Mc lc Li ni u Chng 1: Tng quan ti liu 1.1. Gii thiu k thut nui cy t bo thc vt 1.1.1. Tng quan 1.1.1.1. Lch s pht trin 1.1.1.2. Nguyn tc 1.1.1.3. ngha 1.1.2. Cc k thut nui cy 1.1.2.1. Nui cy phi 1.1.2.2. Nui cy m v c quan tch ri 1.1.2.3. Nui cy huyn ph t bo 1.1.2.4. Nui cy chi bt nh 1.1.2.5. Nui cy to r 1.1.3. Mi trng nui cy 1.1.4. Cc cht iu ha sinh trng thc vt 1.1.4.1. Auxin 1.1.4.2. Cytokinin 1.1.4.3. Gibberellin 1.2. Gii thiu sm ngc linh 1.2.1. Phn loi 1.2.1.1. Lch s pht hin 1.2.1.2. Phn loi 1.2.2. c im hnh thi 1.2.3. Sinh thi v phn b 1.2.3.1. c im sinh thi 1.2.3.2. Khu vc phn b 1.3. Gii thiu hot cht Saponin 1.3.1. Gii thiu hot cht Saponin 1.3.2. Tch chit Saponin 1.3.2.1. i tng1

1.3.2.2. Phng php Chng 2: Vt liu v phng php 2.1. Mu th nghim 2.2. Mi trng nui cy 3.3. m bo iu kin v trng Chng 3. Cng ngh nui cy t bo sm ngc linh 3.1. Quy trnh nui cy 3.2. Thuyt minh quy trnh 3.2.1. Nui cy m so 3.2.1.1. Nguyn liu 3.2.1.2. Mi trng 3.2.1.3. Thit b v dng c 3.2.1.4. Quy trnh thc hin 3.2.1.5. iu kin nui cy 3.2.2. Nui cy r 3.2.2.1. Nguyn liu 3.2.2.2. Mi trng 3.2.2.3. Thit b v dng c 3.2.2.4. Quy trnh thc hin 3.2.2.5. iu kin nui cy 3.2.3. Nui cy sinh khi 3.2.3.1. Nguyn liu 3.2.3.2. Mi trng 3.2.3.3. Thit b v dng c 3.2.3.4. Quy trnh thc hin 3.2.3.5. iu kin nui cy 3.3. iu kin nh hng n nui cy sm ngc linh 3.3.1. nh hng ca mt s cht kh trng 3.3.2. nh hn ca cc hoocmon cho s pht sinh hnh thi 3.3.3. nh hng ca auxin ln kh nng tng sinh m so 3.3.4. nh hng ca BA ln kh nng tng trng chi2

3.3.5. nh hng ca than hat tnh n kh nng tng trng 3.3.6. nh hng ca IAA, IBA, NAA n kh nng ra r bt nh Chng 4: Kt lun Ti liu tham kho

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Li ni u............................................................................ Nhn sm Panax ginseng C. A. Meyer l cy dc liu c s dng t hng ngn nm nay. Nhng tc dng y hc ca Nhm Sm, c dng trong y hc c truyn, hin c chng minh theo nguyn l ca y hc hin i. Nhm Sm t cy thuc huyn thoi ca phng ng xut hin rng ri phng Ty, vi nhng sn phm thng mi a dng. Nhn Sm ngy nay l cy thuc mang li gi tr kinh t to ln. Nhn Sm ca Vit Nam, Triu Tin, Trung Quc l ba trong nm loi sm qu c th gii cng nhn (hai loi cn li l sm M (Panax quinquefolius v Panax trifolius mc vng Bc M) v c hp cht c trng saponin. Vit Nam t ho c c mt loi Sm c hu l Sm Ngc Linh (hay cn gi l Sm Khu Nm) Panax vietnamsis Ha & Grutzv., c bit n trn th gii vi tn gi Vietnamese ginseng. D ch mi bit n t nm 1973 nhng qua nghin cu, cc nh khoa hc nhn thy sm Ngc Linh khng ch c tc dng dc l c trng ca chi Nhn Sm m cn c nhng tc dng in hnh nh chng stress, chng trm cm, tc dng ln s chng oxy ha invitro v in vivo... Nhm cht c tc dng quyt nh nht n tc dng dc l ca loi sm ny l cc saponin triterpenoic. Sm Ngc Linh l mt trong nhng loi sm c hm lng saponin khung dammaran cao nht (khong 12 15%) v lng saponin nhiu nht so vi cc loi khc ca chi Panax trn th gii (Dong v cng s, 2007). Vi nhng c im , sm Ngc Linh khng ch l loi sm qu ca Vit Nam m cn ca c th gii. Hin nay sm Ngc Linh ch phn b trn vng ni Ngc Linh thuc hai tnh Kon Tum v Qung Nam cao 1.500 2.200m v thi gian trng t ht cho n khi thu c c ln n 5 6 nm. Do tnh trng khai thc qu mc, sm Ngc Linh hin ang nm trong 250 loi qu him, ang c nguy c b tuyt chng trong Sch Vit Nam. Do yu cu tm c nhng k thut gip tng sinh khi sm mang dc tnh nhanh v hiu qu ang tr nn cp thit. Vi mong mun trn nhm xin thc hin ti: Nhn sinh khi sm Ngc Linh chit xut hot cht saponin.

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CHNG 1: TNG QUAN TI LIU 1.1. Gii thiu k thut nui cy t bo thc vt 1.1.1. Tng quan 1.1.1.1. Lch s pht trin Nm 1665, Robert Hooke quan st c t bo di knh hin vi v a ra khi nim t bo. Nm 1838, Matthias Schleiden v Theodore Schwann xng hc thuyt t bo. Nm 1902, nh thng thi Haberlandt ln u tin a ra tng cy m ca sinh vt ra ngoi c th nhng ng dng t bo qu chuyn bit nn khng thnh cng. Nm 1904, Haning nui cy phi u tin cc loi h ci. Nm 1934, White thnh cng trong vic pht hin ra s sng v hn ca vic nui cy t bo r c chua. Nm 1951, Skoog v Miller pht hin ra cc hp cht c th iu khin s phn chi. Nm 1962, Murashige v Skoog ci tin mi trng nui cy nh du mt bc tin trong k thut nui cy m. Mi trng ca h c dng lm c s cho vic nui cy nhiu loi cy v vn cn c s dng rng ri cho n ngy nay. Nm 1964, Guha v Maheshwari ln u tin thnh cng trong to c cy n bi t nui cy bao phn ca cy c rt. Nm 1960 1964, Morel cho rng c th nhn ging v tnh lan bng nui cy nh sinh trng. T kt qu , lan c xem l cy nui cy m u tin c thng mi ha. T n nay, cng ngh nui cy m v t bo thc vt c pht trin vi tc nhanh trn nhiu cy khc v c ng dng thng mi ha. Nm 1994, thng mi ha ging c chua chuyn gene FlavrSavr. 1.1.1.2. Nguyn tc Nui cy t bo thc vt da trn hai nguyn tc: Da vo tnh ton nng ca t bo Mi mt t bo bt k ca mt c th sinh vt a bo u c kh nng tim tng pht trin thnh mt c th hon chnh khi gp iu kin thun li. Gia ng vt v thc vt khc bit v t bo thc vt c tnh ton th. Thc vt c kh nng tr li hon

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ton mt c th mi t c quan sinh dng trong khi ng vt th khng c kh nng ny. Da vo kh nng bit ha v phn bit ha ca t bo: Bit ha l s bin i ca t bo t trng thi t bo phi cho n khi th hin mt chc nng no . Phn bit ha l qu trnh t bo bit ha v cu trc v chc nng tr li trng thi t bo u tin sinh ra chng t bo phi. t bo thc vt c c kh nng bit ha v phn bit ha. 1.1.1.3. ngha Nhn nhanh ging cy trng. Bo qun ngun gene. To cy sch bnh, cy n bi kp, cy khng ht. To ht nhn to. Sn xut cc hot cht sinh hc. Lai to ging cy trng.

1.1.2. Cc k thut nui cy 1.1.2.1. Nui cy phi Phi pht trin qua hai giai on t dng v d dng. giai on d dng cn c cc cht iu ha sinh trng pht trin. Trong nui cy phi, ng ng vai tr rt quan trng, ng sucrose cho nhiu kt qu tt hn. Ngoi ra mt s cht t nhin nh nc da, dch chit malt, sacein thy phn l nhng cht rt cn trong nui cy phi. i vi cc cht iu ha tng trng, auxin thng dng nng thp, kinetin c vai tr c bit cho s pht trin ca phi. Nhit , nh sng cng nh hng n s pht trin ca phi, thng phi nui cy cn nhit , nh sng thp hn phi pht trin t nhin. Nhit 25 2 C thch hp cho sinh trng v ny mm ca phi. Cc phi th cp rt kh sinh trng hot ng khi chng c tch ri nui iu kin chiu sng 4.000 lux hoc hn trong 4 gi chiu sng sut 4 ngy nui u tin.

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1.1.2.2. Nui cy m v c quan tch ri Trong nui cy m v c quan tch ri, khu chn mu c tm quan trng c bit. Kiu di truyn quyt nh n nng sut ca cy trng cng nh kh nng ti Cc b phn ca cy non d ti sinh hn, khi cy gi i, kh nng ti sinh ca sinh trong mi trng in vitro. chng cng gim theo nhng i khi qua nhiu ln cy chuyn mu gi tng bc c tr ha. Nhng m cn non v mm thng d nui cy hn nhng m non th d b Thng thng cc b phn ca cy trong giai on sinh dng d ti sinh hn Nhng chi ban u c tch t v tr thp trn cy pht trin trong mi tn thng hn trong qu trnh kh trng. trong giai on sinh sn. trng in vitro tt hn v chi gc tng trng nhanh hn chi nch. Nhu cu dinh dng khi nui cy cc b phn khc nhau ca cy l khc nhau nhng c mt s yu cu chung nh ngun carbon, ngun ng, nguyn t a lng, vi lng v cc cht iu ha sinh trng. Trong trng hp nui cy m, cc cht iu ha sinh trng c vai tr quan trng hn v cc m tch ri khng c kh nng tng hp cc cht ny. 1.1.2.3. Nui cy huyn ph t bo Mu cy trn mi trng lng c kh nng tng trng nhanh hn so vi mi trng c. C th do mu cy c tip xc hon ton trong mi trng nn hp thu u cc cht dinh dng t mi trng. Mun nui cy huyn ph t bo u tin ta phi to c m so Ngun nguyn liu quan trng sn xut cc hot cht M so l mt khi m khng bit ha, c c tnh phn chia mnh v thng to ra do s xo trn trong qu trnh to c quan nht l qu trnh to r. Trong nghin cu sm Ngc Linh, m so cn l giai on trung gian kch thch to r, mt c quan cha nhiu hot cht saponin. Qu trnh to thnh m so c th c cm ng trong mi trng c t l

A/C~1.

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Thnh phn dinh dng cng l mt yu t quan trng nhm ti u ha qui trnh sn xut cc hp cht th cp. Hin nay, mi trng ti u cho mt loi tho dc no u phi c rt ra t thc nghim. 1.1.2.4. Nui cy chi bt nh C th kch thch s thnh lp chi bt nh t cc mu cy l v cung l ca nhiu loi thc vt. Qu trnh sinh to hnh thi c quan t cc mu cy l rt khc nhau ty thuc vo s kt hp ca cc cht iu ha tng trng thc vt trong mi trng nui cy. BAP kch thch cho s thnh lp chi trn l ca Petunia, Nicotiana, S ti to chi t cc a l Betula pendula c adenine v zeatin, kt hp vi L lan H ip (Phalaenopsis) c kh nng ti to protocorm trc tip trn b S thnh lp chi l ca Vitis vinifera trn mi trng MS c 2 mg/l BA.

Anthurium, Orchile, Sainpaulia,... -

chiu sng. mt ca l trn mi trng c BAP 10mg/l v NAA 1 mg/l. Kh nng ti to chi cn ph thuc vo kch thc ca mu cy. Mu cy qu nh khng th p ng li c vi cc iu kin nui cy v s ha nu. 1.1.2.5. Nui cy to r Giai on to r trong nui cy in vitro l giai on to cy con hon chnh c y thn, l v r chun b ra vn m. Cc cht c tc dng to chi c loi b, thay vo l cc cht kch thch qu trnh to r. S ra r ph thuc vo nhiu yu t: hm lng auxin ni sinh, t l C/N, nh sng, s tr ha ca mu, kiu di truyn. Ngi ta thng b sung auxin kch thch qu trnh ra r in vitro. 1.1.3. Mi trng nui cy Trong tt c cc mi trng nui cy u bao gm nm thnh phn chnh sau y: Cc mui khong a lng Cc mui khong vi lng Cc vitamine ng lm ngun carbon Cc cht iu ha sinh trng8

Ngoi ra, ngi ta cn b sung thm mt s cht hu c c thnh phn xc nh nh acid amine, EDTA, hoc khng xc nh nh nc da, dch chit nm men... vo trong mi trng ty nhu cu ring ca tng mi trng nui cy. Trong hng trm mi trng do nhiu tc gi ngh cho nhiu loi cy khc nhau, c th chia lm ba loi: Mi trng ngho cht dinh dng: White, Knop. Mi trng c hm lng cc cht dinh dng trung bnh: B5, Gamborg. Mi trng giu cht dinh dng: MS (Mura shige Skoog).

Thnh phn mi trng nui cy t bo v m thc vt thay i ty theo loi v b phn nui cy. i vi cng mt mu cy nhng ty theo mc ch th nghim th thnh phn mi trng cng s thay i ty theo giai on phn ha ca mu cy. T bo m thc vt i hi pH ti u cho sinh trng v pht trin trong nui cy. pH ca mi trng dinh dng nh hng trc tip ti qu trnh thu nhn cc cht dinh dng t mi trng vo t bo. V vy cn iu chnh pH mi trng thch hp trc khi nui cy. pH ca mi trng nui cy thng khong 5,8 6 th tt trong nui cy m. Nu pH mi trng thp hn 4,5 hoc cao hn 7 u c ch s pht trin ca m (Nguyn Vn Uyn, 1993 v Bi B Hng, 1995). Thng s dng NaOH hay HCl long iu chnh pH. 1.1.4. Cc cht iu ha sinh trng thc vt Cc cht iu ha sinh trng l nhng cht vi liu lng thp nhng hiu ng sinh hc cao, c tng hp ti mt c quan v gy nh hng iu tit n cc qu trnh sinh l, trao i cht no trong nhng c quan khc. N ng vai tr ch o trong qu trnh sinh trng, pht trin v nhng qu trnh sinh l, sinh ha khc cng nh trong phn ng thch nghi ca thc vt i vi iu kin mi trng (Bi Trang Vit 2000). 1.1.4.1. Auxin Auxin l nhm cht iu ha sinh trng c s dng rng ri trong nui cy m thc vt. IAA l auxin t nhin c trong m thc vt, cn NAA, IBA, 2,4-D l cc auxin nhn to, thng th cc auxin nhn to c hot tnh mnh hn.

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Auxin hot ha s phn bo, sinh trng ko di, cn cho s to mch dn v ra r, tng trng ca qu, to qu khng ht, kch thch sinh trng ca ng phn (Bi Trang Vit 2000) S vn chuyn auxin c vai tr quan trng trong s tng trng v phn ha, auxin gip ko di v phn chia t bo, cc hin tng hng ng, u th ngn, lo suy, rng, u, tng trng v chn qu... (Nguyn Vn K 2000) Trong nui cy m thc vt th auxin c vai tr kch thch to chi bt nh ( nng thp), to v nhn nhanh m so (callus) v to phi soma (2,4-D). Nhng vai tr quan trng v chin lc ca auxin l kch thch s phn chia t bo v s hnh thnh r (Trn Vn Minh 2004). S p dng loi v nng auxin trong mi trng nui cy ph thuc vo: Kiu pht trin cn nghin cu. Hm lng auxin ni sinh ca mu cy. Kh nng tng hp auxin t nhin ca mu cy. S tc ng qua li gia auxin ngoi sinh v auxin ni sinh. c tnh ca auxin.

1.1.4.2. Cytokinin Cc cytokinin l dn xut ca edenine. Cc cytokinin c s dng thng xuyn nht l BAP, Zeatin v 2-iP l cc cytokinin t nhin, cn BA v kinetin l cytokinin nhn to. Chng thng c ha tan trong NaOH hoc HCl long. Cc cytokinin kch thch s phn chia t bo vi iu kin c s hin din ca auxin. Cytokinin cng gip s gia tng kch thc t bo v sinh tng hp protein. Cytokinin ngn cn s lo ha m, thc y s hnh thnh chi non nhng li c ch s to r. T l auxin/cytokinin rt quan trng trong s hnh thnh v pht sinh hnh thi trong cc h thng nui cy. T l auxin/cytokinin cao c tc dng gip to m so v r i vi s pht sinh phi. Ngc li, t l auxin/cytokinin thp s dn n s sinh sn chi v chi nch. Vai tr ca cytokinin: Kch thch phn chia t bo. To v nhn m so.10

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Kch thch pht sinh chi trong nui cy m. Kch thch pht sinh chi nch v km hm nh hng u th ca chi nh. Lm tng din tch phin l do kch thch s ln ln ca t bo. C th lm tng s m ca kh khng mt s loi. To chi bt nh nng cao. c ch s hnh thnh r. c ch s ko di chi. c ch qu trnh gi (ho vng v rng) l, kch thch to dip lc.

1.1.4.3. Gibberellin GA3 l cht c s dng nhiu nht trong 20 cht thuc nhm gibberellin. Chng c to ra ti nh r, chi, l non v ht. C tc dng kch thch ko di chi v ny mm ca phi v tnh. C kh nng ha tan trong nc. Vai tr ca gibberellin: Kch thch ko di chi do tng cng phn bo v ko di t bo. Ph ng ht ging hoc c ging. Kim sot s ra hoa ca cc cy 2 nm tui. c ch s hnh thnh r bt nh. Kch thch sinh tng hp ca -amylase ht cy ng cc ny mm. c ch tng hp kch thch qu trnh to c. Kch thch s ny mm ca phn hoa v sinh trng ca ng phn. C th gy to qu khng ht hoc lm tng kch thc qu nho khng ht. C th lm chm s ho gi l v qu cy c mi.

1.2. Gii thiu sm ngc linh 1.2.1. Phn loi 1.2.1.1. Lch s pht hin Vit Nam c nhiu cy thuc c gi l Sm. Nhng ch c bn loi Sm thuc h Nhn Sm (Araliaceae). Chi Panax gn gi vi cy Nhn Sm Panax gingseng C. A. Meyer l Sm Ngc Linh, Sm Tam Tht, Sm Nam v Sm V Dip, u l cc cy thuc qu. Nm 1968, KS.V c Minh tm thy cy Sm Ngc Linh ti vng ni Ngc Linh v tm t tn n l Sm Khu 5. Nm 1973, on iu tra cy thuc ca Ban dn11

y Khu 5 do DS. o Kim Long dn u pht hin mt loi Panax mc trn vng ni Ngc Linh v tm t tn l Sm t Trc vi tn khoa hc Panax articulatus, h Nhn Sm (Araliacea). Tn khoa hc ca cy c cng nhn l Panax vietnamensis Ha et Grush., h Nhn Sm Araliaceae, cng b ti Vin thc vt Kamarov (Lin X trc y) nm 1985 do H Th Dung v I. V. Grushvistky t tn. 1.2.1.2. Phn loi ng). Tn nc ngoi: Vietnamese ginseng. 1.2.2. c im hnh thi Cy tho, sng lu nm nh thn r, cao khong 40 80 cm, c trng hp cao trn 1m. Thn r nc, mc b ngang nh c gng, nhiu t, khng phn nhnh, mang nhiu r nhnh v c, c nhiu vt so do thn kh sinh hng nm li, mt ngoi nu nht, rut trng ng, phn cui ui i khi c c hnh cu. Thn r mang nhiu r ph. Hnh dng rt thay i, c bit i vi dng sm trng, r c rt pht trin, tng hng nm rt r rt, thng chng c 3 dng chnh: dng c c rt, dng con quay v dng b c (dng rt ph bin). Sm Ngc Linh trng thng cao n 1 m, mang nhiu l khp kn, c khi n 7 l cht to hn bnh thng. Thn kh sinh mnh, mc thng, mu xanh hoc hi tm, mang 2 4 l kp chn vt mc vng. Gii: Plantae Ngnh: Magnoliophyta B: Apiales H: Cam Tng (Araliaceae) Chi: Panax Loi: Panax Loi: Panax vietnamensis Ha et Grush Tn khc: Sm Vit Nam, Sm Ngc Linh, Sm Khu Nm, Thuc Du X

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Hnh 1.1: R c Sm Vit Nam

Hnh 1.2: Cy Sm Vit Nam 5 tui

Hoa t, cm hoa mc thnh tn n ngn thn, cung da 10 20 cm, c th kem 1 4 tn ph hay 1 hoa ring l bn di tn chnh. Hoa nhiu, mu lc vng, hoa hnh tn n mc di cc l thng vi thn. Mi tn 60 100 hoa, cung hoa ngn. L kp chn vt mc vng vi 3 5 nhnh l, cung l kp, mang 5 l cht hnh phin bu dc,trng ngc hoc hnh mc, di 10 14 cm, rng 3 5 cm, gc hnh nm, mp kha rng ca, chp nhn, c lng c 2 mt. Qa hch, hnh trng, khi chn c mu hay vng nht, sau en, ht hnh thn mu trng, b mt ht c nhiu ch li lm, c vn, qu mc tp trung trung tm ca tn l, di 0,8 1 cm v rng khong 0,5 0,6 cm, mi qu cha mt ht, mt s qu cha 2 ht v s qu trn cy bnh qun t 10 30 qu. Ma hoa: thng 4 thng 6. Ma qu: thng 7 thng 9.

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Hnh 1.3: Sm Ngc Linh 1.Ngn thn mang hoa; 2. N; 3. Hoa; 4. Qa; 5. Ht; 6. Thn r (c). 1.2.3. Sinh thi v phn b 1.2.3.1. c im sinh thi Sm Ngc Linh l loi cy thn tho a m v a bng, sinh trng cao t 1.200 2.100 m so vi mt bin, mc ri rc hay tp trung thnh tng m nh di tn rng. Mi trng rng ni c Sm mc lun m t, thng xuyn c my m, nhit khong 15 - 180C, lng ma khong 3000 mm nm. t rng y c to thnh do l cy mc lu ngy, c mu nu en, ti xp, hm lng mn cao v cha nhiu nc.14

Sm Ngc Linh sinh trng mnh trong ma xun h . Ma hoa qu t thng 5 thng 10, cy ra hoa qu tng i u hng nm. Sau khi qu chn rng xung t, tn ti qua ma ng khong 4 thng v s ny mm vo u ma xun nm sau, Sm c kh nng ti sinh t nhin t ht kh tt. Sm c phn thn trn mt t li hng nm, li cc vt so r. Cn c vo vt so trn thn r tnh tui ca Sm. 1.2.3.2. Khu vc phn b Trong s hn mi loi v di loi bit ca chi Nhn Sm Panax), Vit Nam c ba loi mc t nhin v mt loi l cy nhp trng. Sm Ngc Linh c pht hin sau cng vo 1973. n 1985 n mi c cng b l hon ton mi i vi khoa hc. n nay Sm Ngc Linh ch mi c pht hin duy nht vng ni Ngc Linh thuc hai tnh Qung Nam v Kon Tum. Trong s hn mi loi v di loi bit ca chi Nhn Sm Panax), Vit Nam c ba loi mc t nhin v mt loi l cy nhp trng. Sm Ngc Linh c pht hin sau cng vo 1973. n 1985 n mi c cng b l hon ton mi i vi khoa hc. n nay Sm Ngc Linh ch mi c pht hin duy nht vng ni Ngc Linh thuc hai tnh Qung Nam v Kon Tum. V gii hn cng nh phn b ca loi sm ny ni Ngc Linh hin nay c nhiu thay i. 1.3. Gii thiu hot cht Saponin 1.3.1. Gii thiu hot cht Saponin Saponin hay saponosid l mt nhm glycosid ln, gp rng ri trong thc vt. Chng c xem l thnh phn hot cht ch yu ca cy Sm Vit Nam cng nh cc loi Sm khc trn th gii. Cc saponin damaran c xem l hot cht quyt nh lm nn cc tc dng sinh hc. T phn di mt t ca Sm Vit Nam hoang di phn lp v xc nh c cu trc protopanaxadiol oxid II v 52 hp cht saponin bao gm 26 saponin bit v 26 saponin c cu trc mi c t tn l vina-ginsenoside-R1-R24 v 20- O-MeG.Rh13,4,5). Cc saponin dammaran c xen l hot cht quyt nh cho cc tc dng sinh hc c ga tr ca Sm Triu tin cng chim mt t l rt cao v hm lng v s lng trong thnh phn hp cht saponin ca Sm Vit Nam 50 52 saponin c phn lp).15

Trong cc saponin dn cht ca 20 S)-protopanaxadiol gm 22 hp cht vi cc i din chnh l: ginsenoside-Rb1, -Rb3, -Rd. Cc saponin dn cht ca 20 S)protopanaxatriol gm 17 hp cht vi cc i din chnh l: ginsenoside- Re, -Rg1, notoginsenoside R1. Cc saponin c cu trc ocotillol gm 11 hp cht vi cc i din chnh l: majonoside R1 v R2. c bit M-R2 chim gn 50% hm lng saponin ton phn t phn di mt t ca Sm Vit Nam v tr thnh 1 hp cht ch yu ca Sm Vit Nam so vi thnh phn saponin trong cc loi sm khc trn th gii v gp 48 ln hiu sut chit c t i dip tam tht. Hai saponin dn cht ca acid oleanolic ch chim mt t l rt thp vi hemsloside Ma3 c pht hin u tin trong mt loi Panax thuc h Nhn sm. Bng 1.1: Hm lng mt s Saponin chnh trong sm Hm lng saponin chnh (%) Nguyn liu G-Rb1 u mm Sm 2 tui Sm 3 tui R c 4 tui R c 5 tui R c 6 tui Thn r 4 tui Thn r 5 tui Thn r 6 tui 0.943 0.979 0.846 0.818 1.721 1.824 1.518 1.565 2.716 GRd 0.898 0.426 0.678 0.396 0.518 0.632 1.778 0.981 0.840 G-Ry1 1.359 1.235 1.419 1.696 2.219 2.285 2.432 1.652 3.648 R2 2.859 1.868 2.409 3.141 3.816 4.166 2.946 4.276 5.342 Tng cng 6.059 4.236 5.352 6.051 8.674 9.474 8.674 8.494 12.546

16

Bng 1.2: Hm lng Saponin ca Sm Ngc Linh so snh vi cc loi Panax spp. trng trt Loi aglycon 20(S)ppd 20(S)ppt Ocotillol Oleanolic acid Hiu sut ton phn (%) Panax Panax Panax Panax

ginseng notoginseng quinquefolius vietnamensis 2.9 0.6 0.02 3.5 2.1 2.4 4.5 2.7 1.2 0.04 0.07 4.0 3.1 2.0 5.6 0.09 10.8

(Ghi ch: 20(S)-ppd: 20(S)-protopanaxadiol; 20(S)-ppt: 20(S)-protopanaxtriol) 1.3.2. Phng php tch chit Saponin 1.3.2.1. i tng Thn r v r c sm Ngc Linh ti c thu hi ti cc vng c trng. Cc chun ginsenosid Rb1, Rd v Rg1 c phn lp vi tinh khit cao (trn 98 %). 1.3.2.2. Phng php Ch bin dc liu Thn c v r c sm Ngc Linh sau khi ra sch, kho st tnh dc liu v mt vi phu, th tinh khit v nh tnh bng sc k lp mng. Mu sm ti c em sy nhit 500C n khi t m quy nh cho dc liu kh theo tiu chun (khng qu 13 %). Mu sm ch bin c chia thnh cc phn em hp 1000C trong cc khong thi gian 2, 4, 6, 8 gi ln lt c nh s S1, S2, S3 v S4. Sau cc mu sm hp c em sy nhit 500C cho n khi t m di 13 %. Chit saponin ton phn Cc mu sm c ct lt v chit bng thit b chit Soxhlet vi dung mi l methanol 100 % thu ly cao ton phn. Cao ton phn c phn tch bng ct pha o Diaion HP-20 vi cc dung mi ra ln lt l nc, methanol 100 % v cloroform.

17

Dch methanol 100 % c c quay thu hi dung mi v sy kh trong t sy chn khng thu c rn saponin ton phn. Phn tch bng sc k lp mng Mu cao saponin ton phn c ha trong dung mi MeOH v c chm vi cng mt lng mu trn bng mng silicagel, s dng h dung mi CHCl3-MeOHH2O vi t l 65:35:10.

Hnh 1.4: Sc k sc k lp mng Phn tch bng sc k lng hiu nng cao Thc hin chy mu HPLC trn my sc k lng hiu nng cao. S dng h dung mi A v nc theo t l: - T 0 10 pht: 15% A + 85% B. - T 11 20 pht: 15% - 25% A + 85% - 75% B. - T 21 30 pht: 25% - 35% A + 75% - 65% B. - T 31 40 pht: 35% - 40% A + 65% - 60% B. - T 41 50 pht: 40% - 45% A + 60% - 65% B. - T 51 60 pht: 45% - 50% A + 55% - 50% B. - T 61 70 pht: 50% - 25% A + 50% - 75% B. - T 71 85 pht: 25% - 15% A + 75% - 85% B. Cn mu chnh xc vi khi lng bng nhau v ha tan trong pha ng, lc mu vi mng lc kch thc 0,45 m trc khi bm. Th tch mi ln bm l 20 l, tc dng 1 ml/pht, pht hin bc sng 206 nm.18

CHNG 2: PHNG PHP V VT LIU 2.1. Mu th nghim Th nghim c tin hnh trn cy Sm Ngc Linh c th l mu l, thn, hoc r... 2.2. Mi trng nui cy Ngoi vic s dng mi trng nn MS, cn b sung thm mt s thnh phn c trng ty theo tng giai on tin hnh. Bng 2.1: Thnh phn mi trng MS (Murashige v Skoog, 1962): Thnh phn NH4NO3 KNO3 Khong a lng CaCl2.2H2O MgSO4.7H2O KH2PO4 MnSO4.4H2O ZnSO4.7H2O H3BO3 Khong vi lng KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O St EDTA Na2.EDTA FeSO4.7H2O Myo-Inositol Thiamin (B1) Vitamin Nicotinic acid Pyridoxine HCl Glycine Nng (mg/l) 1650 1900 440 370 170 23,3 8,6 6,2 0,83 0,25 0,025 0,025 37,3 27,8 100 0,1 0,5 0,5 2

19

Mi trng nui cy lun c iu chnh pH = 5,8. Mi trng c hp kh trng bng autoclave nhit 1210C, p sut 1 atm. Bng 2.2: Thi gian hp kh trng mi trng nui cy m 1210C Th tch mi trng (ml) 20-25 50 100 250 500 1000 2000 3000 4000 2.3. m bo iu kin v trng Mi trng nui cy c cha ng, mui khong,... rt thch hp cho cc loi nm v vi khun pht trin v vi tc pht trin nhanh hn rt nhiu ln so vi t bo thc vt. Dng c thu tinh: Ra sch, hp trong ni p sut 121 oC/15 pht. Nt y: S dng nt cao su. Nt c nht bng gn khng thm nc bi Mu nui cy: Cc mu ly ngoi t nhin nh cy, l, hoa, cnh cn ra sch S dng cc cht c hot tnh dit khun nh cn chlorua, nc javen,... V trng ni thao tc v t cy. Mi trng: Mi trng thung c hp kh trng 121 oC, 1atm trong 30 Thi gian ti thiu (pht) 24 26 28.5 31.5 35 40 48 55 63

v khng kh khng c i qua. Sau em i hp trong ni hp 121 oC/15 pht. bng x bng di vi nc nhiu ln hn ch s tp nhim. pht.20

C mt s cht trong mi trng nui cy khng bn nhit (vitamin, khng sinh,..), nhng cht ny c lc v trng sau c thm vo mi trng sau khi c hp, lm ngui n khi cm trn tay c. Bng 2.3: Nng v thi gian s dng 1 s cht x l m cy thc vt Thi gian kh Stt Cht kh trng Nng trng (pht) 1 2 3 4 5 6 7 Hypochlorite calcium Hypochlorite sodium Nc bromine Oxy gi Chlorua thy ngn Nitrate bc Khng sinh 9-10% 0,5-5% 1-2% 3-12% 0,1-1% 1% 4-50mg/l 5-30 5-30 2-10 5-15 2-10 5-30 30-60 Rt tt Rt tt Rt tt Tt Tt Tt kh Hiu qu

21

CHNG 3: CNG NGH NUI CY T BO SM NGC LINH 3.1. Quy trnh nui cy Mu Sm Ngc Linh

Kh trng mu cy

Nui cy to m so

Nui cy to r

Tng sinh sinh khi

Chit xut hot cht Saponin 3.2. Thuyt minh quy trnh 3.2.1. Nui cy m so (Dng Tn Nht v cs) 3.2.1.1. Nguy n liu: Cc mu l v cung l. 3.2.1.2. Mi trng: S dng mi trng nn MS. Ngoi ra cn b sung: 1.0 mg l 2,4-D v 0.2 mg l TDZ. Mi trng nui cy c iu chnh pH = 5.8. Mi trng c hp kh trng bng autoclave nhit 1210C, p sut 1atm. 3.2.1.3. Thit v dng c: Phng chun b mi trng, kh trng mi trng nui cy, bo qun dung dch m. Gm c ni hp mi trng, t lnh, bp in, cn phn tch, ng ong, pipet, my o pH.22

-

Phng cy v trng gm t cy, n UV, cc dng c hp v trng. Phng lnh nui cy gm k st, n chiu sng, nhit k, my iu ha. Cc dng c gm n cn, a, dao ct mu, ko, bng gn, chai ng nghim

v trng loi 250 ml, 500 ml, giy v trng, giy thun. 3.2.1.4. H a cht:-

Cn 960, 700. Nc ct v trng. Dung dch javel. Dung dch x phng long. ui tr nh thc hin Chn mu. Ct mu l c kch thc khong 1.0 1.0 cm. Cung l b dc v ct on Ra nh mu bng bng tm nc Javel. Ra sch s b mu bng nc. Lc mu trong cn 700/ 30 giy. Ra bng nc ct 4-5 ln. Lc mu trong dung dch HgCl2 0.1 % c nh vi git Tween-20 trong 5 pht. Ra li bng nc ct v trng 5-6 ln. Kh trng dng c. t mu vo mi trng y np, bao gi ghi tn mi trng, mu cy, ngy thng, em vo phng

-

3.2.1.5. -

khong 1.0 cm. nui. 3.2.1.6. iu in nui cy: Nhit : 25 20C. Cng chiu sng: 2.500 3.000 lux. Thi gian chiu sng: 16 gi ngy. m: 75 - 80 %.

23

Hnh 3.1: M so Sm Ngc Linh

Hnh 3.2: Cc giai on ca Sm Ngc Linh ti sinh m so c ngun gc t l. (a). cy ly mu l, (b). s hnh thnh m so, c v d). qu trnh tng sinh ca m so, (e). ti sinh chi bt nh, (f). s hnh thnh r, g). cy con a ra vn m, (h).cy con pht trin trn ni Ngc Linh, tnh KonTum sau 2 thng.24

3.2.2. Nui cy r (Dng Tn Nht v cs) 3.2.2.1. Nguy n liu: S dng cc m so hnh thnh. 3.2.2.2. Mi trng: S dng mi trng nn MS. Ngoi ra cn b sung: 3.0 mg l NNA. Mi trng nui cy c iu chnh pH = 5.8. Mi trng c hp kh trng bng autoclave nhit 1210C, p sut 1atm. 3.2.2.3. Thit v dng c: - Phng chun b mi trng, kh trng mi trng nui cy, bo qun dung dch m. Gm c ni hp mi trng, t lnh, bp in, cn phn tch, ng ong, pipet, my o pH. - Phng cy v trng gm t cy, n UV, cc dng c hp v trng. - Phng lnh nui cy gm k st, n chiu sng, nhit k, my iu ha. - Cc dng c gm n cn, a, dao ct mu, ko, bng gn, chai ng nghim v trng loi 250 ml, 500 ml, giy v trng, giy thun. 3.2.2.4. H a cht: Cn 960, 700. Nc ct v trng. Dung dch javel. ui tr nh thc hin

3.2.2.5. nui.

Kh trng dng c. Ct nh m so khong 0.5 0.5 cm t vo mi trng. y np, bao gi ghi tn mi trng, mu cy, ngy thng, em vo phng

3.2.2.6. iu in nui cy: Nhit : 25 20C. Cng chiu sng: 2500- 3000 lux. Thi gian chiu sng: 16 gi ngy. m: 75- 80 %.

25

Hnh 3.3: R Sm Ngc Linh 3.2.3. Nui cy sinh khi (Dng Tn Nht v cs) 3.2.3.1. Nguy n liu: Cc m so hnh thnh 3.2.3.2. Mi trng: S dng mi trng nn MS. Ngoi ra b sung 1.0 mg l 2.4 D v 0.2 mg l TDZ. 3.2.3.3. Thit v dng c: Phng chun b mi trng, kh trng mi trng nui cy, bo qun dung dch m. Gm c ni hp mi trng, t lnh, bp in, cn phn tch, ng ong, pipet, my o pH. Phng cy v trng gm t cy, n UV, cc dng c hp v trng. Phng lc nui cy gm h thng bnh tam gic 250 300 ml, hay cc h

thng nui cy bioreactor, my lc, my lnh, n chiu sng. Phng thu, tch chit hot cht. Cn 960, 700. Nc ct v trng. Dung dch javel. ui tr nh thc hin: 3.2.3.4. H a cht: -

3.2.3.5.

- Ct nh cc m so - Cho vo mi trng. - y np chai, bao giy. Ghi ngy thng, tn mi trng, ging. - em i lc lng 3 thng). - Sau thi gian ny ta thy khi lng t bo c nhn ln rt nhiu v c th em i lc ly t bo nhm thu sinh khi hay nhn sinh khi.26

3.2.3.6. iu in nui cy: Nhit : 25 20C. Cng chiu sng: 2.500- 3.000 lux. Thi gian chiu sng: 16 gi ngy. m: 75- 80 %. Thi gian lc lng: 3 thng. S vng: 100 120 vng/ pht.

Hnh 3.4: Nui cy trong Bioreactor 3.3. iu kin nh hng n nui cy sm ngc linh 3.3.1. nh hng ca mt s cht kh trng Thit k th nghim: Chn chi mc thng hay chi bn lm nguyn liu nui cy c chiu di 2-3 cm, ct b ht l, x l bng cn 70 % trong 1 pht t cy v trng, sau trng li 3 ln bng nc ct v trng, sau x l mu bng dung dch Ca(OCl2) hay HgCl2 sau trng bng nc ct v trng nhiu ln cho sch ht cht kh trng. Sau khi kh trng xong ct phn u c mu xanh, chiu di 0,4 cm t vo mi trng. Bng 3.1: Kt qu kho st nh hng ca cc loi nng cht kh trng: Mu Sm Ngc Linh (Mm, thn, Thi gian (pht)27

S mu 10 5 0,2 % 10 15 20

HgCl2 0,3 % 5 5 15 20 0,4 % 5

100% 100% 100% 20% 40% 40% Cht Cht

Cht

r)

Mu Sm Ngc Linh (Mm, thn, r)

S mu 10 5 Thi gian (pht) 0,2 % 10 15 20

Ca(OCl)2 0,3 % 5 5 15 20 0,4 % 5

100% 100% 100% 40% Cht Cht Cht Cht

Cht

Kt qu: Sm Ngc Linh c x l bng HgCl2 vi nng 0,2 % trong 20 pht c t l nhim thp (khong 20 %). Cn x l bng Ca(OCl)2 vi nng 10 % trong 5 pht th t ln nhim thp (khong 10 %). 3.3.2. nh hng ca cc hoocmon ln s pht sinh hnh thi Sm Ngc Linh Thit k th nghim: S dng cc mu mm, thn, r Sm Ngc Linh. Nui cy phi hp trn cc t hp mi trng khc nhau nh gi s pht sinh hnh thi. Bng 3.2: nh hng ca cc hoocmon ln s pht sinh hnh thi Sm Ngc Linh i tng phn cy Hoocmon Kt qu pht sinh h nh thi IBA Mm, Ngc Linh thn, r 0,2 0,5 IAA NAA K 0,2 0,5 2iP G Cht Cht

Sm

28

0,5 0,2 0,5 0,5 0,5 8 1

1 0,5 0,25 1 3 0,5 3 1 0,5

M so M so R Phi soma Phi chi R Chi

Kt qu: - Hoocmon IBA (0,5 mg/l) + 2iP (1 mg/l): Sm Ngc Linh pht sinh phi soma. - Hoocmon NAA (1 mg/l) + G (3 mg/l): Sm Ngc Linh pht sinh phi chi - Hoocmon NAA (8 mg/l) + 2iP (0,5 mg/l): Sm Ngc Linh pht sinh r. 3.3.3. nh hng ca auxin ln kh nng tng sinh m so Sm Ngc Linh Thit k th nghim: Cc mu m so c to t giai on khi to c cy vo mi trng MS b sung 0,2 mg/l TDZ v cc loi auxin 2,4-D, IBA v NAA vi nng thay i t 0,5; 1; 2; 3 v 5 mg l trong iu kin chiu sang 16 gi/ ngy. Bng 3.3: nh hng ca auxin ln kh nng tng sinh m so Sm Ngc Linh Nng Auxin (mg/l) 0,5 1 2,4-D 2 3 3,73 2,65 3,18 4,56 T l tng sinh hi kh

29

5 0,5 1 IBA 2 3 5 0,5 1 NAA 2 3 5

2,50 3,45 3,56 3,63 3,10 2,53 2,81 3,33 3,37 3,38 3,77

Kt qu: Sau qu trnh tng sinh, m so nui cy trn mi trng b sung 1 mg/l 2,4-D c t l tng sinh khi kh cao nht. S kt hp gia auxin v cytokinin lm tng kh nng thu nhn ng v cc cht dinh dng, ko theo s tng sinh ca m so. 2,4-D li l cht cho t l tng sinh khi cao nht. c bit, m so trn mi trng b sung 2,4-D c hnh thi tt nht v c kh nng ti sinh cao. 3.3.4. nh hng ca A l n qu tr nh tng trng chi Sm Ngc Linh Thit k th nghim: Nhng chi tt nht sau khi thu nhn c th c tch v chuyn vo mi trng MS c b sung 1 g/l than hot tnh, 30 g/l sucrose, 0,5 mg/l NAA v BA ( 0,5; 1; 2; 4 mg/l). Bng 3.4: nh hng ca BA ln qu trnh tng trng chi Sm Ngc Linh BA (mg/l) 0,5 Trng lng ti (g) 0,61 Chiu cao chi (cm) 5,66 S lng l chi 330

1 2 4

0,87 0,72 0,71

6,16 4,11 4,33

3,3 4 3,9

Kt qu: Trong cc nng BA c s dng, nng 1 mg/l BA kt hp vi 0,5 mg/l NAA cho kt qu tng trng chi cht nht vi trng lng ti ca chi l 0,87 g v chiu cao chi l 6,16 cm. Nh vy mi trng nui cy c b sung 1 mg/l BA v 0,5 mg/l tt nht cho qu trnh tng trng chi. 3.3.5. nh hng ca than hot tnh n kh nng tng trng chi Sm Ngc Linh Thit k th nghim: Nhng chi tt nht trong th nghim c tch v cy chuyn sang mi trng MS c b sung 0,5 mg/l NAA, 1 mg/l BA, pH = 5,7 vi cc nng than hot tnh ln lt l 1; 2; 3 v 4 g /l. Bng 3.5: nh hng ca than hot tnh n kh nng tng trng chi Sm Ngc Linh Than hot tnh (g l) 0 1 2 3 4 Trng lng chi (g) 0,53 0,61 1,01 0,97 0,94 Chiu cao chi (cm) 3,3 4,6 5,3 6,8 8,5 S lng l chi 3,6 3,7 3,3 2,7 3,1

31

Kt qu: Than hot tnh khng phi cht iu ha sinh trng thc vt, nhng c kh nng thay i thnh phn mi trng. Than hot tnh iu chnh pH mi trng, trng, hp th cc cht lm cn tr s pht trin ca m. Kt qu thu c cho thy khi nng than hot tnh tng th c s thay i r rt v trng lng cng nh chi cao ca chi. Nng 2 g/l than hot tnh l ph hp nht cho qu trnh tng sinh chi Sm Ngc Linh. 3.3.6. nh hng ca IAA, I A, NAA n kh nng ra r bt nh t m so Bng 3.6: Thit k th nghim: M so c cy vo mi trng ra r c cha cc auxin (NAA, IBA, IAA) cc nng ln lt l 1; 3; 5; 7 mg/l. Nng Auxin (mg/l) 1 3 NAA 5 7 1 3 IAA 5 7 1 IBA 3 5 10 0 70 80 100 1,6 0,1 4 0,3 4,8 0,3 7,83 5,21 15,81 16 21 18 0,2 0,2 70 50 0 0 2,6 0,1 2,1 0,1 6,23 12,21 9 8 (%) 30 100 r mu 3 0,3 8,7 0,1 T l ra r S lng T l hi lng r mu ( ) 5,98 21,88 Chiu di ti a ca r (mm) 18 13

32

7

60

3,5 0,1

8,06

1,7

Kt qu: D dng nhn thy IAA khng thch hp cho qu trnh ra r Sm Ngc Linh t m so. NAA v IBA th ngc li. Nng 3 mg/l NAA cho kt qu t nht vi t l ra r ln n 100 %, IBA nng 5 mg l cng cho t l ra r t 100 %. iu ny c th gii thch auxin tng hp c hot tnh cao hn auxin t nhin.

33

CHNG 4: KT LUN Sm Ngc Linh c cng nhn l mt trong nhng cy Sm c hm lng saponin cao v vi s lng nhiu nht so vi cc loi khc trn th gii. V th vic nui cy m t bo thc vt gp phn khng nh n vic bo tn ngun dc liu qu gi ny. T qu trnh kho st cho thy nng 3 mg/l tr nn ca 2,4-D l khng ph hp cho s pht sinh m so ca Sm Ngc Linh. i vi qu trnh ra r t m so, m so c nui cy trn mi trng MS b sung NAA cho t l cao nht.

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Ti liu tham kho [1]. Dng Tn Nht v cng s, ti Hot cht saponin t cc nguyn liu nui cy invitro ca cy Sm Ngc Linh (Panax vietnamensis Ha et Grushv.), Trung Tm Sm v Dc Liu TP. HCM. [2]. Nguyn c Lng, (2002), Cng ngh t bo, NXB HQG TP. HCM. [3]. Bi Trang Vit, (2000). Sinh l thc vt i cng. i hc quc gia Thnh ph H Ch Minh. [4]. Duong Tan Nhut, (2006), Primary study on multiplication of adventitious roots of Panax vietnamensis A valuable material source for saponin isolation, Ho Chi Minh City. [5]. Nguyn Th Liu, Nguyn Trung Thnh, Nguyn Vn Kt, (2011), Nghin cu kh nng to r bt nh ca Sm Ngc Linh (Panax vietnamensis, Ha et Grushv.) trong nui cy in vitro, Khoa Nng Lm, i Hc Lt. [6]. Nguyn Trung Thnh, Nguyn Vn Kt, Paek Kee Yoeup, (2007), nh hng mi trng nui cy n s tng trng sinh khi v s tch ly sn phm ginsenoside trong nui cy t bo lng ca Sm Ngc Linh (Panax vietnamensis Ha et Grushv.) [7]. TS. Trn Cng Lun, Sm Vit Nam, Trung tm Sm v dc liu TP H Ch Minh. [8]. Nguyn Vn Thun, Nguyn B Hot, Nguyn Vn Bt, Bc u nghin cu trng cy Sm Ngc Linh (Panax vietnamensis) di tn rng t nhin [9]. L Th Hng Vn, Nguyn Ngc Khi, Nguyn Minh c, (2010), Nghin cu s nh hng ca qu trnh ch bin ln s thay i thnh phn ha hc saponin v tc dng tng lc ca Sm Vit Nam.[10]. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays

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