HQI NGH! KHOA HOC C O N G NGHE SINH HOC TOAN QUGC 2013
NHAN NHANH HOA LAN NGOC DIEM {RHYNCHOSTYUS GIGANTEA) BANG KY THUAT PHpJ SOMA
Nguy§n Thien Quang*, Tran Van Minh
Tru-dng D^i hgc Quic te, Truimg Bai hgc Quoc gia Tp. Hd Chi Minh
TOM TAT
Nuoi cdy 14 non tao PLB tren moi tniong MS, TDZ (0,1 mg/l). PLB dugc sir A ^ lam nguyen liSu nuoi cay va ducfc ck dianh lal mong nufii cay tao mo seo tren moi truong MS, IAA (0.) mg/I) va lang sinh tren moi truong MS, 2,4-D (0,5 mg/l), kinetin (1 mg/l) Dich huyen phii te b^o soma dugc hinh thinh va nuoi dy tang sinh tren moi tnrong MS, 2,4-D (0,5 mg/l), kinetin (1 mg/I). Tl bao soma dugc nu6i cay bi^t hoa thanh phoi soma tren moi tnrang MS, BA (0.3 mg/l). Djch huyk phii tl bao phoi dirge trai tai sinh tren moi tucmg ban rin MS, NAA (0,1 mg/l), BA (0,5 mg/l). Vi nhan gifing hoa lan ngoc dilm bang ky thuat phfii soma thu nhan 4.400 choi tren mgt lit te bao phfii
Tli- khoa mo seo. dich huyln phii tl bao phoi, the phfii PLB, tai sinh, djch huyln phii tl bao soma MdDAU
Hg hoa lan chiem khoing 10% cac loSi thu-c v|t hgt kin. Lan la loai Id-n, da dang v l hinh dgng, kich thu-oc, mau sic, thich i>ng v6i vung smh thai rgng, quan trgng trong nganh cong nghifip hoa vo-i 20-30 000 ioki kinh ti (Griesbach, 2003) Nu6i cay mo ti bao da dugc irng dung nhan nhanh cac loai hoa lan, cac loai thich hop vol dilu kifin nu6i cay khSc nhau. va thi phot gik (PLB-protoconn like body) lk the phat sinh ph6i dku tien. Hoa lan ngpc diem ik loai do-n than c6 lilm nang kinh te du-g-c tim thiy if vung Dong nam a (Vifit nam, Iko. Thai Lan). Nhan giong hoa lan ngoc diem bkng prolocorm qua nu6i ciy chli, la tiln sinh loii Rhynchoslylis gigantea, ti Rhynchostylis retusa, nuoi cay tao PLB tryc tiep ti> lal mong t l bao (Bui Van Le et al., 1999)
Phoi soma 1^ hg thing thich hg-p de nhkn nhanh ckc loai hoa lan qu;^ hilm (Li vk Xu, 2009), C6 it bko cko ve nuoi cay phat sinh-phlrbvc-trep va gian li^p u- liua lati iiliO'i'vQ nO- (Chen et Sf:, 19!JSJ; Chen va Chang, 2ou^; Uhen va Uhang.
2004). dia tan (Cheng va Chang, 1998), ho digp (Ishii e( ai. 1998). Da lai sinh thanh c6ng phoi soma Rhynchostylis gigantea (Lindl) RidI hoang dgi co cinh hoa b^ng diem tim (U vk Xu (2009). loai Rhynchostylis retusa (Thomas va Michael, 2007) Nghien CLPU nuoi cay tgo mo sgo, tgo phoi, va tai sinh phoi la rao can dau tien b-ong ky thugt phoi soma (Jain e( ai., 1995). Trong bki bko nay chiing t l i nghifin cini nhan nhanh hoa lan nggc diem bang ky thugt null cay phii soma,
NGUYEN Lieu vA PHlfCNG P H A P Nguyen lieu
Nggc dilm ru-ng Vifit nam c6 miu do huylt du. Mlu nuli cly: (i) L i non chli in vilro 20 ngiy tuli. (iii) PLB du-g-c cat lal ming
Mfii bv£mg dinh du-o-ng Idioang: MS (Murashige v i Skoog, 1962) co bo sung: BA (6-ben2ylaminopurine), 2,4-D (2.4- dichlorophenoxy acetic acid), TDZ (thidiazuron), kinetin (6-furfurylaminopurine). IBA (indol-3-acetic acid), NAA (a- naplhalene acetic acid), nu-oc dOa (10%), du-o-ng sucrose (30 g/1), than hgal tinh (1 g/l)
Bilu kien nufii cly: nhigt dg phdng 26+2 "C. am d l 65 %, ci/ong d l chieu sang 22,2 pmol/m^/s. thcri gian chilu sing 10 gio/ngay, v l friing mil tru-o-ng nufii ciy d 121 "C (1 al) trong 20 phOl, toe d l Ilc 100 vong/phiit
Phutyng phdp
Thi nghifim b l tri theo khli dly du ngau nhien, 3 lan lap Igi. moi lan igp lai nuli ciy 3 binh tam giic (chCra 60ml mli I r u ^ g bin rin hay 50ml mli tru-ong long). Sinh khoi l l bao ban dau nuli cay tgo djch huyln phii 10 g/IOOmI mfii tnrt-ng. S l lifiu du^c phan tich bing phim mim MSTATC (p=0.05)
Thiet ki thf nghiim Nuli ciy tgo va lai sinh mo sgo
Anh hucmg cua moi tarfi-ng khodng da li«?-ng den tgo PLB: Chli non hoa lan nggc dilm in vitm 20 ngiy tuli du'gc duac si> diuig lim nguyen lifiu nuli ciy. Mau nufii ciy l i l i non, dugc nufii ciy trfin mli tafo-ng khoang tgo phii PLB: MS, VW, i/2MS CO b l sung N/W^ (0.1 mg/l) va TDZ (0,1 mg/l). Thfi'i gian nuli cay 30 ngiy.
Anh hudng cua TDZ v i NAA din tgo PLB: M6i tnffi'ng MS co bo sung NAA (0,1 0,5 mg/l) va TDZ (0,1 0.5 mgfl). Thl-i gian nudi dly 30 ngay.
NuOi d y tgo mfi sgo: PLB du-gc clt lit mong dgt trfin mfii tnro'ng nufii ciy tgo t l bao m l sgo MS, nu-6-c di>a (10 %) cd b l sung IAA (0,1 0,5 1 2 mg/l). Thoi gian null cly 30 ngay.
Nuli cly tang sinh mfl sgo: M l sgo li> nghiin ci>u bfin du^c si> dung frong nghien cuoi nuli cly tang sinh. Sinh khoi m l sgo dira vao nufii cly ban diu 500 mg/mSu. Mli bu-l'ng nuli cly tang sinh tfi bio m l sgo MS co bo sung 2,4-D (0,1 0,2 0.5 0.7 mg/I) va kinetin (0,1 0,5 1 2 mg/l). Thdi gian nufii cay 45 ngay.
HOl NGHj KHOA HOC CONG NGHE SINH HQC T O A N Q U 6 C 2013
Nuoi cay tai sinh mfi seo: Mo seo tir nghien ciJu tren, dirge si> dyng lim nguyen lifiu nuli c l y tai sinh. Khoi lu-gng mfi seo du-a vio nufii cly l i 500 mg/miu. Mil tnro-ng nuli ciy tai sinh t l bio mo seo iwis co bo sung NAA (0 0,1 0,5 mg/l), BA (0,1 0,5 1 mg/l), TDZ (0,1 0,5 1 mg/l). Thdi gian null cay 45 ngay
Nuli cly lao va lang sinh djch huyln phu le bio m l sgo
Null ciy lao dich huyln phu t l bao mfl sgo: Mfi seo di/oc dung lam nguyfin lieu nufli cay tao dich huyln phCi. Khli lu-gng mfl seo du-a vao nuli ciy long la 500 mg/50ml mfli tru-o-ng nuli cay. Mli tru-o-ng nufii c i y tgo dich huyln phO mfi seo MS CO bo sung 2,4-D (0,5 mg/l), NAA (1 mg/l). BA {1 mg/l) va kinetin (1 mg/l). Tho-i gian nuli c i y 20 ngay Null ciy tang sinh dich huyln phu t l bao mfi seo' Djch huyen phu t l bao mo sgo cly chuyin l i n thCr 6 du'gc si> dgng lam nguyfin lifiu nufii cly. Khoi lu-gng mfl sgo du-a vao nufii ciy 1 g/50ml moi trmrng. Mfii tnj-fi'ng nuoi c i y tang sinh d]ch huyen phii t l bao m l seo MS co bo sung NAA va kinetin. Thfl'i gian nufii ciy 20 ngay
Tai sinh dich huyln phu t l bao mfi seo
Nufli cky cim i>ng phit sinh phfii soma: Djch huyln phii tfi bao c i y chuyen fin thCr 6 du-gc si> dyng lam nguyfin ligu nufii ciy. Khfil lu-gng l l bio du-a vao nufli ciy l i 10 g/50ml mfli tru-o-ng Mfii tru-o-ng nuli cly cam Crng phai sinh phii MS co bo sung BA (0 0,1 0,3 0,5 mg/l). Thai gian nuli cly 45 ngay
Trii l l bao phfii soma trfin mfii tru-fl-ng agar Dich huyln phu t l bao phii o- thi nghigm trfin du-gc su- dung l i m nguyen ligu nuli cly T h I tich bai tl'bao 5 ml/60ml mli tm-fl'ng ban ran. Mli tru-fi-ng nufii c l y trai dich huyen phii phoi soma MS c l bo sung NAA (0,1 0.5 mg/l), BA (0,1 0,5 1 mg/l) Tho-i gian nufii ciy 45 ngay.
K^T QUA VA THAO LUAN Nuoi cay tao v i t i i sinh mo seo
Anh hu-fl-ng cCia mfli lru-6-ng khoang da lu-gng din tao PLB: Trfin mfii tru-o-ng khoang MS cfi b l sung NAA (0,1 mg/l) va TDZ (0,1 mg/l) klch thich phat sinh Ihl phfli PLB co ty le 78%. PLB hinh thanh Iheo cym v i cfi hai hinh thai PLB khae nhau la Irfin to miu xanh va Iron nho miu trlng irtrng c i vfi-i li lg l i i sinh chli cao (4,6 PLB/mSu). Vdi moi tru-dng khoing 1/2MS, PLB cd dgng trfin to mau tring v i ll lg lai sinh ehli thip (Bang 1). Moi tardng khoang MS du-gc si> dyng cho cac nghien cu-u sau.
Bang 1. Anh hu-o-ng cua khdang da lu-g-ng d^n t^o the phoi PLB Khdang da lu-g-ng Ty lg tgo PLB (%)
1/2 MS Vacln-Went CV(%)
M h hu-dng ciia TDZ va NAA din tgo PLB: Tit ea cae nghifim thde dfiu co ty lfi hinh thinh PLB eao. La non du-gc nufii ciy tgo Ihl phfii PLB trfin moi tardng MS. TDZ (0,1 mg/l), eo li Ifi tgo PLB 100% va dgt 8,6 PLB/mau. Tuy nhien, da s l PLB if dgng trin lo miu xanh. chi cfi d nghigm thi>c TDZ (0,1 mg/l) xuil hien mgt hinh thii khae l i PLB trdn nhfl miu trang tning c i . Oay l i logi PLB eho ti l§ t i i smh chli cao (Bang 2)
Bang 2. Anh hu-6-ng cua chat dieu hoa sinh tru-dng d^n tao the phdl PLB ChJt a i u hfia sinh tm-dng Ty 1$ tgo PLB (%) S l PLB Irwku N/\A(0,1mg/l) 100 3 2
TDZ(0.1mg/l) 100 Bfi NAA (0.1 mg/l) ••• TDZ (O.lmgfl) 60 4 2
NAA (0,5mg/l) * TDZ (0.1mg/l) 74 T'S NAA (0,1mg/l) t TDZ (0,5mg/l) 80 36 CV(%) 14 10_4
Nufli ciy^lgo mfi sgo: PLB du-gc lal mong du-gc su- dung nufii eiy. Mfl seo xuil hign trfin mlu null cly trfin mfii tru-dng MS cfi bo sung IAA (1 mg/l), dat du-dng kinh m l seo 3,2 mm (Bang 3). PLB xult hifin it trfin be mgt m l seo sau 45-50 ngay nufli eay Mfi sgo du-ge tach rdi va nuli cay trfin mfii tru-dna tang sinh m l sgo. Do PLB l i the giong phfli, mil phmyng thdc lai sinh eiia cac loai hoa lan nufii cay In vitro, nen t l bio mfi seo phat sinh c6 nguln gfic td tfi bio the gilng phii.
Bang 3. Anh hu-dng cua IAA den tao mo sf o
IAA (mg/l) Ty If tgo mo seo (%) Ouimg kinh mo sgo (mm) 06i chdng
IAA(0,1mg/l)
lAA(0,5mg/l) 18 2 2
IAA(1,0m9fl) 74 28 IAA(1,0mg/l) 100 z]2 IAA (3,0mg/l) 68 3 0
CV(%) 10,6 g'z
HOI NGH! KHOA HOC CONG NGHE SINH HOC TOAN QUOC 2013
I
ciy tang sinh mfi seo; Mfi sgo lang sinh nhanh bfin mfii tnidng ban rin MS co b l sunp kinehn (0,5 mg/l) va 2,4-D ng/l) dgt sinh khli 2.377 mg/khli, cfl hg s l tang sinh 4,6 (Bang 4). Mfi seo cfi mau trang nga, cfi nhan to, tfi bio j k n dac CO cau true ciia le bao tlln phfiin 0cr2;iFO"a6rrtatig Sirth tt
0,5 CV(%)
Kinetin
0.1 0.5 1.0 2.0 0.5 1,0 2,0
(mg/l) Sinhk 512 652 924 1.642 726 946 1.346 1.684 1.168 2.145 2.377 1.958 10,4
Sinh khfil mo s^o (mg) He so tgng sinh khfii
Nufii cay tai sinh mo sgo: Mfii trydng t i i sinh MS co bo sung to hgp NAA (0.1 mg/l) v i BA (0.5 mg/l) dgt higu suit tai sinh chfii cao 96 % va dgt 3,6 choi/cym (Bang 5). Phln lfi bao ehu-a tai sinh cd miu xanh, hinh Ihinh hgt nhi PLB. Mfi sgo cd nguon gfic tir PLB co hifiu suit tii sinh cao, hinh thanh chli va PLB Uong qua trlnh tai sinh sinh Kfit qua nghien cdu duvc su- dyng lam co sd eho qui binh l i i sinh t l bao djch huyln phii mfi seo sinh phfii.
Bang 5. Anh hu-crng cCia N/U^ va BA den tai sinh mfi s f o
Ty I f tai sinh mo s§o (%) S6 chfii /cum
CV(%)
Nufii c l y tao va tang sinh dich huyen phu te bao md seo
Nuli ciy tgo djch huyen phu t i bao mfi seo: Dich huyln phii tang sinh khli sau 30 ngiy nufli cly tren mfli tardng MS cfl b l sung 2,4-D (0,5 mg/I) va kinetin (1 mg/l), cfi sinh khli 2 186 mg/ci,im, he sfl tang sinh khfii la 4,2 (Bang 6). Te bio tao djch huyln phu tu-ang dli ding nhit. Bfi sung NAA, BA vao mfii hirdng nufii cay MS cho t l bao m l seo frong djch huyln phu ph^n hfia khfing theo quy lugt nhat ^nh, bieu hign qua nhilu hinh dgng t l bao khic nhau, cfi nhieu cym te bio xual hifin, h§ s6 ting sinh thap so vdi mfii tnfdng MS co b l sung 2,4-D (0,5 mg/l) va kinetin (1 mg/l). B l sung t l hgp kinetin v i 2,4-D friich hgp cho qua trinh nuli eay tgo djch huyfin phu mo sgo so vdi BA kit hgp vdi NAA hoac 2,4-D.
BSng 6. Anh hu-dng cua chat dieu hoa slnh tru-dng den tao dich huyen phit te bao mo seo Chat sinh tnrdng Sinh khoi mfi seo (mg) He s6 tang smh khfii 2.4-D (0,5 mg/l) + kinetin (1 mg/l) 2.186 4,2 2,4-D (0,5 mg/l) * BA (1 mg/l) 542 1.4 N/W, (1 mgfl) t kinefln (1 mg/l) 1 106 2.2 N/y^(1 mg/l)+ BA(1 mg/l) 744 1,5
CV(%) 10^6 1 0 ^
Nufii cly ting sinh djch huyen phii l l bao m l sgo: Mfli tnj-dng null ely MS cfl b l sung 2.4-D (0,5 mg/l) va kinetin (1 mgfl) kich thich ting sinh dich huyfin phii t l bao m l sgo. cd sinh khli 8.256 mg/cym va h$ so tang sinh 8,2 (Bang 7). T l bio CO hinh dgng dong nhll, ting sinh nhanh, it co cym, co clu Imc 16 bio Hin phii. Bo sung vio mfii tru-dng nufli ciy MS to hgp kinetin va 2,4-D cho thiy thich hgp klch thich lang sinh dich huyln phu.
B i n g 7. Anh hu'dng cua 2,4-D vii kinetin den tang slnh djch h u y l n phit te bao mo seo Chit d i l u hda sinh tru-dng (mg/l) Sinh khfii mfi sgo (mg) Hf s l tang smh khfii 2,4-0 (0,5 mgfl) + kinelin (0,5 mg/l) 7 642 7,6 2,4-0 (0,5 mg/l) + kinetin (1 mgfl) 8.256 8,2 2.4-D (0.5 mgfl) + kinetin (2 mgli) 6.284 6.2 C V ( % ) 1 0 4 8^2 Tai sinh djch huyen phij te b i o m l sgo
HOl NGH! KHOA HQC C O N G NGHE SINH HOC T O A N Q U O C 2013
N u l i c l y c a m d n g phat sinh p h & s o m a : Trong nufii c i y dieh huyfin p h u . tru-dc khi nufii c l y tai s i n h , djch h u y l n phii du-gc kich thich cam d n g tai sinh ngay frong moi t a r d n g nufli c i y long hay tren m l i tm-dng ban r i n . Q u i trlnh kich thich c a m d n g c i n b l sung cytokinin va g i a m auxin. T D Z khfing kich thich phat sinh phfii (Nguyfin T h i e n Q u a n g , 2011). Moi tm-dng Ihich h g p c h o c a m d n g phat sinh phfii M S co b f i sung BA (0,3 mgfl). T l b i o m f l seo bigt hfla h i n h thanh phfli trong mfli tru-dng nuoi cay, c o m a t d o hogt hoa (1,3 x 10"* lb/ml) v a higu s u i t c i m d n g 82 % ( B i n g 8). T o e d o l a n g sinh giam nhanh, ngu-oc lai higu s u i t h g a l hfia t a o phfli tang n h a n h . Djch h u y l n p h i phan hfia phfii h o a n l o a n s a u 4 5 ngay nufii c i y .
Bang 8. Anh hu-d-ng cua BA d i n cam u-ng phdt sinh phfii
BA (mg/l) Mat dfi t l b^o hogl hoa (CFU/ml) Hi6u s u i t hogl hoa (%) tgo phfii 0,0 0 , 4 x 1 0 ' 26 0,1 1,1 x l O ' 72 0,3 1,3x10* 82 0,5 1 , 0 x 1 0 ' 68 CV(%) 10.8 10,8
Tai sinhtfi b i o phfli soma fren mfii fru-dng agar: Mfii l a r d n g nufii c i y M S c f i b l sung N A A (0.1 mg/l) va BA (0,5 mg/l) thich h g p t i i sinh mo sgo, Chfii t i i smh khoe, khfing x u i l hifin bifin dj, co ty lg l i i sinh 8 6 %, s l c h l i dgt 2 2 ch6i/5ml djch h u y l n phu l l b i o phfii soma (Bang 9 ) . Thu n h i n 4,400 e h l i tren m f l l lit dieh huyen phu l i ban phfli
Bang 9. Anh hirang cua N/VA va BA d i n tai sinti m d s f o
NAA (mg/l) BA (mg/l) Ty I? tai smh mfi seo (%) S6 chfii /5ml dhp t l bao phfii
K ^ T L U A N
Nufii c i y t g o v i tai sinh mo s g o : La non du-o-c n u l l e i y l a o PLB tren m l i fru-dng moi tru-dng M S , T D Z (0,1 mg/l). PLB c l t lal mfing du-gc nufii e l y trfin mfli tru-dng tgo mo sgo MS, IAA (0,1 mg/l), m o sgo tang sinh tren mfli tru-dng M S , 2,4-D (0,5 mgfl), kinetin (1 mgfl). v i tai sinh fren mfli tru-dng M S , NAA (0.1 mgfl). BA (0,5 mg/l) dgt hieu s u i t l a i sinh chfii c a o P h i n t l b i o chu-a tai sinh e h u y i n qua m i u x a n h , hinh thanh hat n h d P L B
Nufli e l y l a o v i lang sinh dieh huyen phu t l bao mfl seo: Nufli c i y t a o dich h u y l n phii s a u 10 n g i y nufli eay tr&n mfli fru-dng M S c o b l sung 2 , 4 - 0 (0.5 mg/l) v i kinetin (1 mgfl), efi h e sfi l a n g sinh khoi la 4.2. T e bao phat s i n h d j c h huyfin phu khflng theo q u y lugt n h i t djnh, bieu hign qua nhifiu hinh d g n g t l b i o k h i c nhau, v i c f i n h i l u c u m t l b i o x u l l hien.
Sau 30 ngay nufli c l y . te b a o cfl hinh d g n g dfing n h i t ; l l b i o l a n g sinh n h a n h , i l co c u m ; co m i l p h l n t i bSo mfl seo bigt hfla t h i n h l l bao phfii.
Tai sinh djch h u y l n phCi t l b a o m f i s e o : Djch h u y l n phu te b a o m f l s g o du-gc nufli c i y c a m d n g phat sinh p h 5 i I r i n m l i t r u d n g M S , BA (0,3 mgfl)^va l a i sinh Iran mfii tru-dng M S , N A A (0,1 mgfl), BA (0,5 mgfl), co l y lg t a i sinh 8 6 %, s l chfli dgt 22 choi/5ml djch huyen p h u l l bao phfli s o m a . D a x i y du-ng du-gc h e t h i n g n h i n nhanh h o a ngpc d i l m bang ky thu§t phfli soma, thu n h i n 4,400 chfli fren mfit lit d|eh huyfin phu t l bao p h i i
T A I L i g u T H A M K H A o
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HOi NGH! KHOA HQC CONG NGH? SiNH HOC T O A N Q U O C 2013
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MICROPROPAGATION OF RHYNCHOSTYLIS GIGANTEA ORCHID BY SOMATIC EMBRYOGENIC CULTURES
Nguyen Thien Quang*, Tran Van Minh
International University. Vietnam National University Ho Chi Minh City SUMMARY
Young leaves were cultured for protocomi performance on the MS medium, TDZ (0,1 mg/l). Prolocorm lilte bodies (PLB) were used as planting materials. PLBs were cut into slices and placed on the medium for callus initiated Callus was initiated on the MS medium, IAA (0.1 mg/I) and was proliferated on the MS medium, 2 4-D (0.5 rag'l), kmetin (\ mg/l). Somatic cell suspension was initiated and proliferated on the MS medium, 2.4-D (0.5 mg/l), kinetin (1 mg/l). Somatic cell suspension was differentiated to embryogenic cell suspension on the MS medium, BA (0.3 mg/l). Embryogenic cell suspension was plated and regenerated on the MS medium, NAA (O.I mg/l), BA (0.5 mgfl). Micropropagation of Rhynchostylis sp. via embryogenic culture was set up to produce 4,400 plantlets per one liter of somatic embryogenesis suspension
Keywords- callus, embryogenic cell suspension, protoconn lilce body (PLB), regeneration, somatic cell suspension
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