 ± 2,52 75.89 ± 2,56 78,70 ± 2,33 76,74 ± 2,32 77.44 ± 1,82 69,00 ± 2,28 62,22 ± 1,58 60,56 ±1,25 Ghi chu: TB: Trung binh; SE: sal so chuan
So lieu o bang 2 cho thay ty le tai sinh eay va sd d moi truong N6 cao hon d moi truong MS tir 2-8,2%.
cay tai sinh/md seo phoi hda d moi trudng N6 cao Ddi vdi moi gidng, trong moi truong N6 ty le tai sinh hon d moi trudng MS. Tong cpng 75-90 mo seo phoi cay va sd cay tai sinh/mo seo cao hon d moi trudng hda cua mdi gidng (25-30 phoi/lan lap lai) dupe nuoi MS. Nhin chung, nhCmg gidng cd ty le tai sinh eay cay trong moi trudng tai sinh chdi. Ty le tai sinh cay cao trong moi trudng cung cd sd cay tai sinh/mo seo
cao hon so vcVi nhirng gidng co ty le tai sinh cay thap.
Sir khac nhau nay cung co tht'' do j'uih hinVng ciia thanh phan chfit khoang da lirmig, vi lumig c() trong m()i truimg nudi c.iy. Co 13 gidng cho ly le tai sinii
^20% trong moi truimg MS, trong khi co 14 gidng cho ty le tai sinh ^20% trong moi truimg N6. So lieu (V
Bang 2. Anh h m m g ciia mdi trudng ddn t.ii sinh cay MOI tnnniK' MS
bang 2 ciing cho tha'y trong cung mdi truimg tai sinh ty le mo seo plioi hda tao chdi d cac- gidng cung khac nhau dang ke, dao dong tir 13,33 - 12,56% (ddi viri moi tririmg N6) va tir 10.0 -37,14% (ddi vin nidi trudng MS).
(lionj,: lua Klwng Dmi IS Iluimg Xuyeii 5 ' {rr.vj
m-v2
riiieu Ihroiig 1 (^Rl
Xi-23
y le nio seo lai sinh chill (X)
(TH I SE) 3(),()() t 1,45
10
1R()4 Bac Thom
:57,14 il.22 21,05 1 O.SM 20,00 ± 1,11
13.33 ±0,25 3(;,(i7 1 1,28^
13,33 i (),(i7 JO,(H|^0,:i:i
2ii,(i(ij_(),9r)
;u),()0 1 i,2r) 11 \'S1
Bac Thcnn 13
14 15 16 17 18
i) S_
XL
LiiaThmii El 2 Lua Tlurm
:^(i,i.5i
;!2.54 ± 34,24 ±
l,4(i 1,3«
E14
LiiaThcmi LTIO Tieu Huong 138 Khang Dan DB IR56
33,33 ± 1,23
So cay lai
•>mh/!iio '^eo (TH I Si:) .5,,% 1 0,20 6,.54 I 0,18 4.:;s 1 o.LM 4,16 ! 0,2S 4,50 I o,:',.s_
8.18 ± 1,22 2jf>±i).\r-> _ 3,:53 1 0,05_
5,0U_i_0.22 J , 5 0 iJ),_lS
6.55 i {),2()_
7,23 i 0,31
y le mo SCO tai siiih ciu I (XJ
(TH SI.) :',4,15 1.54
4 2,.50 2 1,2S 25,r,4 A 1,15 24.22 i 1,21
\(iju 1 (),f)4
Mid Innnig \ i ) Sii eay lai sinh/mo seo j ; r B ^ S I j
0,2l_r 0,42 7.24 -. 0,35
1.3S t^O.32 1,15£0.:;4_
4,7N 0,2.s 0,85
74 _ 10,45 13.;',3±0.45 _31,1S
:5S,21
0,02 ± 1.25 3,20 ^ 0.25 , 15 r 0.15 J_,12_
3(),S2^ 1,.55
' 1 t 0,38
37,24 ± 1,62 7,85 . 0 , 4 2 3S,f^2^ 1,24
5,IS -0.22 7,14 . o,::t() 7 1.1 r (I.2S S,.4 ::: 0.4.1 6,50 I 0,2,3
31,.50± 1,36 16„55± 1,05 22,S3± 1,44 lS,^^5i 1,27
6,25 ±0.L'.') 3,05 ±0,11 4,55^ 1,14
;57,-i5^ i,:;(;
ilii.-lO : 1,7S l S , 4 5 x l , 4 4 1
7,44 I 0.>uS 7.00 ' 0,32 3,1(1^0.15
2,05 ± 0,12
-'0.4 1 -1 Eivt 20,8:.' 1,47
4,78^ 1,24 3.42 X 1,15 Ghi chii: TB: Trung binh: SE: s:u .SY) chuiin
2. Anh hudng cua chat di6u hda sinh trudng dcfn tao md seo va tai sinh cay
Ket qua nghien ciru cr bang 1 va bring 2 cho thay moi trudng N6 co anh h m m g tdt hem den tao md seo va tai sinh cay so viri mdi Irmmg MS. De nghien ciru anh hmmg cua chat didu hoa sinh truimg den tao md seo phdi hda, da sir dung mdi truimg N6 bd sung 2,4- D va kinetin vdi ndng dd khac nhau vao moi truimg nudi cay. Da su dung phdi non cua 5 gidng liia co kha nang tao md seo va tai sinh cay cao trong sd 18 gidng da khao sat la IR64, VSl, Bac T h o m sd 8, Lua Thom LTIO, Phieu Huong 1 de nghien cuu anh hudng cua chat dieu hda sinh trudng den tao md seo va tai sinh cay. Ket qua nghien cuu anh hudng cua chat dieu hda sinh trudng den tao md seo phdi hda va tai sinh eay dupe the hien d bang 3 va bang 4.
Trong mdi thi nghiem, tdng cdng 100 phdi dupe
truimg 2,4-1) (2,5 mg/1) va kinetin (0,25 mg/1) trong mdi truong duov sir dung Uun i\in chirng so sanh. Mo seo phdi hoa co mau vang, xop cd kha nang tai sinh chdi cao hon cac loai md seo kliac. Do dd, de cd the ket luan to hi.ri) chat dieu hda sinh tru(Vng nao thich h(ri) nhfit cho tao md seo phdi hoa. da bd sung nong dd chat dieu hda sinh truimg khac nhau vao moi truimg. Klii khdng bd sung 2,4-D va kinetin vao moi truimg, mo seo phdi hda k h d n g hinh thanh. Trong mdi truimg dui.n: bd sung 2,4-D (1,0 mg/1) va kinetin (0,1 mg/1) ty le md seo phdi hda tao thanh d ca 5 gidng giam so xin ddi chung. Ty le md seo phdi hoa cua ca 5 gidng hinh thanh d mdi trudng dupe bo sung 2,4-D (2,0 mg/1) va kinetin (0,1 mg/1) dat cao nhat va cao hon so vdi ddi chiing khoang 5-8%. Trong khi trong mdi trudng bd sung 2,4-D (3,0 mg/1) va kinetin (0,1 mg/1) ty le md seo phdi hda d eac gidng nay thay ddi k h d n g dang ke so vdi ddi chimg. Nhu nudi cay trong 3 lan lap lai. Td hpp chat dieu hda sinh vay, cd the ket luan mdi trudng N6 bo sung sucroza
6 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2012
KHOA HOC C 6 N G NGHE
(30 g/1), cazein hydrolysat (300 mg/1), Dprolin (300 trudng tliich hop nhat clio tao mo seo phdi hda tu mg/1) 2,4-D (2,0 mg/1), kinetin (0,1 mg/1) la moi phiMnon.
Bang 3. Anh hudng ciia chg[t di^u hda sinh trudng ddn tao md seo phdi hda tir phdi non Ndng dd
2,4-D+kinetin (mg/1)
0,0 + 0.0
1.0-1-0,1 2,0 + 0,1 3,0-H 0,1 2,5 +0.25 (Ddi chung)
IR64 (TB ± SE)
0 ± 0
65,67 ±1,35 78,(i7± 1.96 72.33 ± 1,48 70.38 ±1.75
Ty le phoi non tao md seo VSl
(TB ± SI-:) 010 72,48^1 ^OA 84.33 ± 2,82 75,(i7± 1,45
77,45 ±2,52
Bac Thoni sd 8 (VB ± SE)
OiJ)
J_\.27 1 2 ^ 81,(i7 t 2,15 74,2S± j,(">S 75.89 1 2,56
)h6i hda (%) Lua 'ITiom
LTIO a n 1 SIX
OiX 72,33 ± 1.76 83.67 ± 1,62
77,41 1 1.82
i^liieu Hucmg 1 CrB ± SE)
OiO 73.26 ±2,18 86.12 ±2..56 75.78 ± 2,67 78.00 ±1,25 Ghi chu: TB: Tnmg binh: SE: sni so chuiin
Tdng cdng 75-90 md seo ])h6i hda ciia mdi gidng (25-30 phoi/lan lap lai) duot.- nuoi cay ti'ong moi trudng tai sinh chdi. Trong moi truimg khdng bd sung chat dieu hoa sinh truimg. chdi khong dui.A:
hinh thanh. Chimg td chat dieu hda sinh trudng BAP va a-NAA ed vai trii quan ti-png. quyet dinh den i)hat sinh chdi tir mo seo phoi hda. Trong ca 3 trmVng hoj):
khi giam ndng do BAP tdi 1.0 mg/1 va giur nguyen ndng dp a-NAA (0,1 mg/1); giCr nguyen ndng dp BAP (2,0 mg/1), tang ndng dd a-NAA tdi 0,5 mg/1; tang ndng dp BAP tdi 3,0 mg/1 va a-NAA tdi 0,5 mg/1, ty le mo seo phoi hda tao chdi d ca 5 gidng deu giam so
mg/1) va (j(-N.'V\ (0.1 mg/1). Nhu vay. khi tang hoac giam ndng do I W va u-NAA trong moi truong da khong CO tac dung tang kha nang phat sinh chdi tir mo seo phdi hiia. Do do. co thi^ ket luan moi truong N(i bd sung sucroza (30 g/1), cazein hydrolysat (2.0 g/1), Dprolin (300 mg/1), BAP (2,0 mg/1), u-NAA (0,1 mg/1) la moi truimg thich hoji nhat cho tao chdi tir mo seo phdi hda cd ngudn gdc tir phdi non (Bang 4). Cho den nay da cd mdt sd tac gia sir dung mdi truimg N6 trong nghien ciru tai sinh cay va bien nap vao phoi non lua (Koetje va cpng sir, 1989; Liu va cpng su, 2004; Hiei va Komari, 2008); vao phoi non ngo (Fu va cdng su, 2011).
vdi ddi chung (mdi trudng dupe bd sung BAP (2,0
Bang 4. Anh hudng cua chat di^u hda sinh trudng den tai sinh cay cua mpt sd gidng liia Ndng do BAP +
a-NAA (mg/1)
0 + 0 1,0 + 0,1 2,0 + 0,5 3,0 + 0,5 2,0 +0,1 (Ddi chimg)
Ty le md seo phdi hda tao chdi (%) 1R64
(TB ± SE) 0±0 20,12 ±1,36 27,67 ± 1,45 24, 33± 1,24 31,18 ±1,12
VSl (TB ± SE)
0±0 24,56 ±1,12 32,26 ±1,66 30,15 ±1,27 36,82 ± 1,55
Bac Thom sd 8 (TB ± SE)
0±0 24,89 ±1,56 31,15 ±1,88 28,55 ±1,42 37,24 ± 1,62
Lua Tilom LTIO (TB ± SE)
0 ± 0 27,25 ±1,67 32,35 ±1,34 29,56 ±1,45 36,66 ±1,78
Phieu Huong 1 (TB ± SE)
0 ± 0 30,78 ±1,75 37,67 ±1,67 34,82 ±1,22 41,74 ±1,55 Ghi chdi: TB: Trung binh; SE: sai so chuiin
Khi chuyen cac md seo ed cae mam chdi dai hon 2 mm sang mdi trudng khdng cd chat dieu hda sinh trudng, chdi dupe keo dai va phat sinh r^. Sau 2 tuan trong mdi trudng nay chdi da phat trien thanh cay hoan chinh cao 3-7 em, mang 1-5 vh. Chiing dupe eay chuyen trdng trong bau dat va luu giir trong nha kinh d nhiet dp 28-35"C. Ty le cay sdng sau 1 thang dat
tren 95%. Tat ca cac cay sinh trudng, phat trien tdt trong nha kinh cho den khi hat chin. Khdng cd bieu hien ve bien di hinh thai dupe quan sat thay d cac eay nay.
Hinh anh mo ta eac bude tai sinh cay tir phdi non lua 12 ngay tuoi cua mdt sd gidng liia dupe the hien d hinh 1.
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Hinh 1. Hmh anh md ta cac buiVc tai sinh cSy tir phdi non lua 12 ngay tudi ciia 5 gidng lua: IR64, VSl, Bac l l i o m sd 8, Lua'IhoTO LTIO, PhiOu Huimg 1.
AI- .1.7 Mo SCO f/io r:i tir phoi lim non trong moi truimg tiio nu) seo •^:iu i' tuiin nuoi cv'iy. lU-Ii'): l;io choi tai sinh s:iii 3 tirin nuoi < .vi • i '1-C5: C Yiy liia tni sinh trong U'ong c/iiiii <> nliii kmh.
IV. KET LOAN
Quy trinh lai sinh cay in vitro tir phdi non lua da dupe nghiiMi ciru de nang cao kha nang lao md seo phoi hda va tai sinh cay.
Ket qua nghien cuu anh hudng eua mdi tnrimg den tao md seo va tai sinh cay tir phdi non cua 18 gidng lua da cho thay mdi truong N6 cd anh huimg tdt hon so vdi mdi trudng MS ve tao md seo phoi hda va tai sinh cay.
Ket qua nghien cuu anh hudng cua td hpp chat dieu hda sinh truimg den tao md seo phdi hda va tai sinh eay tir phdi non ciia 5 gidng lua khac nhau cho thay td hpp chat dieu hda sinh trudng 2,4-D (2,0 mg/1) va kinetin (0,1 mg/1) cho ty le tao md seo phdi hda cao nhat. Td hpp chat dieu hda sinh trudng BAP (2,0 mg/1) va u-NAA (0,1 mg/1) cho ty le tai sinh cay cao nhat.
Tao md seo tir phdi non dupe thue hidn trong mdi trudng N6 chira 2,4-D (2,0 mg/1) + kinetin (0,1 mg/1), sucroza (30 mg/1), cazein hydrolysat (300 mg/1), Dprolin (500 mg/1) dudi anh sanh lien tue 2000 lux d32"C.
Tai sinh md seo phdi hda thanh chdi dupe thue hien trong mdi trudng N6 bd sung BAP (2,0 mg/1), a- NAA (0,1 mg/1), cazein hydrolysat (2,0 g/1), I^prolin (500 mg/1), sucroza (30 g/1) d 26"C, chu ky 16 h sang: 8 h tdi. Chdi dupe keo dai va tao eay hoan chinh khi nudi eay md seo ed choi dai hon 2 mm
trong moi truimg N6 khdng bd sung chat dieu hoa sinli truimg.
TAI LJ$U THAM KHAO
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AN IMPROVED PLANT REGENERATION SYSTEM DERIVED FROM IMMATURE EMBRYOS OF INDICA RICE CULTIVARS FOR RICE TRANSFORMATION
Nguyen Van Khiem, Nguyen Van Cuu, Phung Thi Phuong Nhung, Luu Thi My Dung, Pham Hong Quan, Do Nang Vinh.
Summary
An improved in vitro plant regeneration system derived from immature embryos of Indica rice cultivars grown in Vietnam has been established. Various factors were tested for callus induction and plant regeneration. It was found that 12 days old immature embryos with the plumule radical axis side placed in contact with N6 basal medium was better than with MS basal medium in response of callus induction and plant regeneration. Callus induction and plant regeneration responses of immature embryo were also affected by the growth regulator levels in the medium. Plant regeneration system of immature embryos of Indica rice cultivars was included steps: Firstly, immature embryos was cultured for callus indution on N6 medium containing 2.4-D (2.0 mg/1), kinetin (0.1 mg/1), sucrose (30 mg/1), casein hydrolysate (300 mg/1), L-proline (500 mg/1) under 2000 lux continuous light, 32"C for 2 weeks. Secondly, embryogenic calli were cultured for shoot induction on N6 medium supplemented with BAP (2.0 mg/1), a-NAA (0.1 mg/1), casein hydrolysate (2.0 g/1), I^probne (500 mg/1), sucrose (30 g/1) at 26"C, 16 h light 8 h dark for 2 weeks.
Thirdly, for shoot elongation and rooting induction, callus appearing shoots more than 2 mm in length was subcultured to N6 medium without phytohormones for 2 weeks. Whole plants were transferred to the greenhouse. This plant regeneration system could be used for transformation various genes in immature embryos of different rice cultivars.
Keywords: Plant regeneration system, in vitro plant regeneration, embryogenic callus, immature embryos, NSmedium.
Nguoi phan bi$n: PGS.TS. Ngd XuSn Binh
