Cdng nghj sinh hpe & Giong cdy trdng
X A Y D t T N G H ? T H 6 N G T A I S I N H B A C H D A N U R O {Eucalyptus urophylla S.T. Blake)
Tir M 6 SEO PHyc vu CHON DONG TE BAO
Nguyen Thj Hworng', Nguyin Van Viet^
'•^Trudng Bgi hoc Ldm nghiep T O M T A T
Tii smh bach d ^ thong qua t?o da ch5i tnrc tiSp t& mo seo da du-<?c xay dvng thanh cong. YJX qui cho th^y, diing doan than mam 8-10 ngay tu6i nu6i tren mfii tnrong khoang MS b6 sung 0,4 mg/l NAA, 0,2 mg/l IB A, 0,2 mg/l BAP, 100 ml/l nu<ic diia, 20 g/l sucrose, 7 g/I agar, nuoi frong t6i 2 tuan, sau do chuyin ra nuoi sang voi cuimg dfi anh sang 2000 lux cho ti 1| tgo m6 seo 97,8%. Tai sinh da choi tryc tifip tCr mfi s?o tren m6i truong khoang MS bfi sung 0,6 mg/l BAP, 0,1 mg/l Kinetin, 0,3 mg/l NAA, 100 rnI/1 nud-c dira, 20 g/l sucrose, 7 g/l agar, nufii dudi anh den neon 2000 lux cho ty le tai smh chfii dgt 71,8% va so chfii trung binh/mlu dat 8,5 sau 4 tuan nuoi cSy. Kich thich ra r l lao cay hoan chinh tren mfii tnrdng khoang co bin MS bo sung 0,3 mg/l NAA, 0,2 mg/l IBA, 20 g/l sucrose,? g/l agar, ty 1? ra rS dat 92.4%. Cay hoan chinh du(jrc chuyen ra trfing trong hhi dit vdi thSnh ph5n rugt biu ia d§t, cat sach (1:1), ty le song-dgt 89%. H§ thfing tii sinh cay bach dan hi^u suit cao c6 the ap dung trong tgo giong bach dan bing phirong phap chon dong te bao mang biSn dj soma c6 kha nang chiu man.
T(r khda: Bach Hia UrS, da zhhi, doan thSn mam, manh \& m^m, mo sf o, tai sinh.
I. D^T VAN DE
Viet Nam la qu6c gia giap bien voi bo bifin dai va tham thuc v^ phong phii. Tnroc ap luc ciia su bien d6i khi hau, cac giong cay ban dia ng^y cang bi thu hep di?n tich song do tinh trang nuoc bifin dSng. Vi vay, cac nha khoa hpc dang huong dfin giai phap t?o ra mot so loai cSy co the s6ng va smh trudng t6t trfin nhiing khu vuc co difiu kien khi hau bat thuan nhu n6ng, l^nh, hgn ho|c nhiing viing dit bi nhilm phen, man.
Bach dan {Eucalyptus) la cay g6 ciing quan trong, CO ngudn gdc tii Uc. Bgch dan dugrc trong riing vol dien tich lon va pho bien nhSt, udc tinh khoang 20 trieu ha trfin toan thfi gidi, dac bi?t trong nhifiu d Trung Qudc, An Dp, Nam My va Dong Nam A (FAO, 2000). Cac chi bgch dan gdm hon 700 loai va cac gidng lai, nhieu loai co gia tri kinh te cao nhu loai E.
Camaldulensis, E. grandis, E. globulus, E.
urophylla vk gidng bach dan lai la cac loai dang dupe gay trong rung chii yfiu.
Trong nhimg nam g§n day, cdng ngh^ nudi cay mo te bao thuc vat da ra ddi va dang khdng ngiing phat triln, thu duac Ax nhieu nhiing
thanh tuu ndi bat, co vi tri quan trpng trong linh vuc sau xuat giong cSy trong. Uu diem noi bat cua phuong phap nay la cd thfi tao duac so luang cay con ldn trong thdi gian ngin, giu dupe dac tfnh di truyfin on dinh, dong dfiu vfi kieu hinh, cac ca the phat triln binh thudng, khde manh (Le Tran Binh va cdng s^, 1997).
Tai sinh cvun chdi in vitro true tiep tCr m6 s?o da va dang dupe nghifin ciiu manh nh4m phuc vy cho cong tac nh§n gidng, chpn tgo gifing cay trong ndng lam nghiep (Duang Tan Nhut va epng sir, 2007). Day la ky thuat giiip dilu khien su phat sinh hinh thai cua thyc vat mpt each cd dinh hudng dya vao su phan hoa va phan phan hda tren co sd tinh toan nang ciia te bao thuc vat nhSm tao ra cay hoan chinh tu vgt iifiu in vitro. Quy trinh tai sinh mpt sd loai bach dan ciing da dupe bao cao:
Bandyopadhyay va cong sy (1999) d3 tai sinh thanh cdng lo^i bach d ^ E. nitens va E.
globules tir than mam; Cid va cdng sy (1999) tai sinh thanh cdng loai bach dan Iai E. grandis X E. urophylla tir vat lieu la mam; Dibax va cdng su (2010) tai sinh loai bgch dan E.
cammaldulensis tir manh la mam; Huang va
26 T4.P CHI KHOA HQC VA CONG NGHE LAM NGHIfiP THANG 10/2017
Cdng nghe sinh hpc & Giong cdy trong epng sy (2010) ciing bao cao tai sinh thanh
cdng loai bach dan Eucalyptus urophylla tu dinh than mam. Cho dfin nay, nhilu loai bach . dan da dupe nghifin ciiu tai sinh thanh cdng
thdng qua t£io da chdi hoac phdi soma. Tuy vgy, cac ket qua nghifin ciiu thu dupe cho th^y d mdi loai bach dan, thim chi d cac ddng trong cung m$t loai thi kha nang tai sinh cd the khac nhau (Alves va cdng sy, 2004; Dibax va cong sy, 2005; Hajari va epng sy, 2006). Kfit qua ciia nghien cuu nay se la co sd khoa hpc vfi xay dung h? thdng tai sinh thdng qua tgo da chdi true tifip tir md s?o cd ngudn gdc khac nhau dgt hieu qua cao phuc vu cho cdng tac tao giong cay trdng cd kha nang chdng chiu cac difiu ki?n bat Igri.
n . PHirONG PHAP NGHIEN CifM 2.1. V4t li^u nghidn cuu
Hgt gidng va vat li?u canh banh te Bgch dan Uro (Eucalyptus urophylla) dupe cung cap tir Vien Nghifin ciiu cSy nguyfin li$u giiy tai xa Phii Ninh, huyen Phu Ninh, tinh Phii Thp.
2.2. Phu-ong phap nghien cihi 2.2.1. Phuangphdp nghien cuu ehung
Bd tri thi nghiem theo phuang phap sinh hpc thyc nghiem, lap lgi 3 lin, mdi lin lap lai cd dung lupng mau ldn (n > 30), kit qua la gia tri trung binh ciia cac lan lap, khii triing mdi trudng nudi cay d nhi?t dO IIS^C, ap suat 1 atm, mdi tmdng co pH = 5,8. Cudng dp chifiu sang 2.000 - 3.000 lux, nhiet dp phong nudi 24
±2"C.
2.2.2. Phuomg phdp bS tri thi nghifm Thi nghiem 1: Tgo mlu sach va tai sinh chdi in vitro
Hat bgch dan da dupe tuyfin lya Iheo tieu chuin TCVN 5378:1991 v l hat gidng lam nghiep; mlu canh banh te dupe cat tii nhiing cay sinh tnrdng tdt, khdng sSu benh, mdi doan canh cd chilu dai 20 - 30 cm, dudng kinh 0,2 - 0,3 cm, logi bd phin qud non. Tiep theo, mau dupe lam sgch bSng xa phong loang (10%) sau
dd sat khuan be mat bSng ethanol 70% trong 1 phiit Khu triing mau bang Javen 6% vdi cac thdi gian khac nhau ( 3 - 1 1 phiit). Sau khi khii trung, mlu dupe cay trfin moi trudng khoang co ban MS bo sung 20 g/l sucrose, 7 g/l agar dfi tai sinh chdi in vitro.
Thi nghiem 2: Cam iing tao md seo tii cac vat lieu khac nhau
Khi hat nay mam tao thanh cay, mau canh da tai sinh choi non. Thu nhan cay mam, la mam va choi non, sau do cat mau thanh cac manh nhd (c5t la thanh manh nhd 0,5 cm ; cit than mim thanh doan 0,5 cm) va nudi cay tren moi trudng khoang ca ban MS-bd simg (0,2 - 1,2 mg/l) NAA, (0,2 - 0,3 mg/l) IBA, (0,1 - 0,6 mg/l) BAP, 100 ml/1 nuac dira, 20 g/l sucrose, 7 g/l agar, nudi tdi hai tuin sau do chuyfin ra nudi sang.
Thi nghiem 3: Tai sinh chdi tryc tilp tir md seo Mo seo dupe tao ra tir cac vkt lieu khac nhau dupe nuoi cay trfin mdi trudng khoang ca ban MS bd sung (0,2 - 1,2 mg/l) BAP, (0,1 - 0,2 mg/l) Kinetin, (0,1 - 0,6 mg/l) NAA, lOOmI/1 nude dira, 20 g/l sucrose, 7 g/l agar.
Thi nghiem 4: Kich thich ra re tao cay hoan chinh
c i t chdi huu hieu (cao 2 - 2,5 cm) nuoi tren mdi trudng khoang ca ban MS bd sung 0,5 mg/l NAA, 0,2 mg/l IBA, 20 g/l sucrose va 7 g/l agar.
2.2.3. Phuffngphdp xu ly sd lieu
Xvr Iy sd heu theo phuang p h ^ thdng ke sinh hpc ling dung cac phan mem da lap trinh tren may tinh dien tur nhu Excel va SPSS.
2.3. Dia diem nghien ciru
Nghifin cihi dupe thyc hien tai phong thi nghiem Cdng ngh? tfi bao, Vien Cdng nghe sinh hpc Lam nghiep - Tnrdng Dai hoc Lam nghiep.
HI. KET QUA VA THAO LUAN 3.1. Tao mau sach va tai sinh choi in vitro
M I U hat bach dan sau khi duac lam sach T ^ CHI KHOA HQC VA CONG NGHE LAM NGHIEP THANG 10/2017 27
Cdng nghi sinh hpc & Giong cdy trong
nudi cay khdi dau, kfit qua dupe trinh bay o bang 1.
dupe khii trung bing dung dich javen 6% vdi 5 cdng thiic thi nghi$m bd tri khac nhau vfi thdi gian. Sau 20 ngay nudi cay tren moi trudng
Bang 1. Ket qu& tao mau sach vk kha nsing tai sinh choi Cong thirc
khu trung KT, KTi KTj.
KT.
KT,"
= 3 - 5 ' - 7 '
= 9'
• u ' Tyl8
MSU hat mlu sach (%)
35,6 90,6 95,0 96,7 99,4 fi.,=
Tyl8
= 8,48> Fus • nay mam
30,0 82,8 61,1 57,2 15,6 '5,99
1 (%) Tyle
Mau canh binh te mau s^ch (%)
5,7 17,3 33,7 47,7 71,3
Frinh ' • 9,02 >
Tf l& man tAi sinh
(%)
5,5 16,8 31,4 18,9 17,8 Fu,'S,99 Ket qua cho thay (bang 1), ty le mlu sach tir
vgt lieu hat dat 35,6 - 99,4%; tir vat Ueu canh dat 5,7 - 71,3%. Kha nang tai sinh choi tir cac cdng tiiuc khii trimg cung khac nhau dang kl, tii vgt Ueu hat va canh non co ty le tai sinh chfii cao nhit lin lut^t la 82,8 va 31,4%. Nhu vay, tir cac ngudn vat lieu khac nhau da cho kit qua sai khac rd ret d cac cong thuc khu trimg, vdi thdi gian khii trimg cang dai thi ty le tao mlu sach cang cao, nhung kha nang tai sinh chdi cang giam. Difiu nay cung cd thi giai thich ring dung dich javen 6% la mdt loai hoa chit diing dfi khu triing lam sgch mau nhung lgi co tinh dpc ddi vdi t l bao thyc vat, nlu khii Iriing thdi gian dai thi hoa chat se ngim vao md ciia tfi bao thyc vat gay dpc cho phoi va lam cho phoi bi chet. Do vgy, trong thi nghiem nay da lira chpn cdng thiic khu trimg dfii vdi vat lieu hat bach dan la KT2 = 5 phiit, vat lieu la canh non la KT3 = 7 phut cho hieu qua tai sinh cao nhat Kit qua phan tich phuong ciing cho thiy Flfnh M t li^u h8t) > Fo,05; Fiinh (vll I j ^ cinh) > ^0.(35, tUC Ik thdi gian khii triing khac nhau anh hudng rd
ret den kha nang tao mau sgch va kha nang tai sinh chdi.
3.2. Anh hvong cua chat dilu hoa sinh trirdng thuc vait den hieu qua tao mo see
Trong giai doan cam ling tao md s ^ , moi trudng dinh duang cd vai trd rit quan trpng dfin kha nang hinh thanh cac khdi md seo, d|c bi?t la chit dieu hda sinh tnidng. Cac nghien ciiu cho thay, auxin va cjliokinin la hai nhom chat difiu hoa sinh tnidng cd anh hudng lon nhat, lupng chat dilu hoa sinh trudng bfi sung ddi vdi vat Ueu nghifin cuu ia la thudng cao hon so vdi than. Cac mlu md seo co chat lupng tdt la nhihig khdi md seo ciing, mau nau dd, khdng bi nhilm, phat triln tfit. Cac tii nghiem tifin hanh dudi day sS giiip tun ra ning dp NAA, IBA, BAP phii hpp cho viec cam iing hinh thanh md seo.
3.2.1. T^o md sff tir vdt lieu doan thdn mam Kfit qua thi nghiem vfi anh hudng ciia to hpp chit dilu hda sinh tnrdng NAA, IBA, BAP dfin kha nang cdm iing t^o md seo cua doan thin sau 4 tuin nudi ciy dupe the h i ^ 6 b ^ g 2 .
28 T ^ CHI KHOA HQC VA CONG NGHfi LAM NGHIEP THANG 10/2017
Bang
D C TM, TMi TMj T R , TMj T H ,
GU chii: +
2. A n h hirong cua chat dieu hoa sinh trudng den tao Chat D H S T (mgfl)
N A A 0 0,2 0,4 0,6 0,8 1,0 1,2
I B A B A P 0 0 0,2 0,1 0,2 0,2 0,2 0,3 0,3 0,4 0,3 0,5 0,3 0,6
Ty 1^ mSu tao m 6 Sfo (%)
7,6 67,8 97,8 83,3 82,2 80,0 74,4 P,.j,= 19,37 mo seo CO mdu nau nhat, ldnh thtt&c khoi mo seo
^ Fo,o nho (
mo s^o tir doan than mam fi^c diem
m 3 s e o
x6p
-
cung cimg Cling
x6p
xop
= 4,60 Suang kinh <1 cmj
Ch^t IiTQng m o s ^ o
+ + +++
+++
-HH- ++
+
; + + ; mo seo co mdu trang, Idch thudc trung binh (1 cm); +++: mo seo co mdu hong nhgt, Idch thu&c ldn (>1 cm); ++++;
mo seo cd mdu hong, kich thudc ldn (>1 cm).
Til kfit qua cua bang 2 cho thay, khi bd sung chat difiu hda sinh tnrdng vao mdi trudng nudi cay, kha nang tao md seo tir vat Iifiu la doan than kha tdt, tit ca cac cdng thiic deu cho ty le md s?o cao han so vdi cdng thiic ddi chiing. O cdng thiic khdng bo sung chat difiu hda sinh tnrdng, khdng cd mau nao tgo duac md s?o.
Khi b6 sung chit dieu hda sinh trudng co ndng dp til (0,2 - 1,2 mg/l) NAA; (0,2 - 0,3 mg/l) IBA va (0,1 - 0,6 mg/l) BAP, kha nang cam ung tao md seo cd su thay ddi rd ret, chat lupng md seo cung cd su khac nhau, ty le tgo md seo cao nhit dat 97,8% (hinh la, b, c) d cdng thiic TM2, khi ndng dd chat difiu hda sinh tnrdng tifip tuc tang, ty le tao md seo cd xu hudng giam din cdn 74,4% (TMe), cimg vdi dd la chit lupng m l u md seo ciing suy giam.
Nhu vgy, cdng thiic mdi trudng TM2 cd thanh phin gom mdi trudng khoang co ban MS bo Bang 3. Anb htfdng cAa chat dieu b6a
sung 0,4 mg/l NAA, 0,2 mg/l IBA, 0,2 mg/l BAP, 100 ml/l nude dira, 20 g/l sucrose, 7 g/l agar cho ty Ifi tao md seo cao nhat va chat lugng md seo tdt. Kfit qua phan tich phuang sai cho thiy Fta± > Fo,os, chiing td mdi trudng nuoi ciy cd bd sung chit dieu hda sinh trudng thyc vat khac nhau cd anh hudng ro ret dfin kha nang tao mo seo tir doan than mam.
3.2.2. Tpo md sfo tur mdnh Id mdm Tfi bao thupc cac md hoac cac ca quan da phan hda ciia cac cay song tu diep thudng phan phan hoa dudi tac dpng cua auxin (rieng re hay kit hpp vdi mpt cytokinin) dfi cho ra md seo (Lfi Hdng Giang va epng su, 2010). Trong thi nghiem nay, chiing toi nghifin ciiu su anh hudng ciia td hpp NAA, IBA va BAP cd ndng dp khac nhau dfin kha nang cam iing tao md seo ciia manh la mim. Sau 4 tuin nudi ciy, kfit qua duac trinh bay d bang 3.
sinh triroug den tao mo seo tir manh Id C T T N
D C LM, LM2 LMj L M , LMs LM.
(rhi chii.
Ch^t D H S T (mg/I) N A A
0 0,2 0,4 0,6 0,8 1,0 1,2
IBA 0 0,2 0,2 0,2 0,3 0,3 0,3
B A P 0 0,1 0,2 0,3 0,4 0,5 0,6
+ ; mo seo CO mdu ndu nhgt, T y
dudng
i | mau tao md seo (%)
3,4 83,3 80,0 94,4 68,9 38,9 30,0 Erinh ^
fi^c diem m d s e o
xop
~
cung cung cimg Xop xop 7,90 > F0.0S = 4,61 kinh khoi mo seo nho (<1 cm) ; ++.
Chat l u v n g m o seo
+ + -t-H- +++
+++
-H- +
mo s?o CO mdu trdng.
dudng kinh trung binh (1 cm); +++: mo seo co mdu hong nhgt, kich thuac ldn (>} cm)
TAP CHI KHOA H Q C VA CONG NGHE LAM NGHIEP THANG 10/2017 29
Cdng nghe sinh hoc & GiSng cdy tron^
Qua kfit qua bang 3 cho thiy, ty 1? tao mo seo ciia cac cdng thiic thi nghiem bd sung chat difiu hoa sinh trudng cd ndng dp khac nhau da cho ket qua khac nhau tuang doi r5 r?t. CJ cdng thiic ddi chiing khong bd sung chit difiu hoa sinh tnrdng, mlu tao md seo rat kem, d cac cdng thiic thi nghiem cd ndng dp chat difiu hoa sinh tnrdng tang din (0,2 - 1,2 mgA) NAA;
(0,2 - 0,3 mg/l) IBA; (0,1 - 0,6 mg/l) BAP. Kit qua cho thiy rit khac nhau, trong d6 ty lfi tgo md seo tang din tif cdng thiic LMi (83,3%) din cong thiic LM3 (94,4%), tilp tuc tang nong dp chat dilu hda sinh trudng thi ty IS tgo md seo giam di rd ret, ty lfi thip nhit d cdng ihiic LM6 (30%), nhu vay cd thfi ndi chit difiu hda sinh tnrdng cd anh hudng ldn den kha nang tao mfi seo, dac biet ndng dp cao co the gay lic che qua trinh hmh thanh md seo. Thi nghifim nay, da chpn dupe cong thiic phii hop cho tao mo seo tir vat lieu manh la mam la LM3 (vdi moi trudng khoang ca ban MS bo sung 0,6 mg/l NAA, 0,2 mg/l IBA, 0,3 mg/l BAP, 100 ral/l nude dira, 30 g/l sucrose, 7 g/l agar), cho hieu
qua cao vfi ty 1§ tao mo s?o dgt 94,4% (hinh Id, e), chit lupng md s?o tdt, mlu nau do.
Cling. Kfit qua phan tich phuong sai cho thiy Ftinh > Fo,o5, chiing td ndng dp cac chat dilu hoa sinh trudng khac nhau anh hudng ro ret din kha Mng tao md s§o tu la mam.
3.3. Anb hirdng ciia chat dilu hoa sinh tnrdng thuc v^t d i n kha nang tai sinh chii
D I CO dupe so lupng cay ldn, doi hoi mo seo cd kha nang tii sinh lupng ldn ch^i, cac chdi sinh trudng va phat trien tot. Trong nghifin ciiu nay, hai nhdm chat difiu hoa sinh tnrdng dupe sii dyng la auxin va cytokinin, cu thfi dd la BAP, Kinetin va NAA. Vat Ueu dupe su dimg^la-md seo co ngudn gdctir doan than mam va manh la mam.
3.3.1. Tdi sinh chdi tir md seo tgo ra tit than mdm
Kit qua nghifin ciiu v l anh hudng nong dg cac chat difiu hda sinh trudng BAP, Kinetm v^
NAA dfin su tai sinh chdi tir mlu mo seo tao ra tir doan than mam sau 4 tuan nudi cay dupe the hien trong bang 4.
Bang 4. Anh birarog ciia chat dilu hda sinh tru&ng den tai sinh cboi tir mo seo CTTN
DC TS, TS, TSj TS4 TS, TSJ
Chat DHST (mg/l) BAP
0 0,2 0,4 0,6 0,8 1,0 1,2
Kinetin 0 0,1 0,1 0,1 0,2 0,2 0,2
NAA 0 0,1 0,2 0,3 0,4 0,5 0,6
Mo seo tu than mam Ty le tai sinh
(%)
17,8 38,9 51,1 71,8 54,4 52,2 41,1
So chdi TB/mau 2,5 3,1 5,6 8,5 6,6 5,5 5,7 Frij, = 64,62 > Foil, = 4,75
M6 seo tir manh la mam Ty le tai sinh
(%)
18,9 34,4 54,4 56,7 67,8 58,9 47,8 FM-33,24 >
SO choi TB/mau 2,9 5,4 6,1 6,8 8,3 6,6 4,9
•F,.i, = 4,7S
Ket qua cho thay (bang 4), d cac cong thuc thi nghidm co hd sung cac chit dieu hoa sinh tuong c6 nong do tang dan tuong ling v6i cong thiic TSi dSn TSj. Sau 4 tuSn thi nghi$ra cho Icet qua co sir Ichac nhau ro r^t, voi cac cong thuc tir TSi d8n TS3, n6ng dp chat diSu hoa
sinh truong tang 16n thi ty le tai sinh choi va s6 choi tjo ra co xu huang tang dfci theo chieu thu$n, tai cong thiic TS3 cho kSt qua cao nhat vS ty le tai sinh choi va s6 lugtng chfli Ito to?!
la 71,8% va 8,5 ch6i/mau (hinh If, g, h). Tiep tuc tang ndng do cac chit dihi hoa sinh truemg
30 TAP c m KHOA HQC VA CONG NGHE LAM NGHIEP THANG 10/2017
Cong nghi sinh hpc & Giong cdy trdng nhir d cdng thiic TS4 din TSg thi kit qua cd xu
hudng tang nghich, cd nghta la ndng dp chat dieu hda sinh trudng tang nfin nhung ty lfi mlu tai sinh choi va s6 lupng chdi tao ra lai cd xu hudng giam di. Cu thfi la d cdng thiic TSg bd sung cac chat difiu hda sinh trudng thuc vat cd ndng dp cao nhit thi ty lfi t^o chfii va sd chdi tnmg binh ciia mpt mau cung cd gia tri thip nhit (trir cdng thiic ddi chiing) d^t lin lugt la 41,1% vk 5,7. Nhu vay, trong pham vi nghien cuu nay da chpn dupe cong thiic TS3, co thanh phan mdi trudng khoang co ban la MS bd sung 0,6 mg/l BAP, 0,1 mg/l Kinetin, 0,3 mg/l NAA, 100 ml/l nude diia, 20 g/l sucrose, 7 g/l agar la cdng thiic mdi trudng phii hpp cho tai sinh chdi tir md s?o cd ngudn gdc la doan than mam. Ket qua phan tich phuang sai cung cho thay Ftinh > Fo,o5, chiing td ndng dO cac chit dieu hda sinh trudng khac nhau cd anh hudng rd ret dfin kha nang tai sinh chdi.
3.3.2. Tdi sinh chdi tdr md spa tpo ra tir mdnh Id mdm
Kit qua nghifin ciiu anh hudng ciia td hpp cac chat dieu hda sinh trudng BAP, Kinetin va NAA cd ndng dfi khac nhau dfin su tai sinh chdi tir mau md seo cd nguon tir manh la mam sau 4 tuin nudi ciy dupe thi hien trong bang 4.
Tii kit qua bang 4 cho thay ty le tai sinh va sd choi trung binh tren mpt mau ciia cac cdng thiic cd bd sung BAP, Kinetin va NAA diu ldn hon cdng thiic ddi chiing (DC) khdng bd sung chit dilu hda sinh trudng cho ty 1$ tai sinh thap (18,9%). C) cac cdng thiic thi nghidm cd bo sung cac chat difiu hda sinh trudng cd ndng dp khac nhau theo quy luat tSng din, cho kit qua thfi hien sir khac biet va chia thanh hai pha rd rang. Tir cdng thiic TSi din TS4, khi tang ndng dp chit difiu hda sinh tmdng tiii cac gia tq vfi ty le sdng ciing nhu sd chdi tang theo. Tuy nhifin, khi tilp tuc tang nong dp cac chat dieu hda sinh trudng (TS5 - T%) thi kfit qua lgi cd chilu hudng ngupc lai (ty 1§ nghich). 6 cdng thiic TS4 cho kit qua tfit nhat vfi ty 1? mau tai
sinh choi cung nhu so chdi trung binh cua mau dat iin luot la 67,8% va 8,3 chdi/mlu (hinh 1 i).
Nhu vay, cfing thiic thi nghipm TS4 cd thanh phin la mfii tnidng khoang co ban MS bd sung 0,8 mg/l BAP, 0,2 mg/l Kinetin, 0,4 mg/l NAA, 100 ml/l nude dira, 20 g/l sucrose, 7 g/l agar 1^ cdng thiic mdi tnidng phu hpp cho giai dogn tai sinh chdi tir md seo cd ngudn gdc la mdnh la mam. Kfit qua phan tich phuang sai cimg cho thiy Fdnh > Fo.os, chiing td, chat dieu hoa sinh trudng khac nhau co anh hudng r5 ret dfin kha nang tai sinh chdi.
3.4. Kich thich r a r^ tao ciy con hoan chinh Cdc chdi phat triln tdt (kich tiiudc 2 - 2,5 cm) duac ciy chuyen Ifin. mdi trudng khoang ca ban MS bd sung 0,5 mg/l NAA, 0,2 mg/l IBA, 100 ml/l nude diia, 20 g/l sucrose va 7 g/l agar. Nudi trong tdi I tuan dau sau do chuyfin ra nudi sang 2 tuan vdi cudng dp chieu sang 2000 lux, nhiet dp phdng nudi 24 ± 2*'C, sau 4 tuin dat hom 92,4% chdi ra rfi. Cay eon dupe chuyfin ra trdng trong biu vdi rapt biu hdn hpp, phdi trOn dat tang mat va cat theo ty le
1:1, dat til? sdng 89,7%.
IV. KET LU4JV
Tai sinh cay bach dan thdng qua tai sinh da chdi true tifip tii md seo dat mdt sd kfit qua sau:
- Dung dung dich javen 6% khu trimg hat trong 5 phiit, 7 phiit ddi vdi mau canh, ty le mau sgch lin lupt la 90,6% va 33,7%; ty le nay mim lin luot la 82,8 va 31,4%.
- Than mam cd ty lfi tao md seo la 97,8%
tren mdi tnrdng khoing MS bd sung 0,4 mg/l NAA, 0,2 mg/l IBA, 0,2 mg/l BAP; manh la mam cho ty le tao md seo dat 94,4% trfin mdi trudng khoang MS bo sung 0,6 mg/l NAA, 0,2 mg/l IBA, 0,3 mg/l BAP.
- Md seo cd ngudn tu than mam cho ty le tai sinh chdi va sd choi trung binh lan lugt la 71,8% va 8,5 chdi/mlu tren mdi tnrdng khoang MS bd sung 0,6 mg/l BAP, 0,1 mg/l Kinetin, 1J<F CHI KHOA HQC VA CONG N G H | : LAM NGHIEP THANG 10/2017 31
Cone nglie sinh hoc & Gione ciy trdng
0,3 mg/l N A A . Vat lieu m o seo tir manh la m i m nuoi c i y tten moi tnrdng khoang M S bo sung 0,8 mg/l BAP, 0,2 mg/l Kinetin, 0,4 mg/l N A A cho ty le tai sinh chai d j t 67,8%, sh choi tnmg binh/mau d?t 8,3.
- Choi h u u hieu d u p e nuoi cay tr^n moi tnrong khoang c o b a n M S bfi sung 0,5.
mg/lNAA, 0,2 mg/I B A , 100 ml/l nuoc dia, 20 g/l sucrose v a 7 g/l agar, t y le ra r l % 92,4%.
<g> (h) fl) Hinh 1. Mot sl Mnh kah trong quy binh tai sinh cSy bach dan Urd
Ghi chii: a, b. c) tao mo s?o tir dogn thdn mdm; d, e) tao mo s^o tit la mdm; f, g, h) choi tdi sinh tit mo seo CO nguon goc tie than mdm; i) choi tdi sinh tir mo s?o co nguon goc mdnh Id mam.
TAI UtV THAM KHAO
1. Duong Tin Nhirt, Nguyen Thanh Hai, Nguyin Diic Huy, Luong Nggc Thuin (2007). 5\t phat sinh phoi ciia cac te bao sinh dudng th\rc vat Tgp chi Cdng ngh4 Sinh hge 5.2: 133-149.
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Cong nghe sinh hoc thuc vgt trong cdi tiin giong cdy trong. NXB. Nong nghi§p Ha NOi: 62-104.
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rao s?o va tai sinh ch^i tir m6 la non ciy Bi ky nam {Hydnophytum formicarum Jack.). Tgp chl Khoa hoc - TruongD?i hpc Can Tha, \6all6-212.
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Organogenese de explante foliar de clones d&Eucal}^tus grandis x Eucalyptus urophylla. Pesq AgropBrasil, 39(5): 421-*30.
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32 T ^ CHI KHOA HQC VA CONG N G i q : LAM N G H I f P THANG 10/2017
Cong nghp sinh hoc & Gidng cdy trong
seedling explants of two commeicially important tenqjerate eucalypt SRecies - Eucalyptus nitens and E.
globules. Plant Science, 140: 189-198.
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and M. Quoirin. (2005). Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis.
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AN EFFICIENT REGENERATION SYSTEM THROUGH CALLUS OF EUCALYPTUS UROPHYLLA S. T. BLAKE FOR CELL LINES SELECTION
Nguyen Tbi Huong*, Nguyen Van Viet^
''^Vietnam National University of Forestry
SUMMARY
Hypocotyls of 8 - 1 0 day seedling were used as explants to establish a regeneration protocol for Eucalyptus urophylla. The explants were cultured in Murashige T. and Skoog F. (MS) medium supplemented with a- naphthalene acetic acid (NAA) 0.4 mg/L, P-indo! butyric acid (IBA) 0.2 mg/L, 6-benzylaminopurine (BAP) 0.2 mg/L, coconut water 100 ml/L, sucrose 20 mg/L, agar 7 g/L, the ratio of callus induction was 97.8% after 4 weeks culturing. Callus were cultured in MS medium supplemented with BAP 0.6 mg/L, Kinetin 0.1 mg/L, NAA 0.2 mg/L, sucrose 20 mg/L, coconut water 100 ml/L and agar 7 g/I, showed high frequency of adventitious buds formation (71.8%), Regenerated shoots rooted m MS medium supplemented with NAA 0.3 mg/L, IBA 0.2 mg/L. Plandets were flien successfiilly transplanted to greenhouse with 92.4% survival.
Generally, Eucalyptus urophylla regeneration protocol via multiple-shoots induction from callus could be used for tit&er studying as choosing salt tolerant lines formed by in vitro somatic mutations.
Keywords: Eucalyptus urophylla, hypocotyls, multiple shoots, regeneration, rooted.
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