B GIO DC V O TO Trng i hc Lt -----------o0o------------

IU TRA V NGHIN CU BO TN NGUN GEN CC GING HOA CC V HOA LAN CT CNH HIN ANG SN XUT TI LT
TI CP TRNG TRNG IM M S: B2005-29-41-T

C quan qun l: B Gio dc v o to C quan ch tr: Trng i hc Lt Ch nhim ti: Nguyn Vn Kt

B GIO DC V O TO Trng i hc Lt -----------o0o------------

IU TRA V NGHIN CU BO TN NGUN GEN CC GING HOA CC V HOA LAN CT CNH HIN ANG SN XUT TI LT

TI CP TRNG TRNG IM M S: B2005-29-41-TD

C quan qun l: B Gio dc v o to C quan ch tr: Trng i hc Lt Ch nhim ti: Nguyn Vn Kt

NHM THC HIN TI

Cn b tham gia thc hin ti: 1. TS. Nguyn Vn Kt 2. Th.S. Cao Th Ln 3. Th.S. Nguyn Thnh Sum 4. NCV. Phng Mai 5. NCV. Trng Th Lan Anh Cc ti sinh vin bo co 1. Kho st hiu qu c ch virus ca ribazole trn nm ging cc nui cy in vitro. inh Th Hnh. Sinh vin Nng hc kha 27 2. Tc ng ca ribazole ln s sinh trng ca cc (Farm trang, Farm tm, N hoang tm, Tho o, Tho vang chanh) in vitro. Nguyn Th Thm. Sinh vin Ti chc Sinh hc kha 13 3. Tc ng ca ribazole ln s sinh trng ca cc (Chrysanthemum sp.) in vitro. Phm Th Hng Hng. Sinh vin Sinh hc kha 27 4. Phc trng ging hoa cc (Farm hng, Pingpong vng, Nt vng, Nt tm, Tia mung vng) bng ribazole. Cao Vit Tun. Sinh vin Nng Hc Kha 27 5. Kho st nh hng ca bin php b tr ribazole trong vic lm sch virus ln s sinh trng v pht trin ca ba ging a lan ( B Mai, Tm ht, Vng Ba Ru) ti Lt. V Th Phng Thanh. Sinh vin Nng hc kha 27 6. Kho st v tnh hnh sn xut cc ging cc ti Lt hin nay. Nguyn Th Nh Qunh. Sinh vin Nng hc kha 28.

TM TT KT QU NGHIN CU

Tm tt Qua iu tra thc t cc loi cc v lan ct cnh ti Thnh ph Lt cho thy hin nay c khong 38 ging lan ang trng vi mc ch ct cnh, trong c khong 14 ging c gi tr v c nhiu ngi tiu dng a chung. Cc ging cc ct cnh nhp vo Lt c trn 72 ging, tuy nhin nhiu ging b lng qun, ch cn li khong 54 ging cn c th phc hi. Nhm bo tn cc ging hoa ny, chng ti tin hnh cc bin php kim tra hin trng ly nhim cc loi virus, v phc trng thnh cng 14 ging a lan, 54 ging cc in vitro. Abstrast Lam Dong Province of Vietnam has an exceptional diversity of cutting flower of Cymbidium and Chrysanthemum with approximately 38 and 72 varieties respectively. Plant tissue culture and micropropagation techniques play an important role in conservation programme and management of botanical collection. Chrysanthemum and Cymbidium plants collected from farmers were identified the highest virus infection rate among of these species. The results obtained by observation samples of 54 varieties Chrysanthemum and 14 varieties Cymbidium. Using meristem culture and treated with Virazol in vitro suggest that the possibility could be obtained virus free plants through cultures.

M u
1. Tnh cp thit ca ti Vic du nhp ngy cng nhiu cc ging hoa ct cnh l mt xu hng cn thit p ng nhu cu thng thc hoa ca ngi dn cng nh xut khNu. Tuy nhin song song vi vic chy ua thay i v ging trn th trng sn xut hin nay th ng thi cng c rt nhiu ging dn dn b lng qun, khng phi v cc ging hoa ny khng m bo cht lng m l do th hiu ca ngi tiu dng. Vic nghin cu cc bin php cng ngh trong lnh vc nhn ging cc loi hoa ct cnh c gi tr kinh t ca a phng nhm mc ch bo tn ngun gen trong tp on hoa ct cnh l mt bin php cn thit cho xu hng pht trin a dng v hoa ct cnh trong tng lai v trnh lng ph mt s ln cc ging hoa m bn thn t nc chng ta hin nay cha th lai to c. 2. Mc tiu ti ti thc hin nhm nhng mc ch sau: - iu tra tnh hnh sn xut hoa ct cnh hin c ti lt. - Su tp v bo tn cc ngun gen qu v hoa cc, hoa lan ct cnh, v.v...c gi tr ti tnh Lm ng bng k thut nui cy in vitro. - Nghin cu cc iu kin mi trng sinh trng thch hp ca cc ging cy con bo tn trong iu kin in vitro. - Nghin cu cc iu kin mi trng gi th sinh trng thch hp ca cc ging cy con vn m. - To mi trng thc tp v nghin cu cho gio vin v sinh vin. 3. Phm vi v gii hn ca ti ti ch thc hin nghin cu bo tn cc ging cc v cc ging hoa lan ct cnh hin cn tn ti v ang sn xut ti Thnh ph Lt.

CC T VIT TT BAP 6-benzyl-aminopurine dSm-1 deciSiemen per meter n v biu th dn in ca dung dch EC IAA IBA KC Kin LM MS NAA PLB VW dn in (Electrical conductivity) Indol-3-acetic acid Indolbutyride acid Knudson (1946) Kinetin (6-furfuryl-aminopurine) Lindemann(1970) Murashige and Skoog (1962) -naphthalene acetic acid Protocorm like body Vacin and Went (1949)

NCV Nghin cu vin TDZ Thidiazuron (N-phenyl-N,-1,2,3-thiadiazol-5-ylurea) 2,4-D 2,4-dichlorophenoxyacetic acid. LSD 0.5: Sai khc ti thiu c ngha P = 0.5 CV% H s sai d FrW: Trng lng ti NTL: ng Nguyn T Lc Lt

Chng 1 TNG QUAN TI LIU

1. S lc v cy m trong cng tc nhn ging cy trng 1.1 Tnh ton nng ca t bo thc vt trong nui cy m Nui cy m thc vt (hay cn gi l nui cy thc vt in vitro) l qu trnh nhn ging v tnh thc vt trong ng nghim. Nui cy m thc vt l phm tr khi nim chung cho tt c cc k thut nui cy cc b phn khc nhau (t bo, m, c quan) thc vt trong mi trng nhn to di iu kin v trng (15, 22, 29).. K thut nui cy m t bo thc vt da trn c s l lun khoa hc v tnh ton nng v kh nng phn ha, phn phn ha ca t bo thc vt. Cui th k 19, nh bc hc ngi c Haberlangt (1902) l ngi u tin xut phng php nui cy m t bo thc vt chng minh tnh ton nng ca t bo. Theo ng, mi t bo ca bt k c th sinh vt no u mang ton b lng thng tin di truyn ca c sinh vt , v vy khi gp iu kin thch hp mi t bo u c th pht trin thnh c th hon chnh (15, 22). Nm 1934, bt u giai on th hai trong lch s nui cy m thc vt, khi White, ngi M, nui cy thnh cng u r c chua (Lycopersicum esculentum) vi mt mi trng lng cha mui khong, glucose v nc chit nm men. Sau White chng minh rng c th thay th nc chit nm men bng hn hp ba loi vitamin nhm B: Thiamin (B1), Pyridoxin (B6) v Nicotinic axit. T vic nui cy u r c tin hnh trn nhiu loi cy khc nhau. Nm 1958, tnh ton nng ca t bo c khng nh bng cng trnh nghin cu ca Stewart v cs. (1958) trn m r cy c rt. Cc tc gi ny nui cy m r c rt trn mi trng c c nc da v thu nhn c khi m so gm cc t bo nhu m. Khi chuyn m so ny sang mi trng lng c cng thnh phn v nui lc th nhn c huyn ph gm cc t bo ring l v cc nhm t bo. Tip tc nui cy trn mi trng lng, khng cy chuyn th thy hnh thnh r. n nhng nm 60, khi ng thi Stewart (1963), Wetherell v Halperin (1963) cng thng bo rng t bo c rt t do khi nui cy trn mi trng thch to thnh hng ngn phi, cc phi ny pht trin qua cc giai on ging nh qu trnh to phi bnh thng c rt, lc ny tnh ton nng ca t bo cng c khng nh. T nhng khm ph trn, hng lot cc bo co v tnh ton nng ca t bo c thng bo, hu nh tt c cc c quan u c th pht trin phi. Phi soma c ghi nhn nhiu ging nh Atrapoda, Begonia, Citrus, Coffea, Cymbidium, Hordeum, Kalanchoe, Nicotiana, Panax, Ranumculus, Solanum, Oryza... Ngy nay bng k thut nui cy nh sinh trng, ngi ta nhn ging v phc trng hng lot cc cy trng c gi tr nh khoai ty, lan, thuc l, da, cc cy lng thc, cy n qu.... Vic nhn ging ny tr thnh cng ngh v c p dng nhiu nc trn th gii (10, 11, 15. 22, 30).

1.2 Cng ngh sinh hc thc vt trong nhn ging cy trng Cng ngh sinh hc thc vt hnh thnh do p lc t nhin ca x hi theo hai khuynh hng: p lc gia tng nng sut v p lc gim nng sut ma mng. Trc nhng xu hng , i hi ngy cng phi a nhanh k thut mi vo cng tc la chn, nhn ging cy trng. S pht trin ca cng ngh sinh hc thc vt theo thi gian c th c tm tt nh sau: t nhng nm 1920 bt u l lai cho, nm 1960 l k thut gy t bin, nm 1970 l s pht trin k thut cy m, n nm 1980 l k thut dung hp t bo trn v t nhng nm 1990 n nay l s pht trin ca k thut gen (10, 12, 15. 22, 30). Nh s kt hp gia nui cy m thc vt v cng ngh gen, ngi ta to ra c nhng thuc tnh mi cho cy trng mt cch c nh hng v hiu qu. Nh thuc tnh totipotent ca thc vt, ngi ta c th hon ton ti to nn mt lot cc cy mi hon chnh t mt t bo mang c tnh mi ch trong mt thi gian ngn. Nh s kt hp ny gip cho cng ngh sinh hc thc vt ni chung v nui cy m ni ring c nhng bc tin mang li hiu qu kinh t rt ln. C th ni mt cch khi qut nhng u im ca cng ngh ny l: - Chn lc nhanh nhng tnh trng mong mun, nui cy m l con ng nhanh nht gip chn lc v biu hin tnh trng. - Nui cy m to ngun nguyn liu thc vt tuyt vi cho qu trnh chuyn gen thc vt. - Nui cy m kt hp vi k thut dung hp t bo trn v t bin c th to ra cc dng lai khc loi. - Gip nhn ging v tnh vi tc v s lng ln. - Gip to c cy sch bnh. - Thc vt nui cy m l ngun nguyn liu tt sn xut cc cht qu him, c bit l cc dc cht. - Cc cy m l i tng tt v hiu qu ca nhng nghin cu v sinh l, sinh ha, di truyn, bnh l, sinh hc phn t. - Cc cy m gip trao i quc t ngun ging thc vt rt d dng. - Cc cy m c th cung cp quanh nm. 1.3 Vai tr ca cht iu tit sinh trng trong nui cy m Cht iu tit sinh trng hay cn gi l cht kch thch sinh trng thc vt l cc yu t ha hc cn thit cho s sinh trng v pht trin ca thc vt. Cht iu tit sinh trng thc vt c th l nhng cht t nhin c sn sinh vi mt hm lng rt nh trong mt b phn no ca c th thc vt hoc l nhng cht c tng hp nhn to. Nhng cht iu tit sinh trng ny c vn chuyn n cc b phn khc ca c th gy nn nhng tc ng iu tit ca cy.

Vai tr iu ha ca cht iu tit sinh trng thc vt l khi ng cho s hnh thnh cc phn ng c bit hay cc qu trnh sinh l nht nh hoc tr hon qu trnh . Cc cht iu tit sinh trng thc vt gm hai nhm chnh l auxin v cytokinin, ngoi ra gibberelin v ethylen cng c vai tr quan trng i vi sinh trng, pht trin v trao i cht thc vt. 1.3.1 Cc auxin Cht auxin t nhin c tm thy nhiu thc vt l indol axetic axit (IAA). IAA c tc dng kch thch sinh trng ko di t bo, iu khin s hnh thnh r. Ngoi IAA, cn c nhng dn xut khc l naphtyl axetic axit (NAA) v 2,4 diclophenoxyl axetic axit (2,4-D). Cc cht ny cng ng vai tr quan trng trong s phn chia m v trong qu trnh to r (10, 12, 40, 41). NAA, IBA, 2,4-D l nhng auxin tng hp, c hot tnh mnh hn auxin t nhin IAA. Chng c vai tr quan trng i vi phn chia t bo v to r. i vi r, v c bn auxin c ch s pht trin ca r, ch nng cc thp (10 -10-12g/ml) th mi c tc dng kch thch sinh trng ca r, vt qua ngng ny (10-8mg/l) th li th hin tc dng c ch.
-9

IAA kch thch s ra r v km hm s pht trin callus. Ngc li, 2,4-D kch thch s hnh thnh callus v km hm s hnh thnh r trong mi trng nui cy. Mc d cng nhm cht auxin nhng hai cht ny li c tnh cht i khng.. NAA c Went v Thimann (1937) tm ra. Cht ny c tc dng lm tng h hp ca t bo v m nui cy, tng hot tnh enzim v nh hng mnh n trao i cht ca nit, tng kh nng tip nhn v s dng ng trong mi trng nui cy (10, 12, 40, 41). 1.3.2 Cytokinin Cytokinin l cht iu tit sinh trng c tc dng lm tng s phn chia t bo. Cc cytokinin thng gp l 6-benzyl aminopurin (BAP), Kinetin. Kinetin c Shoog pht hin ngu nhin trong khi chit xut axit nucleic. Kinetin l dn xut ca base nit adenin. BAP l cytokinin tng hp nhn to nhng c hot tnh mnh hn kinetin. Kinetin v BAP cng c tc dng kch thch phn chia t bo, ko di thi gian hot ng ca t bo phn sinh v lm hn ch s ha gi ca t bo. Ngoi ra cc cht ny c tc dng ln qu trnh trao i cht, qu trnh tng hp ADN, tng hp protein v tng cng hot ng ca mt s enzim (10, 40, 41). Benjamin v cs. (1987) cho rng nng BA cao (1-5ppm) kch thch s pht trin ca chi nh v u r ca cy Atropa belladona. Lat v cs. (1988) cho rng khi s dng kinetin nhn nhanh cy Picrohiza kurroa phi dng nng t 15mg/l. Nhng nghin cu ca Miller v Skoog (1963) cho thy khng phi cc cht iu tit sinh trng ngoi sinh tc dng c lp vi cc cht iu tit sinh

trng ni sinh. Tc ng phi hp ca auxin v cytokinin c tc dng quyt nh n s pht trin v pht sinh hnh thi ca t bo v m. T l auxin/cytokinin cao thch hp cho s hnh thnh r, t l ny thp s kch thch qu trnh pht sinh chi, nu t l ny cn bng th thun li cho s pht trin m so. Barna v Wakhlu (1998) ch ra rng tc ti sinh chi ca cy Plantago ovata tng cao khi trn mi trng s dng KIN 4-6 M phi hp vi NAA 0,05M. Das (1958) v Nitsch (1968) khng nh rng ch khi tc ng ng thi ca auxin v cytokinin th mi kch thch mnh m s tng hp ADN, dn n qu trnh mitos v cm ng cho s phn chia t bo. Skoog v Miller (1957) khng nh vai tr ca cytokinin trong qu trnh phn chia t bo, c th l cytokinin iu khin qu trnh chuyn pha mitos v gi cho qu trnh ny din ra mt cch bnh thng (10, 12, 40, 41). 1.3.3 Cc nhm cht khc Ngoi nhng nhm cht iu tit sinh trng k trn, trong nui cy in vitro cn s dng mt s nhm cht khc nh Abscicis acid, ethylen, cc amino acid... Abscisis acid s dng nng cao l cht c ch sinh trng v trng thi ngh bt buc. Ethylen c sinh ra trong qu trnh nui cy thc vt. Ethylen c th km hm s sinh trng t bo v Ny nhanh s lo ha trong qu trnh nui cy m. Silver nitrat c th c s dng ngn chn ethylen hnh thnh trong qu trnh nui cy m (40, 41). Amino acid c th nng cao s tng trng ca t bo v s ti sinh cy. L. Glutamine c th c xem nh l ngun nit. Enzim hydrolyzed protein N-Z amin Type A nh casein hydrolyzate c th c s dng hiu qu n 2g/l. Thm vo malate, citrate, pyruvate v acid v c tng t c th c hiu qu trong mi trng nui cy protoplast v c th lm gim bt c hi ca mui amonium (Gamborg & Shyluk, 1970; Gamborg, 1986). Ngoi ra, cc cht chit ca cy nh coconut milk ca cy qu hch xanh c th rt hiu qu trong vic cung cp phc hp cht dinh dng v c khng xc nh v cc nhn t sinh trng. 1.4 K thut vi nhn ging (micropropagation) Trong 5 thp k qua c nhiu bo co v k thut vi nhn ging (Arditi v Krikorian, 1996; Chakrabarty v cs., 2001; Rasai v cs., 1995; Rout v cs., 2000). Vi nhn ging l s nhn ging v tnh vi s lng ca cy trng thng qua nui cy m in vitro. K thut nui cy ny c ng dng rng ri trong vic nhn ging cc loi cy cnh v nhng loi khc trn ton th gii (Chu 1992; Huetteman v Preece 1993; Mantell v cs., 1985; Prerik, 1987). Mt trong nhng dng th v v quan trng ca k thut nui cy m in vitro l kh nng ti sinh v sinh sn ca cy t nui cy t bo v m. Dng n gin nht ca ti sinh cy in vitro l kch thch s pht trin ca chi. K thut ny tn dng s pht trin c th cho s pht trin chi bng nh sinh trng hoc chi bn. Trng thi ng ca nhng chi ny b ph hy v s pht trin ca cc chi non c kch

thch bng t hp cc cht iu tit sinh trng (thng s dng cytokinin) ti sinh ra chi con. Cc chi con ny sau c tch ra cho pht trin r ti sinh cy con. S ti sinh cy t nui cy m c th c thc hin bng cch nui cy t nh sinh trng, t callus v t t bo, hoc chi nch v chi nch l tin thn ca nh sinh trng. Ngc li, s ti sinh bt nh din ra ti nhng v tr khc nh l cc t thn, t l mm, vng ko di ra ca r. S ti sinh chi bt nh thng ph thuc vo s c mt ca gen cu trc. Nhng s ti sinh cy t callus v t t bo thng khng c mt ca gen cu trc. S pht sinh c quan l s hnh thnh cc c quan ring bit nh chi hoc r. S ti sinh phi soma l s hnh thnh cu trc lng cc cha c chi v r. Hu ht cc cy trng c th ti sinh cy theo cch pht sinh c quan hoc phi soma nhng rt t loi c th c c hai cch. Mt s loi c th ti sinh mt cch d dng t nui cy m v t bo trong khi mt s loi ch c th ti sinh bng qu trnh bt nh. S ti sinh cy t chi nch hoc chi nh c ng dng rng ri trong vi nhn ging do c s bin d t nht. Ngc li s pht sinh chi bt nh v s ti sinh cy t callus bi s pht sinh c quan hoc bi phi soma c bin d cao nh tc ti sinh ca n. 1.4.1 Cc bc thc hin trong k thut vi nhn ging Trong k thut vi nhn ging c 4 giai on c trng (Murashige 1974, George v cs. 1984). Giai on 1: La chn mu cy v nui cy khi u. Giai on 2: Nhn nhanh chi con hoc chi mm t mu ban u. S ti sinh chi bt nh l k thut nhn nhanh c s dng thng xuyn nht trong vi nhn ging (Chu 1992). Mi trng nui cy v iu kin sinh trng trong giai on ny c cung cp ti u t l nhn nhanh chi t ti a. Giai on 3 : l giai on ti sinh r t chi con ti sinh cy con. Giai on ny cn mi trng c trng hoc khng, iu ny ph thuc vo kiu gen ca loi. Giai on 4: hon chnh cy con cho thch nghi trn iu kin trng trn hn hp t trong nh knh sau chuyn ra ng rung. 1.4.2 Cc nhn t trong mi trng nui cy invitro Mc thnh cng ca bt k mt k thut nui cy m t bo hoc nui cy c quan u lin quan n nhiu nhn t. Nhn t quan trng l la chn thnh phn dinh dng v cht iu tit sinh trng. Trong hai n ba thp k qua c nhiu bo co v thnh phn ca cc mi trng c bn (Sheet v Shillito, 1997; Perik 1987; Torres, 1989). Mi trng nui cy C hn 50 cng thc mi trng khc nhau c s dng cho nui cy in vitro nhiu loi cy khc nhau (Gamborg v cng s, 1976; Huang v Murashige, 1977). Trong mi trng MS c s dng ph bin nht, c ci bin bng s thay i thnh phn cc vi lng (Chand v cs., 1977; Jha v Sen, 1985; Rout v cs., 1999; Saxena v cs., 1988).

Mi trng dinh dng bao gm cc loi mui v c, ngun cacbon, vitamin v cht iu tit sinh trng. Mt s thnh phn khc c th c b sung thm nh nit v c, acid v c v cht chit cy trng (Gamborg, 1986). Cc mui khong c s dng nh l thnh phn ca mi trng nui cy m t bo. Hu ht cc mi trng cha t nht 30mM nit v c v kali v c. Mui amonium c th c s sng t 2-20mM. nh hng ca mui amonium c th thay i t c ch n hiu qu. Nng Ca, SO4, photphat v mui Mg t 1-3 mM l thch hp. Ngoi ra trong mi trng cng cn b sung thm mt s khong vi lng. Mi trng MS (1962) hoc Linsmainer v Skoog (1965) (LS) c s dng rng ri, c bit l trong qu trnh ti sinh cy. Mi trng B5 (Gamborg v cs., 1968), N6 (Chu, 1978), Nisch v Nitsch (1969) (NN) v cc dn xut t cc mi trng ny c th c s dng rng hn cho nhiu loi cy. Theo Huang v cs., Indra D. Bhatt v Uppeandra Dhar (2000), cy du ty pht trin thch hp trn mi trng MS. 1.4.2.2 Cc cht iu tit sinh trng. Liu lng cht iu tit sinh trng ln mi trng nui cy quyt nh rt ln n s thnh cng ca nui cy m thc vt. Mor v Zieslin (1987), Rout v cs. (2000) nghin cu nh hng ca cc cht iu tit sinh trng v mi tng tc gia chng ln vi nhn ging ca nhiu loi cy khc nhau, trong c bo co rt r v nh hng ca auxin v cytokinin ln s nhn nhanh chi ca nhiu loi cy khc nhau. Cytokinin l cht iu tit sinh trng thch hp cho vic nhn nhanh chi. Theo Indra D. Bhatt v Uppeandra Dhar khi s dng BA, Kinetin, 2-ip trn mi trng MS, tc nhn nhanh tt nht khi b sung BA. Theo kt qu nghin cu ca Kang v cs. (1994), BA l cht iu tit sinh trng thch hp nht cho s nhn nhanh chi t mu l ca cy du Fragaria x ananassa. Loi du ny s cho s lng chi cao, hiu qu nht trn mi trng MS c b sung 4,0 M BA kt hp vi 0,1 M NAA. Tc ng kt hp gia BA v auxin s lm tng s lng chi (Uppadhaya v Chandra, 1983). Hu v Wang (1983) cho rng nng cytokinin cao s lm gim s lng chi sinh sn, nhng khi s dng nng BA cao s cho cht lng chi khng tt, chi hnh thnh dng hoa th hoc nhng nt nh cui gc. Trong nui cy r in vitro, s c mt ca cytokinin trn mi trng thng hn ch kh nng ra r. Cy s hnh thnh r trong mi trng c b sung auxin hoc khng cn b sung thm auxin ph thuc vo kiu gen ca cy trng (Rout v cs., 2000). 1.4.2.3 Ngun cacbon v nhng c cht khc Ngun cacbon c s dng trong mi trng thng t 2-3 % ng saccaro hoc glucose (nhng rt t). Nhng ngun cacbon khc c th l lactose, maltose, galactose nhng rt t s dng. Ngun cacbon ny ng vai tr trong s pht trin t bo. Hu ht cc cy trng c th tng hp cc vitamin cn thit cho s sinh trng v pht trin nhng vi nhng vitamin c trng nh vitamin B1), vitamin B3, vitamin B6 v myo- inositol cn c b sung vo mi trng trong nui cy in vitro nng cao s sinh trng v s phn ha (Gamborg v cs., 1968).

Agar c s dng rng ri trn cc mi trng c v bn c, nhng mt vi egllin agent khc c th s dng nh gelatin, agrose, aglinate v gebrite. Than hot tnh c b sung vo mi trng cc giai on khc nhau ca qu trnh nui cy in vitro. Than hot tnh khng phi l cht iu tit sinh trng, n ng vai tr nh l cht ht Nm ca mi trng. Than hot tnh c kh nng hp th mt s cht khng c li cho s pht trin ca cy nh cc cht c sn sinh ra trong qu trnh kh trng mi trng nui cy hoc mt s cht do cy trng tit ra. i lc than hot tnh cng ng vai tr nh cht iu tit sinh trng (Ebert v cs., 1993; Eymar v cs., 2000; George v Sherrington, 1984). B sung thm than hot tnh vo mi trng c th c li cho vic hnh thnh r ca cy do than hot tnh c tc dng hn ch mc chiu sng hoc than hot tnh c kh nng hp th cc cht c ch s ra r c trong mi trng nui cy. (Dumas v cs.,1995; George v Sherrington, 1984). 1.4.2.4 Nhn nhanh chi y l giai on ct li quyt nh n s thnh cng trong nui cy in vitro. C 3 cch thng c s dng trong vi nhn ging : thng qua callus, s hnh thnh chi bt nh v chi nch (Bhojwani v cs., 1996). Trong trng thng qua nui cy chi nch, tc nhn nhanh ph thuc vo nng cytokinin thch hp trn mi trng v s cy chuyn trong qu trnh ti sinh cy. Trn cy du ty Fragaria x ananasa, tc nhn nhanh chi t kt qu tt nht khi b sung vo mi trng MS cc cht iu tit sinh trng BA (4,0 M) v NAA (0,1 M) sau 60 ngy nui cy. Nhng khi b sung vo mi trng nng BA cao hn 4,0 M cng vi 0,1 M NAA s lm cho chi sinh trng khng tt v cht lng chi xu, do tc nhn nhanh cng gim. 1.4.2.5 S hnh thnh r S hnh thnh r ca nhiu loi cy c th khng cn b sung thm cht iu tit sinh trng (Saxena v cs., 1998). R ca nhiu loi cy c th c hnh thnh d dng khi nng cc mui khong gim i mt na hoc gim hn, v nng ng gim t 2 hoc 3% n 0.5%. (Webb v Street, 1977). Theo Indra D. Bhatt v Uppeandra Dhar khi nghin cu nh hng ca auxin (IBA, NAA) ln s hnh thnh r ca loi Fragaria x ananasa, cy ra r tt nht khi s dng NAA vi liu lng thp (0.1mg/l). R ca mt s loi cy c th c hnh thnh d dng hn khi b sung thm than hot tnh vo mi trng, i khi c b sung thm auxin. Than hot tnh lm nng cao s pht trin ca r khi r u tin va c hnh thnh (Marthur v cs., 1999). S pht trin ca r tt hn khi trong mi trng c cht ht cc cht c ch ra r hoc lm gim mc chiu sng bng cch b sung than hot tnh (Druart v cs., 1993; George v Sherrington, 1984). S pht trin sung mn ca h r ca cy con trong nui cy in vitro l iu kin thit yu cho s pht trin tt ca cy con trong iu kin nh knh v trn ng rung.

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2. S lc v lch s nghin cu h lan (Orchidaceae) v chi a lan (Cymbidium sw.) 2.1 Khi qut chung Lan c bit n u tin phng ng vo 551-479 trc cng nguyn bi Khng T. Cy lan c bit n u tin Trung Quc l Kin Lan (tm ra Phc kin) l Cymbidium enrifolium. Theo Phrastus c xem l ng t ca thc vt hc v cng c th ni l cha ca ngnh hc v lan. Orchis c ng dng ch nhng cy lan tm thy vng a Trung Hi. Th k th nht sau cng nguyn, Dioscorides dng Orchis m t 2 loi a lan trong quyn sch v dc liu ca ng v c Linnaeus ghi li trong sch Cc Loi Cy C (Species Plantarum) vo 1753, ri John Lindley s dng u tin t tn cho h lan l Orchidaceae t nm 1836 v tn ti cho n nay. Vit Nam s nghin cu v lan cha r rt lm nhng c l ngi u tin tm hiu v nui trng lan l vua Trn Anh Tng, k n l Joanis Loureiro, l nh truyn gio ngi B o Nha (1789) trong cun Thc Vt Chng Nam B (Flora Cochinchinesis) cc cy lan trong cuc hnh trnh n nam phn Vit Nam v c Bentham v Hoocker ghi li trong Cc Ging Cy C (Genera Plantarum)(1862 1883). F. Gagnepain v A. Guillaumin m t 101 ging gm 636 loi lan cho c 3 nc ng Dng trong b Thc Vt Ch ng Dng do Hlecomte ch bin. ng k nht l quyn II b sch Cy C Min Nam Vit Nam ca gio s Phm Hong H vi 289 loi lan c m t v v hnh. Mi dy gio s b sung thm 364 loi lan trong quyn III tp 2 ca b sch Cy C Min Nam Vit Nam xut bn 1993. Trong chi Cymbidium.sw c 15 loi: C.aloifolium (Lindl.).sw, C.atropurpureum (Lindl.) Rolfe., C.banaense Gagn, C. bicolor lindl, C.dayanum Reich.b.f, C.devonianum Paxt., C.enrifolium (Lindl.).sw, C.erythrostylum Rolfe, C.filay sonianum Lindl., C.inrigne Rolfe, C.lancifolinum Hookf, C.lowianum Reich.b.f, C.schroederi Rolfe., C.sinense (Jacks)Willd., C.macroshizon Lindl.. Theo Trn Hp trong cun Phong Lan Vit Nam tp 1 v 2 xut bn 19881990 th h lan Vit Nam c khong 368 loi, trong chi Cymbidium c khong 10 loi sau: C.aloifolium (Lindl.).sw, C.dayanum Hookf, C. devonianum Paxt., C.enrifolium (Lindl.).sw, C.evrardii Guillaum, C.grandiflorum Griff, C.inrigne Rolfe, C.lancifolinum Hookf, C.parishii Reich.b., C.poilanei Gagnep. Theo V Vn Chi v Dng c Tin trong cun Phn Loi Hc Thc Vt Thc Vt Bc Cao xut bn 1978 th h lan Vit Nam c 91 chi 463 loi, trong chi Cymbidium c khong 12 loi sau:

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C.aloifolium Swartz., C.cyperifolium Wallex Lindl, C.dayanum Reich.b.f, C. devonianum Paxt., C.ebarneum Reich.b.f, C.enrifolium Swartz., C.erythrostylum Lindl., C.giganteum Wall., C.inringne Reich.b.f., C.lancifolinum Hookf, C.polanei Gagn., C.munronianum kinget plant. 2.2 Mt vi nt v a lan Lt. Hin nay, nhu cu hoa ti ni chung v hoa lan ni ring trn th gii v trong nc ang ngy cng tng. Trong nm 2000 kim ngch xut khNu hoa lan ct cnh v ging cy lan trn th gii t 150 triu USD, trong ch ring hoa lan ct cnh chim 128 triu USD. Do vic trng lan tr thnh mt hng pht trin kinh t ca nhiu nc trn th gii v ang pht trin mnh m khu vc ng Nam . Thi Lan tr thnh mt nc xut khNu hoa ct cnh v cy ging lan nhiu nht trn th gii. t 1990 n 1995 s lng lan ct cnh xut khNu Thi Lan tng 1.7 ln, t 15.6 triu cnh ln 26.5 triu cnh. Chnh ph Malaysia thy hiu qu kinh t ln ca vic trng lan nn giao 300 ha t cho hip hi hoa lan Malaysia, vi mc ch quy hoch vng ny thnh trung tm sn xut hoa xut khNu. Trong nhng nm gn y Nh nc ta cng nh hng pht trin vic trng lan khng ch ph v cho xut khNu m cn phc v cho ngnh du lch. Cc cp lnh o nh nc t Trung ng n a phng bt u thy c tm quan trng ca ngnh xut khNu hoa lan nn c nhng ch trng khuyn khch pht trin. Nm 1978, ngnh xut khNu thnh ph Lt xut khNu l u tin sang cc nc Chu u vi 3000 cnh lan., cho n nay th s lng ny tng ln ng k. Hin nay vic trng lan em li hiu qu kinh t cao, y l mt mt hng thu nhiu ngoi t, nhng bn thn n li chng yu cu phi c vn u t qu cao. iu kin t nhin ca Lt ph hp vi vic trng nhiu loi hoa, c bt l hoa lan. Trong a lan lt vn ni ting t lu v s phong ph v v p c o m ch c a lan Lt mi c c. Nhng nm gn y, ngnh trng lan Lt c nhng bin chuyn ng k. Din tch trng hoa thay th cho trng rau ngy cng tng. Hin nay Lt c 400 ha trng hoa vi 300 triu cnh hoa xut khNu hng nm. Hng pht trin n nm 2010 tng din tch trng ln 450 500 ha trong u tin pht trin trng lan. Thm vo , vi s tr gip ca cng ngh nui cy m, vic nhn ging v to ra nhng ging mi do lai to cng d dng hn, t m thi gian trng lan c rt ngn li. Hin nay sn lng hoa lan ct cnh c bit l a lan vn cha cung cp cho nhu cu rng ln ca th trng. Trong khi dch bnh thi gi hnh, v mt s bnh do virus honh hnh lm gim mt lng ln s chu a lan c Lt. Do vy ngnh trng lan trong Cymbidium l ch o s cn tip tc pht trin. T mt vi dn liu nu trn, ta c th mnh dn kt lun vic nui trng Cymbidium Lt c mt u th tuyt vi. V y l mt tim nng to ln m nu chng ra bit cch tc ng s em li li nhun cao. H lan l h c s lng loi ln th 2 vi khong 20000 - 25000 loi. Trong Cymbidium c mnh danh l n hong ca cc loi hoa, Cymbidium c nhng

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c im ni bt c v gi tr thNm m ln gi tr khoa hc. V tao nh, hi ha ca chng t lu hin din trong vn hc ngh thut v gn lin vi i sng vn ha ca con ngi Phng ng. 2.3 Hnh thi bn ngoi ca a lan. V hnh thi bn ngoi a lan l nhng cy thn tho, a nin, nhnh hng nm to thnh nhng bi nh. Thn ngm ca a lan (cn hnh) thng ngn ni nhng c vi nhau, cc c lan thc cht l nhng cnh ngn ca cn hnh. C gi, khi b tch khi cn hnh c c th pht sinh on cn hnh mi, t mc ln nhng cy con, do ngi ta xp Cymbidium vo nhm lan a thn. C lan (gi hnh) thng c dng con quay hay dng ht xoi. ng knh ca gi hnh t 1cm n 15cm. Gi hnh c bao bc bi cc b l xp xt nhau, do gi hnh ca a lan khng r rng nh mt s loi lan khc nh denrobium, catleya. R ca mt s loi a lan b sinh hay ph sinh thng mc bm trn v cy, mt t. Mt s loi khc c r n su trong bng cy, trong t mn (a sinh hay thc sinh). Nhng r mi thng ch mc cy con, cn cy m t khi hnh thnh r mi m ch thy nhng r ph phn nhnh t r c. L a lan thng c hai dng, dng vy nh theo mt on cn hnh v dng thc nh trn gi hnh. L dng thc nh trn gi hnh thng c cung l, gia b l v cung l c mt phn phn cch, khi phin l rng vn cn on b m ly gi hnh, mt s loi khng c cung l. Ty theo tng loi m hnh dng ca phin l, gn l rt khc nhau, gn dc ni r hay gn chm trong tht l. Mt s loi t chu rp c phin l mu vng xanh, cn li thng l mu xanh m. Bn l v dy ca l thay i ty theo tng loi, cc loi sng trng trng c l hp v dy hn cc loi a bng rp. L c dng di, dng mi mc hay dng phin. u l thng l nhn, hay chia thnh hai thy. Kch thc ca l bin ng t 0.5 cm 6cm, chiu di thay i t 10 cm 150 cm. Chi hoa thng xut hin bn di ca gi hnh t cc nch l, tch cc b gi m ra ngoi. Chi hoa thng xut hin cng vi chi thn mc m ra hai pha hnh ui c, nhng chi hoa cng trn hn, trong khi chi thn th hi dt. Cc l u tin chi thn mc m ra hai pha hnh ui c, cn cc l bao chi hoa th lun m cht quanh pht hoa. Cnh hoa khng phn nhnh, c th dng ng hay bung thng. Cnh hoa c th mang t vi hoa n vi chc bp hoa xp lun phin theo hnh xon c. Bp hoa khi ln bt u dang xa khi cnh hoa, xoay na vng trn a cnh mi xung di, sau bp hoa bt u bung cnh. Hoa Cymbidium thot nhn c 6 cnh vi 5 cnh gn ging nhau v 1 cnh mi. Thc ra 3 cnh bn ngoi chnh l 3 l i, c mu sc ging 2 cnh hoa trong. Cnh hoa cui cng bin i thnh cnh mi c mu sc sc s hn. Hoa Cymbidium l hoa lng tnh, c bit nh c v nhy ci cng gn chung trn mt trc hp nhy (tr nh - nhy). Trn trc hp nhy, th nh nm trn cng mang hai khi phn mu vng, c gt dnh nh keo. Khi phn c y bi mt np mu trng ng, np ny d m ri v cch bit vi num nhy bi mt cc m ni ln. Do hoa c cu trc c bit nh vy nn Cymbidium trong t nhin qu

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trnh th phn ch xy ra c nh cn trng. Tn cng pha bn trong hoa c da mt v i khi cn c tuyn tit mi thm. Sau khi th phn xong, bu hoa xoay dn v v tr c, bu phnh ln v to thnh qu. Qu ca lan thuc dng qu nang c ba gc, bn trong qu cha hng trm ngn ht. Ht lan c kch thc rt nh trng nh bi phn, mu vng la. Khi chn, qu m theo 3 ng gc v pht tn ht theo gi, ht ri vo ni c iu kin thch hp nh Nm , nh sng, th ht s ny mm thnh cy con. Trong t nhin t l ht lan ny mm l rt thp, do khi cha c cng ngh nui cy m th vic trng v nhn ging lan cn thi gian lu v gp nhiu kh khn. 2.4 Phn loi a lan. Trong h thng phn loi h lan l mt h ln th hai vi 20.000- 25.000 loi ch sau h cc (A.L. Takhtajan). Loi a lan thuc Ngnh: Magnoliophyta- Angiospermae Lp: Liliopsida- monocotyledemes B: Orchidales H: Orchidaceae Chi: Cymbidium. Theo cc tc gi V Vn Chi v mt s tc gi khc th trong t nhin c chi Cymbidium c khong hn 50 loi, v Vit Nam c 15 loi trong ch ring Lt c ti 12 loi. - Cymbidium aloifolium Swartz (Hay Cymbidium aimulans Rolfe; Cymbidum Pubesecens Lindl). - Cymbidium cyperifolium. - Cymbidium dayanum Reichb.F - Cymbidium devoniaum Paxt - Cymbidium eburneum Reichb - Cymbidium ensifolium Swartz (hay Cymbidium sudaicum Schltr) - Cymbidium erythrostylum Lindl (hay Cymbidium longifolium Don; Cymbidium Limodorum Angustiflorum Ham) - Cymbidium gagiteum Wall (hay Cymbidium gradnifolium Griff; Cymbidium hookerianum. R) - Cymbidum insigne Rolfe - Cymbidium Lancifolium Hook. F (hay Cymbidium gibsonii Paxt; Cymbidium javancum Hook. F.) - Cymbidium polanei Gagn. - Cymbidium munronianum King et Plan.

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 Lt ngoi 12 loi trn cn c nhiu bin chng khc, nhng hin nay vn cha xc nh r loi, th. T lu ng bo dn tc Lt dng t Tong Plng c ngha l lan s gi nhng loi lan ny. Nhng cho ti khi ngi Php v ngi Kinh n Lt th phong tro trng lan v pht trin lan Lt mi bt u ph bin v ngy cng pht trin. Cui nhng nm 50 ca th k ny, mt s ging lan lai di nhp vo Lt tho mn v nhu cu ging mi v th hiu ca mt s ngi chi lan giu c. Trong qu trnh nui trng lan lai, gii chi lan tch lu dn nhng kinh nghim trong vic xc nh cc yu t gi th, gin che, cch ti nc bn phn, ph hp vi iu kin kh hu Lt. Nhng cy lai pht trin tt v cho ra hoa u n hng nm. T phng php trng lan ngoi c hnh thnh v c ph bin nhng cng ch dng li trong phm vi hp ca nhng ngi trng lan tiu khin. Trong my nm gn nay, phong tro trng lan ca ngi dn Lt ang c chiu hng pht trin mnh cc loi a lan, c bit l cc loi lan nhp ni nh: Tm Ht, Miretta xanh, Xanh Chiu, Trng bt, Trng Bt, Hng Bt, Miss Kim, B Mai, Vng Ba Ru, Miretta xanh, V cc ging lan ny cho hoa rt p, mu sc sc s, hng thm v c bit l lu tn nn lm thu ht cc nh vn, cc ngh nhn chi hoa, h ch nui trng v nhn ging ngy cng nhiu. 2.5 Virus hi thc vt Virus thc vt c pht hin vo cui th k 19 v n u th k 20 c rt nhiu virus gy bnh cho thc vt ln lt c pht hin nh virus khm thuc l da chut, virus thoi ha khoai ty, virus hi c chua,Virus thc vt gy nhiu thit hi nh lm gim cht lng nng sn, gim nng sut v c khi l mt 100% nng sut. Trn th gii hng nm bnh virus gy thit hi khong 70 t USD (Nguyn Vn T, 2002). Ti nc ta, nhiu bnh virus c truyn t nm ny qua nm khc gy thit hi mt vi phn trm cho n 80- 90% nng sut cy trng, c bit virus gy thoi ha nghim trng cc cy trng nhn ging v tnh. Hin nay, cc cy ang trng cng nh cy m Lt u khng hon ton sch bnh, v virus l nguyn nhn gy bnh nghim trng nht cn tr s pht trin ca sn xut trng trt hin i. Vic nghin cu v phng bnh virus gip gim thit hi do bnh virus gy ra l rt cn thit, c bit l nhng nc c nn kinh t da vo nng nghip l chnh nh nc ta. Ivanopski l ngi u tin khm ph ra th gii virus khi nghin cu bnh trn cy thuc l vo nm 1892. Virus c pht hin u tin gy nhng vt khm trn l cy thuc l l Tobaco Mosaic Virus v y cng chnh l loi virus c ph cy ch ln nht hin nay. Cho n nay, vi s pht trin ca sinh hc v s tr gip ca trang thit b, phng tin hin i, con ngi pht hin c trn 4 ngn loi virus, trong c hn 100 loi l virus hi thc vt v c ti hn 25 loi virus gy hi trn cy lan. Nh vy, virus thc vt l nhng vi sinh vt c kch thc v cng nh b v c nhng c im sau y (PGS.TS V Triu Mn v PGS.TS L Lng T, 2002).

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Virus l nhng nucleoprotein rt nh b do phi quan st chng trn knh hin vi in t. Virus c cu to rt n gin: c hai phn chnh l li axit nucleic v v protein. Thng thng virus thc vt c vt cht di truyn l ARN, mt s t cn li c vt cht di truyn l ADN. Virus l vi sinh vt k sinh mc t bo. Virus thc vt c th nhim bnh cho mt hay nhiu loi cy, v mi loi cy c th nhim mt hay nhiu loi virus. Virus thc vt cng nh virus ng vt, sau khi xm nhim vo t bo cy ch chng bt u tc ng n s trao i cht ca t bo, virus s dng vt cht ca t bo to thnh v s virus con mi. Do vy c th thc vt b kit qu dn dn, thoi ha v suy tn c khi b cht. Khong thi gian t lc virus bt u xm nhim n khi cy thoi ha v cht ph thuc vo c im gy bnh ca cc loi khc nhau v ph thuc vo tng loi cy cng nh sc chng chu ca tng cy. 2.5.1 Thnh phn cu to Thng thng mi virus c to t hai thnh phn chnh l protein v axit nucleic, mt s virus c bit cn c c polyanin, lipit, hoc mt s men c hiu. Da vo nhng hiu bit v c im cu ta ca virus m cc nh khoa hc tm ra nhng cht ha hc c tc dng lm gim s nhn ln ca virus trong t bo cy ch. Protein ca virus thc vt c tc dng bo v, bm gi v c vai tr quan trng trong vic virus truyn bnh qua mi gii. Protein ca virus thc vt cng c to thnh t nhiu axit nh alanin, acginin, glixin, triptophan, valin,Cc virus (ARN hay ADN) s quyt nh bn cht protein ca chng. bn ngoi t bo cy ch c nhng im c bit hay nhng th th virus nhn bit m bm vo, t mi bm nucleic vo t bo. Do t kt qu phn tch v protein ca virus v bit c trnh t nhn bit th s tng hp c nhng cht ha hc c cu to ging cc th th gim tc lan truyn virus trong cy ch. Axit nucleic ca virus gi vai tr ch yu quyt nh tnh di truyn, kh nng truyn nhim v gy bnh. Phn ln cc virus thc vt c vt cht di truyn l ARN, mt s t l ADN. C ARN v ADN u l nhng i phn t cha hng trm ngn cc n v nh c gi l cc nucleotit. Mi nucleotit c cu to t mt gc photphat, baz nit v ng 5 carbon. C 2 nhm baz nit l purin v pirimidin vi 5 loi l uracil, adenin, guanin, xitozin v timin. Da trn cu to ca cc nucleotit m con ngi tng hp nn nhng dn xut ca chng hoc nhng ha cht m v mt ha hc tng t cc nucleotit ny. Cc ha cht ny s cnh tranh vi cc nucleotit tham gia to thnh cc axit nucleic, v do lm sai khc trt t nucleotit v lm gim tc ti sn xut ca virus. 2.5.2 V cch gi tn: tn virus thng c t theo qui tc sau Tn virus = Tn cy ch + c im bnh + Virus

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V d : Tobaco Mosaic Virus: y l virus gy hi trn cy thuc l, c im bnh l gy khm l. Odontoglossum Ringspott Virus: virus ny gy hi trn cy lan thuc chi Odontoglossum, l cy b bnh c cc vt bnh hnh m vng. Virus l mt loi k sinh phn t khng c cu to t bo, nhng cng khng sinh sn bng cch phn i nh vi khuNn, virus sinh sn bng cch k sinh vi t bo sng v bt t bo cung cp vt cht cn thit cho virus tng hp thnh protein v vt cht di truyn ca virus, sau cc thnh phn ny lp rp vi nhau to thnh virus con. Phng thc sinh sn ny ngi ta gi l sinh sn ti to (multiplication). Nh vy s sinh sn ca virus hon ton da vo vt cht ca t bo, lm cho t bo kit qu v dn n cht. Virus thc vt mun k sinh v gy bnh cho cy ch th virus phi bm c vo thnh t bo v xm nhim vo trong t bo. Bn thn virus khng ch ng di chuyn v xm nhp vo bn trong t bo c. S xm nhim ca virus thc vt vo cy ch c th thng qua cc vect truyn bnh nh cn trng, bt nhn, hoc qua cc vt thng c gii do con ngi gy ra trong qu trnh ct ta cy trng. Sau khi xm nhp c vo bn trong t bo, s ly lan ca virus t t bo ny sang t bo khc c th qua cu nguyn sinh gia cc t bo, hoc qua dng vn chuyn vt cht. Ngi ta cng pht hin ra rng trn t bo c mt s im nhn bit c bit m virus c kh nng bm vo t xm nhp vo t bo, gi l cc th th. Sau khi xm nhim vo t bo cy ch, virus s gii phng vt cht di truyn (RNA, DNA) qu trnh ny gi l s lt o (uncoating). Tip l qu trnh ti to vt cht di truyn v biu t gen. i vi virus c vt cht di truyn l ARN (ARN virus), qu trnh ti to ARN gm ti to ARN ca virus v mARN protein ca virus. ARN virus li dng ribosom ca cy ch phin dch ra l enzym ARN- ph thuc ARN (ARN- dependent ARN polymerasa, RdRp). Di tc dng ca RdRp t bo s tng hp ra hng lot ARN con ca virus v ARN t gen ca virus, lc ny ARN t gen s tin hnh phin dch ra protein v o. Cui cng ARN con v protein v o lp vo nhau to th virus con hon chnh. V s virus con c to thnh sn sinh ra enzym phn gii mng t bo k ch phng thch virus con ra ngoi, t tip tc xm nhim sang cc t bo bn cnh. i vi virus c vt cht di truyn l ADN, s ti to li vt cht di truyn c th xy ra theo con ng sau: u tin l t DNA s phin m ra RNA , sau xy ra qu trnh phin m ngc t RNA qua DNA v qu trnh tng hp protein t RNA. Cui cng l protein v DNA s kt hp vi nhau to thnh virus mi hon chnh. Nh vy virus s dng vt cht ca t bo sinh sn. Do virus cng gy ra nhng bin i v sinh l v hnh thi bn ngoi ca cy b bnh. 2.5.3 Virus gy bin i v sinh l cy ch. Virus thc vt k sinh bn trong t bo v s dng vt cht ca t bo cy xy dng c th virus mi do gy ra nhng tc ng ln qu trnh tng hp v trao i cht ca t bo cy ch. Virus c th km hm s tng hp, trao i cht ny trong khi li kch thch s tng hp v trao i cht khc.

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Virus tc ng ln qu trnh hot ng ca h thng men trong t bo km hm enzym phn ng to nn mt s cht ny hay kch thch to ra nhiu cht c li cho virus. Trong cy b bnh virus, hot ng mt s men oxi ha c tng cng nh oxidaza (Woods, 1899), peroxidaza (Kokin, 1937), tirodinaza, xitocromoxidaza (Martin, 1954), polyfenoloxida (Van Kammen v Drouwer, 1964), ngc li th hot ng ca mt s men khc b km hm nh catalaza (Vager, 1955). Virus gy bnh cho cy cng tc ng ln qu trnh h hp ca cy. Nhng thay i ny khng n nh (c th cao hay thp) ty thuc vo giai on sinh trng ca cy, iu kin thi tit hay tnh trng sinh l ca cy trng. Bnh virus thng lm gim s lng dip lc trong lc lp, do vy lm gim hiu qu quang hp ca cy. Cng vi s gim s lng dip lc th cc sc t nh antoxianit, carotinoit s tng ln, lm cho l cy nhim bnh virus chuyn mu t mu xanh sang mu vng hay nu . Bn cnh , virus thc vt cn lm thay i tc trao i gluxit trong cy. Ban u cy b bnh b phn hoa, l cy s tch ly cc cht ng v tr nhim bnh. Nhng v sau hm lng ng cc l b bnh s thp hn so vi cc l khe. Nguyn nhn l do virus ban u lm cn tr qu trnh vn chuyn cc b phn b bnh n cc b phn khc dn n vic tch ly ng cc b phn ny. Sau , virus s dng vt cht ca t bo, thc Ny qu trnh h hp nn hm lng ng b phn b bnh s thp hn cc b phn khc trong cy. 2.5.4 Virus gy bin i v hnh thi. Thng thng cy ch ch c biu hin bnh ra bn ngoi khi m b nhim bnh kh nng. Mt s triu chng v hnh thi do virus gy ra: Triu chng khm l: L triu chng rt ph bin ca nhiu bnh virus mi hi thc vt. L cy loang l ch xanh m, ch xanh nht, ch bin vng (Mosaic - khm l). Khm l thng xy ra cc bnh nh: Virus khm thuc l, khm t, da chut, khm l u, khm l to ty. m cht hnh nhn: Thng gp l khm v to ra m cht loi hnh nhn (m vng) nh bnh m hnh nhn cy u , cy mn, cy thuc l, cy hoa cNm chng. Triu chng hi gn l: L hin tng bnh ph hoi gn l dn n gn sng, gn cht, gn bin dng nh virus Y hi cy thuc l to gn sng, virus Y hi mt s ging khoai ty (to gn bin dng). Khm l, ln cy: L hin tng khm l km theo cy ln nh Maize mosaic dwarf virus hay khm sc l (Maize strerk trip mosaic virus) ng, la, v cc cy n t dip. Xon, cun l: Bin dng l nh xon l c chua, cun l khoai ty, xon l cy t, h tiu. Bin mu v bin vng: L nhng triu chng gy bin vng ( la), gy ho xanh ( cam, chanh).

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Ln bi, tn li: L hin tng kh ph bin nh ln bi cy lc, cy chui rt, cy cam b bnh tritera virus. Vt cht thn cy, vt lm thn nh hin tng sng cnh to, vt lm thn cy cam, chanh. Bin dng c, qu: Nh to, mn, nho b nhim virus, qu c chua b m ho, c khoai ty cy b bnh vng ln.

Cc triu chng bnh trn cn ph thuc vo ging cy k ch, iu kin mi trng v chng loi virus gy bnh m c s bin i. Cng mt loi virus m ba nhm chng khc nhau c th th hin thnh nhiu nhm triu chng khc nhau. Bn cnh cc triu chng bnh ny khng mang tnh c th, v d nh virus bnh m vng th vt bnh cng tng t nh bnh m do mt s loi nm gy nn. Do bnh virus khng ch kh pht hin khi mi chm b, m khi b nng v c biu hin bnh ra bn ngoi th cng rt d nhm vi bnh do bnh sinh l, bnh nm, hay mt s tc nhn khc gy ra. Tm li virus gy bnh hi thc vt c mt s c im sau: Cy b bnh virus hin nay th khng th cha c m ch c th lm c ch s sinh sn ca virus trong cy. Bnh virus gy thit hi ln cho ngi trng. i vi nhng cy ngn ngy virus gy thit hi nhanh chng. V d nc ta hin nay, bnh vng ln, ln xon l cy la do virus gy ra v lan truyn nh ry nu ang bng pht, gy hu qu nng n cho b con vng ng bng Sng Cu Long. Nhng thit hi nng n hn c l virus khng lm cho cy trng b cht ngay khi cn nh m lm thoi ho, gim sc sng, dn dn dn n tn li sau khi ngi trng u t rt nhiu vo vic chm sc v nui dng cy. V d Lt, c nhiu gia nh trng a lan mt 70% n 100% chu a lan ang trong giai on c hoa. Bnh virus tn ti lu, kh pht hin v triu chng bn ngoi khng c th d gy nhm ln. Vic chuNn on bnh th phc tp, i hi k thut cao. Biu hin bn ngoi ca cy b bnh virus c chu k bin i theo chu k pht trin ca virus, theo tc ng ca yu t mi trng, c im sinh l ca cy ch. Do nhng biu hin bnh bn ngoi ca cy cng thay i gy nhm ln cho ngi trng. V n khi cy ch khng cn kh nng phc hi theo chu k bnh th cy s hon ton tn li. V d bnh virus hi a lan ti Lt, trong iu kin ma kh, nhit cao din bin bnh virus chm chp, nhng vo ma ma li bng pht. 2.6 Virus hi a lan Nhng triu trng bnh virus u tin c miu t vo nm 1943 trc khi Ivanopski pht hin ra s tn ti ca th gii virus. Virus thc vt gy hi trn rt nhiu loi cy trng, c th thy trong t nhin a s cy u b nhim virus. nc ta m c bit lt, trong nhng nm gn y cng vi s gia tng v nhu cu v qui m trng lan th bnh virus hi lan cng bng pht v gy ra nhiu thit hi nng n v kinh t cho ngi trng lan. Theo c tnh th n nay c khong 25 loi virus hi lan. Nhng triu chng biu hin bnh virus gy hi trn lan rt a dng v khng theo qui tc no c. a s virus thc vt gy vng, cht m hay hoi m (thi nhn) v biu hin ban u ca

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virus nhiu khi ging vi bnh sinh l. V d: Bnh trn a lan do Cymbidium Mosaie Virus gy ra, ban u ging vi biu hin khi bn qu nhiu ur cho cy. Trong h lan th a lan l cy c th ca x lnh v c trng kh lu lt v c gi tr kinh t cao. Ngy nay, di s pht trin ca k thut nui cy m, vic m rng qui m sn xut lan ang gp nhiu thun li, bn cnh th vic ly lan bnh virus cng ang l nguy c s mt cho ngi trng lan nu cy lan khng sch bnh virus. Trong s nhng loi virus hi a lan hin nay th ti Lt c mt s loi gy hi ch yu: Cymbidium Mosaic Virus (CyMV), Tobaco Mosaic Virus (TMV), Tomato Spotted Wilt Virus (TSWV), Cybidium Ringspot Virus (CyRV), Odotoglosum (ORSV)... 2.6.1 c im v hnh thi ca bnh do virus Cymbidium Mosaic Virus trn cy a lan Trn l cy nhim bnh ban u c nhiu chm en nh, sau pht trin thnh vt mu en. Nhng vt bnh ln lin kt vi nhau to thnh nhng mng en c mt trc v mt sau. Khi bnh nng th nhng chm en ban u pht trin trn c phn b l, ton b l kh i nhanh chng v gy ra hin tng kh xm gi hnh. Trn nhng cy lan nhiu nm tui, trong cng mt chu th tt c gi hnh, chi non v pht hoa u b bnh. CyMV ngoi tc ng lm kh l th cn gy thi theo m t gn l ra ngoi mp l. 2.6.2 c im v hnh thi ca bnh do virus Tobaco Mosaic Virus orchid trn cy a lan TMV orchid theo dng vn chuyn vt cht ly lan khp h thng cy trng v gy ra hin tng khm l, ln cy, cy ci cc. Vt bnh thng xut hin phn l, ban u trn l non xut hin mt vi m vng xanh, sau cc m xut hin ngy cng nhiu v gy hoi l. Vi cc l b nhim nh, phin l nh hp v mt l sn si. c im ca vt bnh l cc t bo biu b trn phn sng ca vt bnh nh hp, xp st nhau, phn xanh lam ca vt bnh t bo biu b ln hn bnh thng. 2.6.3 c im v hnh thi ca bnh do virus Tomato Spotted Wilt Virus trn cy a lan Cy nhim virus TSWV c biu hin bnh r rt phn hoa, trn c n non v hoa n, nhng ch yu l hi phn n hoa. Cc vt bnh thng to v c mu en.

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2.6.4 c im v hnh thi ca bnh do virus Cybidium Ringspot Virus (CyRV) trn cy a lan Phn l trn cy b bnh do virus CyRV thng c nhiu sc vng nht phn bit r vi cng xanh ca phin l. Cy b bnh nng th cc vt bnh lin kt li vi nhau lm cho cy b vng, cc l xp st vi nhau v dng thng ng. Bnh thng xut hin cy con 6 thng tui vi b l dy c mu vng nht. Trn cy ln th bnh khin cho pht hoa thp ngn, tuy c n hoa nhng li khng n c. 2.7 Mt s bin php c p dng c cy sch bnh virus Ngy nay, vic to ra cc cy ging sch bnh cung cp cho ngi trng l v cng quan trng v cn thit. Hin nay ch c hai phng php c s dng c cy trng sch bnh l cy gieo t ht khng b tn thng v phng php phc trng ging bng cch nui cy nh sinh trng. 2.7.1 V phng php gieo ht Phng php nhn ging bng ht hin nay l phng php nhn ging thng c s dng cho lan. y l phng php to ra c s lng ln cc cy con v ch yu p dng vi nhng cy m thi gian to cy con bng phng php nhn ging v tnh l rt lu v kh ny mm trong iu kin t nhin. Bn cnh nng cao t l ht ny mm cho nhng cy c th ny mm trong iu kin t nhin nh lan, ngi ta gieo ht trong iu kin phng th nghim. Vn quan trng nht trong phng php ny l tm ra mi trng gieo ht ti u rt ngn thi gian v tng t l ht ny mm. 2.7.2 V phng php nui cy nh sinh trng y l mt trong nhng phng thc nui cy m trong phng th nghim (nhn ging in- vitro). Nhn ging in- vitro hay nui cy m u l thut ng m t cc phng thc nui cy cc b phn thc vt trong ng nghim c cha mi trng xc nh iu kin v trng. Mi trng c cha cc cht dinh dng thch hp nh mui khong, cc hormone tng trng, carbon, v mt s cht b tr khc. K thut nui cy m cho php ti sinh chi hoc c quan (s pht sinh c quan) t cc m nh l, thn hoc r. 2.7.3 Mt s bin php b tr khc 2.7.3.1 Phng php nhit Thng thng, c ch s ti bn ca virus th cc mu thc vt c x l vi nhit 30 37 oC trong thi gian t 10 - 14 ngy hay 40 55 oC trong 5 - 10 pht (Nguyn Vn T, 2002). Tu vo loi mu cy, kch thc mu cy m c thi gian v nhit x l thch hp. Sau y l mt s th nghim tin hnh ca nhiu tc gi vi mc ch c ch s nhn bn ca virus bng nhit l: c ch virus PVY - Potato Virus Y trn cy khoai ty, nm 1956 Thomson, nm 1998 Leonhardt v cng s, tin hnh x l mu 35 38 oC trong 30 - 40 ngy.

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Walkey v Freeman x l mu vi nhit 40 oC trong 16h c chiu sng v 22 oC trong 8 h ti, trn cy Nicotiana Rustica, c ch virus CMV - Cucumber Mosaic Virus. 2.7.3.2 Phng php ho hc Mt trong nhng phng hng khc trong vic c ch s ti bn virus trong t bo l s dng ho cht m l nhng dn xut halogen tin thn ca s trao i cht axit nucleic. Trong giai u ca qu trnh tm kim, cc nh khoa hc tm thy mt s hot cht nh: Iot deoxyuridin, 2 - thiouracil, y l nhng cht ng dng vi purin, pirimidin. Nhng cht ny c tc dng lm ri lon s tng hp bnh thng ca cc axit nucleic ca virus. Bn cnh nhng hot cht c tm thy trong thi gian ny tuy c tc dng c ch tc ng ca virus nhng ng thi cng gy c cho t bo. Hp cht c s dng sm nht trong l 2 thiouracil, c tm ra vo nm 1952 do hai nh khoa hc l Commoner v Mercer. T lu ngi ta bit a s virus c vt cht di truyn cha uracil l mt phn ca acid nucleic. Do Commoner v Mercer c ch s ti bn ca virus trong cy thuc l bng cch s dng 2 thiouracil. Vo nm 1953, Jeener v Rosseels nh vic s dng ng v ca sulfua (S35) ca 2 - thiouraci v chng minh c rng S35 tham gia vo acid nucleic ca virus. Sau hng lot nhng cht ho hc khc thuc nhm ny c nghin cu. Mt s cht cht khc nh 5- Fluorouracil (Heidelberger v cng s, 1957), 5 Chlorouracil, Azaguanin (Matthews, 1953), 8 - Azaguanin l mt hp cht khng c trong t nhin v c tng hp do Matthews vo nm 1953. Matthews s dng 8 - Azaguanin trong th nghim c ch s nhn bn ca virus CMV (Cucumber Mosaic Virus) v virus TMV (Tobaco MosaicVirus). V thc t l vic s dng 2 - thiouracil v mt s cht ho hc khc thi k ny u khng hiu qu (Tam, 1959). Nhng cht ny khng ch c ch s ti bn ca virus m ng thi c ch s sinh trng v thm ch gy cht i vi t bo. Nhng d sao, 2 - thiouracil khi s dng nng ph hp cng thu c mt s thnh cng trong vic c ch c s ti bn ca virus (Holmes,1955). Ngy nay, ribazole l mt ha cht do con ngi tng hp c s dng rng ri trong c ch virus ng vt v c thc vt m t gy nh hng xu ln t bo vt ch nht. Ribazole l hp cht thuc nhm khng chuyn ho nucleotide ca virus do c tc dng c ch s nhn bn ca virus trong t bo. Ribazole c cng thc ho hc l C18H12N4O5 v cu to ho hc nh sau:

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Mt s ch phNm c cha ribazole l: Copegus, Rebetol, Ribasphere, Vilona, v Virazole. Ribazole l cht ho hc c tng hp nhn to, hin cha tm thy trong t nhin, v ln u tin c tng hp vo nm 1970 do nh ho hc Joseph T. Witkowski (Lau, 2002). Vo nm 1972 Sidwell chng minh rng ribazole c th c ch hot ng ca mt s loi virus ng vt m t gy c cho t bo. T cho n nay, ribazole c chng minh l khng ch c ch hot ng ca rt nhiu loi virus ng vt m cn c tc dng vi c virus thc vt. Theo Simpkins v cng s (1981), theo Vicente De Fazio (1987), Chen v Sherwood (1991) v gn y l Fletcher cng cng s (1998) cho thy ribazole c tc dng c ch virus thc vt nhn bn. Vic ng dng ribazole trong nui cy in- vitro c ch hot ng ca virus thc vt trong vic lm sch bnh virus c bt u t nhng nm 1980 v c p dng cho mt s i tng thc vt. a s virus thc vt c vt cht di truyn l ARN, mt s t cn li c vt cht di truyn l ADN. C ADN v ARN u c cu to t cc axit nucleotic. Mi nucleotid u gm c 3 phn l nhm photphat, phn t ng 5 carbon v Baz nit. C 5 loi baz nit l adenin, guanin, xitozin, timin, uraxin. Trong ARN c 4 loi l adenin, uraxin, guanin, xitozin. Cn ADN c 4 loi l adenin, timin, guanin, xitozin. Ribazole c cu to v tnh cht ho hc tng t c adenosin ln guanosin. Bn cnh ribazole c th hot ng c lp v tun hon v l nguyn nhn m ribazole tham gia ng ho vo ARN v ADN ca virus. T ribazole c mt s tc ng chnh sau: Ribazole c th ghp i vi xitozine v uridine, t gy ri lon bin i s tng hp RNA ca virus. Thm vo , dn xut ca ribazole l Ribazole 5 monophotphat, Ribazole 5 diphotphat, Ribazole 5 triphotphat c tc dng c ch hot ng ca RNA polymerase ph thuc vo ARN ca virus. Cui cng ribazole c th cnh tranh vi Guanosin triphotphat, c tc dng c ch hot ng gen ca virus thc vt. Ribazole c p dng vi rt nhiu cy trng nh khoai ty, thuc l, v mi loi cy trng thch hp vi nhng nng ribazole khc nhau.

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V d nh i vi cy Allium Sativum L. th nng ti thch ca ribazole trong cc th nghim ny l 205 m (Hector Loszoya v Saldana; William. O Dowson; T Murashige, 2006) i vi cy Oxalic Tuberosa Molina, Ullucus Tuberosus Caldas, Arraccia Xanthorrhiza, th nng ribazole ti u l 50 mg/l mi trng nui cy (Fletcher, 2001). i vi cy khoai ty, nng ribazole c s dng trong khong 20- 30 mg/l mi trng MS c b sung kinetin, NAA v GA3. Khi s dng ribazole trn cy chui th nng ribazole l 50 mg/ l nhng khi s dng cho Nicotiniana Rustica th nng ribazole li cao hn l 100 mg/ l mi trng nui cy. 3. S lc v lch s nghin cu, c im chung v phn loi v h cc 3.1. Tnh hnh sn xut hoa cc 3.1.1. Trn th gii Trong ngnh sn xut hoa ton cu, hoa cc l loi quan trng th hai sau hoa hng. Cc cn c xem nh l mt trong nhng loi hoa trang tr c a chung nht trn ton th gii. Nht Bn, hoa cc l quc hoa t nm 910 (L.Naeve, v D. Nelson, 2005), nn ngnh sn xut hoa cc mang v li nhun tng hn hai ln, ch trong vi thp nin gn y, nh p dng cc tin b khoa hc k thut, cng nh do nhu cu thng thc cuc sng ca ngi dn tng nhanh. Hoa cc Nht Bn chim n 35% tng sn phNm hoa ct cnh trong nc (Boase v cs, 1997). Trn th gii, hng nm, Nht Bn cng l quc gia sn xut hoa cc nhiu nht, vi 2 t cnh, tip theo l H Lan vi 800 triu; Colombia 600 triu; Italia: 500 triu; Hoa K:300 triu (Boase v cs, 1997). nc Anh, hoa cc l loi hoa ct cnh quan trng ng th hai trn th trng (Flowers and Plants Association, 2001). Ti Nht Bn, hoa cc ct cnh c s dng kh ph bin: 40% c dng lm qu tng; 25% c dng trang tr ti cc khch sn hay trong cc l hi; 25% c dng trang tr trong gia nh v cng theo o Pht; v 10% phc v trong vic ging dy nghin cu (Jaime, 2003). Ci cc (C.coronarium L.) c trng rt ph bin Nht Bn, Trung Quc v vng ng Nam , nh mt loi rau n hng ngy kh ph bin (Oka v cs., 1999). Mt s loi cc khc c cha mt hm lng tinh du ng k, nn mt s loi c trng khai thc tinh du (Schwinn v cs., 1994). 3.1.2 Ti Vit Nam nc ta, c nhiu loi cy di thuc h cc mc nhiu ni. Mt s loi c dng lm thuc trong y hc c truyn, v d nh b cng anh (Lactuca laciniata L.). Hoa cc lm cnh c a vo trng nc ta vo khong t th k XV n u th k XVI. Lc ch yu dng lm cnh. Mi n sau ny, cc mi c trng nh mt loi cy thng mi. nc ta, H Ni l ni c din tch trng hoa cc nhiu nht, vi din tch 450 ha, sau l Thnh Ph H Ch Minh (370 ha). Lt l vng

24

c din tch trng hoa cc ln th ba, vi din tch 160 ha ((N.Q. Trch v .V.ng, 2002). 3.1.3 Ti Lm ng Hu ht din tch trng hoa cc Lm ng tp trung thnh ph Lt, cc huyn n Dng, c Trng, Lc Dngdin tch khng ng k. Mc d c trng t kh lu, nhng hoa cc tr thnh mt sn phNm thng mi mi t nm 1995. Din tch trng hoa cc Lt tng nhanh trong nhng nm gn y. Din tch trng hoa cc chim n 40-50% din tch trng hoa ni chung. Trong vic trng hoa cc, b con nng dn p dng nhiu bin php k thut, nh dng h thng n chiu sng, h thng ti phun sng, nh knh a s hoa cc Lt c trng vi mc ch ct cnh. Mi nm, Lt cung cp cho th trng t 10-15 triu cnh. Ti Lt, hin c khong 70 ging, c du nhp ch yu H Lan, theo nhiu con ng khc nhau: chnh thc v khng chnh thc. Do , vic xc nh tn thng phNm v chng loi cho tng chng loi cc l rt kh khn. 3.2 c im sinh hc h cc (Asteraceae hay Compositae) : 3.2.1 Ngun gc v v tr phn loi Theo V Vn Chi v Dng c Tin, 1978, h cc thuc: -Gii thc vt -Ngnh Magnoliophyta (Angiospermae) - Ngc Lan (ht kn) -Lp Magnoliopsida (Dicotyledonea) - Ngc Lan (hai l mm) -B Asterales (cc). B cc c mt h duy nht l h cc (Asteraceae hay Compositae) c xem l h ln nht ca ngnh ht kn v gii thc vt ni chung. Bao gm gn 1000 chi v hn 20 000 loi, c nhng chi c ti 1000 loi. H cc phn b trn khp ni trn Tri t, sng c trong nhiu iu kin kh hu, mi trng, t ai khc nhau. Dng sng ch yu l thn tho, cy bi, him khi thn g, nhng thn g thp b. (V Vn Chi v Dng c Tin, 1978; H.T.Sn v P.N.Hng, 1986). Trong h cc c chia thnh hai phn h: (Asteroideae=Tubiliflorae) v tha la (Cichorioideae=Liguliflorae). hoa ng

Phn h hoa ng c c im l tt c hoa trong cm hoa u tin hnh ng (ng phu, ng si, ng hnh chung), hoc hoa hai mi, hoc hoa c hoa hnh ng gia v hoa tha la gi xung quanh. Trong phn h hoa ng c gn 740 chi, v rt nhiu loi vi cc cy quen thuc nh l: ngi cu (Artemisia vulgaris L. var.indica (Willd.) DC.), thanh hao (Artemisia carvifloria Wall.), nh ni (Eclipta prostrata L.) thc dc (Dahila pinnata Cav.) Trong , chi Chrysanthemum c trng ph bin nh mt loi hoa trng chu hay hoa ct cnh. Hoa cc l mt loi hoa ct cnh ph bin trn ton th gii, n a dng v cc th v c hng ngn kiu dng khc nhau (Cockshull, 1985). Ngoi ra mt s loi khc trong chi ny cn c dng lm rau n, lm thuc an thn, thuc

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cha bnh nh l: ci cc (C.coronarium L.), bch cc (C. moriforium Ramat), kim cc (C. indicum L.). Phn h tha la c c im l tt c hoa trong cm u tin l hoa tha la. Phn h ny c t loi hn. Mt s loi rt quen thuc nh l rau dip, rau x lch (Lactuca sativa L.), rau dip xon (Cichorium endivia L.), b cng anh (Lactuca laciniata L.) (V Vn Chi v Dng c Tin, 1978). 3.2.2. c im thc vt hc Theo V Vn Chi v Dng c Tin, 1978; H.T.Sn v P.N.Hng, 1986; N.Q. Trch v .V.ng, 2002 th cc c cc c im sau: R - Cc c h r chm, mc cn, theo chiu ngang, m su khong 10-20 cm. r cc c kch thc kh u nhau, vi s lng r ln, nn kh nng ht nc v cht dinh dng rt mnh. Do cc c nhn ging bng phng php v tnh, nn r mc ngang t cc mu thn gn mt t. Thn - Thn hoa cc l thn tho nh, nhiu t, mng nc, gin, d gy. Trn thn non mt s loi c ph mt lp lng t. Mt s loi c dng thn b. Chiu cao thn tu loi. Nhng a s cc ging nhp c thn to, thng, gin. Cn cc ging ni a c thn nh, mnh, cong. L - L cc thuc loi l n, khng c l km, mc so le. Bn l c x thu hnh lng chim. Phin l mng, mt di c ph mt lp lng t, mt trn nhn. Gn l hnh mng. Mi cy c khong 30 50 l. Hoa, qu - Hoa cc v c bn l hoa lng tnh. Hoa cc c nhiu mu sc khc nhau, thch nghi vi th phn nh su b. Hoa nh, st nhau v lun lun tp hp thnh cm hoa u, mt su b th phn c cho nhiu hoa cng mt lc. hoa li ln. Hoa gia l hoa hnh ng, hoa ngoi l hoa tha la gi. cc, qu l qu b. Ch c mt ht mm nm trong khoang ca qu v i khi dnh vi v qu. V ht rt mng, phi ln v thng khng c ni nh. Qu pht tn nh gi v ng vt.

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Hnh 1.1: Cc dng hoa cc (Ngun:RHS A-Z Encyclopedia of Garden Plants (c) DK Images) 3.2.3. c im sinh trng v pht trin Kh hu - a s hoa cc thch hp vi iu kin kh hu n ho, mt m, lng ma y , nht l nhng hoa c nhp t vng n i. Vi nhit trn 250C, cc sinh trng v pht trin km (L.Naeve, v D. Nelson, 2005). t ai - Cc t i hi v iu kin t ai. Thch hp vi t acid nh, pH khong 6.0 - 6.5. Nhn chung, cc s pht trin tt trn t c Nm tt, thong kh, giu cht hu c, c bit l c phn chung. Trc khi trng, cn lm t tt, cy su 20-40 cm m bo thong kh v bn lt y phn hu c (M.N. Dana v B. R. Lerner, 1996; M.Top v B. Tatura, 2002; L.Naeve, v D. Nelson, 2005). Phn bn - Cc l cy i hi mt lng phn bn kh ln, v vy, cn bn 2 ln NPK, vi t l 10:10:10. Mi ln bn khong 25 kg/ 100m2. Tt hn l bn lt trong giai on chuNn b t mt lng phn hu c (M.N. Dana v B.R.Lerner, 1996; M.Top v B. Tatura, 2002). Cn ch khng bn phn cy bc vo giai on ra hoa (L.Naeve, v D. Nelson, 2005). Nc ti - Cc ch c th sng st trong thi gian kh hn ngn, nu kh hn ko di s nh hng n sinh trng pht trin ca cy. V vy, ti nc rt quan trng i vi cc, nht l trong ma kh (L.Naeve, v D. Nelson, 2005). Cn ti mt cch u n v k lng, m bo nc phi thm su xung t t nht 12-15cm. Cn ch trnh lm tn thng cy do nhng tia nc mnh qu. Nhng tn thng ny l nguyn nhn cc virus xm nhp, cc vi khuNn v nm pht trin (M.N. Dana v B. R. Lerner, 1996). Su bnh hi - Mt s cn trng gy hi trn cc thng xuyn l rp mui, su v ba, nhnCc bnh thng gp l: m l, thi thn r, mc sng(M.N. Dana v B. R. Lerner, 1996; L.Naeve, v D. Nelson, 2005). Trong trng hp su hi khng nhiu lm th khng cn thit phi s dng thuc ho hc. Nn p dng bin php phng nga tng hp, bng thin ch, bng by cn trng (M.N. Dana v B. R. Lerner, 1996)

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4. Virus v phng php ELISA Virus rt nh b, ch c th quan st c di knh hin vi in t. Chng cha c cu trc t bo nn bt buc phi k sinh ni bo trn c th mt sinh vt khc (vi khuNn, nm, ng vt, thc vt). Chng ch c cu to bi mt v protein bao bc bn ngoi mt li acid nucleic. Chng c y cc hot ng sng nh di truyn, bin d, tng trng, sinh sn truyn nhim mc d cc hot ng ny u phi gn lin vi t bo vt ch. Virus thc vt c pht hin cch y trn mt th k v thu ht c s quan tm ca nhiu nh sinh hc. Ngnh nng nghip ht sc lng tng trc cc bnh nghim trng do virus gy nn v cho n nay vn cha c thuc phng tr c hiu (bnh khm thuc l, bnh xon l c chua, bnh vng ln, ln xon l la). Cy b bnh virus c th tn li, cht. Mt s b phn ca cy nh thn, hoa, c, qu, l b hy hoi, cy pht trin km dn ti nng sut gim, cht lng nng sn v dinh dng, cm quan km. Mt s trng hp bnh hi cy c th sinh ra nhng c t gy nh hng xu, trc tip n sc kho v i sng ca con ngi v ng vt. Do tc hi ca virus v cng to ln nn cn c nhng bin php chuNn on v pht hin sm c bin php s l cho thch hp. Hin nay c nhiu phng php chuNn on bnh virus thc vt nh phng php huyt thanh (trong phng php DAS-ELISA l phng php huyt thanh hin i nht), phng php cy ch th, phng php chuNn on bng hin vi in t, chuNn on phn t (RT-PCR). Phng php ELISA ti nay vn l phng php pht hin virus hiu qu, nhanh chng c s dng rng ri. thc hin c phng php ny cn phi tch c protein ca virus thc vt, bng cch nghin mu thc vt c cha virus trong dung dch m nhm gi pH khng i dch chit v thc hin quy trnh lm tinh khit virus. ELISA l k thut c s dng ph bin nht xc nh khng th c hiu chng virus thi im xut hin triu chng hoc ngay sau khi xut hin triu chng. K thut cho php xc nh khng th nng rt thp (khong 0,1 g/ ml). Nguyn l phng php ny l khng th c nh du bng cch gn vi enzym. Khi c cht thch hp enzym s phn gii, c cht cho sn phNm c mu. o cng mu bit mc phn ng xy ra. K thut ELISA c dng nhiu l k thut bnh kp(sandwich), theo khng nguyn ca cy bnh (virus) c phn ng vi khng th s cp bit, c xc nh khi cho thm khng th th cp c gn enzym. Cc bc thc hin nh sau: - C nh khng th s cp ( c bit) ln thnh ging - Np dch virus (khng nguyn) vo ging ri . Nu khng nguyn ph hp s to phn ng vi khng th s cp. Ra nh loi b khng nguyn tha. - Thm khng th th cp c gn enzym. Ra nh loi b khng th tha.

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- Thm c cht thch hp vi enzym. Cng mu t l thun vi lng khng nguyn trong dch chit virus. 4.1. Bnh khm thuc l TMV (tobacco mosaic virus) TMV l bnh virus ph bin cc vng trng thuc l trn th gii. Vit Nam bnh gy hi nng trn vng trng thuc l cc tnh ng bng v trung du Bc b. Cy b nhim bnh c th gim nng sut 35%, cht lng thuc l cng gim mnh do hm lng nicotine gim, l d b gy nt. Virus c kh nng truyn bnh cao. Bnh truyn ch yu qua con ng tip xc c hc, cc vt thng do xy xt. Virus vn chuyn trong cy theo h thng mch dn. Bnh Khm thuc l c kh nng gy hi cho nhiu cy khc ngoi thuc l nh cy t, c chua, a lan, hoa cc... 4.2. Bnh khm thuc l trn a lan Cy bnh nhim h thng, gy hin tng khm ln cy, cy ci cc, xut hin cc vt cht hoi phn l, trn l non xut hin cc l xanh, vng phin l nh hp, ,mt l sm si. Cc t bo biu b trn phn sng ca vt bnh nh hp, xp st nhau, phn xanh lam ca vt bnh t bo biu b ln hn. 4.3. Bnh khm thuc l trn cc Trn l non xut hin cc l xanh, vng xen k nhau, gn l nht nht, l n hp, cy ch bng cy khe, cy mt triu chng khi nhit gim xung 110C. Cy nhim bnh h thng. Gy bnh cho Cc, Lan bao gm nhiu yu t v nhiu nhm vi sinh vt khc nhau. Trong bo co ny chng ti ch tin hnh test v nh gi tc hi ca virus TMV. Bi v, TMV l mt virus ph rng, ng thi khng phi l virus gy hi chnh cho cc i tng trn. Do nu cc i tng trn nhim TMV th xc sut nhim cc bnh virus khc rt cao.

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Chng 2 I TNG V PHNG PHP NGHIN CU

2.1 i tng nghin cu: 2.2.1 Vt liu ban u c th ca ti l nghin cu bo tn ngun gen cc ging cc v hoa lan ct cnh hin ang sn xut ti Lt l s a dng v ging, ring ging cc nhp vo lt c hn 70 ging. Chnh v vy, trong phng php thc hin nghin cu chng ti ch nu nhng phng php chung v trn mt s ging lm chuNn, cc ging cn li u tun th theo cc bc sau y: a. Thu thp mu b. Tch v nui cy m phn sinh. c. Nhn nhanh protocorm (i vi a lan), nhanh nhanh cy cc in vitro d. Chuyn protocorm v cy con sang mi trng x l virus e. Tch li m phn sinh f. Nhn nhanh v test virus 2.2 Phng php nghin cu A. Phng php p dng trn i tng lan Cymbidium Cc th nghim tin hnh s dng protocorm ca ba ging a lan sn c trong phng th nghim. Ba ging l Trng bch, Tm ht, v Miretta xanh. C ba gjng ny u cho hoa rt p v cc ging m c trng rng ri Lt. 1. Mi trng nui cy Trong cc th nghim thc hin vi mi trng khong Knudson C, MS v 1/2MS c b sung thm cc cht khc l: Vitamin: cc vitamin l myo - inositol, acid nicotinic, pyridoxine HCl, Thiamin HCl. y l cc vitamin c s dng trong mi trng MS. Peptone c b sung vi hm lng 2g/l. Agar c b sung 8g/ l. Cht iu ha sinh trng b sung l BA: 0.5 mg/ l. ng saccharose vi hm lng 30g/l. 2. Phng php b tr th nghim 2.1. Nghin cu mi trng dinh dng thch hp v s dng ribazole h tr vic loi b virus kt hp vi vic tch m phn sinh nh

Cc protocorme hnh thnh sau khi qua bc nui cy nh dinh trng, chng ti tip tc b tr nui cy trong cc mi trng c hm lng khong MS, 1/2MS v mi trng Knudson C. Cc th nghim v tc ng ca ca than hot tnh, cytokinin (chn BA), cytokinin/auxin (chn BA v NAA) u s dng mi trng khong c bn l MS. Cch b tr th nghim nh sau: - Th nghim nh hng ca BA: Chng ti b tr 5 nng khc nhau: 0, 0.5, 1.0, 1.5 v 2 mg/l. - Th nghim nh hng phi hp gia BA v NAA: ny c thc hin bi cc protocorme nui cy trong mi trng MS, c than hot tnh l 1g/l, nng BA l 1ml/l v c b sung thm cc nng NAA t 0 2mg/l. Chn mi trng thch hp t cc th nghim trn tip tc nghin cu tc ng ca ribazole ln sinh trng ca cy con. Chng ti s dng ribazole trong cc th nghim vi cc nng 0 mg/l, 50 mg/l, 75 mg/l, 100 mg/l v 150 mg/l. Dng ribazole s dng l ch phNm ca Getz Pharma. Cc protocorm ti sinh trn mi trng x l ribazole, tip tc c cy chuyn theo di kh nng phc hi v test virus. Cy sch bnh tip tc c nhn ln gi ging cng nh chuyn ra kho nghim vn m. Cc mu th nghim trn c nui trong iu kin phng nui in- vitro vi: Thi gian chiu sng: 10h/ ngy. Cng chiu sng: 45 micro mol m-2 s-1 Nm trung bnh: 75- 80 % Nhit : 25- 27oC Ch tiu theo di: Cc ch tiu c theo di l s mu cy sng st, s lng protocorm mi pht sinh sau 60 ngy nui cy. Bn cnh mt s ch tiu khc thng xuyn c theo di l thi gian bt u pht sinh protocorm mi, mu sc mu cy. Sau giai on x l, theo di cc ch tiu sinh trng ca cy con (chiu cao, s l, s r, trng lng ti sau ba thng cy chuyn) 2.2. Nghin cu gi th thch hp cho cy lan con khi ra vn m Cy con sau khi rt khi mi trng in vitro cn ra sch agar bm vo r, nhng r cy con vi Mancozeb ri mi trng vo gi th. Khay trng: Khay kch thc 30 45 5cm Vi th nghim gi th: chng ti b tr mt s nghim thc sau: K hiu loi gi th I II 3T Loi gi th Dn mt

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III IV V VI VII VIII

3T/dn xay (1:1) X da Dn xay/X da (3/1) Dn xay/X da (2/1) Dn xay/X da (1/1) Dn xay

S lng cy con/ khay: 100 cy Nm gi th: 70 80% Nm khng kh: 90 95% Nhit nui trng: 20-25oC nh sng: che sng > 70% S ln ti/ngy: 3-5 ln Bn phn: N-P-K t l 3:1:1 pha long c EC 1.0 ds/m phun mi tun 2 ln (tr tun u tin sau khi mi ra cy) 2.3. Nghin cu ch phn bn cho cy con giai on ra vn m Cy con sau khi rt khi mi trng in vitro cn ra sch agar bm vo r, nhng r cy con vi Mancozeb ri mi trng vo gi th. Gi th c chn ra t nhng kt qu nghin cu gi th b tr th nghim. Khay trng: Khay kch thc 30 45 5cm S lng cy con/ khay: 100 cy Nm gi th: 70 80% Nm khng kh: 90 95% Nhit nui trng: 20-25oC nh sng: che sng > 70% S ln ti/ngy: 3-5 ln Bn phn: N-P-K t l 3:1:1 pha long c EC thay i t 0; 0.5; 1.0 v 1.5ds/m phun mi tun 2 ln (tr tun u tin sau khi mi ra cy) B. Phng php p dng trn i tng hoa cc Vi cc ging cc chng ti chn cc ging hoa cc c trng kh ph bin Lt farm hng, pingpong vng, nt vng, nt tm, tia mung vng. Cc ging thu thp cn li u theo mt qui trnh mu tng t cc ging nu ra y. Trong ti chng ti s dng vt liu ban u l cc hoa, c ly t cc vn sn xut ca b con nng dn trong a bn thnh ph Lt. Quy trnh x l mu nh sau:

32

Chn hoa t cy trng trn vn. Ra bng x phng v ra li bng nc ct nhiu ln, m bo sch ht x phng. Kh trng mu bng TTCA vi nng 15g/l, trong 10 pht. Trong qu trnh kh mu lun lc u n, nh nhng m bo cc mu c kh trng u. a vo iu kin v trng ra li nhiu ln bng nc ct (4-5 ln) , trc khi a vo nui cy trong mi trng MS+0.2mg/l IBA+0.5mg/l BA chuNn b sn.

hoa sau khi nui cy 30 ngy, c th tip tc cy chuyn trong mi trng nhn nhanh (MS+0.2mg/l IBA+0.5mg/l BA) m bo s lng nh chi cn thit cho th nghim.

33

Mi trng nui cy Mi trng nui cy l mi trng MS (Murashige Skooge, 1962), b sung 30g/l ng, 8g/l agar, cht iu ho sinh trng gm c: 0.2mg/l IBA, 0.5mg/l BA; v c b sung Ribazole cc nng khc nhau. Mi trng nui cy c iu chnh v pH =5.8 bng KOH (1N), hoc HCL (1N). Mi trng nui cy c un cho ho tan cc thnh phn trc khi kh trng bng autoclave nhit 1210C, 1 atm trong 30 pht. Sau khi kh trng, mi trng c vo bao nilon 500ml, cha 100ml mi trng. Bao nilon cng c hp v trng trong autoclave nhit 1210C, 1 atm trong 30 pht. Qu trnh mi trng vo bao nilon c tin hnh trong t cy iu kin v trng. Bng 2.1 :Thnh phn c bn ca mi trng MS Thnh phn CaCl2.2H2O KH2PO4 Mui khong a lng KNO3 MgSO4.7H2O NH4NO3 MnSO4.4H2O ZnSO4.7H2O H3BO3 KI Mui khong vi lng Na2MoO4.2H2O FeSO4.7H2O Na2EDTA Cu SO4.5H2O CoCl2.6H2O Glyxin Myo-inositol Vitamin v cc thnh Acid nicotinic (Niaxin) phn hu c Thiamin HCl (vitamin B1) Pyridoxine HCl (vitamim B6) Nng (mg/l) 440 170 1900 370 1650 22.3 8.6 6.2 0.83 0.25 27.8 37.3 0.025 0.025 2 100 0.5 0.1 0.5

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Bng 2.2 :Thnh phn c bn ca mi trng Knudson C (1946)

Ca(NO3)2 * 4 H2O KH2PO4 MgSO4 * 7 H2O (NH4)2SO4 FeSO4 * 7 H2O MnSO4 * 4 H2O Sucrose Agar 1000 mg 250 mg 250 mg 500 mg 25 mg 7.5 mg 20 g 12-15 g

Phng php b tr th nghim Cc th nghim c b tr ngu nhin hon ton. Chn mu cy ban u ( hoa) c kch thc tng i ng u. Mi bao nilon c cy 9 mu, lp li 3 ln. Sau khi cy, cc mu cy c b tr nui trong phng cy, m bo nh sng ng u vi thi gian chiu sng 16 gi. Nhit trong khong 18-250C. Phng php ly ch tiu v x l s liu: Sau thi gian nui cy 30 ngy, chng ti tin hnh ly s liu gm cc ch tiu v s chi/cm chi; chiu cao chi (tnh t gc n ngn) nhn xt v mc sinh trng v pht trin ca cy di nh hng ca Ribazole. S liu c tnh ton trn phn mm EXCEL phin bn 2003, ca Microsoft. Gm c tnh cc s liu trung bnh trong mi nghim thc v dng cng c data analysis phn tch s liu bng bng ANOVA. Cc th nghim - nh hng ca Ribazole ln s sinh trng pht trin in vitro Cc cy in-vitro c nui cy t ban u, c dng lm vt liu cho th nghim. T cc nh chi tch ra t cy in-vitro, theo tng ging, s c cy vo cc l nghim thc khc nhau. Trn mi ging, cc l th nghim bao gm: L nghim thc Nng (mg/l) R0 0 R1 50 R2 75 R3 100 R4 150

Sau 30 ngy nui cy, cc cy s c o c cc ch tiu: s chi/cm, chiu cao chi v t l sng. S phc hi ca cy sau khi x l ribazole Cc cy sau khi x l ribazole, chng ti tin hnh tch nh chi, cy chuyn li trong mi trng nhn nhanh bnh thng, kim tra s phc hi ca cc sau khi x l ribazole.

35

Do mt s iu kin khch quan, chng ti tin hnh kim tra s phc hi sau ca cy cn sng tt c cc nng i vi ba ging (tia mung vng, farm hng v pingpong vng); i vi hai ging cn li (nt vng v nt tm) chng ti kim tra l nghim thc R2 so snh vi l i chng. Cc ch tiu quan st cng bao gm: s chi/cm, chiu cao chi v t l sng ca mu cy. Phng php test virus i tng l cc cy hoa a lan, hoa cc c triu chng nhim bnh virus TMV in hnh theo m t trn v cc mu cy trong ng nghim c s l loi virus. Thi gian: t 1: 5//2006 6/2006 t 2: 9/2007 a im: + Th nghim c tin hnh ti phng th nghim Cng ngh sinh hc thuc khoa Sinh hc- i hc Lt + Mu vt ly ti mt s vn trng Cc v lan trn a bn Phng 8 Tp Lt v mu PNCM khoa Nng Lm B kt v my ELISA - Mondel 680 Microplate Reader S/N/14172 - Kit. name : Full kit 1000 Tests (Bio rad) Qu trnh tin hnh b. X l mu Bc 1: Lm sch mu Ra mu sch bng nc lau qua bng cn 700C ngm Javen (4%) khong 5 pht (ch trnh mu b xy st nhiu) Ra bng nc ct v trng 3 ln. Dng giy thm v trng thm kh. Ci s: ngm Javen (4%) trong 5 pht Bc 2: Tch chit Mu thc vt: Cn 2 g mu l cho mi ging trc khi em x l. Nghin mu trong ci s c cha dung dch m chit (lng m chit tu thuc vo lng mu). * Cch pha m chit : 1g mu : 0,5 ml dch m chit : 9,5 ml nc ct v trng Chit ly dch ni, bo qun vo 2 -80C dch chit lng cn. Sau ht ra 2 ng eppendorf (1ml/ng) Votext Ly tm 4500vng /pht trong 5 nhit 60C Ra li bng nc ct v trng 3 ln.

36

c. Test ELISA Bc 1: Ph khng th s cp m ph (mu xanh) pha trong nc ct vi t l 1/5 Khng th s cp (Ab1) ha trong m ph vi t l 1/100. Vortex trc khi s dng. Np vo mi ging 100l. y Microplate bng mng film, tin hnh hn hp trn trong 2 gi 370C. Ra 3 ln bng PBS Tween c pha sn. Qu trnh ra s gip loi b khng th tha v thm kh ging bng giy thm. Bc 2: Np mu Mu: + Mu dch chit (M) c chuNn b phn tch chit virus. + Chng dng (+), chng m (-) c pha bng nc ct v trng. + Chng trng (0): Nc ct v trng + m c cht (khng mu): pha long theo t l 1:19 Vortex trc khi s dng Tin hnh np mu vo cc ging trong Micrplates mu 2 80C trong 12 h (ging c y bng mng film) Ra mu: sau khi v ry kh ging. Ra 3 ln bng PBS- Tween, 2 ln u ra ngay, ln 3 sau 3 pht mi . Sau mi ln thm kh bng giy thm. m cng hp (mu ) c pha long trong nc ct vi t l 1/5. Khng th th cp (Ab2) pha long trong m cng hp vi t l 1/100. Vortex trc khi s dng Np vo mi ging 100l dung dich trn. y mng film v trong 2h 370C. Ra 3 ln bng PBS Tween c pha sn. Qu trnh ra s gip loi b khng th tha v thm kh ging bng giy thm Pha long 2ml m c cht trong 8ml nc ct tit trng (t l 1/5). Dng 2 vin c cht pha vo dung dch m trn. Np vo mi ging 100l hn hp trn. y ging bng mng film v 370C trong 30

Bc 3: Ph khng th th cp -

Bc 4: Np c cht -

Bc 5: c kt qu

37

Sau khi , a mu vo my ELISA, c kt qu ln th nht. c kt qu 3 ln, mi ln cch nhau 30. Ln 3 cch ln 2 l 3h. d. Phn tch kt qu t my c ELISA

- Kt qu th nghim ELISA l sch (m tnh) khi gi tr thu c ca mu bnh 0.5 gi tr i chng sch bnh (chng trng hoc nc) - Kt qu ELISA l b bnh (dng tnh) khi gi tr thu c ca mu kim tra 1.5 ln gi tr i chng sch bnh. - Kt qu th nghim ELISA l nghi ng b bnh khi gi tr thu c ca mu kim tra ln hn gi tr i chng sch bnh t 0.5 1 ln. * Ch : trong bo co ny, mu ghi ng b nhim bnh s b coi l c bnh X l s liu S dng phn mm Excel 2003 phn tch v tng hp cc s liu th thu thp c t cc th nghim. Dng phn mm MSTATC phn tch thng k cc s liu bng ANOVA vi mc sai s c ngha l 0.01.

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Chng 3 KT QU V THO LUN

A. KT QU NGHIN CU TRN CC GING NA LAN CT CNH 3.1 Kt qu iu tra cc loi a lan hin c ti lt Bng 3.1. Danh mc cc loi a lan ct cnh hin ang sn xut ti Lt STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 TN THNG MAI Hng bch Trng Balkis Trng vn Trng l la Trng tm hoa c Trng bo trng Bch hng hong Trng neon Liu r Vng da beo BGH Vng chm Vng Ba Ru Vng la Vng hi Xanh chiu Miretta xanh Vng trng Xanh olive STT 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 TN THNG MAI Tm hng vng Hng cnh sen Hng phn Tm ht Hng nm ngha b mai Tm vit quang ton Xanh oliu Cam la Trng bt Xanh li chm Xanh li Xanh ngc Xanh cnh nhn Vng rt Vng chim Hng c Hng nht

3.2 Kho st mi trng nhn ging K thut nui cy m t bo thc vt hin nay xut hin v pht trin nhiu phng th nghim trn khp th gii. Nhiu phng php nui cy c p dng nhm mc ch nhn ging cy trng, bo tn ngun gen qu, lai to, a dng ho cc kiu di truyn thc vt. Phng php nhn ging v tnh in vitro c s dng trong ti nghin cu nh hng ca khong, vitamin, v cht iu ho sinh trng thc vt trn s tng trng ca ba ging a lan: Trng bt, Tm ht, Miretta xanh. Nhu cu dinh dng, iu kin l ho vi mi i tng thc vt khc nhau l khng ging nhau. Do khng th p dng quy trnh nhn ging ca i tng ny ln i tng khc. Khi nghin cu trn ba ging a lan ni trn, vic tm ra phng php v trng mu cy, mi trng nui cy thun li cho s ti sinh chi, s tng trng v kch thc, trng lng, chiu cao cng nh s phn ho ca cc c quan gp phn xy dng quy trnh nhn ba ging a lan ni trn.

Thng qua vic b tr 4 th nghim kho st nh hng ca mi trng nhn ging in vitro ln s pht trin ca a lan, chng ti thu c kt qu sau. Th nghim 1: nh hng ca mi trng ln qu trnh pht sinh chi kho st cc protocorme c nui cy trong cc mi trng c hm lng khong khc nhau, mi trng MS, 1/2MS v mi trng Knudson C, kt qu sau 2 thng nui cy chng ti ghi nhn kt qu trong bng 3.2. Bng 3.2. nh hng ca mi trng ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh i tng Mi trng MS 1/2MS Knudson C MS 1/2MS Knudson C MS 1/2MS Knudson C T l chi hnh thnh (%) 20 12 16 19 12 16 20 10 11

Trng bt

Tm ht

Miretta xanh

Trong th nghim ny chng ti kho st tng loi mi trng c nh hng nh th no s lng chi trn tng loi ging. Theo kt qu bng 1, biu 1 v biu 2 chng ti thy rng: Vi loi mi trng MS th c ba ging u cho s lng chi khng cao, chng rt hn ch trong s pht sinh protocorme nhng cn i vi loi mi trng Knudson C th s lng chi c pht trin nhiu hn so vi mi trng MS s lng protocom pht sinh ng k nhng protocom c nhiu lng ht. Cn loi mi trng MS th li l kh thi nht loi mi trng ny cho s lng protocom nhiu nht, t lng ht v protocom pht trin u hn, sinh trng mnh hn. Do vy, chng ti s dng mi trng MS lm mi trng c bn trong qu trnh nhn nhanh Cymbidium. Th nghim 2: nh hng ca than hot tnh n qu trnh pht sinh chi ca Lan Cymbidium Than hot tnh khng phi l cht kch thch sinh trng nhng khi b sung vo mi trng nui cy s c li ch v tc dng kh c trong mi trng, c bit l vi cc cy h lan c bit l n c vai tr quan trng trong s to r bt nh. Vic hp thu cc cht c ch trong mi trng nui cy hoc t m cy to iu kin thun li cho s sinh trng ca Cymbidium sp.. Ngoi ra nhng nghin cu gn y cng ch ra rng qu trnh thc Ny c quan bit ha to r cng chu nh hng ca than hot tnh. Kt qu nghin cu trn ba ging Trng bt, Tm ht, Miretta xanh sau 60 ngy cy c th hin bng 3.3.

40

Bng 3.3. nh hng ca than hot tnh ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh. i tng Hm lng than (g/l) 0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 T l chi hnh thnh (%) 11 13 21 22 19 11 12 18 15 2 8 8 18 10 11

Trng bt

Tm ht

Miretta xanh

th nghim ny chng ti quan st s nh hng ca than hot tnh n qu trnh pht sinh chi cho thy: Vi mi trng MS khng b sung than hot tnh th s lng chi khng nhiu, hnh thi cy sinh trng rt km: cy, r c pht trin nhng li cn rt nh nn chng s b nh hng trong qu trnh sinh trng v pht trin giai on ex-vitro. Mi trung MS c b sung 0.5 g/l than hot tnh th cng nh mi trng MS khng b sung than hot tnh s lng protocorme cn rt thp, nhng hnh thi c thay i hn l cy c pht trin, r cng pht trin rt mnh. Nh vy th vi nghim thc ny th ch yu l r pht trin ch cha c s phn chia mnh to lng protocorme thch hp. Cn mi trng MS c b sung 1.5 g/l than hot tnh v 2g/l than hot tnh th lng protocorme to ra nhiu hn 2 nghim thc va ni trn nhng cy to ra cng cha nhiu lm v protocorme cng khng ng u. Vi mi trng MS c b sung 1 g/l than hot tnh th lng protocorme to ra nhiu v vt tri hn cc nghim thc trn, cy, r pht trin mnh v rt u. Qua th nghim ny chng ti thy than hot tnh cng gp phn vo s phn chia t bo to ra s lng protocorme thch hp, gp phn nhn nhanh Cymbidium ng vi mt lng nht nh.

41

Th nghim 3: S nh hng ca cc nng BA n qu trnh pht sinh chi BA l mt cht iu tit sinh trng nhn to thuc nhm Cytokinin, l nhm c s dng rng ri nht trong k thut nui cy m hin nay. Tc dng ch yu ca n l kch thch s phn chia t bo, thc Ny s hot ng ca chi bn. Nng s dng khc nhau tu i tng thc vt nui cy, chng hn nng 22M thch hp vi s pht trin chi Cymbidium aloifolium, nhng i vi Dendrobium aphyllum v Dendrobium moschatum nng thch hp nht cho s pht trin ca chi l 44M (N.R.Nayak v cng s, 1997). tm nng ph hp ca BA ln s to protocorm ca cc ging Cymbidium sp. trong th nghim ny chng ti b tr 5 nng khc nhau: 0, 0.5, 1.0, 1.5 v 2 mg/l. Sau 12 tun nui cy, chng ti quan st v ghi nhn c kt qu bng 3.4. Bng 3.4. nh hng ca BA ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh i tng Hm lng than (g/l) 1 1 Trng bt 1 1 1 1 1 1 Tm ht 1 1 1 1 Miretta xanh 1 1 1 Nng BA (ml/l) 0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 T l chi hnh thnh (%) 8 10 20 14 12 10 13 28 16 16 6 7 20 14 9

Vi th nghim ny th ging Tm ht l loi thch hp nht cho s to chi, t ti 41,5% nhng s cy hon chnh th li rt t, cn Trng bt v Miretta xanh th c 2 loi ny chim t l t hn nhng li va to protocorm va to cy, Trng bt chim 31,7% cn Miretta xanh chim khong 26,8%. Vi MS + 1g/l than hot tnh v khng b sung BA v mi trng MS + 1 g/l tan hot tnh + 0,5ml/l, vi 2 nghim thc ny th s thch nghi ca cc protocorme cn rt km, chng t y khng phi l nng thch hp ch cc protocorme ca a Lan pht trin. Ti mi trng MS + 1g/l than hot tnh + 2ml/l BA th cc protocorm pht trin cng khng nhiu, to cy v r li cn pht trin rt mnh. mi trng MS + 1g/l than hot tnh + 1,5ml/l BA th s lng protocorm c pht trin hn nhng cng

42

ch yu l to cy v r. Nen y cng cha l mi trng thch hp cho qu trnh nhn nhanh cy a Lan. Mi trng MS + 1g/l than hot tnh + 1ml/l BA li cho s lng protocorm l nhiu nht, mt s to cy, r v mt s khc to protocorme cng mnh , u. Do mi trng MS + 1g/l than hot tnh + 1ml/l BA l mi trng thch hp tip tc phc v cho qu trnh nhn nhanh cy a Lan. Th nghim 4: S nh hng BA + NAA n qu trnh hnh thnh chi Th nghim ny c thc hin bi cc protocorme nui cy trong mi trng MS, c than hot tnh l 1g/l, nng BA l 1ml/l v c b sung thm cc nng NAA khc nhau. Sau 2 thng nui cy, quan st, ghi nhn c kt qu nh bng 3.5 sau: Bng 3.5. nh hng ca BA + NAA ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh i tng BA (mg/l) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 NAA (mg/l) 0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 T l chi hnh thnh (%) 11 15 22 16 17 9 11 17 12 12 12 21 27 23 24

Trng bt

Tm ht

Miretta xanh

Ngc li vi th nghim 3: Miretta xanh nhng nh khng thch hp vi cc nng ca BA nhng khi phi hp vi NAA th cc protocorme ca Miretta xanh li pht trin rt tt, tt hn c Trng bt v Tm ht, chim ti 41,5% so vi t l chi. Vi s nghin cu ca cc cht iu ha sinh trng th Trng bt l loi n nh nht , vi th nghim nh hng ca BA th Trng bt chim 31,7% cn th nghim n hng ca s phi hp ca BA + NAA cng chim vi t l tng ng l 32,7%. iu ny cho ta thy c i vi cht iu ha sinh trng nh BA hay NAA th cc protocorme ca Trng bt u pht trin. Tm ht vi th nghim ny ch chim 26,8%. Qua th nghim ny ta thy c s thch hp ca cc loi khc nhau ng vi tng cht iu ha sinh trng l khc nhau.

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Nng BA + NAA 1: 0ml/l mi trng MS + 1g/l than hot tnh th lng protocorm to ra l km, nng BA + NAA 1: 0,5ml/l protocorme c pht trin hn BA + NAA 1: 0ml/l nhng cng khng ng k, cng cn hn ch v lng protocorme. nng BA + NAA 1:1,5ml/l v nng BA + NAA 1: 2ml/l th va to protocorm li va to cy. 2 nng ny lng protocorm to ra khng hn km nhau bao nhiu. Cn nng BA + NAA 1:1ml/l hu ht cc loi l thch hp, s lng protocorme l cao nht, mt s to cy, r. 3.3. Kho st tc ng ca ribazole ln s sinh trng ca protocorm Th nghim 1: Nghin cu nh hng ca ribazole ln s sinh trng v pht sinh protocorm mi ca ba ging a lan tm ht, Trng bt v Miretta xanh ca Lt Ribazole l hp cht thuc nhm khng chuyn ho nucleotide ca virus. Ribazole c cu to v tnh cht ho hc tng t c adenosin ln guanosin. Bn cnh Ribazole c th hot ng c lp v tun hon v l nguyn nhn m ribazole tham gia ng ho vo ARN v AND ca virus v c ch s nhn bn ca virus thc vt trong t bo. Bn cnh tc dng c ch s nhn bn ca virus th bn thn ribazole cng gy c ch ln s sinh trng v pht trin ca t bo thc vt. Trong th nghim thc hin vi ba ging a lan Trng bt, v Tm ht cho thy, nghim thc i chng c t l mu sng t 100 %, trong khi khi nng ribazole tng dn t 50 mg/l, 75 mg/l, 100 mg/l, 150 mg/l c t l sng gim dn so vi i chng khng s dng ribazole (Bng 3.9 v 3.10). c bit nng 100 mg/l v 150 mg/l th t l sng st gim r rt. cc nghim thc i chng s protocorm mi c to thnh cao hn hn cc nghim thc c s dng ribazole, nng ribazole cng cao th s protocorm mi pht sinh trn nhng mu cn sng cng thp dn. Nu ta so snh h s nhn tnh theo nhng mu cn sng th nng 50 mg/l; 75 mg/l v 100 mg/l l kh cao. Tuy nhin khi ta so snh h s nhn ny tnh theo tng s mu ban u a vo nng 100 mg/l ca hai ging Trng bt v Tm ht th s protocorm mi thu c thp hn s tng mu ban u. Bng 3.6. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca Trng bt Nng (mg/l) T l sng st H nhn (trn tng mu) 0 (1) 100 2,9 50 (2) 94,4 1, 6 75 (3) 83,3 0,9 100 (4) 61,1 0,6 150 (5) 44,4 0.5

Trong qu trnh theo di chng ti nhn thy, thi gian bt u pht sinh protocorm mi trong nghim thc i chng c ba ging l sm hn so vi cc nghim thc cn li. nghim thc i chng, ging Trng bt bt u pht sinh

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protocorm mi sau 28 ngy; ging Miretta xanh - 22 ngy; ging tm ht th sau 21 ngy. cc nghim thc s dng nng ribazole cng cao th thi gian bt u pht sinh protocorm cng di. nghim thc (4) v (5) s pht sinh protocorm mi th hin khng r rng nh nghim thc i chng, c bit l nghim thc (5). ging Miretta xanh, cc mu nghim thc i chng bt u pht sinh protocorm sau 22 ngy, nghim thc (2) (3) th sau 31 ngy, nghim thc (4) th sau 37 ngy. Cc mu protocorm nghim thc i chng c mu xanh m nht cn cc mu cn sng nghim thc (5) chuyn sang mu vng nu v hu nh khng cn mu xanh. Nh vy, ribazole khng ch c ch s ti bn ca virus, m qua cc th nghim thc hin chng ti nhn thy: Ribazole c ch s sinh trng v pht trin ca protocorm, nhng nghim thc c nng cng cao th s c ch ny cng th hin r rng. Khng ch c ch s sinh trng v pht trin ca cc protocorm m ribazole cn c th gy cht mu, nng ribazole cng cao th t l mu cht cng cao. Ribazole c mc tc ng ln ba ging a lan l khng ging nhau. Ging Tm ht c h s nhn trong cc nghim thc c ribazole gim r rt so vi i chng hn hai ging cn li. Ribazole gy tc ng c ch vi mc khc nhau cho nhng cy trng khc nhau. iu ny cng ng vi mt s kt qu ca mt s tc gi khc nh: i vi cy khoai ty, nng ribazole c s dng trong khong 20- 30 mg/l. Khi s dng ribazole trn cy chui th nng ribazole l 50 mg/ l nhng khi s dng cho Nicotiniana Rustica th nng ribazole li cao hn l 100 mg/ l mi trng nui cy. Trong khi i vi cy Oxalic Tuberosa Molina th nng ribazole ti u l 50 mg/l mi trng nui cy (Zapta v cs 1995). Bng 3.7. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca Tm ht Nng (mg/l) T l sng st H nhn (trn tng mu) 0 (1) 100 3,4 50 (2) 91,6 0,9 75 (3) 77,8 0,6 100 (4) 55,5 0,4 150 (5) 50.0 0,2

Qua th nghim tin hnh vi ba ging a lan chng ti thy trong khi nng ribazole tng dn th h s nhn ca tng ging c s gim vi mc khc nhau so vi i chng. ging Tm ht nghim thc (2) cho thy s gim h s nhn so vi nghim thc i chng l cao hn hn so vi hai ging Miretta xanh v Trng bt. th nghim vi ging Trng bt v Tm ht, nghim thc (4) v (5) cho h s nhn trn tng s mu ban u l m, iu ny cho thy tng s protocorm thu c

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sau 80 ngy nui cy trong mi trng cha 100 mg/l; 150 mg/ l th s protocorm thu c thp hn tng s mu ban u a vo. Nh vy trong c 3 ging th hai nghim thc (2) v (3) c b sung ribazole m vn thu c s protocorm sau th nghim nhiu hn s protorme mu ban u. Bng 3.8. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca ging Miretta xanh. Nng (mg/l) T l sng st H nhn (trn tng mu) 0 (1) 100 3,3 50 (2) 97,2 2,6 75 (3) 94,4 1,9 100 (4) 77,7 0,2 150 (5) 33,3 0,1

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Th nghim 2: Nghin cu nh hng ca ribazole tch lu ln s sinh trng v pht sinh protocorm mi ca ba ging a lan Tm ht, Trng bt v Miretta xanh ca Lt Cc mu protocorm sau khi qua mi trng i chng v mi trng c ribazole vi cc nng 50 mg/l, 75 mg/l, 100 mg/l trong 40 ngy c chuyn sang mi trng hyponex c bn (mi trng s dng cho nghim thc i chng). theo di s nh hng ca ribazole tch lu trong mu cy ln s sinh trng v pht trin ca cc mu ny trong ln cy chuyn sang mi trng khng cha ribazole ln th nht. Bng 3.9. nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Trng bt Nng (mg/l) T l sng st H nhn (trn tng mu) 0 (1) 100 1,9 50 (2) 100 3,6 75 (3) 100 3,8 100 (4) 100 4,7

T kt qu thu c th nghim 2 sau 60 ngy theo di chng ti nhn thy. Cc mu protocorm sng st sau 60 c nui cy trong mi trng c cha ribazole vi cc nng 50 mg/l; 75 mg/l; 100 mg/l c chuyn qua mi trng khng cha ribvirin th 100 % mu sng st. Bng 3.10: nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Tm ht Nng 0 50 75 100 T l sng st H nhn (trn tng mu) 100 1,2 100 1,9 100 2,7 100 3,2

Trong ln cy chuyn u tin ny, s lng protocorm cng nh h s nhn ca cc nghim thc (2), (3), (4) c ba ging a lan u cao hn so vi nghim thc i chng (1). Tuy nhin, cc mu protocorm nghim thc i chng tuy c s lng t hn nhng u c kch thc ln hn hn cc mu protocorm trong cc nghim thc khc. Nhng mu qua mi trng c ribazole vi nng 100 mg/l th kch thc nh hn cc mu qua mi trng ribazole nng 50 mg/l. Bn cnh mu qua mi trng c ribazole vi nng 100 mg/l v 75 mg/l th khng c mu xanh r rng nh cc mu nghim thc i chng v cc mu qua mi trng ribazole nng 50 mg/l. Vi ging Tm ht, th nghim trc cc mu trong mi trng c ribazole gim s sinh trng r rt so vi nghim thc i chng hn hai ging cn li, th trong th nghim 2 ny h s nhn ca cc mu ny cng thp hn hn hai ging cn li. Qua th nghim ny chng ti nhn thy:

47

Cc mu sau khi qua mi trng c cha ribazole th ln cy chuyn ln th nht ny c s khc bit so vi nghim thc i chng. Hay c s tch lu ribazole trong cc mu qua ribazole. S sinh trng ca cc mu c trong cc nghim thc (2), (3), (4) cng khng ging nhau. Tc l s khc bit v nng ribazole trong th nghim mt nh hng ln s sinh trng v pht trin ca cc mu ny trong ln cy chuyn th nht. Cc mu nng ribazole th nghim mt cng cao th s lng protocorm mi pht sinh trong th nghim hai cng tng nhng kch thc th li gim so vi i chng. Bng 3.11. nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Miretta xanh Nng (mg/l) T l sng st H nhn (trn tng mu) 0 (1) 100 1,7 50 (2) 100 1,9 75 (3) 100 2,2 100 (4) 100 2,6

Bng 3.12. S sinh trng ca cy con ging Trng bch sau khi x l ribazole Mi trng MS 1/2MS Knudson S l 4.25 2.05 2.10 Di l (mm) Di r (mm) S r 42.50 33.90 2.50 40.40 25.20 1.7 50.05 28.75 2.45 FrW (g) 11.15 7.35 10.20

Bng 3.13. S sinh trng ca cy con ging Tm ht sau khi x l ribazole Mi trng MS 1/2MS Knudson S l 3.83 2.80 2.30 Di l (mm) 37.35 40.40 45.05 Di r (mm) 28.40 10.50 29.85 S r 2.90 1.15 2.35 FrW (g) 10.25 7.15 11.20

Bng 3.14. S sinh trng ca cy con ging Miretta xanh sau khi x l ribazole Mi trng MS 1/2MS Knudson S l 2.85 1.90 2.25 Di l (mm) 42.50 40.40 50.05 Di r (mm) 9.40 4.45 6.45 S r 2.50 1.7 2.45 FrW (g) 10.30 8.25 10.00

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3.4 nh hng ca cc loi gi th v EC giai on vn m 3.4.1 Nghin cu mt s gi th vn m Cy lan con ra r tt trong iu kin in vitro trn loi mi trng knudson C b sung NAA 1 mg/l v than hot tnh 1g/l mi trng, chng ti dng lm th nghim tip theo. Chng ti s dng 8 loi gi th khc nhau lm th nghim tm ra loi gi th thch hp cho cy lan con. Theo cc nghin cu trc y (2, 8, 53), Cymbidium sinh trng thch hp trn cc gi th c tnh acid, thng thong v gi c Nm cao. Gi th thch hp trng lan thay i tuy theo loi v tui. Nhiu bo co cho thy hn hp v cy nghin nh kt hp 20 25% cc loi gi th nhn to khc nh perlite, than bn th, v cy, v bo u c s dng nhm ci thin thng thong v Nm trong gi th. V cy nghin nh vi kch thc 0.25-0.5cm thng s dng trng lan con. V cy kch thc ln hay cc loi gi th qu thng thong i hi phi ti nc thng xuyn duy tr Nm, trong lc cc loi gi th qu mn nh bt x da, than bn li gi nc qu ln, km thng thong nn cy d b thi r. iu ny cng ph hp vi nhng kt qu trn 7 loi gi th a ra phng php th nghim, cy con u c kh nng sng c nhng t l sng v kh nng sinh trng pht trin khc nhau (bng 3.15).

Bng 3.15. T l sng ca cy lan Cymbidium sp. trm cc loi gi th khc nhau sau 3 thng chuyn ra vn m
K hiu loi gi th I II III IV V VI VII VIII 3T 3T/dn xay (1:1) X da Dn xay/X da (3/1) Dn xay/X da (2/1) Dn xay/X da (1/1) Dn xay Loi gi th Dn mt T l sng 65/70 50/70 57/70 59/70 53/70 63/70 56/70 52/70 % cy con sng st 92,85 71.43 81.43 84.29 75.71 90.00 80.00 74.29

Trn loi gi th dn xay, 3T c t l sng ca lan con l thp nht do loi gi th ny khng c cht dinh dng cung cp cho cy trng ban u, ng thi kh nng gi nc cng km hn cc loi gi th phi trn khc, cy sinh trng pht trin km. Ngc li trn gi th ch c bt x da cy c t l cht cao, mt phn do thng thong ca gi th thp, ng thi kh nng gi nc qu cao, gy hin tng thi r. Trn loi gi th truyn thng dng trc y l dn mt cho kt qu t l sng cao nht nh nhng tnh cht vt l vt tri hn cc loi gi th khc, loi gi th ny gi Nm tt, c thong kh cao.

49

Bn cnh loi dn mt, cc loi gi th phi trn nh 3T phi trn x da t l 1/1, dn xay v x da t l 2/1 v 1/1 cho t l sng kh cao (trn 80%) nh ci thin c thng thong cng nh kh nng gi Nm ca n. Nu tnh hiu qu kinh t, vic s dng cc cng thc phi trn 3T phi trn x da t l 1/1, dn xay v x da t l 2/1 v 1/1 vn cho kt qu cao, cc gi th ny c th thay th dn mt (loi gi th hin nay rt t v ngy cng khan him) v ph hp vi yu cu sinh trng ca cy con lan con giai on vn m (bng 3.15). Trong th nghim tip theo, chng ti s dng 3 loi gi th (I, III v VI) tip tc theo di cc ch tiu sinh trng ca cy lan con sau 12 thng, kt qu thu c cc bng 3.16; 3.17; 3.18. Bng 3.16. S sinh trng ca cy con ging Trng bch trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m Mi trng I III VI S l 3.83 2.80 2.30 Di l (mm) 37.35 40.40 45.05 Di r (mm) 28.40 10.50 29.85 S r 2.90 1.15 2.35 FrW (g) 10.25 7.15 11.20

Bng 3.17. S sinh trng ca cy con gingTm ht trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m Mi trng S l Cao cy (mm) 42.50 40.40 50.05 Di r (mm) 33.90 25.20 28.75 S r FrW (g) 11.15 7.35 10.20

I III VI

4.25 2.05 2.10

2.50 1.7 2.45

Bng 3.18. S sinh trng ca cy con ging Miretta xanh trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m Mi trng I III VI S l 2.85 1.90 2.25 Di l (mm) 42.50 40.40 50.05 Di r (mm) 9.40 4.45 6.45 S r 2.50 1.7 2.45 FrW (g) 10.30 8.25 10.00

Qua kt qu cho thy, s sinh trng, mc hnh thnh r th cp, chiu di l cng nh trng lng ti cc cng thc gi th khng c sai khc ng k. Cc cy con sinh trng kh tt trn cc loi gi th chn thc hin nghin cu ny.

50

3.4.2 Nghin cu nh hng ca EC ln giai on u ca cy ra r Da vo kt qu nghin cu v loi gi th ni trn, chng ti chn mt loi gi th l Dn xay/X da (2/1) tip tc nghin cu nh hng ca nng cht dinh dng nhm tm ra nng thch hp nht cho sinh trng ca cy lan con trong nm u sau khi ra vn m. nh hng ca nng cht dinh dng ln sinh trng ca cy lan con c nh gi (Bng 3.19). Kt qu tt nht c ghi nhn chiu cao cy, chiu di r, trng lng ti, trng lng kh khi s dng nng EC t 1.0 -1.5 dS/m. R rng trong giai on cy con, nng EC cao s lm gim s pht trin ca cy v cng ph hp vi cc nghin cu trc y trn cc ging lan (15,27,42). Nng mui cao l nguyn nhn lm gim s sinh trng ca cy mc d c th c nhng tc ng lin quan khc lm nh hng n kh nng sinh trng ny. Mui khong tch tr li trong nn c cht gy nn mn thng xy ra khi nng cht dinh dng (EC) c cung cp cao. S tch ly ny gy nn s thiu ht nc cy trng do n hn ch kh nng ht nc ca b r (33). Cy trng phn ng li liu lng EC cao ca cht dinh dng bng cch gim tc hp thu dinh dng v nc, gim tc pht trin, gim kh nng thNm thu ca l v din tch l (53, 54). Trong th nghim ca chng ti cng c nhng kt qu tng t, qua kho st nh hng ca EC ln sinh trng cy lan con (Tm ht) trn cc nn gi th khc nhau sau 12 thng (mi thng ti dung dch dinh dng 2 ln) cho kt qu nh sau cc bng 3.19; 3.20; 3.21.

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Bng 3.19. nh hng ca EC ln sinh trng cy lan con ging Trng bch trn cc nn gi th khc nhau sau 12 thng EC S lng r s cp 5.1 0.5 6.2 0.6 6.4 0.5 6.1 0.3 6.0 0.4 S lng r th cp 9.2 0.7 13.2 1.3 15.5 1.5 15.5 1.2 13.6 0.9 Chiu di l trung bnh (cm) 13.2 1.3 15.7 1.4 16.6 1.7 15.3 1.5 14.3 1.2 Trng lng ti (g/cy) 194.2 13.3 225.4 12.6 237.6 11.7 228.3 12.5 203.3 11.6

C 0.5 1.0 1.5 2.0

Bng 3.20. nh hng ca EC ln sinh trng cy lan con gingTm ht trn cc nn gi th khc nhau sau 12 thng EC S lng r s cp 5.2 0.7 6.1 0.5 6.2 0.3 6.1 0.3 6.2 0.3 S lng r th cp 10.2 0.7 15.2 1.3 15.3 1.5 15.8 1.2 14.8 0.9 Chiu di l trung bnh (cm) 13.2 1.3 15.7 1.4 16.6 1.7 15.3 1.5 14.3 1.2 Trng lng ti (g/cy) 190.2 13.3 221.4 12.6 235.6 11.7 227.1 11.8 200.4 9.6

C 0.5 1.0 1.5 2.0

Bng 3.21. nh hng ca EC ln sinh trng cy lan con ging Miretta xanh trn cc nn gi th khc nhau sau 12 thng EC S lng r s cp 5.0 0.4 6.2 0.6 6.4 0.4 6.1 0.6 6.2 0.3 S lng r th cp 10.2 0.9 12.3 1.1 14.5 1.3 14.4 0.9 13.7 0.8 Chiu di l trung bnh (cm) 12.4 1.4 14.6 1.3 16.5 1.8 15.8 1.2 14.3 1.0 Trng lng ti (g/cy) 194.2 13.3 228.4 12.6 235.6 11.7 228.3 10.5 193.3 10.6

C 0.5 1.0 1.5 2.0

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Danh sch cc ging a lan (Cymbidium sp) c phc trng v bo tn Bng 3.22.Cc ging a lan lit k sau y c thu thp v phc trng thnh cng. STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 TN KHOA HC Cym. anita st. Helier Cym. York ever blooming Cym. Bautista de auzaLa Tullerie Cym. Bengal Bay Golden hue Cym. Sayonara Rakitan Cym. Madrid forest king Cym. Miretta cherob Cym. Fanfarest. Francis Cym. Labell anna belle Cym. Featherhill heritage Cym Wallacia Burnt Orange Cym. Mighty tracey Moon walk Cym. Paros paradise haruka Cym. Enzan spring in the mood TN THNG MI Hng bch Liu r Vng da beo BGH Vng Ba Ru Xanh chiu Miretta xanh Miretta xanh Tm ht b mai Cam la Trng bt Hng c Hng nht

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B. KT QU NGHIN CU TRN CC GING CC 3.5 nh hng ca ribazole ln s sinh trng v pht trin in vitro, i vi cc ging khc nhau S c mt ca ribazole c tc dng c ch s phn chia t bo ca virus, n hn ch mt lng virus c trong mu cy. ng thi n cng nh hng n s sinh trng v pht trin ca cc, trong qu trnh nui cy in-vitro. C th ni, ribazole c nh hng c hi n cy in-vitro. Mt s ging c mc nhy cm vi ribazole hu ht b cht nng 100-150mg/l. Cc ging c kh nng thch nghi tt hn vn sng st vi mt t l no . Ribazole li nhiu du hiu nh hng trn cy: sinh trng pht trin chm, kh nng pht sinh cm chi km, l v thn c biu hin b c ch: l nh, thn nh, chiu di ca t ngnThi gian m cc du hiu ny th hin ra cng khc nhau tu theo ging. Ging pingpong vng Pingpong vng l ging rt nhy cm vi ribazole. 100% mu cy l nghim thc R3 v R4 (c nng 100 v 150mg/l) u cht. Trong nhng ngy ca tun u tin, cc mu cy sinh trng v pht trin nh nhau, khng c biu hin g c bit. n tun th hai, sau khi hnh thnh c 2 l l th nghim R3, v 3 4 l l R4 , chng ti nhn thy cc mu trong l th nghim ngy c du hiu ngng sinh trng. Ring l th nghm R4, chi ngn c biu hin nh b cht (thi hng) dn dn, bt u xut hin tun th ba. i vi l nghim thc R1 v R2, trong hai tun u, hu ht cc mu sinh trng khng khc bit ln so vi l i chng, ch c mt vi mu c du hiu ngng sinh trng, v c cc du hiu khng th sng st ni. n khong tun th ba, chng ti nhn thy s pht trin chm li so vi l i chng. V mt s mu b cht vi triu chng nh l nghim thc R3 v R4. T l cht tng theo nng x l. Cc mu cy trong l nghim thc R1 v R2 mang c im ca s c ch sinh trng, nh trnh by trn. Bng 3.23. nh hng ca ribazole i vi ging pingpong vng L nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 3.15 2.19 100 R1 2.89 0.84 72 R2 2.45 0.33 44 R3 0 0 0 R4 0 0 0

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Hnh 3.1 Cc cy ging pingpong vng sau khi x l ribazole 30 ngy Ging tia mung vng y l ging c kh nng chu ng tt ribazole, hu ht cc mu cy u sng st ( l R4, t l sng t n 81%). Trong thi gian u sau khi x l, cc nghim thc, cc cy pht trin kh u, t thy s khc bit, nht l nghim thc R1, R2 so vi R0. hai nghim thc cn li, cy pht trin chm hn. n tun th hai, s pht trin ca cy trong cc nghim thc c s khc bit ln: l i chng R0 cy tip tc sinh trng tt, cn cc nghim thc cy gn nh ngng sinh trng. S pht sinh cm chi cc nghim thc din ra chm dn v yu t. Cc chi sinh trng chm, nn c chiu cao hn ch. Do nh hng ca ribazole, l cc l th nghim nh. Bng 3.24. nh hng ca ribazole i vi ging tia mung vng L nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 4.51 2.16 100 R1 2.70 1.04 100 R2 2.52 0.74 100 R3 2.41 0.57 100 R4 1.9 0.43 81

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Hnh 3.2 Cc cy ging tia mung vng sau x l ribazole 30 ngy Ging farm hng Farm hng cng l ging chu c ribazole nng kh cao. nng 100mg/l, m t l sng vn t mc 100%. y c l l ngng ca ging ny. V nng 150mg/l, t l sng gim xung cn 44%. Kh nng pht sinh cm chi ca ging farm hng khng mnh so vi ging tia mung vng. Di tc ng ca Ribazole, cm chi pht sinh trong ging farm hng thp (2-3 chi l R1 v R2). Tuy nhin chng ti nhn thy chiu cao chi vn c duy tr so vi R0. i vi l vn b c ch, khin cho l khng pht trin. Bng 3.25. nh hng ca ribazole i vi ging farm hng L nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 3.15 2.72 100 R1 2.63 1.95 100 R2 2.22 1.3 100 R3 1.85 0.9 100 R4 1.58 0.47 44

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Hnh 3.3. Cc cy ging tia farm hng sau x l ribazole 30 ngy Ging nt vng Chng ti nhn thy ribazole nh hng mnh n chiu cao cy, trong khi kh nng pht sinh cm chi ca cy t b nh hng hn. nng 50mg/l, hu nh kh nng pht sinh cm chi khng i, nhng chiu cao gim mnh:t 2.06cm l nghim thc R0 xung ch cn 0.8 cm l nghim thc R1. Tuy kh nng pht sinh cm chi t b nh hng, nhng chng ti nhn thy cc chi km pht trin, biu hin qua ch tiu v chiu cao, kch thc l nh, sinh trng chm T nng 75mg/l, chng ti cng thy xut hin cc cy b cht do nh hng ca ribazole, v tng dn nng cao hn. Bng 3.26. nh hng ca ribazole i vi ging nt vng L nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 2.78 2.06 100 R1 2.6 0.8 100 R2 2.31 0.52 78 R3 1.52 0.24 63 R4 1.36 0.29 44

Hnh 3.4. Cc cy ging nt vng sau x l ribazole 30 ngy

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Ging nt tm y l ging kh nhy cm vi ribazole so vi cc ging trong t th nghim ny. Ch nng 75mg/l bt u c nhng cy b cht. nhng cy b cht ny ch sau 1-2 tun nui cy. Ngay c l th nghim R1, chng ti thy du hiu nh hng ca ribazole t rt sm, ngay tun u tin kh r rt. Nhng du hiu l: sinh trng chm, l nh. Tuy nhin, chng ti nhn thy rng nh hng ca ribazole khng mnh lm n qu trnh pht sinh cm chi ca cy (trung bnh 3.48 chi l R0 , v 2.85 l R1). Chiu cao ca chi cng b nh hng kh ln, l nghim thc R1, chiu cao ch bng mt na so vi chiu cao cy l i chng. Cng chnh v s nhy cm vi ribazole, nn ging ny, chng ti nhn thy c nhng du hiu nh hng rt r rng ca ribazole, v s c ch n qu trnh sinh trng ca cy. Bng 3.27. nh hng ca ribazole i vi ging nt tm L nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 3.48 2.46 100 R1 2.85 1.03 100 R2 2.27 0.68 96 R3 1.95 0.36 74 R4 0 0 0

Hnh 3.5. nh hng ca Ribazole i vi ging nt tm 3.6 S phc hi ca cy sau khi x l ribazole Sau khi x l ribazole, cc cy con sinh trng sau thi gian mt thng s c cy chuyn tr li trong mi trng nhn nhanh bnh thng. Mc ch giai on ny l nhn nhanh cc cy c x l v gip cy phc hi kh nng sinh trng. Lc ny, ribazole vn cn tn d trong cy, nn cng cn nh hng n s sinh trng v pht trin ca cy. S nh hng ny tu theo lng tn d (ph thuc vo nng x l) v tnh nhy cm ca mi ging vi ribazole.

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Ging tia mung vng Ging ny chu ng kh tt vi Ribazole, cng c ngha l t chu nh hng ca Ribazole, do , khi cy chuyn tr li, kh nng phc hi ca cy trong cc nghim thc kh tt. Chng ti nhn thy rng s pht trin ca cy gn nh l ng u nhau. Bng phn tch ANOVA cng cho ta thy c iu : s khc bit gia cc nghim thc l khng c ngha (i vi bng phn tch chiu cao chi, F tnh l 0.0125; F bng mc ngha 95% l 2.39). S khc bit v s pht sinh ca cm chi cng tng t, s lng chi trung bnh trong cc nghim thc khc bit nhau mt cch khng c ngha. (bng phn tch ANOVA Ph lc). Bng 3.28. S sinh trng ca cc cy ging tia mung vng trong mi trng nhn nhanh, sau 30 ngy nui cy T l nghim thc R0 R1 R2 R3 R4 S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) 4.41 2.25 100 4.04 2.25 100 3.96 2.24 100 3.74 2.45 100 3.55 2.23 100

Hnh 3.6: Cc cy ging tia mung vng 30 ngy sau khi cy tr li trong mi trng nhn nhanh

Ging farm hng Do l ging chu kh trc nh hng ca ribazole, nn khi cy chuyn tr li trong mi trng nhn nhanh, s c mt ca mt lng nh ribazole tn d trong mu ch tc ng rt nh n s sinh trng ca cy. Chng ti ch theo di quan st s sinh trng ca cc cy trong qu trnh nui cy, v nhn thy s sinh trng kh u v s pht sinh cm chi cng nh v chiu cao ca chi. Ngoi ra, t nhng kt qu phn tch s liu cho thy s khc nhau gia cc nghim thc l khng c ngha.

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Bng 3.29. S sinh trng ca cc cy ging farm hng trong mi trng nhn nhanh, sau 30 ngy nui cy T l nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) Ging pingpong vng Cc cy trong ging pingpong vng chu nhng tc ng kh ln ca ribazole trong qu trnh x l, nhng khi cy chuyn tr li, cc cy nhanh chng phc hi s sinh trng v pht trin. Chng ti quan st thy trong tun u, cc mu l R1 v c bit l nghim thc R2 pht trin tng i chm so vi cc mu l i chng. iu ny c l gii l do nh hng ca ribazole trong mu vn cn tc ng c ch ln mu, nn mt thi gian, cy mi phc hi tr li c. Bng 3.30. S sinh trng ca cc cy ging pingpong vng trong mi trng nhn nhanh, sau 30 ngy nui cy T l nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 3.74 2.17 100 R1 3.74 2.09 100 R2 3.66 2.01 100 R0 3.22 2.62 100 R1 3.15 2.57 100 R2 3.11 2.48 100 R3 3.07 2.46 100

Hnh 3.7. Ging pingpong vng nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh Ging nt vng V mt s iu kin, chng ti ch cy chuyn li cc cy l nghim thc R2 v l i chng R0. Trong qu trnh x l ribazole, ging nt vng b c ch mnh v s pht trin chiu cao ca chi. Chng ti tm cho rng ribazole nh hng mnh n s pht trin v chiu cao ca chi. Tuy nhin, kt qu thu c t th nghim ny cho ta thy s phc hi kh tt v yu t chiu cao ca chi.

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Chng ti cng quan st thy cc cy l R2 vn cn b c ch trong thi gian u nui cy, nhng ri cng pht trin gn bng vi cc cy l i chng. Bng 3.31. S sinh trng ca cc cy ging nt vng trong mi trng nhn nhanh, sau 30 ny nui cy T l nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 2.81 2.05 100 R2 2.67 2.03 100

Hnh 3.8 Ging nt vng nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh Ging nt tm Cng do mt s iu kin chng ti ch nui cy c cc cy trong hai l nghim thc R2 v l nghim thc i chng. Ging nt tm l ging chu nh hng nng n nht bi tc ng ca ribazole. Chnh v th, qu trnh phc hi ca cy trong mi trng nhn nhanh l kh chm. Do , cc cy nghim thc R2 pht trin chm hn kh r rt so vi cy l i chng. Bng 3.32. S sinh trng ca cc cy nt tm trong mi trng nhn nhanh, sau 30 ngy nui cy T l nghim thc S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) T l sng ca chi(%) R0 3.63 2.46 100 R2 3.37 2.06 100

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Hnh 3.9 Ging nt tm nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh 3.2.1 Ging tuapin hng Ging ny c kh nng chu ng kh tt vi ribazole, do khi cy chuyn tr li kh nng phc hi ca cy kh tt. l nghim thc R1 va R2 (nng 50 v 75 mg/l) 100% cy sng st. Chng ti nhn thy rng s pht trin ca cy gn nh l ng u nhau hai ch tiu chiu cao trung bnh ca chi v s chi trung bnh/cm.Tuy nhin l nghim thc R3 c hin tng mu b cht v s pht trin ca cy khng ng u. Bng 3.33. S sinh trng ca cc cy ging tuapin hng trong mi trng nhn nhanh, sau 30 ngy nui cy S chi trung bnh/cm Chiu cao trung bnh ca chi(cm) R1 R2 R3 CV% LSD0,05 3.5a 3.4a 1.8b 13.86 0.61 4.6a 4.0a 2.0b 13.58 0.73 100 100 89 T l sng ca chi(%)

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Hnh 3.10. Cc cy ging tuapin hng sau x l ribazole 60 ngy 3.2.2 Ging nt ngh: L ging c kh nng chng chu tt, cng c ngha nt ngh l ging t chu nh hng ca ribazole. cc nng , cc mu u sng vi t l 100%. Nhng s pht trin ca cy khng c ng u, th hin bng s liu v ch tiu s chi trung bnh/cm, chiu cao trung bnh ca chi. Bng 3.34. S sinh trng ca cc cy ging nt ngh trong mi trng nhn nhanh, sau 30 ngy nui cy. S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.9b 1.9b 2.4a 9.61 0.29 Chiu cao trung bnh ca chi(cm) 6.5a 7.1a 3.1b 7.46 0.62 T l sng ca chi(%) 100 100 100

Hnh 3.11. Cc cy ging nt ngh sau khi x l ribazole 60 ngy

63

3.2.3 Ging tiger ng T l sng ca cc cy trong ging tiger ng l 100% l nghim th R1 v R2. V cht nhiu nghim thc R3. nng ribazole cao cc cy ging tiger ng chu nh hng kh ln. Cc cy sinh trng v pht trin khng ng u. Bng 3.35. S sinh trng ca cy ging tiger ng trong mi trng nhn nhanh, sau 30 ngy nui cy S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.3b 1.9a 0.3c 25.34 0.45 Chiu cao trung bnh ca chi(cm) 4.6b 7.1a 1.0c 19.42 1.24 T l sng ca chi(%) 100 100 16

Hnh 3.12. Cc cy ging tiger ng sau khi x l Ribazole 60 ngy 3.2.4 Ging pha l xanh Ging ny kh nhy cm vi ribazole, cc mu cy c t l sng st khng cao cc nng . Cy sinh trng v pht trin khng ng u. Chng ti quan st thy rng cc mu cy cc l nghim thc pht trin tng i chm. iu ny c l gii l do nh hng ca ribazole trong mu vn cn c ch ln mu, nn mt thi gian cy mi phc hi tr li c .

64

Bng 3.36. S sinh trng ca cc cy ging pha l xanh trong mi trng nhn nhanh, sau 30 ngy nui cy S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.2a 0.9a 0.8a 39.74 0.60 Chiu cao trung bnh ca chi(cm) 5.9a 3.3b 3.5b 32.03 2.02 T l sng ca chi(%) 95 79 74

Hnh 3.13. Cc cy ging pha l xanh sau khi x l ribazole 60 ngy 3.2.5 Ging pha l cam Trong qu trnh x l ribazole, ging pha l cam b c ch mnh v s pht sinh chi. T l sng ca mu khng cao. Chng ti quan st v nhn thy rng cy ging pha l cam phc hi trong thi gian kh nhanh.

65

Bng 3.37. S sinh trng ca cy ging pha l cam trong mi trng nhn nhanh, sau 30 ngy nui cy S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.1b 2.1a 1.3b 23.72 0.53 Chiu cao trung bnh ca chi(cm) 4.8a 3.2b 4.6ab 23.64 1.50 T l sng ca chi(%) 89 84 95

Hnh 3.14. Cc cy ging pha l cam sau khi x l ribazole 60 ngy Ging tuapin hng Cc cy ging Tua pin hng c s chi trung bnh/cm thp v kh ng u nhau. Tuy nhin chiu cao ca chi li chnh lnh nhau, c nhng mu pht trin kh cao. Bng 3.38. S sinh trng ca cy ging tuapin hng trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.4a 1.6a 1.2a 22.45 0.47 Chiu cao trung bnh ca chi(cm) 7.6a 2.6c 4.6b 9.72 0.72 T l sng ca chi(%) 100 100 100

66

Ging nt ngh L ging chu ng tt cc nng ribazole. Nn khi cy chuyn li vo mi trng nhn nhanh th lng ribazole tn d trong cy t, khng cn nh hng n cy. Cc cy sinh trng v pht trin kh ng u nhau. V t l sng vn t mc 100%. Bng 3.39. S sinh trng ca cy ging nt ngh trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.3a 1.4a 1.5a 26.84 0.56 Chiu cao trung bnh ca chi(cm) 3.5b 5.2a 5.1a 5.50 0.38 T l sng ca chi(%) 100 100 100

Ging tiger ng Cc mu cy trong cc nghim thc sinh trng v pht trin bnh thng. Cy khe mnh v ng u nhau v cc ch tiu s chi trung bnh v chiu cao trung bnh. Bng 3.40. S sinh trng ca cy ging tiger ng trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.6a 1.7a 1.4a 18.58 0.44 Chiu cao trung bnh ca chi(cm) 4.2a 4.3a 4.2a 14.20 0.90 T l sng ca chi(%) 100 100 100

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Ging pha l xanh Pha l xanh l ging cng chu c ribazole nng kh cao. Kh nng pht sinh chi cm khng mnh. Bng 3.41. S sinh trng ca cy ging pha l xanh trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo. S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.8a 1.9a 1.3b 13.55 0.35 Chiu cao trung bnh ca chi(cm) 2.8a 3.1a 2.8a 8.25 0.36 T l sng ca chi(%) 100 100 100

Ging pha l cam Cy ging pha l cam c nhy cm kh cao vi ribazole. Khi em cy chuyn li th cy c cc ch tiu v s chi trung bnh v chiu cao trung bnh ca chi thp. Bng 3.42. S sinh trng ca cy ging pha l cam trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo Nghim thc S chi trung bnh/cm R1 R2 R3 CV% LSD0,05 1.3a 1.3a 1.4a 24.60 0.50 Chiu cao trung bnh ca chi(cm) 4.0a 4.0a 4.3a 7.48 0.50 T l sng ca chi(%) 100 100 100

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C. Th nghim th nghim Test ELISA mt s ging hin ang sn xut v mt s ging qua phc trng Sau khi test ELISA cc mu trn, chng ti tnh ton s liu v so snh vi cc chng (+) v chng (-), chng trng, nc ct thu c nhng kt qu sau t 1 Kt qu test TMV trn mt s ging cc c triu chng bnh virus. Sau thi gian tnh ton v phn tch chng ti c c bng sau: Hoa cc Bng 3.43: Kt qu test ELISA cc ging cc STT 1 2 3 4 5 6 Tn i tng Cc nt, l bnh Cc i a Cc th vng Cc vng Cc pha l, mu l thng Cc vng cha ra hoa, l bnh Ngun test Mu l bnh Mu l bnh Mu l bnh Mu l bnh Mu l bnh Mu l ngu nhin Kt qu ln 1 + + + -

* Mu (+): l b bnh * Mu (-): L khng b bnh T bng trn, ta thy c du hiu dng tnh vi virus mt s cy cc (cc nt v cc i a). M ngun cc kim tra ly t khu vc NTL, nn kh nng ly nhim l rt ln sang nhng ging cha c b nhim bnh nh cc vng. Cc pha l bt u c giu hiu nghi nhim. Hoa lan Bng 3.44. Kt qu test ELISA cc ging lan Stt 1 2 3 4 5 6 7 8 Tn i tng kho st Lan tm ngha, 383 NTL Lan tuyt ngc xanh 382B, NTL Trng bch 397/57 NTL Trng B Ra, 397/57 NTL Miretta xanh, 387 NTL Tm ht, 382 B NTL Tm ht 396 V Trng Ton Lan vng trng, 403/57 NTL Ngun test L bnh L bnh L bnh L bnh L bnh L bnh L bnh L bnh Kt qu ln 1 + + + -

Cam la, 403/57 NTL

L bnh L bnh L bnh L bnh L bnh

+ + + -

10 Tm ht 397/57 NTL 11 Trng bch 382B NTL 12 Trng bch, 396 V Trng Ton 13 Tm ht, 383 NTL

i vi hoa lan, tnh hnh nhim bnh nhiu, sau hai ln test nhng ging lan b nhim bnh v nghi nhim chim 62%, y l mt t l kh cao. Cc ging lan khng nhim bnh cng nm trong khu vc ca cc ging lan b bnh, nn kh nng b nhim virus l rt cao. Cn phi c cc hnh thc test li cc ging lan hin ang sn xut ti cc c s c cc hnh thc bo v ban u thch hp. t 2: Kho st mt s mu cy sau khi tch v x l mu phc trng li ging Bng3.45. Kt qu test t 2 ca 2 ging cc v lan Stt Tn i tng kho st Hoa cc 1 2 3 4 5 Cc nt Cc i a Cc th vng Cc pha l Tiger ng Hoa lan 6 7 8 9 Trng bch Tm ht Miretta xanh Lan vng trng L L L L L L L L L Ngun test Kt qu ln 1

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10 Cam la 11 Bay Golden Hill (BGH) 12 Xanh chiu

L L L

Cc mu hoa lan v hoa cc trong PTN sau khi c s l tch virus khng c trng hp no test c nhim virus r rng. Tuy nhin kt qu ny cng cha nh gi ng hon ton v cn nhiu nghuyn nhn khc anh hng kt qu trn. Bng 3.46. Danh sch cc ging cc ct cnh c gi tr c phc trng v bo tn STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Tn ging Nt tm Nt vng Farm tm Tia mung vng Pingpong vng Th xanh Tia mung hng o trng o ng a hng Farm vng Th vng Th tm Pha l Tm hu Pingpong trng Nt tm Tiger ng o vng Lys Lisa trng Nt ngh Nt trng Tison Tuabin hng nh Saphia vng STT 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Tn ging Saphia ngh Caraven Farm trng Th mi Th vng ngh Th vng chanh Th vng nh xanh Phal trng Phal cam Phal xanh(tua xanh) Vng ng Tia trng Tia ng Tia mung vng ti Tia mung trng Tia mung tm Mai xanh Mai Mai vng Ngc bch hng Ngc bch vng Indo cam Indo hng N hong N hong tm N hong vng N hong hng trng

71

Qui trnh ngh s dng trong nhn ging a lan

Qui trnh nhn ging a lan sch bnh hin nay tr thnh ph bin vi nhng thnh cng nht nh v khng sai khc nhau nhiu ti nhiu phng th nghim nhn ging. Trong vic p dng bin php b tr trong vic loi b virus ca a lan thng ch s dng x l nhit. Trong iu kin Vit Nam, cha thy c ti liu s dng ha cht b tr, v vy trong qui trnh ny, nt khc bit l p dng ha cht (ribazole) trong vic to cy con sch bnh. nh chi (1) nh sinh trng (2) To th protocorme (3)

To cm chi (4)

Tch chi (5)

Cy trng thnh (8)

Trng lan con trong v xp (7) Qui trnh nhn ging in vitro cy a Lan

Cy con (6)

(1) Ly chi t cy lan trng thnh (Kh trng theo phng php thng qui v vo mu trong mi trng MS + 0.1mg/l BA + 1gAC + 75mg virazole), khi mu phc hi c th x l nhit tng dn t 25 n 37oC v gi nhit ny trong vng 3-5 tun. (2) Tch li m phn sinh (0,25-0,5mm) v cy li vo mi trng bc 1 khng c virazole. (3) V (4) Protocorm hnh thnh trong bc (2) tip tc cy chuyn sang mi trng mi (3 thng/ln) (4) Cy chuyn tip tc tng s lng protocorm cng nh chi cy. (5) Tch cc chi t cm chi cy sang mi trng Knudson C + 0,5-1mg/l BA + 0,5-1mg/l NAA + 1mg AC. (6) Bc chuyn cy con ra vn m m np bnh cy v trong iu kin vn m t 5-7 ngy cy thch nghi vi iu kin t nhin. (7) Trng cy con vo khay hay v xp S dng gi th l dn mt hay dn xay + x da (1:1), x l qua thuc dit nm (Mancozeb) v sau ngm vo nc sau mi cho gi th vo khay hay v. Rt cy con ra khi bnh cy, ra nh nhng qua nc my loi b thch khi r, sau trng vo khay vi mt 55cm. khay cy con trong iu kin Nm cao ( 90%), che sng ( 70%) v phun sng u t 3-4 ln mi ngy.

72

QUI TRNH CHUYN CY LAN CON IN VITRO RA VN M

Cy con sau khi rt khi mi trng in vitro cn ra sch agar bm vo r, nhng r cy con vi Mancozeb ri mi trng vo gi th. Khay trng: Khay kch thc 30 45 5cm Gi th: Dn mt; 3T/dn xay (1:1) hoc Dn xay/X da (2/1). S lng cy con/ khay: 100 cy Nm gi th: 70 80% Nm khng kh: 90 95% Nhit nui trng: 20-25oC nh sng: che sng > 70% S ln ti/ngy: 3-5 ln Bn phn: N-P-K t l 3:1:1 pha long c EC = 1,0ds/m phun mi tun 2 ln (tr tun u tin sau khi mi ra cy Phng tr su bnh hi lan: Nhn: Kasuarane (mi tun phun 1 ln) Nm: Mancozeb (mi tun phun 1 ln) Su xanh: Semi-alpha (mi tun phun 1 ln)

73

Qui trnh ngh s dng trong nhn ging cc

Qui trnh nhn ging cc sch bnh hin nay tr thnh ph bin vi nhng thnh cng nht nh v khng sai khc nhau nhiu ti nhiu phng th nghim nhn ging. Trong vic p dng bin php b tr trong vic loi b virus ca cc thng ch s dng x l nhit. Trong iu kin Vit Nam, cha thy c ti liu s dng ha cht b tr, v vy trong qui trnh ny, nt khc bit l p dng ha cht (ribazole) trong vic to cy con sch bnh. nh chi hoa (1) nh sinh trng (2)

To cy con (3)

Cy chuyn (4)

Cy trng thnh (7)

Trng cy con trong v xp (6) Qui trnh nhn ging in vitro cy Cc

Cy con (5)

(1) Ly chi t cy cc trng thnh hoc hoa (Kh trng theo phng php thng qui v vo mu trong mi trng MS+0.2mg/l IBA+0.5mg/l BA + 5075mg virazole), khi mu phc hi c th x l nhit tng dn t 25 n 37oC v gi nhit ny trong vng 3-4 tun. (2) Tch li m phn sinh (0,25-0,5mm) v cy li vo mi trng bc 1 khng c virazole. (3) Cy con hnh thnh trong bc (2) tip tc cy chuyn sang mi trng mi (mi thng/ln) (4) Cy chuyn tip tc trong mi trng MS+0.2mg/l IBA+0.5mg/l BA tng s lng cy con in vitro. (5) Tch cc t t cy con cy sang mi trng MS+0.2mg/l IBA+0.5mg/l BA khong 1 tun trc khi a cy con ra vn m. (6) Trng cy con vo khay hay v xp S dng gi th l peatmoss, x l qua thuc dit nm (Mancozeb) cho gi th vo khay hay v. Rt cy con ra khi bnh cy, ra nh nhng qua nc my loi b thch khi r, sau trng vo khay vi mt 33cm. khay cy con trong iu kin Nm cao ( 90%) trong 3 5 ngy, che sng ( 70%) v phun sng u t 3-4 ln mi ngy.

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KT LUN V KIN NGHN

1. KT LUN: - Cc ging lan ct cnh hin nay ti Lt c khong 38 ging, trong c khong 14 ging c gi tr v c nhiu ngi tiu dng a chung. - Cc ging cc ct cnh nhp vo Lt c trn 70 ging, tuy nhin nhiu ging b lng qun. Qua iu tra v thu thp ch cn khong 54 ging v trong s ch c mt s ging hin ang c sn xut vi s lng ln l: Cc nt, cc i a, th vng, pha l, cc vng, tuapin hong, nut nghe, tiger ong, pha le xanh, pha le cam. Cc th nghim kho st mi trng nhn ging lan cho thy, mi trng MS + 1 g/l than + 1 ml/l BA + 1ml/l NAA th chng ti thy s pht trin ca cc chi i theo chiu hng rt thun li, to ra mt lng protocorm cng rt nhiu. - Ribazole gy c ch qu trnh sinh trng v pht trin ca cy trong iu kin nui cy in-vitro. Cy sinh trng km, chiu cao ca chi nh, l km pht trin. V nng cao hn dn n cy b cht. - S c ch gy ra bi ribazole ln cy cc theo cc mc khc nhau, ty theo ging khc nhau. Nng x l ribazole tng i thch hp i vi nm ging cc trong th nghim ny l trong khong 75-100mg/l. - Cy con c cc ging lan cng nh cc ging cc trong nghin cu ny sau khi qua x l ribazole kt hp vic tch nh sinh trng cho kt qu tt. 2. KIN NGHN: - Tip tc kim tra cc ging cc v lan khc. - Ngoi virus TMV, ta cn phi test cc loi virus khc gy hi cho cc v hoa. - Nh nc cn c s h tr kinh ph hng nm duy tr v bo tn cc ging hoa ct cnh ca Lt, y l mt ngun ging c gi tr trong vic pht trin nn nng nghip rau hoa ca Tnh nh ni ring v trong c nc ni chung

75

TI LIU THAM KHO

TI LIU TING VIT

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33. Ket, N. V, E., J. Hahn, S. Y. Park, D. Chakrabarty and K. Y. Paek., 2004. Micropropagation of an endangered orchid Anoectochilus formosanus. Biologia Plantarum 48.,3: 339-344. 34. Knudson, L., 1946. A new nutrient solution for germination of orchid seed. Am. Orhid Soc. Bull. 15:214-217. 35. Linda Naeve, and Diane Nelson, 2005, Growing Chrysanthemum in the garden, plant new divition lowa State University o Science and Techonology, Ames, lowa. 36. Margit Nothnagl, 2006, Interaction between greenhouse grown Chrysanthemum and Frankliniella occidentalis, Doctoral thesis Swedish University of Agricultural Sciences. 37. Michael N. Dana and B. Roise Lerner, 1996, Chrysanthemums, Press of Department of Horticuture, Purdue University, West Lafayette, IN 38. Morel, G. and Martin, C. 1952. Virus-free Dahlia through meristem culture. C.R. Hebd. Seances Acad. Sci. Paris 235: 1324-1325. 39. Murashige, T. 1980. Plant growth substances in commercial uses of tissue culture. In: Plant Growth Substances 1979. Skoog, F. (Ed.) Springer-Verlag, Berlin. Pp. 426-434. 40. Murat Top and Bill Tatura, 2002, Growing Chrysanthemum, Agriculture Note Farm Diversiffication Service (Bendigo) March, 2002. 41. Nayak, N.R., S.N. Patnaik, and S.P. Rath. 1997a. Direct shoot regeneration from foliar explants of an epiphytic orchid, Acampe praemorsa (Roxb.) Blatter and McCann. Plant Cell Reports 16:583-586. 42. Nayak, N.R., S.P. Rath, and S.N. Patnaik. 1997b. In vitro propagation of three epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobidium aphyllum (Roxb.) Fisch. and Dendrobidium moschatum (Buch-Ham) Sw. through thidiazuron-induced high frequency proliferation. Scientia Hort. 71:243-250. 43. Nyland G. and Gohen AC., 1969. Heat therapy of virus disease of perennial plants. Annual Review of Phytopathology 7: 331-354 44. Oka S, Muraoka O, Abe T, Nakajima S., 1999. Adventitious bud and embroid formation in garland chrysanthemum leaf culture. J Jpn Soc Hortic Sci. 68:70 2. 45. Pan, M.J. and J. van Staden, 1999. Effect of activated charcoal, autoclaving and culture media on sucrose hydrosis. Plant Growth Reg. 29:135-141. 46. Paek, K.Y. and Yeung, E.C., 1991. The effects of 1-naphthaleneacetic acid and N6-benzyladenin on the growth of Cymbidium forrestii rhizomes in vitro. Plant Cell Tiss. Org. Cult. 24:6571. 47. Pierik, R.L.M., 1987. In vitro culture of higher plants. Martinus Nijhoff Publishers, Dordrecht. The Netherlands.

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48. Powell, W. and Uhrig, H., 1987. Anther culture of Solanum genotypes. Plant Cell Tissue Organ Culture. 11: 13-24. 49. Prakash S., M.I. Hoque, T. Brinks, 2002, culture media and containers, Proceedings of a Technical Meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agricultureand held in Vienna, 26 -30 August 2002 p.29-40. 50. Rajapakse, N.C. and Kelly, 1995. Spectral filters and growing season influence growth and carbohydrate status of Chrysanthemum. J. Amer. Soc. Hort. Sci. 120:78-83. 51. Rout GR, Das P., 1997. Recent trends in the biotechnology of Chrysanthemum: a critical review. Sci Hortic; 69:239-56. 52. Schmidt J., E. Wihem, V.A. Savangikar, 2002, Disease detection and elimination, Low cost options for tissue culture technology in developing countries, Proceedings of a Techniques in Food and Agricultureand held in Vienna, 26-30 August 2002 p 55-62. 53. Van Huylenbroeck, J.M. and P.C. Debergh. 1996. Physiological aspects in acclimatization of micropropagated plantlets. Plant Tiss. Cult. Biotech. 2:136141. 54. Verdonck, O., D. Vleeschauwer, and M. De Boodt. 1981. The influence of the substrates on plant growth. Acta Hort. 126:251-258. 55. Yogesh Parmessur and Asha Saumtally, 2001. Elimination of sugarcane yellow leaf virus and sugarcane bacilliform virus by tissue culture, Food and Agriculture Research Council, Reduit, Mauritiu p. 127-134. 56. Zapata, C., Miller Jr., J.C. and Smith, R.H. 1995. In vitro eradication of viruses from potato. In vitro Cell Dev. Biol. Plant. 31:153-159.

79

Ph lc v hnh nh minh ha

(a)

(b)

(c)

(d)

(e)

(f)

Hnh (a, b, c, d, e, f). Minh ha cc cy lan con nui cy in vitro

80

(g)

(h)

(i)

(j)

(k)

(l)

Hnh (g, h, I, j, k, l). Minh ha cc cy lan con trng trong vn m

81

Hoa BGH

Hoa Xanh chiu

Hoa Miretta xanh

Hoa Trng bch

Cnh hoa Tm ht

Cnh hoaTrng bch

MC LC
M u ........................................................................................................................... 2 Cc t vit tt ................................................................................................................. 3 Chng 1. Tng quan ti liu ........................................................................................ 4 1. S lc v cy m trong cng tc nhn ging cy trng ........................................... 4 1.1 Tnh ton nng ca t bo thc vt trong nui cy m ............................................ 4 1.2 Cng ngh sinh hc thc vt trong nhn ging cy trng ....................................... 5 1.3 Vai tr ca cht iu tit sinh trng trong nui cy m ........................................ 5 1.4 K thut vi nhn ging (micropropagation)............................................................. 7 2. S lc v lch s nghin cu h lan (Orchidaceae) v chi a lan (Cymbidium sw.)11 2.1 Khi qut chung ..................................................................................................... 11 2.2 Mt vi nt v a lan Lt................................................................................. 12 2.3 Hnh thi bn ngoi ca a lan.............................................................................. 13 2.4 Phn loi a lan. .................................................................................................... 14 2.5 Virus hi thc vt ................................................................................................... 15 2.6. Virus hi a lan .................................................................................................... 19 3. S lc v lch s nghin cu, c im chung v phn loi v h cc ................ 24 3.1. Tnh hnh sn xut hoa cc .................................................................................. 24 3.2 c im sinh hc h cc (Asteraceae hay Compositae) : .................................... 25 4. Virus v phng php ELISA.................................................................................. 28 Chng 2. i tng v phng php nghin cu ..................................................... 30 2.1 i tng nghin cu: ........................................................................................... 30 2.2 Phng php nghin cu ....................................................................................... 30 Chng 3. Kt qu v tho lun................................................................................... 39 A. Kt qu nghin cu trn cc ging a lan ............................................................. 39 3.1 Kt qu iu tra cc loi a lan hin c ti lt ................................................ 39 3.2. Kho st mi trng nhn ging........................................................................... 39 3.3. Kho st tc ng ca Ribazole (Ribazole) ln s sinh trng ca protocorm .... 44 3.4. nh hng ca cc loi gi th v EC giai on vn m ............................ 49 B. Kt qu nghin cu trn cc ging cc ................................................................... 54 3.5. nh hng ca Ribazole ln s sinh trng v pht trin in vitro, i vi cc ging khc nhau ................................................................................................... 54 3.6. S phc hi ca cy sau khi x l Ribazole ......................................................... 58 Kt lun v kin ngh ................................................................................................... 72 Ti liu tham kho........................................................................................................ 76 Ph lc v hnh nh minh ha ...................................................................................... 80

ii

DANH MC CC BNG BIU

Bng 3.1. Danh mc cc loi a lan ct cnh hin ang sn xut ti Lt ...... 39 Bng 3.2. nh hng ca mi trng ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh ...................................................................... 40 Bng 3.3. nh hng ca than hot tnh ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh. ..................................................................... 41 Bng 3.4. nh hng ca BA ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh ......................................................................................... 42 Bng 3.5. nh hng ca BA + NAA ln qu trnh pht sinh chi ca ba ging Trng bt, Tm ht, Miretta xanh ...................................................................... 43 Bng 3.6. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca Trng bt ............................................................................................................. 44 Bng 3.7. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca Tm ht ................................................................................................................ 45 Bng 3.8. nh hng ca ribazole ln s sinh trng v pht sinh protocorm ca ging Miretta xanh. ............................................................................................ 46 Bng 3.9. nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Trng bt .................................................................................... 47 Bng 3.10: nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Tm ht ....................................................................................... 47 Bng 3.11. nh hng ca ribazole tch ly ln s sinh trng v pht sinh protocorm ca Miretta xanh .............................................................................. 48 Bng 3.12. S sinh trng ca cy con ging Trng bch sau khi x l ribazole .. 48 Bng 3.13. S sinh trng ca cy con ging Tm ht sau khi x l ribazole ........ 48 Bng 3.14. S sinh trng ca cy con ging Miretta xanh sau khi x l ribazole 48 Bng 3.15. T l sng ca cy lan Cymbidium sp. trm cc loi gi th khc nhau sau 3 thng chuyn ra vn m ...................................................................... 49 Bng 3.16. S sinh trng ca cy con ging Trng bch trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m .......................................................... 50 Bng 3.17. S sinh trng ca cy con gingTm ht trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m .................................................................... 50 Bng 3.18. S sinh trng ca cy con ging Miretta xanh trn 3 loi gi th khc nhau sau 12 thng chuyn ra vn m .......................................................... 50 Bng 3.19. nh hng ca EC ln sinh trng cy lan con ging Trng bch trn cc nn gi th khc nhau sau 12 thng ........................................................... 52 Bng 3.20. nh hng ca EC ln sinh trng cy lan con gingTm ht trn cc nn gi th khc nhau sau 12 thng .................................................................. 52 Bng 3.21. nh hng ca EC ln sinh trng cy lan con ging Miretta xanh trn cc nn gi th khc nhau sau 12 thng.................................................. 523 Bng 3.22. Cc ging a lan c thu thp v phc trng .............................. 524 Bng 3.23. nh hng ca ribazole i vi ging pingpong vng .......................... 54 Bng 3.24. nh hng ca ribazole i vi ging tia mung vng ......................... 55 Bng 3.25. nh hng ca ribazole i vi ging farm hng ................................. 56 Bng 3.26. nh hng ca ribazole i vi ging nt vng .................................... 57

iii

Bng 3.27. nh hng ca ribazole i vi ging nt tm ....................................... 58 Bng 3.28. S sinh trng ca cc cy ging tia mung vng trong mi trng nhn nhanh, sau 30 ngy nui cy .................................................................... 59 Bng 3.29. S sinh trng ca cc cy ging farm hng trong mi trng nhn nhanh, sau 30 ngy nui cy ............................................................................. 60 Bng 3.30. S sinh trng ca cc cy ging pingpong vng trong mi trng nhn nhanh, sau 30 ngy nui cy .................................................................... 60 Bng 3.31. S sinh trng ca cc cy ging nt vng trong mi trng nhn nhanh, sau 30 ny nui cy ............................................................................... 61 Bng 3.32. S sinh trng ca cc cy nt tm trong mi trng nhn nhanh, sau 30 ngy nui cy ................................................................................................. 61 Bng 3.33. S sinh trng ca cc cy ging tuapin hng trong mi trng nhn nhanh, sau 30 ngy nui cy ............................................................................. 62 Bng 3.34. S sinh trng ca cc cy ging nt ngh trong mi trng nhn nhanh, sau 30 ngy nui cy. ............................................................................ 63 Bng 3.35. S sinh trng ca cy ging tiger ng trong mi trng nhn nhanh, sau 30 ngy nui cy .......................................................................................... 64 Bng 3.36. S sinh trng ca cc cy ging pha l xanh trong mi trng nhn nhanh, sau 30 ngy nui cy ............................................................................. 65 Bng 3.37. S sinh trng ca cy ging pha l cam trong mi trng nhn nhanh, sau 30 ngy nui cy ............................................................................. 66 Bng 3.38. S sinh trng ca cy ging tuapin hng trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo .............................................................. 66 Bng 3.39. S sinh trng ca cy ging nt ngh trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo ........................................................................... 67 Bng 3.40. S sinh trng ca cy ging tiger ng trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo ........................................................................... 67 Bng 3.41. S sinh trng ca cy ging pha l xanh trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo. ............................................................. 68 Bng 3.42. S sinh trng ca cy ging pha l cam trong mi trng nhn nhanh, sau 30 ngy nui cy tip theo .............................................................. 68 Bng 3.43: Kt qu test ELISA cc ging cc........................................................ 69

iv

DANH MC CC HNH NH
Hnh 3.1 Cc cy ging pingpong vng sau khi x l ribazole 30 ngy .................. 55 Hnh 3.2 Cc cy ging tia mung vng sau x l ribazole 30 ngy ....................... 56 Hnh 3.3. Cc cy ging tia farm hng sau x l ribazole 30 ngy.......................... 57 Hnh 3.4. Cc cy ging nt vng sau x l ribazole 30 ngy .................................. 57 Hnh 3.5. nh hng ca Ribazole i vi ging nt tm ........................................ 58 Hnh 3.6: Cc cy ging tia mung vng 30 ngy sau khi cy tr li trong mi trng nhn nhanh ............................................................................................ 59 Hnh 3.7. Ging pingpong vng nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh ..................................................................................... 60 Hnh 3.8 Ging nt vng nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh ............................................................................................ 61 Hnh 3.9 Ging nt tm nghim thc R0 v R2 30 ngy sau khi cy trong mi trng nhn nhanh ............................................................................................ 62 Hnh 3.10. Cc cy ging tuapin hng sau x l ribazole 60 ngy .......................... 63 Hnh 3.11. Cc cy ging nt ngh sau khi x l ribazole 60 ngy ......................... 63 Hnh 3.12. Cc cy ging tiger ng sau khi x l Ribazole 60 ngy ...................... 64 Hnh 3.13. Cc cy ging pha l xanh sau khi x l ribazole 60 ngy .................... 65 Hnh 3.14. Cc cy ging pha l cam sau khi x l ribazole 60 ngy ..................... 66