HOl NGH! KHOA HOC CONG NGHE SINH HOC TOAN QUOC 2013
N H A N N H A N H H O A LAN H 6 D I E P (PHALAENOPSIS SP.) BANG CONG NGHE PHOI SOMA
Nguyen Van Nam, Triin Van Minh
Tnfdng B^i hoc C}u6c te, Tnrang Dai hgc Quoc gia Tp. Hd Chi Minh TOM TAT
Mo la non in viirodtsgc nuoi cSy t?o PLB tren moi tnrong MS co b6 sung td hop TDZ (3 mg/l) va NAA (0,1 mg'l). PLB c5t lat moni;
dirge nuoi ciy phat sinh va tang sinh mo seo iren moi tnrong MS, BA (0,1 mg/l) co bfi sung NAA (1 mg/l). Dicli huyen phii te bjin mo s?o duqc nuoi ciy phat sinh va tang sinh tren moi trudng MS, BA (0,1 mg/l), NAA (0,5 mg/l) Djcli huy6n phii te bao mo seo duQc nuoi cSy biet hda phat smh ph6i tren moi tnrcmg MS, BA (0,5 mg/l), NAA (0,1 mg/l). Dich huyen phii tc bao phdi dui^c nu.ii cSy trai tren moi trvong lai sinh l)an rin MS, BA {I mg/l), NAA (0,1 mg/l). K.k qua nghien cthi da xay dyng dirge h? thdng nhiin nhanh hoa lan hi diep bSng cong nghe phdi soma, diu nhan 5,800 ch6i hoa lan h6 dtep tren I lit djch huyen phu te bao phoi Tir khoa: mo seo, Ui^ phoi PLB, tang smh, choi in vitro, te bao soma, te bao phoi
MttOAU
Hg lan la m6t ho Idn nhat hijng nhihig hp c6 hoa co khoang 800 gi6ng va 25.000 loai phan bd tren khIp th^ giiri bao gom nhO'ng loai hoa lan thuang mgi nhu' i^a lan, hoang lan, ho diep, vu nif...Trong do, hoa lan ho di^p la loai hoa dep nhlt vk CO gia tn kinh t l cao tren thj tnrd'ng hoa lan the gifri (Park et al., 2002). Vi nhSn gidng h-uyin th6ng trSn loai iio diSp hi§n nay din din m6l vin d^ ma cac phdng thf nghiem vi nhan giong thu'O'ng gap phai do la cay cly mfl thuong sinh trirdng ch|m vk ton nhilu chi phi lao dgng de -san xult cay con v6i khdi lupng Id'n khi dua ra thj trudng vai gia thanh cky con cao vk vl nhkn giong bang ky thuat nuoi cly te bao hi^u qua hon nhSn gilng truyln thing (Chugh ef al., 2009) H& thing nhfin gilng bang phfli soma giai quylt dup'c rko can n§u tren vdi cAc Ig'i the: nhkn nhanh dudi dgng t l bao, ph6i soma la mflt the bi?t h6a co he s l tai smh cao. tin it chi phi lao dong va gifi thanh hg (Aitken-Christie ef ai.
1994). Vgt li?u khdi diu h-ong nuoi cay phoi vo tinh co vai t r i dam bao dugc dac dilm di truyln b l me va duy tri he s l tai sinh cao treing thdi gian nudi cay keo dai trSn cac loai hoa lan (Lin et al., 2000). Moi tardng nufli cay phoi soma thich hg'p chiem vai tro quan Ugng trong qua trinh sinh trudng va bi?t hda t l fcifio phoi hoa lan (Arditii vfi ErsnL 2009) va dilu khiln qufi hinh bi|t hda vfi tfii sinh phoi soma dudi tfic d$ng cua cac chat dilu hda sinh trudng da trd thanh quy lual (Ishii etal. 1998 ; Huan etal., 2004). The giong phoi (PLB) Ifi mgt the phoi vo tinh dgc bigt phat smh trong nufli cly cfic loai hoa lan (Park e( al., 2000) chju nhieu tac dgng ciia cytokinin nhu BA, 2iP va TDZ (Park ef a/., 2002). Tuy nhiSn, chi cd hai loai hoa lan nufli cfiy phfit sinh phoi soma thanh cong (Chen vfi Chang, 2004). Nghien CLPU tao mfl seo cd kha nang phfit sinh phfli, nufli cay tao phoi soma, vfi tfii sinh phdi soma Ifi rfio can dfiu tifin trong cong nghe phoi soma (Wu e( aL, 2004). Bai bao nay nghifin cdu nhfin nhanh hoa lan ho difp bfing cflng nghe phfli soma
NGUYEN Lieu VA PHU'ONG PHAP Nguyen li^u
Giong hoa lan ho digp (Phalaem^sis var White Giant) nhfip nfli tCr Singapore. Mlu nufli cay: (i) Conn dinh sinh trudng chli in wtro. (ii) La non chli In vitro, (iii) The gilng phfli (PLB-protocorm like body) dugc cfit lat mong
MOI tn/dng dmh dudng khoang: nufli cfiy Ifi MS (Murashige va Skoog. 1962), cd b l sung: BA (6-benzylaminopurine), 2iP (2-isopentyl adenine), TDZ (thidiazuron). IBA (indol-3-butyric acid). N/\A (a-napthaleneacetic acid), adenine, pepton (1 g/I), than hgat tinh (1 g/l), nude dira (30 %). Bl (5 mg/l). sucrose (30 g/l)
Dllu kifin nuoi cay: nhifit do phdng 26+2 °C. Im do 65 %, thdi gian chieu sang 10 gid/ngay, cudng dfl chilu sfing 33,3 pmol/m Is, khi> trung moi trudng nufli cay d 121 °C (1 at) trong 20 philt, tic dfl Ific 100 vdng/phut
PhuoTig phap
Thi nghiem b l tri theo khli day du ngau nhifin, 3 lan lap Igi, moi lan Ifip lai nufli cay 3 binh tam giac {chija 60 ml mfli tm-dng bfi^n ran hay 50 ml mfli trudng long). Slnh khoi t l bfio ban dau nufli cly tao dich huyln phu 10 g/IOOml moi trudng. So lieu dugc phfin tich bfing pham mem MSTATC (t=0.05)
Thl4tkB thf nghifm _ ^ Nufli cly tgo va tfii sinh mo seo:
Nufll cly tgo thi phfli PLB: Mfiu nufli cly la la non cay cly mfl (2-3 mm), Moi trudng nufli cay tao PLB: MS c6 b l sung NAA (0 0.1 1 mgll) vfi TDZ (0 0,1 1 3 mg/l).
Nuoi cly tgo mfl sgo sinh phfli: PLB dug'c clt Ifit mdng (dfiy 0,5 mm), dfit tren mfli trudng nufli cly tgo te bfio md sgo sinh phii: MS, BA (0.1 mgfl) cd b l sung NAA (0,1 0,5 1 3 mg/l) va 2.4-D (0,1 0,5 1 2 mg/l).
Nufli cly tfing sinh khli md seo sinh phoi: Mfl sgo sinh phfli cd mfiu trang nga ti> nghien cdu trfin dug-c si> dyng lfim nguyen lifiu nuoi cfiy. Khfli lugng mfl seo dua vao nuoi cfiy Ifi 1 g/cym mfl sgo. Mfli trudng nudi cly tang sinh khli mo sgo MS, BA (0,1mg/l) cd bo sung NAA (0,1 0,5 1 3 mg/l) vfi 2,4-D (0,1 0,5 1 2 mg/l).
,Tfii sinh mfl seo sinh phfli Mo s?o trang nga tu nghidn cdu tren dugc sd dung lfim nguyen Ii$u nudi cfiy. Mdi trudng nufii cly tfii sinh mfl sgo sinh phfli: MS cd bo sung NAA (0 0,1 0,3 0.5 mg/l) va BA (0 0,1 0,3 0,5 mg/l).
HOI NGH! KHOA HQC C O N G NGHE SINH HOC TOAN QUOC 2013
Nudi cay tao va tfing sinh djch huyen phu te bfio md seo sinh phdi:
Nudi c l y tao djch huyfin phO t l bao mfl seo smh phdi: T l bao soma (md sgo trfing nga) dugc si> dung lam nguyen ligu nuoi cay L;io aicn nuyen pnu. Smh khoi nu6i"cay ban dfiu lfi 1 g/50ml the tlch moi trudng nuoi cfiy. Tdc dfl lac 100 vdng/phut). Mdi trudng nufli c l y tgo dich huyen phu te bao soma: MS, BA (0,1mg/l) co b l sung NAA (0,1 0,5 1 3 mg/l) va 2,4-D (0.1 0.5 1 2 mg/l).
Nuoi cay tang sinh khli dich huyln phii t l bfio mo seo sinh phdi Djch huyen phii t l bao soma ftiu dugc tir nghien ci>u tren dugc s u dyng lfim nguyen ligu nufli cfiy tang sinh khfli dich huyln phii. Sinh khli nudi cay ban dfiu la 1 g/50ml the tich mfll trudng nufli cfiy. Mdi trudng nuoi c l y tfing sinh khli dich h u y l n phu le bfio soma: MS, BA (0,1 mg/l) co bd sung NAA (0,1 0.51 3 mg/l). . J H
Tfii sinh djch h u y l n phCi t l bao mfl sgo sinh phdi:
Nufli cay kich thfch cfim img tao phfli djch huyen phii phdi soma- Djch huyln phu t l bfio soma thu dugc tip nghien cuu tren dugc su' dung lam nguyfin ligu nufii cay kfch thich cam irng tgo phoi t l bfio djch huyen phii. Sinh khli nufli cfiy ban d i u la 10 g/50ml t h i tich mdi trudng nufli cly. Mfli trudng nufli cay phat sinh phfli soma: MS cd bo sung N/\A (0,5 mg/l) vfi BA (0,1 mg/l)
Trai vfi tfii sinh djch huyln phii phfli soma. Djch huyen phu t l bfio phdi soma thu dugc tir nufli cay kich thich cam ung phfit sinh phdl soma nghien cdu trfin dugc s d dyng lam nguyfin lifiu nudi c l y trai va tai sinh dich huyln phii. T h I lich trfii Ifi 5 ml/60ml mfli tardng ban rfin: MS cfl bo sung NAA (0,1 0.5 1 3 mg/l) va BA (0,1 0,5 1 3 mgfl).
K!£T QUA VA T H A o LUAiM r^udi cay tao vfi tfii sinh mo seo
N u d i c l y t a o the phdi PLB: Nufli cay la non c h l i / n wfro tren mfli tordng MS cfl bo sung to hgp TDZ (3 mgfl) va NAA (0,1 mg/l). cd ty Id hlnh thfinh phdi cao (48 %) vfi tgo 12,2 PLB/mlu sau 45 ngfiy nufli cay (Bang 1). Cho t h i y TDZ dflng vai trfl quan trpng trong qua trinh phfit sinh phfli PLB (Bang 1) N/\A kich thich qufi trinh phfin chia va phan hfla t l bao vfi TDZ Ifi mpt cytokinin kfch thich qufi trinh bifit hfla phoi
Bfing 1, Anh hud'ng ciia TDZ va NAA d4n tao thS phoi PLB
M6i irudng nuoi cay MS bfl sung (mg/i) j ^ |^ ^ ( , pi_B Nufli cay tao mfl seo sinh phfli: T h I phfli PLB cd TDZ NAA ("'''') ° "^ " tic dfl nhan in vitro chfim do PLB cfl kfch thudc ngfiy cfing to, han c h l d i n qufi trinh vi nhfin 0.0 0.0 gilng. PLB lat mflng day 0.5 mm dugc nuoi cay
^•^ tren MS, BA (0,1 mgfl) cfl bo sung NAA (1 mg/l) -'^ kfch thfch tgo md sgo 96 % vfi phat triln dudng Q., Q'Q . kinh md sgo 6,2 mm (Bfing 2) sau 45 ngfiy nufli ' o'l 2,6 1,2 ^^V- SLP dMrig 2,4-D nudi cay tao mfl seo d l dfin l|o 1,4 1,0 d i n blln dj t l bao soma, chpn NAA trong nufii 3,0 0,6 0,8 c l y la Ihich hpp. Nuli c l y mfl sgo dudi finh 1.0 0,0 - - sfing, mfl sgo cfl mfiu xanh; ngugc Igi, d l h^ong
0.1 6,2 1,4 ^nh sang khulch tan co mau trang ngfi. Tfi bfio '[•^ ^'^ ^'^ mfl sgo trang ngfi cfl cau taic dfic, trdn, phfin
^'^ ' ; chia nhu t l bfio t i l n phfli thfch hgp ch^o qua trinh (.'., 48 .,2 2 nhan smh khli trong Idng (tren mfiy lac) vdi anh I'o 36 8,6 sfing khulch tfin.
Nufli c l y tang smh khli md seo sinh phdi: Md seo trang ngfi duoc nufli cay trfin mdi tardng M tang sinh khli mfl sgo sinh phdi MS, BA (0,1 mg/l) cfl bo sung NAA (1 mgfl). D l tranh biln dj l l bao soma khi nufli cfiy keo dfii, bo sung NAA (1 mg/l) to ra thich hgp kich thich tang sinh k h l i dat 5,46 mg/cym vfi chi s l tang sinh 5,4 sau 45 ngay nuoi c l y (Bfing 3).
CV%
Bang 2, Anh hud'ng ciia 2,4-D va NAA d i n tgo M6i tnrcrng
2.4-D (mg/l)
: nuoi c i y NAA (mg/l)
Ty 1$ tgo mfi s?o
(%)
18,6 m o s ^ o
16,4
Dudng kinh mo sgo (mm)
HOl NGH! KHOA HOC CONG NGHE SINH HQC I O A N Q U 6 C 2013
Bang 3- Anh h u d n g ciia 2,4-D va NAA den tang sinh mo si Moi truong nuoi c l y co t)6 sung Smh khli sau 45 ngay n
(mg/cym) Chi sfi tang SInti khdi
3.16 6,20 5,25 4.27 1.95 3.20 5,46 5.16 4,05
Tai Sinh mfl seo smh phfli Trfin mdi tm-dng tfii sinh md seo smh phfli MS cd bo sung to hop N/\A (0,1 mg/l) va BA (0,5 mg/l) cho ty le tai sinh chdi hoan chinh cao sau 2 thfing nudi cay. Hifiu suat tai sinh dgt 98 % mau dua vao nufli cfiy, co so chli phfit smh 5.6 chfli/cum. va tgo 1,4 PLB/cum (Bang 4). Md seo trfing nga cd c l u true tien phoi bifit hoa thanh PLB cd kich thudc nhfl vfi cac PLB nfiy tilp tyc tai sinh tare tilp hinh thanh choi in vitro hoan chinh
Bknq 4. Anh h u d n g cua BA, 2,4D va N/W den tai sinh mo seo
Mfil tnrdng nuoi cay c6 bo sung Ty ik lai sinh (%) S6 choi /cym S6 PLB /cum
Nudi cly tao vfi tfing sinh djch huyen phii t l bao mo seo sinh phdi
Nuoi cly tgo djch huyln phii l l bfio mfl sgo sinh phor Dich huyln phii t l bao soma hinh thfinh tren mdi trudng nufli cly MS, BA (0,1 mg/l) cd b l sung UAA (1 mg/l) sau 30 ngay nudi cay (Bang 5). N/VA (1 mgfl) thich hgp trong nuoi cly lang sinh khli dgt 2 742 mg/50ml vfi chi s6 tfing sinh khdi 2,7 vfi BA kich thich biel hda hinh thfinh t l bao «ln phdi. Dph huyln phCi cd mfiu trfing nga, dgp, l l bfio cfl hinh dgng vfi kfch thudc dfing kfnh.
Bang 5. Anh h u d n g cua 2.4-D va NAA den tao djch huyen phii te bao m d s f o MOI taroTig nuoi cay co bo sung
2.4-D (mg/l) (mg/SOml)
luoicly 1.244 1 632 3 264 2646 2,262 1.458 1.608 2.742 2 625 11.8
Nufll cly tfing sinh khdi d|ch huyln phu t l bao md seo smh phfli: D]ch huyln phii t l bfio soma hinh thanh trdn mfli tardng tfing smh djch huyen phii MS. BA (0.1 mg/l) cfl' b l sung NAA (1 mgfl) sau 20 ngay nufii cly (Bfing 6), Dgt sinh khli 12.452 mg/50ml va tic dO tang sinh nhanh 12,4 sau 30 ngay nudi cly. Ojch huyln phii cd mfiu trfing ngfi dgp, t l bao cd hinh dgng vfi kich thudc dfing deu. BA va N/\A thfch hgp cho qufi trinh Ifing sinh khoi t l bfio sinh phfli.
HOI NGH! KHOA HOC CONG NGHE SINH HOC TOAN QUOC 2013
• a n g 6. Anh h u d n g cua NAA den tang sinh djch huyen phii te bao mo seo
-KlQl tru'gng.Buou:fiyx6J>dSuag-NAA- Rmh khAi .;ai i ?n n^Ay m.ni ray (mgfflnml) Chi ^ft lang smh khfil (mg/ll
5.434
0.5 10,862 1.R 12.452 2,0 8 244
3-0 6 432
CV% 12.4 Tai sinh djch huyln phCi t l bao mfi sgo sinh phfii
Nufll cly kfch ihich cam ung lao djch huyln phO phfli soma: Dich huyln phii t l bfio soma dugc nuoi cay kich thich tgo phfli soma sau 6 luan nufli cay trfin moi laj-dng MS cd bo sung BA (0,5 mg/l) vfi NAA (0,1 mg/l) Hifiu sufit kich thich tgo phdi tfing din theo thdi gian nufli cly lir luin thu 3-4 sau nudi cay. vfi giam dan sau 4 tufin nufli cly do t l bfio soma lang sinh nhanh hon phoi. Higu suat kich thich cam dng dgt mgt dfl t l bfio 1,2 x 10* va tgo phoi 78,4 % (bao gim t l bao hinh clu, hinh lim. thuy ldi) d thdi diem thich hgp sau 4 tuan cly chuyin lai sinh (Bang 7) T l bao phoi cfl mfiu trfing vang nhal, cd kich thudc Idn, t l bao chat dgm dfic
Bang 7. Anh h u d n g ciia thoi gian nuoi cay den kich thich cfim u n g phat sinh phoi
Thdi gian nufii cay (lufin) MSI do te bao hogt hok (CFU/ml) sau 4 luan Hi6u suit hogl hoa (%) so vdi 16 bfio ban d i u i 0,7 X 1 0 ' ' 50^0
2 0 , 8 x 1 0 ' 58.3 J 1 , 1 x 1 0 ' 72,6 4 1,2x10' 78.4 5 0,7x10' 52,8 6 0.6x10' 44,6 CV% 14,4 10,2 Trfii VO lai sinh djch huyln phii phoi soma' Sau 30 ngay nudi cay Idngidjcb-buyen phii l l bfio phdi soma dugc Irai tren mfll Inidng tfii smh ban rln IVlS cd bo sung N/\A (0,1 mgfl) vfi BA (1 mg/!). Te bao di vfio qufi Irinh bifit hda sinh phdi soma ding nhll, tao Ihanh mpi idp tfi bfio phoi cfl mau xanh, xull hign nhdng PLB nho, va lai sinh Ihanh chli sau 60 ngfiy nufli cly, Ihu nhfin 29 chli/5ml djch huyln phii t l bfio phdi soma (Bang 8). Thu nhfin hifiu suit trong phflng thf nghifim 5.800 choi hoa lan ho digp tren 1 lit djCh huyln phii t l bfio phdi
Bdng 8. Anh h u o n g cua chSt d i l u hoa sinh t r u d n g den tai sinh djch huyen phii phdi soma Mfll Irudng nuSi cSy
BA NAA (mg/l) (mg/l)
Sfi choi lai sinh /5ml dfch huyln phii t l bao sinh phoi
K^T LUAN
Nufll cly igo vfi tfii sinh mo seo: Nufli cly mfl la non in vitro tao PLB Irfin moi trudng MS cd b l sung to hgp TDZ (3 mgfl) vfi NAA (0,1 mg/l). PLB c l l Ifit mflng dugc nudi cly tgo vfi tang sinh mo seo tren mfli trudng MS, BA (0,1 mg/I) cd b l sung NAA (1 mg/l). Trfln mdt Irudng lai sinh md sgo sinh phdi MS, NAA (0,1 mg/l}, BA (0,5 mg/l) cho ly Ifi lfii sinh choi cao dgt higu suit 100 % mau dua vfio nufli cly, s i choi tfii sinh dat 3-5 choi/cum. Md seo sinh phoi bi§t hfla thfinh PLB cd kich thudc nho vfi cac PLB nay tilp tyc lai sinh tn/c tilp hlnh thfinh chdi in vitro hoan chinh
Nudi cly tgo vfi tfing sinh dich huyln phii t l bfio md sgo sinh phfli: Djch huyln phO t l bao soma dupe nudi cay phfit smh Irfin mfli tn/dng MS, BA (0,1 mg/l), N/\A (1 mgfl) sau 20 ngfiy nuoi cay DjCh huyen phii t l bfio mfl sgo^smh phfli sau 30 ngfiy nudi cly cfl tic dp tfing sinh nhanh dgt 12,4 lln. T l bao djch huyen phii cd mau trang ngfi dgp, t l bao cd elu true tiln phdi, cd hinh dgng vfi kfch thudc ding diu. Mdl phln djch tiuyln phu t l bao soma biel hfla tgo phdi soma.
Tfii sinh djch huyln phii t l bfio md sgo sinh phdi: Djch huyln phii t l bfio md seo sinh phdi dugc nudi cay bigt hda tgo phdi h^n mfii tn/dng MS. BA (0,5 mgfl), NAA (0,1 mgfl), vfi tfii sinh trfin mfli trudng ban rln MS, BA (1 mg/l), NAA (0,1 mgfl) sau 60 ngfiy nuoi cly. Kfil qufi nghien ciru da xfiy dyng dugc hfi thdng nhfin nhanh cay hoa lan ho di^p bfing cdng nghd phdi soma, thu nhgn 5.800 chli hoa lan ho ai$p trfin 1 lit djch huyen phu t l bao phdi
929
HQI NGH! KHOA HOC CONG NGHE SiNH HOC TOAN QUOC 2013
TAI UEU THAM KHAO
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MICROPROPAGATION OF PHALAENOPSIS SP. ORCHID BY SOMATIC EMBRYOGENiC CULTURES
Nguyan Van Nam, Tran Van Minh
Intemational University, Vietnam National University, Ho Chi Minh
SUMMARY
Young leaves were used as materials for protoconn like bodies (PLBs) perfomed on the medium MS supplemented with TDZ (3 rag/1) and NAA (0.1 mg/l). PLBs were cut into slices and placed on the medium for callus initiation Callus was initiated and proliferated on the medium MS, BA (0.1 mg/l) supplemented with NAA (1 mg/l). Somatic celt suspension was initiated and proliferated on the medium MS, BA (0.1 mg/l) supplemented with NAA (0.5 mg/l). Somatic cell suspension was differentiated lo embryogenic cell suspension on the medium MS supplemented with NAA (0.1 mg/l) and BA (0.5 mg/l). Embryogenic cell suspension was plated and regenerated on the medium MS supplemented with NAA (0 1 mg/l) and BA (1 mg/l). Micropropagation of phalaenopsis orchid via embryogenic technique was set up to produce 5,800 plantlets per [iter of somatic embryogenesis suspension
Keywords: callus, protocomi like body (PLB), proUferation, plantlet, somatic cell, embryogenic cell
• Author for correspondence. Tel 84-98556980; Email: nvnam0202iaQmail com
