Thi nghi~mkhOi d(lng milu ban dllu.
Khao sat anh hllOng cua phylohormon (BA, IBA, NAA) len cay con. Xac dinh moi lrllang nhan gi6ng thich hdp. Phlldng phap nay cho phep lao ra m(lt IlIdng cay dung cho cac thi nghiem xae djnh d(lc tinh vanadium va <lnh bllong cua chilosan chieu xa len cay
cue lrongdi~u kil$nin vitro.
2. Khao sat anh hlldng chilosan dt mach b~ng bac x~ gamma (200 kGy va 400 kGy) len cay con trong di6u kienin vi/roo
3. Khao sat d(lc linh vanadium len cay con in vitro, xac djnh n6ng d(l
vanadium gay d(lc ma tai d6 acche 50% sinh tru'ong cay (LG 50). 4. Khao sal lac dung ket hdp giiia chitosan phan giai b~ng bac xa
gamma (li6u cbieu thich h<;lp) va vanadium (li6u gay d(le 50%).
PMn thl1 hai. Do'i tw!ng va phuangI'MI'nghien cl1u
C6 lhi khai qual cong viec theosdd6 sau:
Cayhoa cllc
Chrysanthemum indiumL.
Khdi d(lngm~m
Nhan nhanh va khaoSalanh huang I - - phytohormon len sl!l<lisinhcay
Khao sat anh Khilo sal ilnh Khilo sal ilnh
hudng cua NAA hudng cua BA hudng cua IBA
Caycon
I
Anh hu'dngchilosanlencfiy r-- Khilo sal doc linh
can vanadium len cay con
~ ~
Chitosan chie'u x0- Chiwsan chi€ux~ Khilo salke,hqp
200kGy 400kGy chilosan 200kGy
va vanadium
33
Hinh 09. Sadr5thi nghi¢m khiJo sal dQc linh vanadium va tacd~ngchitosan
chie'uX(llenCelYhoacuein vitro.
Phan th'i hai. EJo'i {'(lng vii phuong phdp nghd!n cttu 34
6 cae Lhinghi~m, chung toi SlYdung moi tnrang eel ban la LS (Linsmaier & Skoog, 1965) (lheo Sahavacharin, 1995) [651. Moi u'lIang nay c6 Ihilllb
ph5n nhl(sau: • E)a IlI<!ng KNo, NH. 0, CaCI2.2H,O MgS04.7H,O KH,P04 • Vi lll<!ng MnS04. 4H20 ZnS04. 7H20 H,BO, KI Na2Mo04.2H20 CuS04.5H20 CoCl2·6H20 Na, EDTA FeS04. 7H20 • Vilamin Thiamine HCl Myo-lnositol • Cac ChaL khae
Agar Saccharose • pH: 5,7-5,8 1900 mgll 1650 mgll 440 mgll 370 mgll 170 mg!1 22,3 mgll 8,6 mgll 6,2 mgll 0,83 mgll 0,25 mgll 0,025 mgll 0,025 mgll 37,3 mg!1 27,8 mg!1 0,4 mg!1 100 mgll 14000 mg!1 30000 mg!1
Tit moi trllang cel ban tren, chung toi bo tri cae moi trllang Ihi nghiem bao g6m:
Khdi dQng mau ban d;lu, cbung toi stl' dung vat lieu la cae dOL ng'in va dOllhan diSnuoi caynh~D!LID! hi<lu anb hlldng cua v~L li~ulen qua trlnb Lai sinh cay, moi trllang Melc stl' dung la: LS + BA AA !beo ty I~
Philn tht( hai. £)6'"lLlC/ng vel phtldng phdp nghien Ct(u 35
0,2/0,05 mg/l theo kinh nghi~m cua Tl1Ing lam Cang ngh~ Sinh hoc
Nang nghicp Dalat (Thi nghiem I).
Xac dinh mai tntong nhan gi6ng, chung lai khao S~l'nh huong clia BA lheo cae n<lng d(l: 0,1; 0,3 va 0,5 mgll (Thi nghi~m 2). Tit lhi nghiem nay, chQn ra n<lng d(l BA thich hdp cho mai tntong nhan gi6ng. Xac dinh h~so' nhan.
Cay con thu dUqc tmng qua trlnh nhan gi6ng se dUdC cify chuy6n hai Iftn sang mai truong LS khang b6 sung kich thich sinh lntong M lam gram luqng phytohormon tmng cbung do qua trlnh nhan gi6ng. C~cthinghi~m lie'n hanh sau nay se Slt dung mh cify la cac d6t ngQn c6 tu 3-4 la, cao 1-1,3 COlM dam baD d(l d<lng Mu v6 tu6i sinh lji, tang d(l chinh xac cua thi nghi~m.
Vdi muc dich lim hieu vai [fa clia NAA va IBA len l~i sinh cay, chung tai khao sat anh hltong clia chung len sinh trudng cay. Mai tntong dUdC dilng nhlr sau:
LS+IBA theo cac n<lng dO: 0,1; 0,3 va 0,5 mgll (Thi nghiem 3). LS+NAA theo cac n<lng d(l: 1,0; 1,3 va 1,5 mgII (Thinghi~m4). DG lim hiGu ,Inh hlrdng clia chitosan chie'u xa len sinh lntdng va pMt
tri6'n diy u'ong di6u kit$n in vitro, chung t6i dung m6i tn(ong c6 b6 sung
chilosan chie'u xa li6u 200 kGy va 400 kGy theo cac n6ng do sau: LS + chitosan 200 kGy: 5; 15; 30; 50; 100 mg/l (Thinghi~m5) LS + chitosan400 kGy: 5; 15; 30; 50; 100 mg/l (Thi nghiem 6) Sau d6 so s~nh, chQn ra chilosan chie'u x~ c6 li6u chie'u thich hdp vdi n6ng d(llhich hqp cho Slt sinh ulrdng va phat triGn cay.
8& tim hi&u dQc tinh vanadium len cay con, chung toi b6 sung vanadium
(V)dudi dang vanadat amon (Nfi,N03) vao mai truong nbu sau: LS + V: 1,0; 2,5; 5,0; 10; 30; 50; 100; 200 mgVII (linh theo V) (Thinghi~m7). DG xac djnh kha nang giai d(lc kim lo~i nang cua chitosan chie'u xa len cay con [fong di6u kien in vilro,chung tai b6 Iri thi nghi~m b6 sung ke't hqp V (li6u gay (rc che' 50%) va chitosan chie'u xa d li6u thich hdp (dudC xac dinh 0 thinghi~m5 va 6) vdi n6ng d(l thay d6i nhu sau:
Phdn /hu hai. EJ6i /ul!ng va phuong phdp nghien cua
LS+LG 50 V+chitosan (200 hoac 400 kGy) thea cae n6ng di): 5; 15; 30; 50; 100 mgll (Thinghi~m8).
n.Tie'n hanhth,nghi~m
a. Khii'trung milu clly
36
ChQn canh hoa hOac ch6i non m~nh khoe, dt bo la, hoa, phiin than gia.
Tien hanh kht'( lrung nhtfsau:
Rtta mau nhi6u liin bang xa phang dlidi vai nlidc may. Rli'a lai mau bang mrdc c:lt v6 trung.
Khd' bang dung djch c6n 70% U'ong Iphul. Rli'a lai mau bang mrdc dlt v6 trung.
Khi'r bang dung dich hypocloride canxi 10% til' 4 - 5 phul.
Rii'a lC;ii mall bangmfdccalVO lIung.
b. Di6uki~nva chi tieu thea doi
De" tang tfnh d6ng Mu, chung t6i b6 tri mili nghiem thuc g6m 40 mau
dy (dlroC chia vao 4 blnh tam grac, mili binh 10 milu). Tilt cii cac blnh tam giac m6i trlidng Mu drr<Jc khi'r U,)ng d nhi~tdi) 121"C, lrong thdi gian 25 phut, ap suilt I atm va drr<JcM ngu6i tnrdc khi cily.
Cac thao tac cily dlrQC tiGn hanh trong di6u kien v6 lrung. Tilt c;i cac blnh m6i tnrdng sau khi cily d6u drrQc dat trong di6u ki~nanh sang den huynh quang vdi IN'li gian chiGu sang 10-12 gid/ngay, Wdng di) chiGu sang 2000-3000 lux, nhiet di) phang nu6i dao di)ng 20-25"C.
Mili thinghi~mdli<Jc Jap lai 3 liin. Cac s61ieu thu dli<Jc sau 30 ngay nu6i kG tii' ngay cily.
c. Can trQng IliQ'ng khO
Mau dli<JC chon can la toan bi) cay can thu dlroC tii' cacnghi~mthuc cda thi nghi~mtim hiGu :lnh hlidng chitosan chiGu xa li6u 200 kGy va 400 kGy len ciiy cucin vitro. Sau 30 ngay nu6i cily, cay dli<Jc lily ra khoi blnh tam giac sao
cho kh6ng dii't 1'6, rli'a sach thach bam vao 1'6 va dem sily kh6 d nhiet d6 40'C cho Mn luc U'ong Ilr<Jng kh6ng d6i. Mau dli<Jc dat vao blnh hut am chan kh6ng va dlrQc can bang can miligam.
Phdn Iha hai. £)6'iluC/ng Viiphuong phdp nghien cau
d, Cac chiti~utheo doi
Chieu cao trung blnheuacay.
Ht% s6 nhan trung blnh cua moi nghit%m thuc.
Ty 11$phan h6a r~ biGu thi bAng phIln tram s6mau U,lO r~. TrQng Illqng kh6 lrung blnh ctla diy,
Thoi gian r~ b~t d~uhlnh thanh, Tlnh tr~ngcay,
37
Cac chi tieu (chi€u cao, he siSnhan, IrQng Illqng kh6) trung blnh cua cay dtrqc tinh theo c6ng thuc (theoNguy~nEJlnh Khoa, 1975) [3]: