M XI + x 2 + + x n ~ ~
KET QUA THljC NGHIEM
Chuang III. Nhlin gh5'ng vD tinh cay hoa cuc Chrysanthemum
CHuCiNG III
NHAN GIONG VO TINH CAY HOA CUC
CHRYSANTHEMUM INDICUML. IN VITRO
I.Giaido~nkhoidQng milm
38
h~m m~c dich khdi di)ng mh, kich thich milu lao callus va tai sinb
ch6i d6ng thO'i lrrn hi~u kha nang tai sinh cua hai IO<.J,i choi, chung t6i thi nghi~1l1 sO' dl;)ng hai IO<.J,i mfru ban d~u de' nuoi dly. MQt la ch6i ngqn (d6t ngqn) con non thu dLrqc tll cae m~m gia, hai iii ch6i nach (Mt than) thu duqc tll cae canh mang hoa manh khoe.
M6i lnrCing caban sli dung trong thinghi~m khdi di)ng m~m nay la m6i U'uCing LS c6 b5 sung BA va AA theo kinb nghi~mda duqc nghien cU"u t~i Trung lam C6ng nghe Sinh hoc Nang nghiep Dalal. Ty I~ n6ng di) BA/NAA la 0,2/0,05 mg/I. M6i lnlCing duqc ky hieu 12. Sau 30 ngay nu6i diy, ke't qua nhl( sau:
I3dng J. Khd nang tai sinh cua chai ngQn va ch6i nach duai tac dl:'ng ktt
hqpciiaBAva NAA (0,2 10,05mgll)
Val lieu Ty I~ lai Ty I~ lao Phat sinh d6ng lhCii Chillllrqng sinh(%) callus(%) callus va ch6i(%) cav
~ng9n 100 12,4 77.6 +++
D6t lhan 100 52,5 47,5 +
(Ghi chu: Kyhi~u -: Cay che't,+: Trung blnh, ++: Kha, +++: T6t) Ke'l qua bang I cho thily:
Tiltellcae milu cily d6u c6 kha nang tai sinh, lUy nhien sli phal sinh hlob thai r6 ding phl.1 thuQc m6i lntong va v~tli~unuoi ca'y ban dau.
77,6% Ml ngqn va 47,5% d6llhan c6 xu hudng lai sinh theo hudng lao d6ng lhCii callus va ch6i, ngb)a la ch6i viln phat lrien d6ng thCii c6 blob thanh callus d g6c milu ca"y. Cay con thu duqc c6 sU"c s6ng rill l6l. Cay map va xanh, la 10 (dlrCing kinb la 0,8-1,2 cm). aMt ngqn c6 hien luang de nhanh. Kh6ng c6 hien tuqng pban h6a re xay ra.
52,5% d6t than va 12,4% d6l ngqn (cae d6l ngqn nay c6 kich thudc ban dfin nho (chi6u dai d6t Mdi 0,5 em) do thao tac ca"y) lai theo hudng phat sinh callus. Callus tao duqc c6 milu xanb non, kh6. Tie'p tuc gii1lhem 2
Chudng III. Nhc7n giang va tfnh cay hoa cue Chrysanthemum 39
luiln nfla, lillcd callus Mu liln IlIQt tai sinh ch6i. So IUQng ch6i ciiy lim dUQc nhi6u. Tuy cay con c6 kich thudc nho nhllng chung c6 suc song rill lot. Cay c6 mau xanhd~rnva k.bong ph an b6ar~.
Giaido~n khdi dong milrn k€t thuc sau 6 llIh nuoi cily. Dudi anb hudng cua moi lruClng, ly I~ BA 'AA, dot ng<;>n c6 xu hudng phal sinh hlnh thai theo hlldng vlia tao ch6i vlia t~ocallus u·ong khi dOL than to ra d6ng d6u hOn trang vi~cphal sinb callus hoac pMl sinh d6ng thCli vlia callus vlia ch6i. San ph5m thu du'Qc la cac ch6i non tao nguyen li~u khdi dilu cho giaido~nnhan nhanh (Hlnb 10, II, 12).
100 80 60 40 20 o 77.6 Ja oPMl sinh ca lIus (%)
IIIPhot sinh d6ng thCli callus va ch6i(%) DOL ngon DOllhiin
13ifu d6 I. Anh huang cita v(1lli¢u ten s¥ pMl sinh hlnh thaiciiyhoa
Cue Chrysanthemum indicwnL.
Hinh 10. PMt sinh d6ng lhOi callus va ch6i a dot ngqn
ciiy hoa cue Chrysanthemum indicum L.
Chudng 1lI. Nhrin giong v6tinh ciiy hoa cuc Chrysanthemum 40
lfinh II. Cal/us cay hoa cuc Chrysanthemum indicumL
c¥'
( .... ~_u.•Q .T
,-\u.; ~ ) .
lfinh 12. PMt sinh ch6i tucal/us cay hoa cuc Chrysanthemum indicumL.
Chudng lIf. Nhlin giong va tfnh cay hoa cue Chrysanthemum 41
Hinh J3. Anh huang cua nang dQ BAkhdc nhau len viec tai sinh
cciy hoa cue Chrysanthemum indieum L.
II. Ciaido~nnhan nhanh
Sau khi I~O dllqC m(H 56 Illong chai, chung loi thily dng cac chai cay mdi tao dlloc nay c6 than, la ph5t trie'n t6t, de nhanh thanh bui day, nhLlng he 56 nhan khong 6n djnh. of! lao Mac Illong Idn ciiy con vdi he 56 nhan 6n dinh, chung loi lie'n hanh dl Ml cac chai cay tao dllqC 0 lhi nghiem khOi dong mUu de' cily chuyen. ~)f! dam bao dang d6u ve tu5i sinh ly cua cay, chung loi chQn mUu diy Ia cac d61 ngQn va Mtlh(1 hai ctla mai cay. Cac d61 nay c6 chieu dai 1,0 cm. s6 d61 ngon va d61 lhu hai dl/OC chia deu cho cac nghiem thuc.
Vdi muc dich kich thich lao chai trLlc tie'p khong qua giai doan lao callus, chung loi Sl1' dung moi trLIong nhan gi6ng co ban LS b5 sung BA theo cac nang d(\: 0,1; 0,3; 0,5 va 1,0 mgJl. Cac nghiem thuc dLlqc ky hieu III, II" 113,
114va II,.
Trong thi nghiem nay, chung loi se chQn fa nang d(\ BA thich hqp cho moi u"L1ong nhan gi6ng va xac dinh he 56 nhan. PhLlong phap nay cho phep lao fa mol ILlqng cay dung cho cac thi nghiem in vitro sau nay. Sau 30 ngay nuoi cily, ke'l qua nhLl sau:
Chl(ang 1Il. Nhan gif/ng vo tinh ciiy hoa cue Chrysanthemum 42
Being 2. Sf! phil!triineua dOl thanciiyhoa CueChrysanthemumindicumL.
lren moi IruongLS c6 b6sung BA.
ghiem N6ng dQ Chieucao Phalsinh Hf$so Chiltlltqng
thti'e BA (m n) TB (em) r6(%) nhan eft
III 0 5,57 100 5,15 ++
112 0,1 7,00 100 6,12 +++
II, 0,3 6,75 90 6,30 +++
II, 0,5 6,50 35 7,43 +++
II, 1,0 4,75 10 5,51 ++
(Chi ehu: Kji hi~ll -: Cay ehe'l,+:Cay trung blnh,++: Cay kha,+++:Cay 161)
E 10~ 8 7.00 6.75 6.50 ~ 8 7.00 6.75 6.50 co 5.57 - r - - 4.75 f- 6 ~ 0 r - " 4 u :J 2 '"..c 0 U . , . . II I fl2 113 fl4 fl5 Nghi~mthire
Bi€u db 2a. Anhhuang BALenchducao trungbrnh cl'iyhoa cue Chrysanthemum indicum L.
10c 8 c 8 <" ~ 6 c '"v. 4 ~. 2 0 II 12 13 7.43 14 15 Nghi~IlltIH?C
Bitu d62b. Anh huang BA lenh~so' nhdn
cliy hoa cueChrysanthemum indicumL.
Chuang Ill. Nhdn gi6ng v6 linh cay hoa cue Chrysanlhemam 43
Qua ke't qua bang 2, chung toi thilyr~ng:
Tren moi ullang khong dung kich thich sinh trtlang,cac ch6i viin c6 kha nang nhan nhanh vdi he 56 nhan la 5,15. Tuy nhien vdi Stt b6' sung BA he 56 nhan tang dang kG, nhilt la aD,S mgtl he 56 nhan dat 7,43, tang 144,3 % so vdi d6i chltng la 100%.
Chieu cao u'ung binh cay a cac nghiem tbuc c6 bd sung BA til 0,1-0,5 mgtl tang hdn so vdi diSi chang va giam a n6ng d(\ BA 1,0 mgt/. 60,1 mgtl BA chieu cao trung blnh dat cao nhilf, dat 7,00 cm. Chieu cao giam diln theo chieu tang n6ng d(\ BA. Chieu cao trung blnh tbilp nhilt la 4,75 cm a n6ng d(\ BA 1,0 mgt/.
Nglrdc lai vdi he s6 nhan, ty Ie phat sinh r6 giam theo chieu tang n6ng do BA.6 nghiem tb(lcII,c6 n6ng d(\ BA la 1,0 mgtl, ty Ie phat sinh r6 la 10% so vdi diSi chUng la 100%. 6 cac nghiem thuc c6 bd sung BA, r6 ng~n, manh va phat triGn kem (chI 1-2 r6tg6c).
Chilt Il!dng cay il nghiem thuc II" 113 va 114rilt t6t, cay xanh dam, la to, than cay map, d6t dai. 6 nghiem th(rc d6i chang cay xanh non, Ia trung blnh, than cay 6m, d6t dUi.6lI" cay xanh non, la nh6, than cay 6m, diSt ng~n. VI vaY, ke't hdp cac tieu chudn he 56 nhan, chieu cao ullng blnh, phat sinh r6 va chiltlLlqng cay, nghiem tbuc1I, vdi n6ng d(\ BA 0,5 mgtl la tbich hdp eho cong viec nhan gi6ng vdi he s6 nhan trung blnh la 7,43 (Hlnh13)
III. Tim hiGu nnh hlidng cua IBA len sl/ l,U sinh dly hoa cue
CItJ}wQllthemum illdicllInL.
6 thi nghiem tim hiGu anh hlrang Clla BA Ien sinh trLlilng cay hoa cue vang, chung toi da xae dinh MdC moi u'llang nhan tbich hdp. Tuy nhien vdi muc dich chI Sll d~ng d6t ngon cho cac tbi nghiem sau nay, chUng toi tie'n hanh tai sinh cay theo con dLiang tai sinh cum ch6i til' callusnh~m muc dich thu dlldC mot 56 ILlqng ch6i nhieu hdn va e6 tu6'i sinh 19 d6ng deu nhau. Chung toi dli xac dinb dLlqc moi trLlang tai sinh ch6i til' callus (thi nghiem I), do d6 chUng toi tie'n hanh khao sat anh hLlilng cua phytohormon nh6m Auxin (IBA va NAA) len phat sinh hinh thai tao callus cua cay trang dieu kien
invitro.
DG dam bao d(\ d6ng deu ve tudi sinh 19 cua cay, cay con sau khi tao alldcb~ngnhan gi6ng vo tinhin vilro, m(\t phUn dLiqc giIT lam cay me dG tie'p tuc nhan, philn khac aLlqc dy chuyen sang moi u'llang LS khong b6' sung
Chuangflf. Nhiin gifing v6 tinh c<1y hoa cuc Chrysanthemum 44
phytohormon hai Iftn M lam giam ILiqng phytohormon u'ong chung. Cay thu
MCic nay goi la cay d5i chung. Sall d6, chung toi chQn milll cify la cac d5t ngon cua cay d5i chltng M thi nghiem. Cac d5t nay c6 chi6l1 dai tff 1,0-1,2 em va c6 til 3-4 la.
Moi U'llong sll dung cCi ban ia LS c6 bi! sung iBA thea cac nang d(l: 0; 0,1; 0,3 va 0,5 mg/I, dllqC ky hi¢u la 1I1J, III" III" III,. Sau 30 ngay nlloi
diy,ketqua nhLt sau:
Biing3. Anh huiJng caaIBA len sinh tn((}ng vel ph/a tri#n cua cay hoa
cueChrysanthemum indicumL.
Nghiem Nang d(l IBA Chi6u cao TB Thai gian bilt dftU Chift Illqng
thuc (mg/I) (em) tao r6 ( ngay ) cay
III, 0 5,5 7 ++
III, 0,1 5,0 7 ++
III, 0,3 4,5 6 ++
Ill, 0,5 4,0 5 ++
(Ghi chu: Ky hieu-: Cay che't, +: Cay trung blnh, ++: Cay kha, +++: Cay t6t) Qua bang 3, chung tOi c6 nhan xet nhLi sau:
S,.l phat sinh hlnh thai thea hLidng tao r6 xay ra d tiftcacac nghiem thuc. Khong c6 Slf hillh thanh callus x:iy ra. Tiftca cay con d6u d Gnh u'~ng kha. Cay c6 mau xanh dam, kich thlldc la u'ung blnh (Mong kinh la 0,8-1,0 cm). Ri) dai (8-10 cm), c6 mau u'ilng, manh.
E 8 ~ 5.5 o:l 6 - f- 0 4 '" u " 2 "~ :2 0 U 1111 5 1112 4.5 1Il3 11I4 Nghi~rnlhlrc
Bitud&3a. Anh huang cualBAlen chdu cao
cayhoacueChrysanthemum indicumL.
Chu(Jng //1. Nhcin gic/ng VD tinh cciy hoa cue Chrysanthemum 45 6 7 " 10 I "" ~ '" >, 7 -"" ""'co.0 C c~ 5 .~l<) co~ :;:; 0 .c g . f- 0
ill1 Il12 UJ3
5
ill4 Ngti~mWe
Biiu(163b. Anh huang cua fBA len IhOi gian btil dilut~orl
cayhoaclkChrysanthemum indicumL.
Chieu cao cay giam dan theo sli lang dan n6ng d(l ISA. Ngli<lC lai, thili gian cay b1l.1 dau laor~ lai ngll.n dan. T6m I~i, ISA lhuc da'y hlnh thanh r~ va lam giam chieu caodciiy hoa cuc vang. Neu vdi muc dfch I~O r~ cho ciiy de' lao cay hoan chinh phuc vu cho cong lac gi6ng ngoai d6ng ru(lng n6ng d(l IBA0,1 mgll d nghi~m thuc III, Ia lhfch hqp nhillVI thili gian laor~ du dai d6'cay khong cao qua cGng kh6ng tllilp qua lao thuan Iqi chovi~cra 111~ ( bitu d6 3a, 3b).
1 2.
Hinhf4a. Anh huang cua fBA len sinh Iruang vapM/lriin cay hoa cuc Chrysanthemum indicum L(f: DOl chung, 2:0,5 mg/l,3:0,3 mg/l, 4:0,1 mg/l).
Chuong III. NhCin gio'ng vo linh ciiy hoa cuc Chrysanthemum 46
Hinh 14b. Anh huang cila IBA len sinh truang va phd/t,.;;n cay hoa cuc
Chrysanthemum indicum L.
IV. Tim hi€u anh hu'dng cua NAA Ien 51/ tai sinh cay hoa cuc
Chrysanthemum illdiclll1l L.
Nh~m tIm hiGu ulc dung NAA len phat sinh callus cay hoa cue chung t6i tiGn hanh thl nghi¢m tIm hiGu anh hlldng cua NAA len phat sinh hlnh thai Clla
diy trong die.uki~n in vitro.
Cay con ch,?n lam milu criy cho thi nghi¢m la cae a5t ng,?n cua cay d5i ch((ng. Cae a5t nay c6 chieu dai tii' 1,0-1,2 cm va c6 tii' 3-4 la. M6i trliilng stl' dung la LS+NAA theo cac n6ng d6:1,0; 1,3 va 1,5 mg/l,all'!C ky hi¢u la IV" IV2, IV" IV,. Sau 30 ngay nu6i diy, ke't qua nhll sau:
Bang4. Anh huang cda IBA len sinh /ruang va phd//riln cila cay hoa
cue Chrysanthemum indicum L.
Nghi<?11l N6ng a(i Cbieu cao TB Phat sinh1'6 Chilt llirlng
th((c NAA(m~lI) (cm) (% ) cav
IV, 0 5,50 100 ++
IV, 1,0 3,00 0 +
IV, 1,3 2,75 0 +
IV, 1,5 1,50 0 +
(Ghi cbtl: Ky hi¢u +: Trung blnh, ++: Kha, +++: T5t)
ChLtdng III. Nhan gifJ'ng vo /[nh cl'iy hoa cue Chrysanthemum 47
Qua bang 4, chung toi c6 nhan XCl nht1 sau:
Trong khi d dOi ch((ng cay c6 xu hudng tao r/; lh. d 3 nghi~m thac c6 b6 sung NAA Str ph,j[ sinh h'nh tMi khong theo htrdng t~ocallus cfing nhu l~O r/;. Mac du su tao r/; c6 xay ra sau tuiln dilu nuoi cily, nhung khi r/; d~t I mm thl ngtrng phar triGn va chuyGn mau den. Till ca cay con d 3 nghi~m thac Mu d t1nh lrang trung blnh. Cay c6 mau xanh nhat, nhilt la philn ngon. Kich thudc hi trung blnh (dltong kinh hi 0,8 - 1,0 cm).
Chleu cao cay giam diln theo su lang diln nOng dt) NAA. T6m lai, AA st'r dung d 3 nOng dt) 1,0; 1,3 va 1,5 mgll (nOng dt) rill thilp) tuy k.bong gay ra hlnh thanh callus nhung c6 racd~ng(lc che' manh me phat sinh r/;, lam giam chleu cao d cay hoa cuc vang.
~ 8 8 E ~ 5.50 ~ 6 ~ 0 4 3.00 2.75 '" ~ u n 1.50 ~ 2 & %- n '~~ ~ ~A· .c AiMiW U 0-
IVI IV2 IV3 IV4
Nghi~mth((c
Bi€u d6 4: Anh huang eila NAA len chi€u cao ccly hoa cue Chrysanthemum indicumL.
Nht1 vaY, d glai doan khdi dt)ng milm, valll~t1d6t than hay d6t ngon deu c6 thG lal sinh t6tM tao Ct.lllJ chOi khi nuoi cily lren moi lnrong LS c6 b6 sung ty I~ BAINAA la 0,2/0,05 mgll. Moi lnrong thich hqp dG nhan nhanh t~o
nguOn nguyen lieu cay can la moi truong LS b6 sung BA nOng dt) 0,5 mgll. H~ s6 nhan duqc dc dinh la 7,43. Chilt k.ich thich IBA vdl nOng do sll' dung tu 0,1-0,5 mg/l c6 tac dt.lng thuc diiy nhanh thai gian lao r/; d cay nhung dOng thai lam cay thilp hdn. Cfing nhu IBA, NAA nOng dt) tu 1,0-1,5 mglliam thilp cay, ngoal ra con gay vang la d ngon va (lc che' r/; phal triGn.
T6m lai, giai do~n vao mh ban dilu, tham do moi truong,t1m hiGu cac anh hudng cua moi truong len qua trlnh ral sinh dly ph~c v~ cho cong tac nhan nhanh lao ra s6 luqng Idn cay con la cong vi~cdn thie't M tie'n hanh cac thinghi~min vilra sau nay.
Chuang l/I. Nhc1n gi{;'ng volinh cay hoa cue Chrysanthemum 48
Hlnh 15a. 711cd~ng NAA len sinh Intdng va phdltri€n cay hoa cue
It 3 1.
Hlnh 15b. Tacd~ngNAA len sinh Intdng va phdltrien cay hoa cue
Chrysanthemum indicumL.(I: D6i chUng, 2:1,0 mg/l, 3:1,3 mg/l,4: 1,5mg/l).
Chuang IV. Toc dung clla chitos(Ul ehiellX(J len ChrysanlshemuIII indicumL. 49
CHU<1NGIV
TAC DUNG CVA CHITOSAN CHII~U X~ U'tN SINH TRUaNG
VAPHkr TRIEN CA Y HOA CUCCHRYSANTHEMUM INDlCUML.
I. Tim hi€u anh hUdng cua chilosan chituXl) lieu 200 kGy len sli lai sinh ella diy hoacueChrysanthemum illdicll1llL.ill vitro.
Chung la da biet chitosan khi dllelc phan giai thanh cac phan dOl)n (fragment) ng~nheln, vdi IrQng Itfling phan llt nha hOn e6 hoal linh sinh hoc cao hOn. acae nghien e(lli trudc day, cac t~c gia Ih,long s,t dung chitosan du'!e dt mach b~ng cae tac nhan h6a hQe hay sinh hQe (enzyme) eho mile dieh nghien e(lu e'la minh. G~n day, nglloi ta da chung minh btl'e Xl) gamma eung la m(lt tac nhan mdi r,fl hi~u qua tmng vi~e depolymer h6a cae polysaccharide. Cho nen, nhiell t~cgia da nghien e(lu tim hieu anh huang eua ehitosan du'!e phan giai h~ngph'lclngph~pehiell xa len s,l sinh tnlang va ph~t Iri.;'n eua Ihlfe val. Tuy nhien, cae nghien e(lu eung chi mdib~1 d~u va chua di denke'llu~nIho'ng nhal. Vi v~y. eh,'ng loi lien hanh Ihinghi~111 ve anh huang eua ehilosan ehie'u xa len su sinh Iflldng va ph~t lrien eiia cay hoa cue
Chrysanthemum indicllnl L. lrang di6u ki~n in vitrod6 tim hitu them tAc dl;lng
Clia chitosan chic'lJ x~ len loai cay nay.
Thi nghi4m tic'n hanh tren moi tnfdng cO hall LS co b6 sung chitosan duqe chiell xa v,1i Iidu chic'u 200 kGy. Cole n~ng dii slr dung la 5, 15,30,50,