Tgp chi Cdng ngh? Sinh hpc 12(4): 659-666, 2014
N G H l f e N C t r u N H A N G I 6 N G IN VITRO C A Y D A N G S A M (CODONOPSIS JAVANICA B L U M E ) T H O N G Q U A N u 6 l C A Y C H O I N G U
Phan Xudn HuySn, Nguyen Lam Thanh
Vi?n Nghien cuu Ichoa hpc Tdy Nguyen, Vi?n Hdn ldm Khoa hpc vd Cong nghe Viet Nam Ngay n h | n bdi: 01,7,2014
Ngay nhan dang: 30.9.2014 T6M TAT
Trong nghien ctm ndy, chung toi nhan giong in vitro cay ddng sam co nguon goc td cao nguyen Lang Biang - Lam Dong, la mpt trong nhirng loai thao dugc qui tot cho sue khoe cua con nguoi Cac choi ngii cua dot thdn in vitro dugc dung lam nguon vat li^u cho cac thi nghiem. Sau 30 ngay nuoi cay, gua khao sat cac nong do BA thi moi trudng MS bo sung 0,5 mg/I BA, 30 g/l sucrose, 8 agar g/l, pH 5,8 la tot nhat len sy tai sinh va sinh truong chii cdy (10,40 choi/mdu, chieu cao 2,20 cm). Sy kit hgp giira BA va NAA thi moi ttuong MS CO chua 0,5 mg/l BA, 0,1 mg/l NAA, 30 g/I sucrose, 8 g/l agar, pH 5,8 la tot nhdt len sy tai sinh va sinh tnrdng chii cay (17,70 chli/mlu, chilu cao 2,78 cm). Kinetin (0,1 - 1 mg/l) kit hgp vdi 0,1 mg/l NAA thi kich thich mau cay tai sinh re in vitro, nhung lai lie che su tdi sinh vd sinh truong choi cay. Tren ciing m6i trudng nuoi cay, moi truong c6 bo sung them 1 g/l than boat tinh kich thich sy hinh thdnh re ciia mau ca^ va choi cdy sinh trudng xanh tot, nhung khong thich hgp cho qua ttinh tai sinh choi cay. Moi truong MS co bo sung 0,1 - 1 mg/l IBA hoiic NAA ho|ic lAA, 30 g/l sucrose, 8 g/l agar, pH 5,8 diu thich hgp cho qud trinh tao rl in vitro sau 20 ngdy nuoi cay. Gid the mun xo dua va gia the 14 mun xo dira + !4 cat deu thich hgp cho su sinh trudng ciia cay con t^i vudn uom sau 20 ngay cham soc, vdi ti 1? song dat 100%.
Tir khda: choi ngii, ddng sdm. dot thdn. sinh trutmg choi cdy, tdi sinh choi
MO DAU
Ngay nay, ciing vdi sy phdt triSn cua xa hgi, con ngudi CO xu hudng quay ve vdi thien nhien, sd dung cdc loai thao dugc de cai thi?n sue khoe vd chua benh.
Trong do, cay dang sam (Codonopsis javanica Blume) Id mOt loEii thdo dugc qui, co tac dyng bo ngu tang, nang cao thi lye, tang sdc deo dai, kha nang miln dich cho ca the, ich huylt, sinh tan dich, chong m^t mdi, gidm sttess, O nude ta, cay ddng sam mgc rai rac tai cac tinh miln nui phia Bdc. CJ phia Nam, cdy ddng sam chi thdy tap tiimg xung quanh chan niii Nggc Linh (Ddc GIdy, Kon Tum, Quang Nam - Da Nang) vd cao nguyen Lang Biang - Lam Dong (Dinh Cong Bay, 2010; Dd Tdt Loi, 2013; Doan Trgng Due, 2014).
Hi^n nay, nan pha rimg lam nuong ray lam cho khu phdn bo cua dang sam ngdy cang thu h?p, them vao do, ngudi dan dia phuong thu hdi dang sam mgt each tan diet tu cu gid den cu non, ddn den ngudn dang sam ttong ty nhiln ngdy cdng giam d ^ va c6 nguy ca tuy?t chung. Bdi vay, vi?c nghien cdu nhdn giong ya phdt trien nguon gen thao dugc qui ddng sam la van de can thiet. Trfin the gidi da cd nhiing cdng bo nhan gidng in vitro cdy ddng sam nhu: (Min et al, 1992) nghien cdu phat sinh phdi xd ma d tan so cao vd tai
sinh cay con td mdu Id mam cua cay Codonopsis lanceolata, (Sheng et a/., 1993) nghien cuu taeh protoplasts td callus cua doan than tru dudi la mam cay Codonopsis pilosula (Franch.) Narmf, (Susumu et al, 1995) nhdn giong cay Codonopsis lanceolata TRAUTV. thong qua nuoi cdy chdi dinh, (Zhang et al, 2011) nghien cdu phdt sinh chii tryc tilp tit chii ngu cda dot than cay Codonopsis pilosula (Franch.) Nannf., (Slupski etal, 2011) nghien cdu nhdn giSng Codonopsis pilosula (Franch.) Nannf. thdng qua nudi cay choi ngu cda ddt thdn. (3 nude ta, hien nay da cd mgt so nghien cdu nhdn giong in vitro cay ddng sam nhu (Dodn Trgng Due, 2014) nghien cuu tai sinh choi cdy ddng sam tir callus. Trong nghien cdu ndy, chiing tdi nghien cdu phat sinh chdi tryc tiep tir choi ngu ciia ddt than. Ket qud cua nghien cuu nay gop phan bao t i n va phat tiiln nguon gen thao dugc qui dang sam ciia Viet Nam.
V A T L I E U V A PHUONG PHAP
Nhijng than day cay dang sdm thu td cao nguySn Lang Biang - Lam Dong dugc rda sach qua nude xa
phong, sau do khu trung qua con 70" ttong 1 phut va cudi cung khd tidng qua dung dieh HgCIp 0 , 1 % ttong 8 phut. Mdu sau khi khd trung xong tien hanh cat cac dot than cdy tten mdi tmdng MS (Murashige, Skoog, 1962) CO bd sung 0,3 rag/1 BA (6-benzyl adenin), 0,1 mg/l NAA (a-napthalene acetic acid), 30 g/I sucrose, 8 g/l agar, pH 5,8 (Slupski et al, 2011).
Cac chdi cay tai sinh td choi ngu ciia dot than sau khi khd trung (Hmh la) dugc diing lam ngudn vat lieu cho cdc thi nghiem.
Mdi trirdng nuoi cay
Moi trudng dugc su dung ttong nghien cim nay la mdi trudng MS, tiiy theo muc dich cua cdc thi nghiem md bo sung dgc lap hay phoi hgp giua cac chdt kich thich sinh trudng BA, Kinetin, NAA, IBA (Indole-3-butyric), lAA (Indonle-3-acetic acid), than boat tinh, sucrose va agar.
Dieu ki^n nuoi cay
Thdi gian chilu sdng 10 gid/ngay, cudng do anh sdng 2.500 lux, nhiet do 25±2°C va do dm phong 75 - 85% .
Phuong p h i p
Khdo sdt dnh hu&ng cua BA ten khd nang tdi sinh vd sinh trudng choi cdy
Moi trudng MS CO bo sung cac nIng do 0; 0,1; 0,3;
0,5; 0,7 va 1 mg/l BA, 30 g/t sucrose, 8 g/l agar, pH 5,8, Chi tieu theo doi la so choi va chilu cao choi cay.
Khdo sdt dnh hudng cda BA vd NAA len khd ndng tdi sinh vd sinh trirdng cua chdi cdy
Moi trudng MS b l sung cac nIng do 0; 0,1, 0,3;
0,5; 0,7 vd 1 mg/l BA kit hgp 0,1 mg/l NAA, 30 g/l sucrose, 8 g/I agar, pH 5,8. Chi tieu theo doi la s6 choi vd chieu cao chdi cay.
Khdo sdt dnh hudng cua Kinetin vd NAA len khd ndng tdi sinh vd sinh trudng choi cdy
Moi trudng MS bo sung cac nong do 0,1; 0,3;
0,5; 0,7 vd 1 mg/l Kinetin ket hgp 0,1 mg/i NAA, 30 g/I sucrose, 8 g/l agar, pH 5,8. Chi tieu theo ddi la s6 choi vd chieu cao choi cay.
Khdo sdt dnh hudng cua than hogt tinh len khd ndng tdi sinh vd sinh trudng choi cdy
Ke thua ket qua cua thi nghiem tten, chgn mdi trudng tot nhdt cho sy tdi sinh vd sinh trudng chdi cay de tien hdnh thi nghiem nay. Thi nghiem gdm co
Phan Xudn Huyen & Nguyen Ldm Thanh hai nghiem thdc, b l sung va khdnp bo sung 1 g/l than boat tinh. Chi tieu theo doi Id so chdi, chieu cao choi cay vd ti le tao r l .
Khdo sdt dnh hudng cua IBA, NAA vd lAA len khd ndng tdi sinh re in vitro
Nhiing chdi cay in vitro (Hinh la) tuang duong ve chieu cao dugc cay tren moi trudng MS co bd sung 0; 0,1; 0,5 va I mg/l NAA, IBA va lAA, 30 g/I sucrose, 8 g/I agar, pH 5,8. Chi tieu theo doi la so re, chilu dai re va ti le tai sinh re.
Khdo sdt dnh hudng cua gid the len khd ndng thick nghi cua cdy con dgiai dogn ex vitro
Nhimg cay con in vitro ddng deu ve chieu cao va so r l dugc ttong tten gia the myn xo dua vd gid the V2 mun xo dda + V2 cdt. Chi tifiu theo doi Id chieu cao cay, chieu dai re va ti le song.
X u ly so li^u
Moi nghidm thdc cua thi nghi?m in vitro dugc cay 15 mau, thi nghiem chuyen cay con ra vudn uom thi m l i gid the dugc ttong 30 cay. So li?u ciia cdc thi nghiem dugc xd ly bdng phdn m l m thing kg SPSS (ban 15,0) theo phuang phap Duncan's test (Duncan, 1995) d l i vdi cdc thi nghidm td 3 nghidm thdc trd len, theo phuang phdp T-test dli vdi nhiing thi nghiem c6 2 nghi?m thdc, muc dg tin cay P < 0,05,
KET QUA VA THAO LUAN
Khao sat anh h u d n g cua BA lln khd ndng tdi sinh va sinh t r u d n g c h i i cay
Khd ndng tdi sinh vd sinh trudng choi cdy tir choi ngu ciia ddt than sau 30 ngay nudi cay dugc the hien tten bang 1. Ket qud thi nghidm cho thay, BA CO su anh hudng tich eye len sy phat sinh vd sinh trudng choi cdy, tuy nhien d cac nong dg khac nhau thi sy phat sinh vd sinh trudng chdi cay khde nhau, Moi trudng co b l sung 0,5 mg/l BA Id tit nhdt, vcri so choi thu duac la 10,40 choi/mdu, chilu cao choi cay 2,20 cm (Hinh 1 b^). Khi tdng ndng dg BA tu 0 - 0,5 mg/l thi su phat sinh va sinh trudng choi cay tdng len (Hinh lb], Ibi, Ibj), nhung khi nIng dg BA tdng len 0,7 vd I mg/i thi so choi tao thanh va chilu cao chdi gidm xuIng (Hinh lbs, Ib6). Dilu nay cho thdy, khi ndng do BA thap thi kich thich sy tao chii va tang tnrdng chieu cao chdi cay, khi ndng dg BA tang cao thi dc che su tao choi va tang trudng chilu cao chdi cay.
Tgp chi Cong nghe Sinh hpc 12(4): 659-666, 2014
Bdng 1. Anh hufrng cOa BA l£n khd nang tdi sinh vd sinh truong choi cay sau 30 ngdy nuoi cay
N&ng d $ B A (mg/l) S6 chli/mlu
3,40'' 4,50'' 7,60"
10,40"
6,60' e.so'
Chieu Ci 1,56"
1,62' 2,15°
2,20' 1,91"
1,25^
)o c h o i cdy (cm)
Chu t h i c h : NhOng ky t g khde nhau (a, b, c, d, e, f) trong c$t the hi§n s y si ph6p t h d Duncan's test
I khde c6!? nghTa vtt\ dg tin cdy P < 0,05 trong
Chat dieu hoa sinh trudng BA Id mgt ttong nhiing chat thugc nhom cytokinin c6 vai ttl quan trgng ttong phdn chia t l bdo vd kich thich sy hinh thanh chdi. Bdi vay, ttong nudi cay rad te bao thyc vat BA thudng dugc su dyng ttong giai doan nhdn nhanh. Nong dg cua BA dugc sd dyng ttong nhdn gilng in vitro a nhiing loai cdy khac nhau Id khac nhau, CO loai cdy thich hgp d nong dg thap, co loai cay thich hgp d nong do cao. Ket qua cua thi nghiera nay phu hgp vdi (Susumu et al. 1995) nghien ciru nhan gidng in vitro cay Codonopsis lanceolata TRAUTV. da sd dung BA d nong dg 0,5 mg/l tao cum choi tot nhat (11 choi/raau),
Nhu vay, qua khdo sdt cac nong dg BA thi moi trucmg MS eo bo sung 0,5 rag/1 la tot nhat len sy tdi sinh va smh trudng choi cay tu choi ngu ciia dot than cay ddng sdm.
Kbao sdt anh hudng cua BA va NAA len kha nang tai sinh va sinh trudng choi cay
Qua khao sat cdc nong dg BA kit hgp vdi 0,1 rag/I NAA, kha nang tai sinh vd sinh trudng chdi cay tu choi ngu cda dot than sau 30 ngay nudi cay dugc the hien tten bdng 2. Ket qua thi nghi|m cho thay, khi BA k i t hgp vdi 0,1 mg/l NAA thi sy tai sinh choi cay tot hon khi su dyng BA dgc lap (Bang I). Ndng dg BA tai 0,5 mg/I kit hgp vdi 0,1 mg/I NAA la t i t nhdt, vdi sd chdi tao thdnh Id 17,70 choi/mdu, chieu cao choi cdy 2,78 cm (Hinh IC4). Trong thi nghiem nay, choi cay bieu hien tuong tu nhu thi nghiem sd dyng BA dgc Idp, khi ndng dg BA tang len 0 - 0,5 mg/l thi kich kich su tao choi va tang trudng chieu cao choi cdy (Hinh Ic], IC2, IC3), nhung khi nong dg BA tdng len 0,7 vd 1 mg/I thi uc chi sy tao choi va sinh trudng choi cdy (Hinh Icj, ICg).
Bdng 2. Anh hu-dng ciia BA vd N/VA Ign khd ndng tdi sinh vd sinh tnrdng c h l l cay sau 30 ngdy nufii cdy N o n g d $
Chieu c a o c h o i cay (cm 3,50^
5,50"
12,40°
17,70' 11,40"
7,50'
1,55' 2,12' 2,40"
2,78' 2,54"
1,75"
C h i i t h i c h : 'Nh&ng ky t y khde nhau (a, b, c, d, e, 0 ' m " g cot the hi?n s y sai khde c6 J nghTa v6'i dg tin cay P < 0,05 trong phdp t h d Duncan's test.
Trong nhdn gilng in vitro, nhieu tac gia da nghien cuu cho thdy, khi ket hgp cac chat dieu boa sinh trudng giiia nhora cytokinin vd auxin thi nang cao hi?u qua tai sinh va sinh trudng chdi cdy. Slupski et al. (2011) cho thdy BA ket hgp vdi NAA ttong
nghien cdu vi nhan giong Codonopsis pilosula (Franch.) Nannf. thdng qua nuoi cay choi ngu vd thu dugc so choi cao nhat la 69 choi/mau sau khi cay chuyen 4 lan tten moi trudng mdi. Zhang et al, (2011) Cling sd dung BA k i t hgp vdi NAA ttong 661
Phan Xuan Huyen & Nguyen Lam Thanh nhan gidng cay Codonopsis pilosula (Franch.)
Nannf., tu m l i c h i i ngu ddt than phat smh 15 - 18 chii, Dodn Trgng Ddc (2014) nuoi cay mo cay dang sam da kit hgp giiia BA va NAA trong nhan nhanh callus va tai smh 3 - 5 chii cay tu nhdng mau callus CO dudng kinh 5 mm.
Nhu vdy, raoi trudng MS cd bd sung 0,5 mg/l BA, 0,1 mg/I NAA la t i t nhdt len su tai smh va smh trudng choi cay td chii ngu cda dot than cay dang sdm.
Khao sat dnh hirdng cua Kinetin va NAA l&n su tai sinh va sinh trirdng choi cay
Qua khdo sat cac nong d6 Kinetin k i t hgp vdi 0,1 rag/I NAA, kha nang tai sinb vd smh tiirdng c h i i cay tu choi ngu cua dot than sau 30 ngdy nuoi cay dugc the hi?n trfin bang 3. Ket qud thi nghiem cho thay, sy tai sinh vd sinh trudng choi cay bi dc che, m l i dot than tai smh mgt chii cay va bieu hien hinh thdi ben ngoai can coi, chieu cao ch6i cay chi dat td 1,21 - 1,61 cm (Hmh ldi, Id2,^ Idj, Id., Id;), Nhung ngugc lai thi kich thich cac choi cdy tai sinh r l manh me, khi nIng do Kinetin tang 0,1; 0,3, 0,5; 0,7 va 1
mg/l thi ttgng lugng tuoi cua re tang Ita, tuong dug la 0,16; 0,17; 0,19; 0,28 va 0,35 g. D i l u nay cho thdy, khi tang ndng dg Kmetin thi kich thich c h i i cdy phat sinh re va tang ttgng lugng tuoi cda bg re, nhung lai uc chfi tdi sinh va tang trudng chdi cay.
Kinetin la mgt chat thugc nhom cytokinin c6 vai tto quan ttgng ttong phdn chia t l bao va kich thich sy hmh thanh chii cay, Slupski vd ddng tdc gia sir dyng Kinetm ttong nhan giong in vitro cdy Codonopsis pilosula (Franch.) Nannf. Qua day cho thay, ddi tugng cay dang sam k h i n g theo nguyen tSc chung ttong nuoi cdy mo te bao thyc vat khi ti Ie Kinetin/NAA ldn hon 1 thi kich thich mdu cay phat sinh chii cay, nhung ngugc lai kich thich mau cdy tai sinh vd tang sinh ttgng lugng tucmg cda rl, KSt qua nay md ra hudng nghien cdu nhdn sinh khoi re IW vitro cay dang sdm.
Nhu vdy, moi trudng MS co b l sung cac nong dg Kinetin k i t hgp vdi 0,1 mg/l NAA thi kich thich raau cdy tdi sinh va tang sinh re, nhung lai dc chi sy tai sinh va sinh trudng c h i i cay.
Bdng 3. Anh hu&ng ciia Kinetin va N/\A len s y tai sinh vd sinh trudng c h l i cdy sau 30 ngay nuoi cay.
N&ng d 6 Kinetin (mg/I) 0,1 0,3 0,5 0,7 1,0
NAA (mg/l) 0,1 0,1 0.1 0,1 0,1
1 , 6 f 1,50=
1,35"
1,30'"
1,21'
Chi4u cao c h o i cay (cm) T r g n g l u g n g tUKri cOa r l (g) 0,16'
0,17' 0,19"
Chii t h i c h : Nhung ky t u khde nhau (a, b, c, d) trong cpt the hign s y ssi khac co y nghTa v&\ dfl tin cdy P < 0,05 trong phdp thip Duncan's test.
Khao sat anh h u d n g cua than hoat tinh ISn s\f tai sinh va sinh t r u d n g c h i i cdy
Cac choi ngu cua dot than dugc nuoi cay tten moi ttudng MS CO chua 0,5 mg/l BA, 0,1 mg/l NAA, 30 g/l sucrose, 8 g/l agar, pH 5,8 va co bo sung 1 g/l than hoat tinh. Kha nang tai sinh va smh trudng choi cay sau 30 ngdy nuoi cay dugc the hien tren bang 4. Kit qua tid nghi?m cho thay, moi trudng co b i sung than boat tinh thi 100% mau cay phdt sinh re, ttong khi do, moi Qudng khong cd than hoat tmh tiii mlu ciy khong tao re (Hinh Ici, le:). S i chii cay tao Ihanh tten m6i tiTidng khdng co than hoat tinh (17,50 chii'mlu) cao hon tten moi hudng co than hoat tmh (2,40 chli/mdu), nhung chieu cao va sy sinh trudng chii cdy tidn raoi
trudng c6 tiian hoat tinh t i t han tten raoi trudng khong c6 than hoat tinh. Dieu nay cho thay, khi bo sung than hoat tinh vao moi trudng nudi ciy thi kich thich mau cay phat sinh r l vd chii cdy sinh trudng xanh tit, nhung lai dc che qua trinh tai sinh chii cdy
Trin the gidi va ttong nude da cd nhirng cong bo cho thay than hoat tinh c6 tac dung kich thich tao re in vitro, ket qud cua thi nghiem nay phu hgp vdi kSt qua ciia Dodn Trgng Ddc (2014) khi nghiSn cuu tai sinh re in vitro cdy dang sdm co bo sung 0,5 mg/l BA, 0,7 mg/l IBA va I g/l than hoat tinh; Pham Thi Thu Hdng et al, (2013) nghien cdu anh hudng cua tiian hoat tinh len khd nang tao re in vitro cay ttdu bd canh phugng va cho thay 100% m l u ciy phat sinh r l khi
Tgp chi Cong nghe Sinh hpc 12(4): 659-666, 2014
bo sung 0,5 - 2 mg/l than hoat tinh sau 4 tudn nuoi cay, Chapla etal. (2009) cho thdy than hoat tinh d ndng do I - 2 mg/l deu thich hgp len su sinh trudng vd tao rl in vitro cay Miltoma flavescens Lindl..
Nhu vay, raoi trudng c6 bo sung 1 g/l than hoat tinh thi kich thich mau cay tdi sinh rl, chdi cay sinh trudng xanh tdt, nhimg Iai uc chi qua trinh phdt sinh chdi cay.
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Hinh 1. Nhdn gilng in vitro cdy dang sdm: a Choi cdy dang sdm m vitro: b,, b?, bj, b^, bs vd be. Choi cay tr§n mdi tnrgng bo sung 0; 0,1; 0.3; 0,5; 0,7 vd 1 mg/l BA, Ci, Cj, Cj, Ca, Cs va Cg Choi cay fr6n moi tn^ong bo sung 0; 0,1; 0,3, 0,5; 0,7 va 1 mg/l 8A k^t hgp vd\ 0,1 mg/l NAA; di, d!,.da, da va dj. Choi cay trfin moi tryong bo sung 0,1; 0,3, 0,5; 0,7 va 1 mg/l Kinetin k^t hgp viti 0,1 mg/l NAA, ei vd 62, Choi cdy trSn moi tryb'ng co vd khong c6 than hoat tinh; fi, fs, U vd U Cay in vitro tren moi trudng bisung 0, 0,1; 0,5 vd 1 mg/l IBA; g,, g?. ga vd g^. Cay in vitro ben moi Inrong bo sung 0; 0,1; 0,5 va 1 mg/i NAA, h,, hj, hj va ht. Cdy in vitro trdn moi trud'ng bo sung 0; 0,1; 0,5 va 1 mg/l lAA, i, va \! Cay dang sdm trSng tren chau gid t h i myn xo dija vd gid t h i Vi myn xo dOa + Vi cat; ki vd kj. So sdnh cay dang sam trong tren gia the myn xo dira va gia the 'A mvn xo dt>a + Vi cdt.
P h a n X u a n H u y e n & N g u y i n L d m Thanh Bang 4. Anh huong cua than hoat tinh I6n s y tdi smh va sinh truo'ng c h i i cdy sau 30 ngdy nufli cay
Moi tru'O'ng nuoi cdy C h i ^ u cao c h i i cay (cm) Ty I f t ^ o re (%)
Co ttian hoat tinh Khong co than hoat tinh
2,40' 17,50"
3,10' 2,75"
Ctiii t h i c h : 'Nhirng ky t y khde nhau (a, b) frong cgt the hi$n s y sai khac c6 y nghTa voi dO tin cdy P < 0,05 trong phdp Ihir T-
Khao sat anh hudng cua IBA, NAA, lAA len qua trinh tdi sinh r l in vitro
Khd nang tai sinh re in vitro cua cac choi cay dang sam sau 20 ngay nuoi cay dugc the hien tten bang 5. Ket qud thi nghidm cho thay, nghiem thuc khong bo sung chdt dieu hoa sinh trudng thi ti le tao re la 33,33%. Cdc nghidm thdc bd sung 0,1 - I mg/l IBA, NAA va lAA thi ti 1^ tdi sinh re dat 100%. Tuy nhien, cac chit dilu hoa sinh trudng khac nhau vd d nhung nong dg khac nhau thi qud trinh tao re khac nhau. Tac dyng cua IBA, khi d ndng do 0,5 rag/1 thi so re phat sinh nhieu nhdt (9,60 re/mau), khi tdng len 1 mg/l thi so re va chieu dai re giam xuong, IBA d nong do 0,1 va 0,5 mg/I thi cho thiy tu re chinh mgc ra nhilu rl phy (Hinh lf2, Ifs), Tac dung ciia NAA, khi ning do tang 0 , 1 - 1 mg/l thi s i re tang len, khi tdng nfing do 0,1 - 0,5 mg/I thi chieu ddi re tang I6n, nhung khi tang len I mg/I thi chieu dai re Igi gidm xu6ng (Hinh lg2, Igj, lg4). Tdc dyng ciia lAA, khi nong do tdng 0 , 1 - 1 mg/l thi so re va chieu dai re gldm xuong (Hinh l h | , Ih^, Ihs), d nong do 1 mg/l
cho thay kich thudc re idn hon d nhirng nIng dg khac.
IBA, NAA va lAA la nhiing chat thugc nhom auxin c6 tdc dung kich thich'tgo r l in vitro, tren tbe gidi vd ttong nude cd rat nhieu cong bo da su dpng IBA, NAA, lAA d l nghien cdu tai sinh re in vitro:
(Slupski et al. 2 0 I I ) cho thay lAA d nong do 0,9 mg/l lAA la thich hgp nhat len su tao r l in vitro cdy Codonopsis pilosula (Franch.) Nannf.; (Zhang etal, 2011) ciing nuoi cay mo cay Codonopsis pilosula (Franch.) Nannf. va cho thay ti 1? tdi sinh re djjl- 100% khi IBA d ndng do 1,5 mg/I; (Doan Trgng Die, 2014) nghiin cdu tao re in vitro cay dang sam da sir dyng 0,7 mg/l IBA kit hgp vdi 0,5 mg/I BA va 1 g/l than hogt tinh, (Pharn Thi Thu Hdng et ai, 2013) da chi ra NAA d cac n i n g dg td 0,25 - 0,75 mg/I deu thich hgp tao re in vitro cay ttdu ba canh phugng, vdi ti Ie tai sinh re 100% sau 4 tuan nudi cay,
Nhu vay, IBA, NAA va lAA d cac nong d | ti 0 , 1 - 1 mg/I deu thich hgp cho qua ttinh tao re in vitro cay dang sam.
Bang 5. Anh h u o n g IBA, HAA. lAA l&n qud Irinh tdi smh re in vitro cdy ddng sam sau 20 ngay nuoi cay.
NAA (mql\) lAA (mg/l) So re/mau C h i l u ddi r i (cm) x y l# t ^ o rS (%) 1,30'
4,50"
9,60' 6.10°
6,50' 14,50' 19,20' 9.00' 8,27"
4,20°
1,56"
0,84"
0,97"
0,46"
0,75' 1.35^
1.10' 0.84"
0.35' 0,32"
C h i i t h i c h : Nhu-ng ky t y khde nhau (a, b, c, d, e, f, g, h) trong c6t t h i hien s y sai khac co y nghTa v6i dO tin cdy P < 0,05 trong phdp t h y Duncan's test.
Tgp chi Cong ngh? Sinh hpc 12(4): 659-666,2014 Khao sat dnh h v d n g cua g i i the l£n kba n a n g thicli nghi ciia c3y con d giai doan ex vitro
Kha nang thich nghi va sinh trudng cua cay con ddng sam d giai doan vudn uom sau 20 ngay ttdng vd cham sdc tren gia the myn x a dua va gid t h i Vi myn x a dda k i t hgp vdi Vi cat dugc thi hien tten bdng 6, Ket qud cho thay, ti le song ciia cay deu dat 100%, nhung cay con ttong tten gia t h i 54 mun xa dua + Vl cdt (chieu cao cay 6,12 cm, chilu dai re 4,60 cm) CO chieu cao cay cao ban va chieu dai r l dai hon nhiing cay trong tten gid t h i mun x o dua (chieu cao cay 5,91 cm, chieu ddi r l 2,72 cm). Dieu nay cho thay, cay dang sam ciy md co sue s i n g manh va thich nghi cao vdi d i l u ki^n tu nhien. Han nda, gid t h i myn xo dda va gia t h i myn xo dda k i t hgp vdi cdt cd dg thdng thoang va giir dm thich hgp cho sy sinh trudng cda eay con ttong giai doan dau d dieu kien vudn uom. Viec chuyen cay con tii Ing nghiem ra vudn uom Id mgt cong doan quan trgng trong nuoi cay m6, cdy con in vitro thudng nuoi cay ttln moi trudng thach nen khi chuyin ra vudn uom
bg re phai thich nghi ttin nhirng gia t h i radi, han nda, do dm d mdi trudng ty nhien thap, ddn den cay thudng bi heo va chet, vi vay, ttong thdi gian dau can phai che chan va phun suong giii am cho cay con. Gid the mun xo dua la mgt ttong nhung loai gia the dugc su dyng ttong cay con d giai doan vudn uora, tten the gidi va ttong nude da c6 nhieu c i n g bd sd dyng gia thi mun xo dua d l t r i n g cdy cay md tai vudn uom: (Stefanello et al. 2009) ttong cay con Miltonia flavescens Lindl. tten gia the mun x a dua da eho thay ti 1| sdng cua cay d^t 100% sau 30 ngdy cham soc; (Yamamoto et al, 2009; Yamakami et al ,2009) ciing su dyng gid the myn x a dua ttong cay con Miltonia sp.; (Pham Thj Thu Hang et al, 2013) da tring cay con trdu bd canh phugng tten gid t b i raun x a dda phdi ttgn vdi trau hun, vdi ti le song cda cay dat 100% sau 4 tuan cham sdc.
Nhu vay, gid the myn x o dda vd gid thi Vi myn x a dua + V2 cat deu thich hgp tting cdy con ddng sam tai vudn uom.
Bdng 6. kn\\ hudng ciia gid the len sy thich nghi ciia cdy con b giai do^n ex vitro sau 20 ngdy chdm s6c Gid t h i t r i n g
Mgn x o dii-a VS myn x o dCfa + Vi cdt
Chieu cao cay (cm) 5,91'•
6,12"
C h i i u ddi re (cm) 2,72"
4.60"
Ty 1$ song (%) 100 100
Chu thich: 'Nhiing ky ty khac nhau (a, b) trong cgt thi hi§n sy sai khde co y nghTa vdi dO tin cgy P < 0,05 trong phdp thip T-test.
KET LUAN
Tu cac ket qud nhdn dugc, chiing t6i riit ra kit luan choi ngu cua dot than la nguon vat lieu thich hgp cho qud trinh nhan gidng in vitro cay ddng sam.
Qua khdo sdt cdc n l n g dd BA lln sy tai sinh va sinh trudng chdi cay thi mdi trudng MS c6 bd sung 0,5 mg/l BA, 30 g/I sucrose, 8 g/l agar, pH 5,8 la tdt nhdt.
Sy phoi hgp giira BA va NAA thi moi trudng MS c6 b i sung 0,5 mg/l BA, 0,1 mg/l NAA, 30 g/l sucrose, 8 g/l agar, pH 5,8 la t i t nhdt len qud trinh tdi sinh va sinh trudng chdi cay. Ndng dg tu 0,1 - 1 rag/I Kinetin ket hgp vdi 0,1 mg/l NAA thi dc che sy tdi sinh va sinh trudng chdi cdy, nhimg lai kich thich mau cdy tdi sinh vd tang sinh r l , kit qua ndy md ra hudng nghiin cdu nhdn sinh k h l i r l in vitro cay dang sdm. Tren cdng rapt radi trudng nuoi cay, m l i trudng cd bo sung them I g/I than hoat tinh thi kich thieh cdc mdu cay phat sinh re va choi cay sinh trudng xanh tdt, nhung lai dc ehe qua trinh tai sinh chii. Mdi trudng ed bd sung 0 , 1 - 1 mg/l IBA hoac NAA hoac lAA d i u thich hgp eho qua ttinh tao r l in
vitro. Gid the myn xo dua vd gid the Vi myn xa dua + Vl cdt dfiu thich hgp chuyen cay con ra vudn uam.
Ldi cam on: Cdc tdc gid xin chdn thdnh cdm on Vien Nghien cuu Khoa hpc Tdy Nguyen da tgo dieu kien cho chdng toi hodn thdnh nghien ciiu ndy.
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Nha xuit ban Hong Ddc: 811-813,
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S T U D Y O N IN VITRO P R O P A G A T I O N O F CODONOPSIS JAVANICA B L U M E VIA A X I L L A R Y S H O O T C U L T U R E
Phan Xuan Huyen-, Nguyen Lam Thanh
Tay Nguyen Institute of Scientific Research, Vietnam Academy of Science and Technology
SUMMARY
In the present study, we propagated Codonopsis javanica Blume plant denved from the Lang Biang highland of Lamdong province, which is one of the valuable herbal plants and has been proved to be good for the human health. Axillary buds of in i iiro stem nodes were used as material source for all experiments. After 30 days of culnue. for Uie BA experiment, MS medium supplemented with 0.5 mg/l BA, 30 g/l sucrose, 8 g/l agar, pH 5 8 was the best for shoot growth and regeneration (10.40 shoots/explant, height of 2 20 cm). For the experiment using BA in combination with NAA, MS medium containing 0.5 mg/l BA, 0 1 mg/l NAA, 30 g/l sucrose, 8 g/l agar, pH 5.8 was the best for shoot growth and regeneration (17.70 shoots/explant, height of 2.78 cm). Kinetin (0.1 - I mg/l) in combination with NAA at O.l mg/l stimulated root regeneration but inhibited shoot growth. The same culture medium with tiie addihon of I g/l activated charcoal stimulated root formation of explants and shoots growth, but was not suitable for shoot regeneration. MS medium containing 0 . 1 - 1 mg/l IBA or NAA or lAA. 30 g/l sucrose, 8 g/I agar, pH 5,8 was found to be the most suit^le for in vitro rooting. The substrate of coconut fiber powder and substtate of half coconut fiber powder in combination with half sand proved the most suitable for plantlet growth in the greenhouse after 20 days of transplantation, with a survival rate at 100%.
Keywords: axillary shoot. Codonopsis javanica Blume, stem node, shoot regeneration, shoot growth i 11
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