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PHNG PHP TO HT NHN TO I.

t vn mt s ging thc vt, vic duy tr ni ging theo phng thc thng thng l kh khn v tn nhiu thi gian, s lng nhng loi ny ngy cng t, i khi c nhng loi i n tuyt chng do iu kin bt li. khc phc vn ny v tin xa hn na l thng mi ha, cc nh khoa hc khng ngng nghin cu, tm ra cc phng php mi nh nui cy m, phi v tnh, ht v tnh, trong bi ny chng ta s cng tm hiu v ht nhn to. II.Cng ngh to ht nhn to 1. Khi nim Nm 1978, Murashige pht hin u tin v k thut ht nhn to. Nm 1987, Redenbaugh v cng s pht trin thnh cng k thut to ht nhn to. V c bn, ht nhn to ging ht t nhin, c cu to l phi v tnh c bc bi lp o bn ngoi l lp gel dinh dng v c kh nng ny mm nh ht t nhin, im khc bit l ht nhn to khng c phi nh.

Hnh: Cu to ht nhn to Nm 1988, McKersie v cng s to thnh cng ht nhn to cy c linh lng, 65% ht bin i sau bc lm kh. Nm 1985, Kitto v Janick bc ht nhn to c rt bng polyxyethylene t t l sng 3%. Janick (1988), to ht nhn to thnh cng trn cn ty. Gray (1987) thnh cng trn nho v cy c nn. Nm 1988, la m c to ht nhn to do Carman v cng s. 2. Mc ch S biu hin thnh cng ca vic ti sinh cy thng qua vic hnh thnh phi v tnh mt loi thc vt m ra mt phm vi ng dng mi l to ht nhn to nhm to mt h thng vn chuyn d dng cho vic gieo trc tip phi ln t trng. Phi v tnh c to thnh invitro thiu mt v bao ngoi v c khuynh hng ny mm ngay tc khc (nu phi trng thnh) di nhng iu kin ti u hoc c th chuyn vo trng thi th ng nu b thiu nc(Litz v Gray, 1992). Ngc li phi hp t vi v bao bn ngoi c mt kh nng chu hn rt cao (senaratna v csv, 1990;TengvHor, 1997). Do , cn c mt h thng cm ng phi v tnh sao cho n hot ng ging phi hp t, v t c th chu ng c nhng tn hi mc ti thiu.

3. ngha Cng ngh ht nhn to s dng ph v tnh c vai tr rt quan trng trong vic trng trt nhng loi thc vt Khng to c ht Ht c to thnh vi mt s lng thp Kh nng ht sng st thp Vic nhn ging thc vt kh khn v cht mm khng th bo qun c 4. Cc nhn t trong to ht nhn to 4.1 phi v tnh a, lch s pht trin -1958, Street v Reinert m t s hnh thnh phi v tnh t cc t bo n ca c rt -1977, Murashige a ra kin to phi v tnh c th tr thnh bin php vi nhn ging -n nay cng ngh phi v tnh c xem l rt c trin vng cho nng nghip th k 21 b,khi nim v phi v tnh Phi v tnh hay phi soma l cc th nhn ging (propagule) c cc tnh bt ngun t cc t bo dinh dng, bao gm c phn m phn sinh ngn v m phn sinh gc, do c th hnh thnh chi v r. Khng ging nh cc t bo eukaryote, hu ht cc t bo thc vt u c kh nng pht trin thnh phi di nhng iu kin xc nh.Williams v Maheswaran (1986) cho rng phi v tnh c th c hnh thnh t mt t bo n hay t c cm t bo phi, tin trnh ny khc vi nhng qu trnh t nhin khc v c gi l qu trnh hnh thnh phi v tnh. Phi v tnh rt ging phi hu tnh hnh thi v sinh l nhng khng c ti t hp di truyn.do tt c nhng cy con ti sinh bng con ng ny th c vt cht di truyn ging ht cc t bo sinh dng sinh ra chng. c tnh ny ca phi sinh dng khng nhng cho php thc hin s

nhn ging v tnh m cn to ra nhng thay i chuyn bit v trc tip a cc tnh cht mong mun vo cc c th bng vic chn thm nhng trnh t gen c nh vo t bo sinh dng. (Saiprasad, 2001). Ging nh cc t bo ca m phn sinh, cc t bo sinh phi c cc c tnh c bn nh sau: t bo nh, c cng mt ng knh, c hot ng bin dng rt mnh m, c cng tng hp ribonucleic acid rt cao, c t bo cht m c, khng bo rt nh, nhn to, d nhn thy, hch nhn rt to v sm mu, c bit l cc t bo ny c mt lng ln cc ribosom, ty th, li ni cht nh v c vch rt dy (Ammirato, 1987; Emons, 1994; Raghvan, 1983; Thorpe, 1988). c. S pht sinh hnh thi ca phi v tnh Tt c cc phi v tnh u c cu trc lng cc, gm c m phn sinh ca c chi v r, do c th hnh thnh mt cy hon chnh trong nui cy in vitro. S pht sinh hnh thi phi v tnh rt ging so vi phi hu tnh. nhng phi pht trin bnh thng s tri qua nhng giai on pht trin ring bit nh: hnh cu, hnh tim, hnh thy li v hnh l mm. C ch pht sinh phi soma Nhng quan st chi tit v qu trnh pht sinh phi pht hin c 4 pha : 0, 1, 2 v 3, c nhn thy trong giai on u ca tin trnh pht sinh phi trong h thng nui cy ni trn (Fujimura v Komamine, 1979). pha 0, nhng t bo n (giai on 0) hnh thnh nhng cm t bo c kh nng pht sinh phi (giai on 1) trn mi trng c auxin. Trong sut giai on ny, nhng cm t bo hnh thnh t nhng t bo n c kh nng to phi khi mi trng nui cy khng c auxin, hnh thnh nhng cm t bo giai on 1. Sau , pha 1 xut hin khi cy chuyn nhng cm t bo giai on 1 qua mi trng khng c auxin. Trong sut pha 1, nhng cm t bo tng sinh chm v dng nh khng bit ha. Sau pha 1 s phn bo xut hin nhanh trn mt phn ca nhng cm t bo, dn n vic hnh thnh nhng t bo phi hnh cu. Pha ny c gi l pha 2.

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Pha tip theo sau, pha 3, cy con in vitro pht trin t nhng phi hnh cu qua phi hnh tim v phi hnh thy li.

Hnh 2.6 Cc giai on pht sinh phi v tnh Cc kiu pht sinh phi soma S pht sinh phi soma bt nh Cc phi v tnh c th pht trin t cc t bo hay cc m so c lin quan ca mt s loi thc vt nhit i, cc phi bt nh ny c th c to trc tip t t bo n trn b mt ca phi non hoc gin tip t b mt ca phi non ny. Phng php ny c s dng trong chng trnh di truyn ci to ging, chng hn nh cu cc phi b cht non do lai to. S pht sinh a phi v tnh Hin tng ny xy ra khi nui cy cc non non ca thc vt ht trn. Cc khi m c kh nng to phi cao khi c cy chuyn sang mi trng mi s pht trin v tng trng thnh phi. M c kh nng pht trin thnh phi c th c phn bit vi m khng c kh nng pht trin thnh phi do mu trng ca phi v ho khi nhum bng acetocarmine. Di nh n t ngoi cc t bo phi c th pht hunh quang mu xanh l cy. S pht sinh phi soma do cm ng Hin tng ny do s nui cy lng cc t bo v m so sau khi cc m ny chu cc x l c bit em li s cm ng kh nng to phi. Ngi ta thc hin nhiu nghin cu trn nhiu loi thc vt cc iu kin nui cy khc nhau quan st kh nng to thnh m so.

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d. Nhng nhn t nh hng n s hnh thnh phi v tnh Vic cm ng to phi v tnh l mt trong nhng cng vic kh mi m v rt kh khn. t c hiu qu cao v thnh cng, vic tm hiu cc iu kin sinh l, ha hc tc ng n s hnh thnh phi l mt cng vic rt quan trng. Mu cy Mi loi mu cy c nhng kh nng cm ng to phi khc nhau. S la chn mu cy rt khc khe, v d, nhiu loi mt l mm i hi mu cy phi mt giai on c th no ca hp t phi non th mi hnh thnh m so t to phi v tnh. Nhng b phn thch hp c s dng nui cy to phi Asparagus officinalis, Lilium nh chi nh, cung l, lng thn, mm, phi cha trng thnh, cn nhng b phn khc th hiu qu to phi rt thp (Hisato Kunitake, 1998). Mi trng nui cy Gn 70% nghin cu thnh cng u nh vo s dng mi trng MS vi hm lng mui khong c bn (Ammirato, 1983; Litz v Gray, 1992; Thorpe, 1988). L do v mi trng ny c cha hm lng mui nitrat v ammonia cao. T l gia mui nitrat v ammonia trong mi trng rt quan trng trong vic cm ng to phi (Niedz, 1993, 1994). Polyamine trong mi trng dinh dng c hiu qu kch thch n vic hnh thnh phi v tnh (Minocha v Minocha, 1995). Nhng cht hu c khc nh nc da, casein thy phn, tinh cht mch nha (malt extract) nhng nng khc nhau cng c s dng v cho thy c nhiu nh hng n cm ng to phi mt s loi thc vt. Bn cnh cng c nhng ngh v vai tr ca nitrogen, amino acid, amid, cng nh ca kali, photphat n s hnh thnh phi( Tisserat v cng s, 1979; Ammirato, 1983). Ngun cacbohydrate Sucrose l mt ngun cacbon chnh trong mi trng nui cy. Tuy nhin trong mt vi trng hp, galactose (Cabasson v cng s, 1997), lactose (Jumin v Nato, 1995), maltose (Dhir v Yavada, 1995), glucose v fructose (Canhoto v Cruz, 1994)

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v manitol (Kunitake v cng s, 1991) khi kt hp vi sucrose y mnh s cm ng to phi cng nh s pht sinh hnh thi bnh thng ca phi. Cht iu ha tng trng Cht iu ha tng trng c chia lm 5 nhm chnh l auxin, cytokinin, acid abscisic, gibberellin, ethylen. Cc cht iu ha tng trng l ti cn thit cho vic cm ng to phi v mi cht iu ha tng trng th li c nhng tc dng ln vic to phi khc nhau trn nhng loi thc vt khc nhau v nhng nng khc nhau. Qu trnh cm ng hnh thnh phi v tnh nhng loi thc vt khc nhau cho thy c 3 kiu nh hng ca cht iu ha tng trng i vi phi: 1) S hin din ca cht iu ha tng trng l cn thit trong tt c cc giai on pht trin cu phi 2) Cht iu ha tng trng cn thit trong giai on cm ng, tuy nhin, sang n giai on pht trin cao hn th cc cht iu ha ny cn phi c loi b 3) S hnh thnh phi v tnh ch xut hin khi c s c mt ca cht iu ha tng trng, nhng khi phi v tnh pht trin sau giai on hnh cu th s c mt ca cht iu ha khng cn tc dng na. Cht iu ha tng trng l auxin, thng l 2,4-D v NAA c s dng trong hn 50% v 25% trng hp cm ng to phi (Evans v cng s, 1983; Litz v Gray, 1992). C nhng s khc bit mc t bo v mc phi trong hot ng phn chia t bo ca cc t bo phi v tnh v t bo m so, cng nh l nhng s khc bit trong s pht trin hnh thi ca phi v tnh c cm ng trn nhng cht auxin khc nhau ( Rodrigeuz v Wetztein, 1998). Auxin c chng t cho thy c mt tc dng tch cc khi c s dng kt hp vi cytokinin v TDZ. Tuy nhin nng auxin cao thng hn ch s pht trin ca phi v tnh trong nhng giai on u tin ca s pht trin phi. Cytokinin cng c nhng tc dng tng i ln s hnh thnh phi, nhng n khng c nhng tc dng ng k. Mc d vy, s kt hp ca c auxin v cytokinin cho thy nhiu thun li t c hiu qu cm ng phi ti a trn mt s loi (Cruz v cng s, 1990; Nishi, 1997). Tuy nhin hin nay, c nhiu nghin cu s

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dng TDZ, cht iu ha tng trng thuc nhm cytokinin, nhm mc ch cm ng to phi v tnh mt s thc vt hai l mm (Murthy v cng s, 1998). V vy trong nhng nm gn y vic s dng TDZ tr nn ph bin. Mi trng khng c hormone thng c s dng cho s pht trin ca phi v tnh t dng hnh cu cho ti khi pht trin thnh cy con. i khi, nhng nng thp ca hormone trong mi trng cn cho s biu hin hoc trong mi trng cn cho s pht trin th c th c li, thm ch l bt buc phi c, ty theo loi, kch thch s pht trin bnh thng ca phi. Tm li, cm ng to mt cu trc lng cc cn phi c tn hiu ca mt loi hormone. Ngc li, cm ng to c quan th phi c hai tn hiu hormone khc nhau: u tin l to chi, sau to r, s dng hai mi trng khc nhau. nhng t bo phi pht trin bnh thng thnh nhng cy con th cn mt mi trng khc, c hoc khng c hormone. Thi gian x l Khong thi gian mu cy c x l vi cht iu ha tng trng l mt nhn t quan trng trong vic cm ng cng nh hnh thnh phi v tnh mt cch bnh thng. Khi mu cy c chuyn sang mi trng khng c nhn t tng trng, mt khong thi gian ti thiu l 8 ngy rt cn thit cho vic cm ng hnh thnh phi vi mt hiu qu rt cao m khng cn quan tm n dng cht iu ha tng trng s dng. S tng quan ga tui ca mu cy v sucrose Thng th tui (Wetzstein v cng s, 1989) hay giai on pht trin ca mu cy (Karunaratne v cng s, 1991) l mt nhn t quan trng trong cm ng to phi. Do , s pht trin ti a hay tui sinh l ca mu cy l mt nhn t ch yu trongcm ng to phi c hiu qu mt s loi thc vt. Hn na, ngun dinh dng c bn cn thit (sucrose/cacbonhydrate) cho cc loi m th khc nhau ng k ty vo tui pht trin (Monnier, 1990). Bn cnh , s nh hng ca cc cht kch thch tng trng khc nhau n cc loi m khc nhau th ph thuc nhiu vo ngn sucrose c trong mi trng nui cy (Thompson v Thorpe, 1987). Mt

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nng sucrose cao (4-6%) c b sung giai on cm ng v pht trin ca phi th rt c li cho vic to phi mt s loi thc vt. Nng ca mi trng Nng ca cc thnh phn trong mi trng c bn c nh hng n vic cm ng to phi v tnh. Hu ht cc nghin cu to phi trn mi trng MS u cho hiu qu to phi cao. Nng mi trng cng long th kh nng to phi cng km. Trng thi vt l ca mi trng S to thnh phi v tnh thng c cm ng trn mi trng bn rn. Tuy nhin, trng thi ca mi trng (bn rn hay lng) th rt quan trng trong vic duy tr v lm tng cng s pht trin ca phi v tnh. S cm ng to phi v tnh ca Gossypium klotzschiapum trn mi trng MS lng c b sung vitamin B, c cha cc cht iu ha tng trng cho thy t hiu qu cao (Yuqiang Sun v cng s, 2003). Lilium longiflorum, kt qu tng t cng c pht hin (Tribulato v cng s, 1997; Duong Tan Nhut v cng s, 2002). Kiu gen Mt vn thng gp trong cm ng to phi na l s ph thuc rt ln vo loi cy trng v kiu gen. Kiu gen c mt nh hng to ln trong vic cm ng to phi ca hu ht cc loi thc vt. Khng phi tt c cc thc vt u c kh nng cm ng to phi. i vi nhng cy t th phn, nhng ging cy khc nhau th kh nng pht sinh phi v tnh cng c th khc nhau. c cc th nghim khc nhau tin hnh trn cc loi c kiu gen khc nhau kho st kh nng hnh thnh phi v tnh. Chng hn nh cy Asparagus, ngi ta quan st kh nng to phi v tnh trn cc ging c kiu gen khc nhau (Hisato v Masahiro, 1998). Delbreil v Julien (1994) m t nhng khc bit quan st c t tn sut to phi t chi dnh ca 12 ging Asparagus c cc kiu gen khc nhau. Nhng khc bit v tn sut hnh thnh phi cc loi a bi ( lng bi, t bi) cng c pht hin ( Kunitake v cng s, 1992). Haensch v cng s (1996) quan

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st kh nng hnh thnh phi v tnh v ti sinh cy nhng ging Lilium c kiu gen khc nhau. Cng chiu sng Cc nhn t khc trong mi trng nui cy nh l cng nh sng cng c nh hng n vic cm ng to thnh phi v tnh. Cc nghin cu cng ba co v nhng nh hng khc nhau ca ch sng v ti. Nhn chung, ch ti cho thy c nhng nh hng tt hn trong giai on cm ng. mt s loi thc vt, s cm ng cn c nhng iu kin trong ti. Nhng iu kin trong ti c th c to bng cch bao mi trng nui cy bng nhng tm nhm mng hoc t chng trong hp carton. Nhng nghin cu to phi v tnh loi Gossypium kltzschianum v L.longiflorum t c kt qu tt khi nui cy trong ti (Yuqiang Sun v cng s, 2003; Tribulato, 1997). e. Nhng vn thng gp trong qu trnh pht sinh phi v tnh Mt vi vn thng gp trong qu trnh pht sinh phi v tnh vi loi thc vt l s xut hin nhng phi d thng. Cc l mm c th b xon li, m phn sinh chi c th c hnh dng khng bnh thng hoc c th thiu. Nhng trng hp ny cn phi c nhng thao tc ph thc y s xut hin bnh thng v s pht trin ca phi bng cch s dng nng t thp n va phi cc cht iu ha tng trng trong mi trng, s dng acid abscisic hoc nhng cch x l thm lc cao, hay bng nhng phng php vt l khc nh s sy kh (Ammirato, 1983; Parrott, 1988). Nhng thc vt c ti sinh t nui cy m so v huyn ph t bo c th b bin d v hnh thi hoc bin d phn sinh dng. Tuy nhin, nhng trng hp t xut hin nhng cy c ti sinh bi s pht sinh phi v tnh hn l nhng cy c ti sinh t s pht sinh c quan (Ammirato, 1987; Reisch, 1983). Nu nhng cy c s dng vi mc ch nhn ln, th vic kim tra li kiu di truyn l rt quan trng bng cch tin hnh kim tra hu th v ch s di truyn cho nhng c im tiu biu. Nu nhng cy c s dng vi mc ch sn xut th nhng kiu bin d nn c b i.

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Hnh 2.7 Phi c hnh dng d thng Mt iu ng ch na l s pht sinh phi v tnh v s pht sinh c quan th khng loi tr nhau. V d, c ba l c th c ti sinh t phi v tnh (Phillips v Collins, 1984). Tuy nhin khi nui cy trn mi trng thun li cho s pht trin chi th cng to ra phi v tnh, sau nui cy trn mi trng thun li cho s nhn ln ca chi th to ra nhiu cy ti sinh hn so vi khi nui cy bnh thng. V vy nhng phng php lm cho pht sinh phi v lm ti sinh c quan c kt hp lm ti u ha kh nng ti sinh cy. Tht th v khi trn cng mi trng thun li cho s pht trin sinh cc chi ca nhng phi v tnh cng l mi trng thch hp ti sinh nhng hp t phi lai khc loi. T cho thy c mt vi c im tng t gia phi hp t v phi v tnh f. ng dng ca vic to phi v tnh Phi v tnh gip cho cng tc vi nhn ging v sn xut vi s lng ln thc vt bng bioreactor, v ng thi m ra mt hng mi trong cng ngh to ht nhn to. phi v tnh cn l nguyn liu cho vic chuyn gen thc vt, cng ngh nui cy t bo trn. p dng cng ngh ng lnh phi v tnh l mt phng php rt hiu qu trong vic bo qun phi mm trong mt thi gian di. Qu trnh to thnh phi v tnh cn gip ch cho vic nghin cu tnh ton th, cng nh tm hiu c ch bit ha t bo thc vt. Vic to thnh cng phi v tnh mt s loi thc vt, c ht trn ln ht kn, cy mt l mm v hai l mm, m ra nhiu trin vng mi cho

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nn cng ngh sinh hc thc vt, tuy nhin y l mt cng vic kh khn v kh mi m.

Hnh 2.8 Chi lan H ip ti sinh t phi f. Mt s thnh tu to phi v tnh Phi v tnh c nghin cu trn hn 130 loi, bao gm cc loi ng cc, cc loi c, cy h u v cc cy c qu hnh nn (Ammirato, 1983; Wann,1988). Trong , vic nghin cu to phi Lan cng rt c cc nh khoa hc quan tm. To phi thng qua nui cy protocorm lan H ip (Jen Tsung Chen v Wei Chin Chang, i Loan): Jen-Tsung Chen v Wei-Chin Chang l 2 nh nghin cu lan ni ting chu . Hai ng tin hnh nui cy Protocorm lan H ip trn mi trng MS khng b sung cht iu ho sinh trng. Sau 45 ngy nui cy, 28.1% mu cy hnh thnh phi, trung bnh 1 mu hnh thnh 1 phi. Khi nui cy th nghim Protocorm lan H ip trn mi trng MS b sung TDZ (0.45M, 4.54M, 13.62M), hai ng thy rng TDZ c nh hng trc tip n s hnh thnh phi v tnh. Trong , mi trng b sung TDZ nng 13.62M cho hiu qu to phi cao nht: gn 100% mu protocorm hnh thnh phi (45NSC). Trung bnh

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13.5 phi/mu cy Ngc li, khi b sung thm NAA (nng 0.54M v 57.37M) s c ch s to phi. Phng php nui cy to phi lan H ip t cung hoa (Ken Tokuhara v Masahiro, Nht Bn) Theo bo co ca hai nh khoa hc ngi Nht ny, khi nui cy mu cung hoa trn mi trng New Dogashima (NDM) c cha (NAA 0,5M + BA 4,4M + sucrose 29,2 mM) th m so pht sinh phi (embryo callus) s c to thnh sau 7 thng nui cy, vi t l 73% mu cy hnh thnh embryo callus. Tc ng ca iu kin nui cy v c quan nui cy ln s hnh thnh phi soma lan Oncidium (Jen-Tsung Chen v Wei-Chin Chang): Theo bo co ca tc gi, s hnh thnh phi c lin quan n v tr mu cy: nhng mnh c v tr pha u l c kh nng to phi cao hn l nhng v tr khc v mi trng tt nht cho vic to phi lan Oncidium l: mi trng 1/2MS (cha 85mg/l KH2PO4) b sung 10-20 g/l sucrose, 170mg/l NaH2PO4 v 0,5 mg/l peptone. Phng php nui cy to phi lan H ip t m so (Nguyn B Hng , Hng Ngc Trm, 2005). Phi v tnh s hnh thnh khi nui cy m so trn mi trng MS b sung NAA 0.5 mg/l + BA 2 mg/l + ng 30g/l sau 11 tun nui cy

h h 4.2 To v bc bng sodium alginate C rt nhiu tc nhn to gel c s dng lm v bc cho phi. c th l agar, alginate, polyco 2133 (Bordon Co.), carboxyl methyl cellulose, carrageenan, gelrite (Kelko. Co.), guargum, sodium pectate, tragacanth gum, dextran, xanthan gum nhng hp cht ny s ng li thnh gel khi c cho vo mi trng c cht in phn thch hp nh ng sulphate, calcium chloride hoc ammonium chloride nh vo nhng lin kt ion. Phng php c s dng rng ri nht to v bc cho phi l s dng sodium alginate (Bajaj, 1995; Jeon v csv, 1986; Redenbaugh v csv, 1993). Alginate c s dng nhiu do n c nhng c tnh rt thun li nh tnh dnh va phi, khng gy c cho phi sinh dng, c cc c tnh tng hp sinh hc, kh nng to thnh gel nhanh, r tin, c lu, cng ca gel va phi c th va to thun li cho s h hp ca phi v va bo v c phi khi nhng tn thng bn ngoi.

Hnh : Cng thc ho hc ca alginate Alginate, cht hu c mch thng, k nc. Nguyn tc chnh trong qu trnh to v bc alginate l sodium alginate cha phi s to thnh tng ht nh, trn v cng khi nh vo trong hn hp NaCl2.2H2O nh vo s trao i ion gia ion Na+ c trong hn hp sodium alginate vi Ca2+ c trong hn hp CaCl2.2H2O. V bc cng nhiu hay t l ph thuc vo s lng ion Na+ trao i vi ion Ca2+. Do nng ca hai tc nhn to gel, sodium alginate v CaCl2.2H2O, v thi gian cho vic to lin kt ion phi tht ti u c th to thnh v bc mt cng thch hp nht. Khng nh phi hp t, phi v tnh thiu lp ni nh cha cht dinh dng bn ngoi nui phi, do bng vic thm vo cht nn gel nhng cht dinh dng, cht iu ha tng trng, carbohydrate s to mt ni nh nhn to thch hp ti a cho tng trng v sng st ca phi (Gray, 1990; Gray v Purohit, 1991; Redenbaugh v Walker, 1990). Ngoi ra nhm trnh cho phi b mt nc, hay cc tn thng c hc, tng sc khng cho phi ngi ta c th thm vo cht nn gel cht khng sinh, thuc tr nm, tr su, vi sinh vt. Mc d vic to ht nhn to t cch bc phi v tnh bng sodium alginate cho thy c mt s thnh cng nht nh trong vic ti sinh cy con, vn cn nhiu vn kh khn khi s dng v bao alginate ny, cht dinh dng c th b mt i khi v bao (Redenbaugh v csv, 1987), s trao i kh km (Redenbaugh v csv, 1993) c mt s ngh cho rng, vic thm vo than hot tnh s gip nng cao kh nng sng st,

pht trin ca phi v tnh hn. Nguyn nhn l do than hot tnh tng cng kh nng h hp ca phi, v n gi li nhng cht dinh dng nhiu hn trong v bc, phng thch chng rt chm dng cho s pht trin ca phi. 5 Quy trnh to ht nhn to

Hnh 2-5: Cc dng phi soma

Phi hnh cu

Phi hnh tim

Phi hnh thu li

Phi hnh l mm

Bc 1: Chun b nguyn liu to ht nhn to Vi nguyn liu l chi (shootbud)

Dng dao ct nhng chi (shootbud) c kch thc 2-3 mm t cy nui cy in vitro.

Vi nguyn liu l phi v tnh

Dng dao tch ri cc phi hnh tim. Bc 2. Dng pince cy gp cc phi/chi cho vo mi trng to v alginate. Bc 3 Dng pipette 10ml ht mi trng alginate c cha chi/phi nh vo dung dch CaCl2.2H2O 100mM trong 15 pht.

Bc 4.Dng pince gp ht vo a petri c cha nc ct v trng lm sch lng CaCl2.H2O cn st li.

Bc 5. Cy ht nhn to vo mi trng nui cy (MS khng b sung cc cht iu ha sinh trng).

Hnh : Quy trnh to ht nhn to

III.

ng dng trong thc t:

Mi nm nc ta sn xut hn 10 triu c khoai ty ging, trong Lt chim qu na bi c iu kin kh hu, t ai ph hp. Tuy nhin, nng sut khoai ty thp, cht lng c ging cn km v t l c mang mm bnh kh cao bi cha c quy trnh sn xut tin tin. nng cao cht lng c ging, chng ti tin hnh nghin cu, sn xut ht khoai ty nhn to; ng thi kch thch sinh trng bng k thut bc x ht nhn.

Ht khoai ty nhn to

Rung khoai ty L Nhn (H Nam) Ht khoai ty nhn to cn c u im kh b tn thng khi vn chuyn v c mt lp da bao bc bn ngoi, c th ct gi kh lu nhit thp (4-6oC). Nng sut tng t 1 n 2 ln Trng khoai ty bng ht nhn to sch bnh, cht lng cao, gi thnh h l hng i mi m ra nhiu trin vng cho nng dn v cc c s sn xut, nht l nhng vng chuyn canh khoai ty ch bin khoai sy kh xut khu. Vic chiu x bng gamma tuy to iu kin thun li ht khoai ty tn ti mi trng bn ngoi vi iu kin thin nhin khc nghit, nhng cng cn mt s bin php b tr bi ht mng manh, d b mt nc v l ming mi bo b ca vi sinh vt. Trc khi mang ht ra trng cn x l lnh t 4 - 6oC trong vng mt tun, sc nhit 38oC trong vng hai ngy hoc x l bng mt s loi axit v cht bo v thc vt.

III.

u nhc im ht nhn to u im Phi pht trin nhanh, s lng nhiu, c th h thp gi thnh v tit kim sc lao ng. Cy trng i sau mang u th ca ging cy c nh, khng h thay i. D dng s dng trong qu trnh vn chuyn, lu tr v trng. Thi gian bo qun lu hn, kh nng pht trin ht ging vn cn tt cho khong thi gian di hn. Sn phm ng nht v mt di truyn ng dng quy m ln, thch hp cho c canh quy m ln. Bo tn ging cy, trnh s tuyt chng ca cc loi c nguy c tuyt chng To iu kin nghin cu v chc nng v ht, hnh thnh ni nh trong pht trin ca phi v ny mm ht ging. Tng nng sut cy trng Nhc im Hn ch khi s dng v bao alginat: cht dinh dng c th i ra khi v bao, s trao i kh km Kh nng sng st v pht trin ca phi v tnh thp. thng mi ha ht nhn to th trc tin i hi qu trnh to phi v tnh trn cc i tng thc vt phi cho ra nhng phi pht trin bnh thng, c kiu hnh ng nht Kh nng ti nh mt s loi thc vt t phi v tnh v s thch hp kh hu sau ca cy cn gp nhiu kh khn

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