Vietnam J Agri. Sci. 2016. Vol. 14, No 6' 921-930 Tgp chi KH Ndng nghiep Viet Nam 2016. tdp 14, sd 6: 921-930 www.vnua.edu.vn
NGHIEN ClJrU CAM UfNG VA NUOI CAY R I BAT DjNH CAY BA KICH {Moriada officiaa/isHovi]
Ninh Thi Thao*, Nguyen Thi Phueng Thao, Nguyen Thi Thuy Linli, Nguyin Tuan Minli, Nguyen Quynh Chi, Tran Thi Anh Dao
Khoa Cong nghe sinh hgc, Hpc vi$n Nong nghiep Viet Nam Email*: ntthao(^vnua.edu.vn
Ngay gui bdi: 11.05.2015 Ngay chdp nhan: 28.06.2016
T O M TAT
Nghien cdu nay dugc tien hanh nhdm tgo ngudn re bat djnh cay ba kich in vitro va budc dau khao sat mpt sd yeu td den su tang tm-dng r l bdt dmh. Dogn than va la cay ba kich duoc nudi cdy tren mdi trudng MS bo sung a- N/y\, lAA va IBA vdi 5 nong do khac nhau (0,1-1,0 mg/1) de cam dng tao r l bat dinh. Kdt qua cho thdy. a-NAA the hign hieu qua tao r i bdt dinh to doan than cay ba kich t6t hon so vdi 1/\A va IBA TE le dogn than tgo re dat cao nhat (100%) trgn mdi trudng bd sung 0,75 mg/I a-NAA. Khdc vdi vat lieu dogn than, mo la cay ba kich hoan toan khdng cam dng tao re bat dmh tren mdi trudng MS + 0,75 mg/I a-NAA sau 4 tuan nudi cdy. Kha nang tang trudng cua r l bat djnh cdy ba kich tren mdi trudng ndn B5 cao hon so vdi mdi trudng nIn MS. Bo sung a-NAA, lAA va IBA vao moi trudng nuoi cay cd tac dyng thuc day su tang sinh khdi r l bat djnh ba kich. Mdi tru'dng 85 + 1,0 mg/1 a-NAA la thich hop nhat cho nudi cdy r l bdt djnh ba kich, cho khoi lugng r l tuoi dat duac cao nhdt (1,264 g) sau 12 tuan nudi ciy.
Td khda- Ba kich, re bat dinh, a-NAA, 1/SkA, IBA.
Adventitious Root Induction and Culture of Morinda officinalis How
ABSTRACT
This paper presents the results on induction of adventitious roots of Morinda officinalis How and effect of some chemical factors on adventitious root growth. Stem and leaf explants were cultured on MS medium supplemented with a-NAA. lAAand IBA at five concentrations (0.1-1.0 mg/1) to regenerate adventitious roots. Among the auxins, a- NAA showed the highest adventitous root formation for stem explants, followed by lAA and IBA. The maximum percentage of stem explants induced root (100%) was obtained when cultured on MS medium supplemented with 0.75 mg/I a-NAA after 4 weeks of culture. However, adventitious root induction was not obsen/ed in leaf explants cultured on MS + 0.75 mg/1 a-NAA Of the two tested media, B5 medium sustained better root growth than MS medium. a-NAA, lAA and IBA had positive effect on adventititous root growth and development. After 12 weeks, the highest adventitious root biomass (1.264 g) was obtained on 85 medium supplemented with 1.0 mg/1 a-NAA
Keywords: Adventitious root, a-N/\A. l/V\, IBA, fiJIorinda officinalis How.
1. D A T V A N D E anthranoid, physcion, rubiadin, daucosterol,..
Dich chidt eua cay ba kich da dflgc chflng minh Cay ba kich (Morinda officinalis How) la cd tac dung bd than, tang cfldng sflc de khang, mdt cay dfldc lieu phan bd tu nMen ci Lao, Vidt giam huyet ap, bd tri nao, giup an va ngd ngon Nam, Trung Qudc, An Do, Trieu Tien. Theo (Li et al, 2003). 6 Viet Nam, ba kich dupc xg'p nghign cdu cua Zhang et al. (2013), cu cay ba dflng dau trong nhom cac vi thudc bo dUdng khi kich chfla nMeu boat chat dfldc ligu quy nhfl (Quyet dinh so' 05/2008/QD-BYT). Do cd gia tri
921
Nghien cdu cam dng va nudi cay re bat dmh cay ba kich (tvlonnda officinalis How)
dfldc lieu va nhu cau sd dung cao, cay ba kich trong tu nhien dang bi khai thac kiet que. Theo Nghi dinh sd 48/2002/ND-CP ban hanh ngay 22 thang 04 nam 2002 cua Chinh phu, cay ba kich dUdc dua vao sach dd Viet Nam cdn phai dfldc bao ve.
Hien nay, nguon cung cap dfldc lieu vdn chu yg'u bdng thu hai ttf nhign va nuoi trdng truyen thdng. Tuy nhien, viec nudi trong lai phu thugc rat nhieu vao cac didu kien sinh thai va trdng trot. Cay ba kich cd thdi gian thu hoach phai tfl 3 - 5 nam. Hdn nfla, viec phdng trd cac loai dich benh, tdn du cua thudc bao ve thuc vat ciing la mdt vdn de khd khan. Vdi cac Uu diem nhu nang cao ham lugng, chu dgng qua trinh san xuat, toi flu hoa qua trinh chiet xuat hdp chdt muc tieu, nhan nudi sinh khoi cay dugc Ueu bdng phUdng phap cdng nghe sinh hgc de thu hdp chat thfl cap la mgt giai phap trien vgng dg khac phuc nhiing ban ehe cua phfldng phap nudi trdng truyen thdng. Tuy nMen, tren ddi tfldng cay ba kieh, eae nghign cflu chi mdi tdp trung vao danh gia tac dung dupc ly (Chen et al, 2013), phan tich thanh phan boa bgc (Feng et al, 2012) hay nhan gidng in vitro (Hoang Thi The va cs., 2013;
Vo Chdu Tua'n va Huynh Minh Tfl, 2010) ma chua cd nghign cflu nao Uen quan den cam dng va nhan nudi sinh khdi trong dieu kien in vitro.
Do vay, co the ndi, day la cong trinh dau tien ci Viet Nam va tren the gidi ap dung cong nghe sinh hgc de cam dng va nhan nuoi r l bdt dinh cay ba kich gdp phan dap flng ngudn nguydn hgu dung ehe bidn san pham sfl dung trong linh vUc y dfldc.
2 .VAT LIEU VA P H U O N G PHAP Nghien cflu sd dung doan than va la tfl cay ba kich in vitro sinh trfldng, phat trien khde manh dg cam flng tao rl bat dinh. Doan than khong mang nach la cd chieu dai khoang 1,0 cm dugc dat theo phfldng ngang tren moi trfldng nudi ca'y. La cd cud'ng va la khong co cud'ng dugc gay vet thfldng bdng each dung dao kMa nhe nhang (khdng lam rach la) 3-5 dudng tren bd mat la, sau do dat be mat la co vet thUdng tiep xdc vdi mdi trUdng nudi cay. Ba chat dieu tiet smh trUdng thudc nhdm auxin gdm a-NAA, lAA
va IBA cl cac ndng do khac nhau (0,1-1,0 mg/1) dfldc bd sung vao mdi trUdng nudi ca'y de cam flng doan thdn va la ba kich ra r l bd't dinh.
Rg bdt dinh ba kich sau khi cam dng dUdc cdt vdi kich thfldc khoang 1,0 cm va nudi cdy tren nen mdi trfldng MS va B5 cd hoac khdng co bd sung chat dieu tiet sinh trudng thugc nhdm auxin (lAA, IBA va a-NAA) b cac ndng do khac nhau (0,1-1,0 mg/1) de khao sat stf tang trfldng cua re ba't dinh cay ba kich.
Mdi trfldng nudi cdy bo simg 30 g/1 sucrose dUdc digu chinh pH = 5,8 trfldc kM hdp khd triing cl nMet do 121''C trong 20 phut, 1,1 atm. Dieu kien nuoi cay in vitro la 16h sang/8h tm, cfldng dp anh sang 2.000-2.500 lux, nMet do 25 ± 2°C.
Cac tM ngMem dfldc bd tri nhdc lai 3 lan mdi cdng thdc, moi cdng thflc 90 mau ddi vdi thf nghiem tao re bat dinh va 27 mau doi vdi thi nghiem nhan nuoi re. Cac cM tieu dUdc theo ddi va do dg'm sau 4 - 1 2 tuan tuy tijfng tM ngMem.
Sd' lieu dflgc xd ly thdng ke bdng chUdng trinh Excel va IRRISTAT 5.0.
3. KET QUA VA THAO LUAN 3.1. Kha n a n g t a o re bat dinh cay ba kich
Stf binb thanh re bat dinh bao gom 4 giai doan: tao te bao boat hda, Mnh thanh viing te bao md phan smh, Mnh thanh sd kb6i rl va keo dai re. Sfl Mnh thanh tg'bao hoat hda xay ra d vung tUdng tang. Ben canh vai trd cua kieu gen, loai md, ban chat va ndng do eiia auxin cd vai trd rat quan trgng trong sfl hinh thanh re bdt dinh. Giai doan boat hda te bao can auxin b ndng do cao, trong kM giai doan keo dai rg can auxin ci ndng do thd'p hdn (Verstraeten etai., 2013).
3.1.1. Anh biidng cua a-NAA den kha nang tao re bat dinh cua doan than cay ba kich
Nhieu nghien cflu da chdng minh a-NAA la auxin cd kha nang cam dng sfl tao re bat dinh in vitro tfl cac loai vdt lieu khac nhau cua nhieu loai thflc vat. Tbeo Trdn Thanh Hfldng va cs.
(2009), trong khi 2,4-D cd vai trd trong giai doan hoat hda te bdo de binb thanh vung tg' bao md phan sinh va tao sd khdi rd cl cay chudi thi 922
Ninh Thi Thao, Nguyin Thj Phuong Thao, Nguyen Thj Thu^ Unh, Nguyin Tuan Minh, Nguyen Quynh Chi. Tran Thj Anh Dao a-NAA kich tMch manh sfl tang trfldng cua
vung te bao keo dai d i n de'n so' r l d trang thai keo dai cao nha't. Trong ngMen cflu nay, a-NAA dfldc bd sung vao mdi trfldng nudi cd'y vdi ndng do dao ddng tfl 0,1 de'n 1,0 mgA de cam dng tao rg bat dinh doan than cdy ba ki'cb. Kdt qua sau 4 tudn theo doi cho thay, doan than ba kich hoan toan khong tao r l tren mdi trfldng MS khdng bd sung a-NAA. Trong khi dd, bo sung a- NAA vao mdi trfldng nuoi ca'y da cam dng doan than ba kich tao re bat dinh vdi ty le mdu tao r l
dao ddng td 44 - 100%. Ty Id mdu tao r l tang dan va dat cao nhat (100%) khi bd sung 0,75 mg/1 a-NAA vao mdi trfldng nuoi ca'y. Tren moi trflcing nay, sd r l ehinh va chieu dai r l cung dat cao nhat, tUdng dng 4,53 r l va 1,58 cm. Tang ndng dd a-NAA bd sung vao mdi trudng nudi cay len 1,0 mg/1, ty le mau ra r l giam xud'ng 81%, tuy nhign ty le mau ra re nhanh lai dat cao nha't (66%). Trong khi do sd'nhanh/mdu cao nha't (7,07 nhanb/mau) ghi nhan dfldc tren mdi trudng bd sung 0,5 mg/1 a-NAA (Bang 1).
LSDflce
cv%
Bang 1. Anh hfldng cua a-NAA tdi kha nang tao re b a t dinh cua doan than cay ba kich sau 4 t u a n nudi ca'y
a-NAA (mgfl) 0 0,1 0,25 0.5 0.75
Ty le mau tao r i
(%)
0 44 69 89 100
S 6 r e chinh/mau ( r l )
2,18 2,89 4,54 4.53
Ch idu 1 dai re (cm)
0.82 1,2 0,94 1,58
chinh T y l e m i u r a r i
(%)
20 60 64 56
nhanh So nh^nh/mau (nhSnh)
2,17 3,10 7.07 5.82
' ' / " ^
H i n h 1. Sfl h i n h t h a n h rd bat dinh t d doan t h a n ba kich t r e n moi trvfdng bo s u n g a-NAA sau 4 t u a n nudi cay
923
Nghien cdu cam dng vd nudi cay r l bat dinh cdy ba kich (Morinda officinalis How)
Quan sat Mnh thai mdu cho thay, sau 5 ngay nuoi cay, cac mdu doan than cd xu hfldng cong len phia tren moi trflcing, hai ddu vdt cdt xua't Men callus va callus xuat Men eang nhieu khi Ifldng a-NAA bd sung vao mdi trUdng nudi cay cang ldn. Tren mdi trfldng bd sung a-NAA, r i bat dinh xua't hien tai vi tri hai ddu vet cat cua doan than va phat trien tren be mat moi trUdng nuoi cd'y, trong khi dd tren mdi trfldng ddi chdng doan than ba kich hoan toan khdng cam dng tao r l (Hinh 1).
Nhu vdy, CO the tha'y a-NAA cd anh hflcing tieh cflc dg'n cam dng tao r l bat dinh tfl doan than cay ba kich. Xet ta't ca cac ehi tieu theo doi, ndng do a-NAA thich hdp de cam flng tao re bat dinh tfl doan than cay ba kich la 0,75 mgA.
3.1.2. Anh biidng cua lAA den kba nang tao rSba't dinh cua doan than cay ba kich
Tudng ttf nhfl a-NAA, bo sung lAA vao mdi trfldng nudi cay cung c6 tac dung kich tMch doan than ba kich tao rd bat dinh, vdi ty le mau tao rl dao dgng tfl 36-67%. Trong do bd sung lAA b ndng do 0,75 mg/1 cho ty lg mdu tao re dat cao nhat (67%). Day cung la cdng thdc cho sd rl/mlu dat cao nha't (3,83 rl). Bd sung LAA b ndng do thap (0,1 mg/1) eho ty le mau tao rg (36%) va s d r l chinh/mdu tha'p (2,28 rg), nhflng chieu dai r l cMnh dat cao nha't (1,99 cm). Tuy nhign, khac vdi trfldng hgp bd sung a-NAA, rg bat dinh tao ra trgn mdi trfldng bo sung lAA khong ra rl nhanh, ty le de r l nhanh la 0% cl tdt ca cac cdng thflc bd sung lAA (Bang 2).
Quan sat hinh thai mdu cho thdy cac doan than ba kich eiing cd xu hfldng cong len va tao eaUus b 2 dau vet cdt sau 3 - 5 ngay nudi cd'y.
Tuy nMen lUdng callus cam dng it hdn so vdi tren moi trudng chda a-NAA. Callus td hai doan than tren moi trUdng chda lAA cd mdu vang nau trong khi callus tfl doan than tren mdi trfldng ddi chdng cd mau xanh. Sau 7 - 1 0 ngay nudi ca'y, r i bdt dinh bdt ddu Mnh thanh td vi tri doan than gan vdi vi tri callus xua't hien. Cac rl bat dinh cd mau trdng, an lan xudng be mat mdi trudng va khdng phan nhanh (Hinh 2).
Ben canh a-NAA va lAA, nghien cdu cdn sd dung IBA de cam dng tao r l bat dinh td doan than cay ba kich. Tuy nhien, IBA hoan toan
khdng cd tac dung cam dng tao re bd't cdy ba kich. Cac mlu doan than ba kich tren mdi trfldng bd sung IBA tao callus d 2 dau doan than nhflng khdng tao r l . Mac dd IBA khdng phai la auxin tMch hdp cho tao r l bdt dinh cay ba kich, nhflng tren mdt so ddi tfldng dfldc ligu khac, IBA lai td ra thich bdp cho sfl hinh thanh va tang trfldng rg bd't dinh nhfl cay Nhan sam (Nguydn Trung Thanh va Paek Kee Yoeup, 2008), cay sam Nggc Linh (Nguyen TM Lilu va cs.,2011).
So sanh anh hfldng cua ba auxin gdm a- NAA, lAA va IBA ddn stf tao re ba't dinh tfl doan than cay ba kich cd the tha'y a-NAA cd anh hflcing manh nhat den stf cam dng tao rl hdt dinh cay ba kieh. Ket qua nay cung trung vdi ket Man cua Verstraeten et al. (2013) khi ngMen cflu sfl tao r l bat dinh cl cay Arabidopsis thaliana Verstraeten et al (2013) da bd sung 5 auxin khac nhau gdm lAA, IBA, a-NAA, 2,4 D va picloram vao mdi trudng nudi cdy de cam dng tao r l bat dinh tfl doan than cay Arabidopsis va nhan thay, d cung ndng do, trong kM lAA, IBA a-NAA cam flng tao r l bat dinh tM 2,4 D va picloram hoan toan khdng cam flng r l ma chl cam flng tao callus tfl m l u cdy. Trong dd, a- NAA cho Meu qua cam dng tao re bat dinh cao hdn so v6i lAA va IBA. Theo Davies (1995), Litwack (2005), Taiz and Zeiger (2005), hgn canh sfl dieu khien bap thu eac chat hda tan trong te' bao, auxin kich tMch sfl keo dai te bdo thong qua sfl tac dgng trflc tig'p lgn vach te bao, do vay kich tMch sfl kdo dai r l bdt dinh. Auxin kich tMch hoat dpng cua bdm proton cl mang nguyen sinh chat, lam tang ndng dd H tJ khoang gian bao, khi dd pH giam. Sfl giam pH cua vach lam cho cau noi giiia extensin, hemicellulose, cac hdp chat pectin vdi cellulose bi pha v3; Ca^* ndi Udn cac chudi hdp chdt pectin bi loai di; mpt so enzym thuy phan dfldc boat boa giup cho stf tdng hdp hoac phan buy polysaccharid va protein dfldc thflc Mdn, gidp duy tri tinh long leo cua vach te bao.
Nhu vdy, cd thd ket ludn, mdi trfldng MS + 0,75 mgA a-NAA la tdi flu dd cam flng tao r l bat dinh td doan than cay ba kich, eho ty le mau t?o r l dat 100% va sd rl/mlu dat 4,53 r l sau 4 tudn nudi cay.
924
Ninh Thi Thao, Nguyen Thi Phuong Thdo, Nguyen Thi Thuy Linh, Nguyin Tuan Minh, Nguyin Quynh Chl, Tran Thj Anh Dao
B a n g 2. A n h hfldng c u a lAA tdi k h a n a n g t a o rd b a t d i n h c u a d o a n t h a n cay b a k i c h s a u 4 t u a n nuoi ca'y Ty 1$ m§u t?o r l
(%)
s6rSchinh/mlu (r§)
Chieu d^i r l chinh Ty i§ mlu ra r l nhSnh (cm) (7o)
LSDo.05 CV%
2,28 2.41 2,21 3.83 2.32 0,76 1.9
1,99 1,79 1,66 1,41 0,98 0,46 2,0
H i n h 2. S d b i n h t h a n h r e b a t d i n h t d d o a n t h a n ba k i c h t r e n mdi trfldng bd s u n g lAA s a u 4 t u a n n u d i cay
B a n g 3 . A n h h d d n g c u a v a t lieu d e n k h a n a n g t a o re b a t d i n h ba k i c h s a u 4 t u a n
Vat lieu Dogn than t.a 00 cu6ng La khong co cuflng
Tyl# mlu
(%)
100 0 0
tao r l So r l chinh/mlu (rl) 4.48
Chilu ddi r l chinh (cm) 1,55
Ty l§ miu ra r l nhanh
(%)
56
So nhinh/mlu (nhSnh)
5,85
925
Ninh Th] Thao, Nguyen Thj Phuong Thao, Nguyen Thj Tbu^f Lmh. Nguyen Tu^n Mmh, Nguyin Qujnh Chi, Tran Thj Anh Ddo
3.1.3. Anb bifdng cua vat lieu nuoi cay den kba nang tao ribat dinh cua cay ba kich
Trong ba loai vat lieu sd dung de cam flng tao r i bat dinh tren moi trfldng MS + 0,75 mg/1 a-NAA (moi trfldng tdi flu cho tao r l bat dinh td doan than) gdm doan than, la c6 cud'ng va la kbdng cd eudng, sau 4 tuan nudi ca'y, chl ed doan than cay ba kich cam dng tao r l bd't dinh vdi ty le 100% vdi sd r l cM'nh/mlu dat 4,48 re, trong kM dd md la cd cuo'ng va la khdng co cud'ng hoan toan khdng tao r l bdt dinh (Bang 3). Quan sat trong qua trinh lam tM nghiem cho thay, cac mau cay Cling cd xu hfldng cong ldn trgn be mat mdi trflcing va tao callus tai vi tri gay vet thfldng, b ddu cudng la va hai dau doan than sau khoang 3 - 5 ngay nudi cdy (Hinh 3). Sau 7-10 ngay nudi cdy, re bdt ddu xuat Men tfl mdu doan than, tai vi tri gin vdi vdi vi tri xud't hien eaUus. Sau 4 tuan nudi cay, 100% mau doan than tao re trong kM md la va la cd cud'ng vin chua cam dng re (Hinh 3A B, C). Tuy nhign, ndu nuoi cay mau la cd cud'ng lau hdn, cu the sau 6 tuan, mdt so' mlu re bdt dinh bat ddu xud't hien tai vi tri dau cudng la ndi gan vdi vi tri cua caUus (Hmh 3D).
Sfl Mnh thanh re bdt dinh khdng cM phu thugc vao kieu gen, Ioai, ndng do chat dieu hda sinh trfldng (Tiberiapop, 2001) ma cdn phu thugc loai md, cd quan, tuoi va giai doan phat tridn eua cay (Vo TM Bach Mai, 2004). NgMgn cdu nay ghi nhan sfl khac nhau trong kha nang tao re bdt dinh gifla cac vdt Ueu nudi cay khac nhau cua cay ba kieh. Kha nang tao r l bdt dinh
cua doan than ba kicb la cao hdn so vdi md la.
Ket qua nay ciing triing vdi ket ludn trong nghign cdu cua Ling et al. (2013). Ling et al nghign cdu tao re bat dinh cay Labisia pumila var. alata tfl vat lieu doan than va la va nhdn thay, kba nang tao re bdt dinh cua doan tiian cao hdn so v6i md la. De giai thich cho dieu ndy, nhdm tac gia cho ring, sfl cd mat cua mo gidng tidn tang sinh gd (procambial-like tissue) b cac cdu trdc xung quanh mach dan trong than ed vai trd ldn trong vigc quydt dinh tao re bd't dinh.
Theo Torres (1989), cac te bao cd ngudn goc khac nhau, trong cac mo khac nhau se eo tiang thai sinh ly, sinh hda khac nhau, do vdy ma kha nang boat hda cua cac te bao de Mnh thanh md phdn sinb r l va keo dai r l la khac nhau.
3.2. Khao sat m o t so y e u to a n h hfldng den sfl t a n g trifdng re b a t d i n h cay ba kicb 3.2.1. Anb biidng cua moi tn/dng nuoi cay den sjt tang tritctngre bai dinh cay ba kicb
Trong hai mdi trflcing nen khao sat la MS va B5, re bat dinh ba ki'ch hoan toan khdng tai^
trfldng tren mdi trUdng nen MS. Sau 6 tuan nuoi cdy, ty le mlu de nhanh la 0%. Trong khi dd, tren moi trfldng nen B5, sau 6 tuan nudi cay ty le m l u r l de nhanb la 44%, so nhanh/re dat 1,88 re va chidu dai re dat 0,32 cm (Bang 4).
Trong qua trinh nudi eay, cae mlu rl ed xu hudng chuyen td mau trdng sang mau vang va trdn mdi trfldng B5, r l bdt dinh cdn tao callus ddng thdi vdi sfl phat smh r l nhanb (Hinh 4).
Hinh 3. Stf h i n b t h a n h r e b a t dinh t d doan t h a n (A), la k h o n g cd cuong (B), la c6 cud'ng (C) sau 4 t u a n nuoi cay va la cd cud'ng sau 6 t u a n nuoi cay (D)
cua cay ba kicb t r e n moi trfldng MS + 0,75 mg/1 926
Ninh Thj Thao, Nguyin Thj Phuong Thdo, Nguyin Thj Thuy Lmh, Nguyen Tuan Mmh.
Nguyin Quynh Chi, Trdn Thj Anh Dao
Hinh 4. Re bat dinh cay ba kich tren moi trfldng MS va B5 sau 6 tuan nuoi cdy Bang 4. Anh bddng ciia moi trddng nuoi ca'y den sd tang trfldng
re bat dinb cay ba kich sau 6 tuan nudi ca'y Moi tnrong Ty 1^ r l de nhanh (%) So r l nhanh (rl) Chieu dai r l nhanh (cm)
LSDoii CV%
Mdi trfldng sd dung trong tM ngMem la moi trfldng MS va B5, day la nhflng mdi trUdng dugc sd dung pho bie'n dg nudi cay re in vitro nMeu ddi tUdng kbac nhau nhfl cay ca rdt (Le Thi Tbuy va cs., 2014); cay Nhan sam (Nguygn Tnmg Thanh va Paek Kee Yoeup, 2008), cay nhau (Nguygn Thi Nggc Hfldng va Vd Thi Bach Mai, 2009).„ Cac mdi trfldng tren chda hdu het cac chat dinh dfldng cdn tMdt, chu ye'u la khoang cho sfl smh trflcing cua rl, tuy nhien, bam Ifldng khoang trong cac mdi trfldng la khac nhau. Trong thanh phdn khoang da Ifldng cda mdi trfldng B5, ham Ifldng NH4* cM bdng 5% so vdi moi trfldng MS, nen tinh ddi khang gida stf ddng boa dam va hap thu cac cation da lupng khdng cao, cd the vi thd ma r l bat dinh cay ba kicb tang trflSng td't bdn tren mdi trudng B5.
Trong tbanh phan vitamin, thyamine ddng vai trd quan trgng trong stf chuydn hoa carbohydrate va trflc tiep tham gia vao qua trinh sinh tong hdp mgt sd loai acid amin, cho ngn thyamine can dfldc cung cap Uen tuc trong
qua trinh nudi eay. Thyamine hien dign trong vitamin B5 vdi ham lugng cao gap 100 lan so vdi trong vitamm MS. \ ^ vay, cd the thyamine cdng da anh hudng den sil tang trudng cua re bat dinh cay ba kich.
3.2.2. Anb bitdng cua a-NAA/IAA/IBA din svt tang sinh khoi re bat dinb cay ba kicb
Nhieu nghien cflu da cho thay cac chd't dieu tig't sinh trfldng, dac biet la nhdm auxin, cd anh hfldng dang ke de'n sfl phat trien cua re bat dinh trong dieu kign in vitro. Cac loai auxin khae nhau vdi ndng do khae nhau anh hflcing dg'n stf tdng trfldng cda r l va sfl tdng hdp cae hdp chat muc tigu (Reis et al., 2011; Amoo et al, 2013).
Trong nghien edu nay ba auxin la a-NAA, LAA va IBA dupc bd sung vao moi trUdng B5 b cac ndng dp khac nhau de khao sat anh hudng cua chung den sfl tang trfldng cua r l ba't dinh ba kich. v a t lieu nudi cdy la doan than ba kich cd kich thfldc 1,0 em. Ke't qua sau 12 tuan nudi cay dUdc trinh bay b bang 5.
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Nghien cdu cam dng va nudi cay r l bat djnh cay ba kich (Morinda officinalis How)
Bang 5. Anb bddng cua a-NAA/IAA/IBA den kha nang tang sinh khoi re bat dinb cay ba kich sau 12 tuan nuoi cay
a-NAA/l/WIBA (mg/I)
0 0.1 0,25 0.5 1.0 LSDoos CV%
d-N/W 0.0853 0.823 0,979 1.106 1.264 0.014 0,9
Kh6i lu'gng r l tuwi lAA 0.0853
0,19 0,181 0,279 0,358 0,017 4.3
(g) IBA 0.0853
0.682 0.573 0,882 1,149 0,03 2,5
Ket qua bang 5 cho thay, a-NAA, lAA va IBA CO tac dung tich cflc trong tang sinh khdi re bat dinh. O tat ca cac cdng thflc bd sung a-NAA lAA, IBA deu cho thay re bat dinh ba kich tang trflcing manh hdn so vdi cong thdc ddi chdng.
Khoi Ifldng r l tfldi thu dfldc trgn moi trfldng ddi chflng dat 0,0853 g sau 12 tudn nuoi cay. Trong kM dd bd sung auxin vao mdi trudng nudi eay lam tang khoi Ifldng rg ba kieh gap tfl 2,12 (moi trfldng chfla 0,25 mg/1 lAA) de'n 15,29 lan (moi trfldng chfla 1,0 mg/1 a-NAA) so vdi cdng thdc doi chflng. Tren mdi trUdng bd sung a -NAA lAA va IBA, khdi lugng re bat dinh tang ty le thuan vdi ndng dp auxin va dat cao nha't khi bo sung 1,0 mg/1. Tren moi trfldng bd sung 1,0 mg/1 a -NAA, lAA va IBA khdi Iflgng r l tfldi dat dugc lan lUdt la 1,264 g; 0,358 g va 1,149 g sau 12 tuan nudi cdy td 1.0 cm ri bat dinh ba kich (Bang 5).
Quan sat hinh thai cho thay, tren mdi trUdng khdng bd sung auxin, sd' Ifldng nhanb thfl cap sinh ra it (khoang 2-3 nhanh), cac nhanh ehd ydu phat trien ve cMeu dai ma khdng phan nhanh phu, cac nhanh cd mau trdng va cM phat trien lan tren be mat chfl khdng an sau xuong mdi trUdng nudi cd'y. Trong kM do, b mdi trfldng bo sung auxin, r i ba kich cd xu hfldng chuyen sang mau vang, de nMeu nhanh thd cap va cd nhidu nhanh phu phat trien tfl nhanh thd ca'p.
Rg nhanh khdng chi phat trien lan rdng ma cdn an sau xuong mdi trfldng nudi cay. Bd sung auxm d ndng do eao, cu the bdt dau td ndng dp 0,5 mg/1 a-NAA; 0,5 mgA IBA va 1,0 mg/1 lAA, callus xuat hien td rl ba kieh ddng thdi vdi sfl xua't Men rd thfl cap (Hinh 5).
Nhu vay, cd the tha'y, bd sung auxin vao mdi trUdng nudi eay da cd tac dung tich cflc
trong viec lam gia tang sd Ifldng r l nhanh, do vay lam tang sinh khdi re bd't dinh ba kieh.
Trong ba auxin sd dung, a-NAA va IBA cd anh hflcing manh me hdn lAA den sfl tang sinh khdi rd bat dinh ba kich. Kim et al (2003) chflng minh IBA cd anh hfldng manh me den su hinh thanh re thd cdp sau 7 ngay nudi ca'y rl bat dinh cua cay sam Panax ginseng. San Jose et al.
(2012) cdng da chdng minh bd sung 0,1 mg/I IBA vao mdi trUdng nudi cay da lam su hinh thanh re thd cap cda cay Alnus glutinosa gia tang ro ret so vdi ddi chdng trdn mdi trUdng khdng CO IBA. Theo AbduUaMl (2010), bd sung a-NAA va IBA vao mdi trfldng nudi edy lam gia tang sinb khoi rg bat dinh cay Morinda citrifolia. Tuy nhien nlu tang ndng dg a-NAA vugt qua mdc 0,5 mg/1 hoac ndng do IBA vfldt qua mdc 1,0 mg/1 tM xuat Men callus td rl. Stf xua't hien cua callus khdng tang kha nang tich luy cac hdp chd't va lam giam tinh ben vflng trong san xuat hdp chat thd cap trong nudi cay r l bat dinh (Abdullahil, 2010).
4. KET LUAN
Doan than Id vdt Udu tM'ch hdp dd tao rl bat dinh cay ba kich. Mdi trfldng tMch hdp nhat dd tao r l bat dinh cay ba kich la MS + 0,75 mg/1 a-NAA, cho ty le doan than tao r l dat 100%, so r l / m l u dat 4,53 r l va chieu dai r l cMnb dat 1,58 cm sau 4 tudn nudi cay.
Moi trUdng thich hdp cho sfl tang tnlbng rl bat dinh ba kich la B5 + 1,0 mg/1 a-NAA. cho sinh khdi r l tfldi dat 1,264 g td 1,0 cm r i sau 12 tuan nudi cay.
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Ninh Thj Thdo, Nguyin Thj Phuong Thdo, Nguyin Thi ThuJ' Linh, Nguyin Tuan Minh, Nguyin Quynh Chi, Tran Thj Anh Ddo
Hinh 5. Re b a t dinh in vitro cay b a kich nuoi ca'y t r d n moi t r d d n g MS bd s u n g a-NAA/IAA/IBA sau 12 t u a n nudi cay
LC)I CAM ON
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