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TRNG I HC NNG LM TP.HCM
B GIO DC V O TO
KHOA: CNG NGH SINH HC
NG TH THANH THY
NHN GING IN VITRO CY GING HNG
(Pterocarpus macrocarpus)
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
Tp.H Ch Minh
Thng 08/2006
2
TRNG I HC NNG LM TP.HCM
B GIO DC V O TO
KHOA: CNG NGH SINH HC
NHN GING IN VITRO CY GING HNG
(Pterocarpus macrocarpus)
Lun vn k s
Chuyn ngnh: Cng ngh sinh hc
GVHD: SVTH:
TS.Trn Th Dung ng Th Thanh Thy
Kha: 28
Tp.H Ch Minh
Thng 08/2006
3
LI CM T
Ti xin chn thnh cm t:
Ban Gim hiu trng i hc Nng Lm Thnh Ph H Ch Minh, Ban
ch nhim B Mn Cng ngh sinh hc, cng tt c qu thy c truyn
t kin thc cho ti trong sut qu trnh hc ti trng.
TS. Trn Th Dung ht lng gip ti trong sut thi gian thc tp tt
nghip.
ThS. Trng Mai Hng cung cp ht ging gip ti thc hin ti.
Cng lao to ln ca cha m khng ngi cc kh nui con khn ln
v cho con c n hc ti ngy hm nay.
Cm n KS. Nguyn Th Thu Hng, KS. Trn Th Bch Chiu v KS. Tn
Bo Linh gip ti rt nhiu v to iu kin thun li cho ti trong
sut thi gian thc tp tt nghip.
Cc bn b thn yu ca ti chia s cng ti bao kh khn trong lc
thc tp.
Sinh vin thc tp
ng Th Thanh Thy
4
TM TT
NG TH THANH THY, i Hc Nng Lm Tp. H Ch Minh. Thng
2/2006. Nhn ging in vitro cy Ging hng (Pterocarpus macrocapus). Gio vin
hng dn: TS. TRN TH DUNG.
Ging hng l mt trong nhng loi cy g qu ang c nguy c b tit chng
do nn cht ph rng ba bi. khi phc li hin trng rng nh trc y phi mt
rt nhiu thi gian. Nhng y l mt vic v cng cp bch cu nguy cho tnh
trng l phi ca hnh tinh ang ngy cng b thng tn. V th, chng ti tin hnh
nhn ging v tnh cy ging hng tm ra quy trnh sn xut ging hng m bo
v s lng v cht lng.
Nhng kt qu t c:
Ht ging hng rt kh nhim khun hay nhim nm so vi nhng b phn
khc nh ht phn, hoa, Tuy nhin, ht ging hng c v bc dy lm hn ch kh
nng ny mm ca ht, dn n gim s lng cy con in vitro.
Mi trng MS c b sung nng BA = 1,5 (mg/l) v NAA = 0,1 (mg/l) thch
hp cho s to chi ca cy ging hng in vitro.
B sung vo mi trng nui cy WPM nng NAA = 2mg/l s to c cy
ging hng in vitro hon chnh vi thi gian to r l nhanh nht (ch trong 4 ngy l
xut hin r).
5
MC LC
CHNG TRANG
Li cm t .......................................................................................................................... i
Tm tt ............................................................................................................................... ii
Mc lc ............................................................................................................................. iii
Danh sch cc bng .......................................................................................................... vi
Danh sch cc hnh ......................................................................................................... vii
Danh sch cc ch vit tt .............................................................................................. viii
1. M U ....................................................................................................................... 1
1.1 t vn .............................................................................................................. 1
1.2 Mc ch yu cu ................................................................................................... 2
1.2.1 Mc ch ......................................................................................................... 2
1.2.2 Yu cu ........................................................................................................... 2
1.3 Gii hn ti ........................................................................................................ 2
2. TNG QUAN TI LIU .............................................................................................. 3
2.1 Gii thiu v cy ging hng ................................................................................ 3
2.1.1 M t cy ......................................................................................................... 3
2.1.2 Sinh hc ......................................................................................... ................. 3
2.1.3 Phn b .......................................................................................... ................. 3
2.1.4 c im g v cng dng ............................................................ ................. 4
2.1.5 Tnh trng ....................................................................................... ................. 4
2.1.6 Gii php bo v ............................................................................ ................. 4
2.2 Nhn ging cy trng in vitro ............................................................... ................. 4
2.2.1 Khi nim nui cy m t bo thc vt ......................................... ................. 4
2.2.2 C s khoa hc chung v nui cy m t bo thc vt ................. ................. 5
2.2.3 Li ch ca nhn ging bng nui cy m t bo thc vt ............ ................. 6
2.2.4 Cc phng php nui cy m t bo thc vt .............................. ................. 7
2.2.4.1 Nui cy nh sinh trng ...................................................... ................. 7
2.2.4.2 Nui cy m so ..................................................................... ................. 7
6
2.2.4.3 Nui cy t bo n ................................................................ ................. 8
2.2.4.4 Nui cy protoplast - chuyn gen ........................................... ................. 8
2.2.4.5 Nui cy ht phn n bi ..................................................... ................. 8
2.2.5 Cc giai on nhn ging in vitro .................................................. ................. 8
2.2.5.1 Giai on 1 ............................................................................. ................. 8
2.2.5.2 Giai on 2 ............................................................................. ................. 9
2.2.5.3 Giai on 3 ............................................................................. ................. 9
2.2.5.4 Giai on 4 ............................................................................. ................. 9
2.2.5.5 Giai on 5 ............................................................................. ............... 10
2.2.6 Cc yu t nh hng n nhn ging in vitro .............................. ............... 10
2.2.6.1 Mu nui cy .......................................................................... ............... 10
2.2.6.2 iu kin nui cy .................................................................. ............... 11
2.2.6.3 nh hng ca mi trng nui cy ...................................... ............... 12
2.2.7 Nhng vn trong nhn ging in vitro ........................................ ............... 12
2.2.7.1 Tnh bt nh v mt di truyn ............................................... ............... 12
2.2.7.2 S hoi mu ............................................................................ ............... 13
2.2.7.3 Vic sn xut cht gy c t mu cy .................................. ............... 13
2.2.7.4 S dng thuc khng sinh ...................................................... ............... 14
2.2.7.5 Hin tng thy tinh th ......................................................... ............... 14
2.2.8 Cht iu ha sinh trng thc vt ................................................ ............... 14
2.2.8.1 Auxin ...................................................................................... ............... 15
2.2.8.2 Cytokynin ............................................................................... ............... 15
2.2.9 Nhng thnh tu v nui cy m cy rng .................................. ............... 16
2.2.9.1 Trn th gii ........................................................................... ............... 16
2.2.9.2 Ti Vit Nam .......................................................................... ............... 17
3. VT LIU V PHNG PHP NGHIN CU ..................................... ............... 19
3.1 i tng th nghim ............................................................................ ............... 19
3.2 Thi gian v a im nghin cu ......................................................... ............... 19
3.3 Vt liu nghin cu ............................................................................... ............... 19
3.3.1 Trang thit b v dng c dng trong nghin cu .......................... ............... 19
3.3.2 Mi trng nui cy ...................................................................... ............... 19
3.4 iu kin nui cy in vitro .................................................................... ............... 21
7
3.5 Phng php kh trng ......................................................................... ............... 21
3.5.1 Vt liu ........................................................................................... ............... 21
3.5.2 Phng php kh trng mu .......................................................... ............... 21
3.5.3 Cy mu ......................................................................................... ............... 21
3.6 Phng php th nghim ....................................................................... ............... 22
3.6.1 Th nghim 1 .................................................................................. ............... 22
3.6.2 Th nghim 2 .................................................................................. ............... 22
3.6.2.1 Th nghim 2a ......................................................................... ............... 22
3.6.2..2 Th nghim 2b ....................................................................... ............... 23
3.6.3 Th nghim 3 .................................................................................. ............... 24
3.6.4 Phn tch thng k ......................................................................................... 25
4. KT QU V THO LUN ..................................................................... ............... 26
4.1 Th nghim 1: nh hng ca nng v thi gian kh trng n t l sng ca
mu cy cy ging hng in vitro. ................................................................................. 26
4.2 Th nghim 2 ......................................................................................... ............... 27
4.2.1 Th nghim 2a: nh hng ca nng BA v NAA ln kh nng to chi
ca cy ging hng in vitro ........................................................................... ............... 28
4.2.2 Th nghim 2b: Kho st nh hng ca mi trng nui cy n kh nng
to chi ca cy ging hng in vitro ............................................................................ 30
4.3 Th nghim 3: Kho st nh hng ca IBA v NAA n s hnh thnh r ca
cy ging hng in vitro .................................................................................. ............... 31
5. KT LUN V NGH ......................................................................... ............... 33
5.1 Kt lun ................................................................................................. ............... 33
5.2 ngh .................................................................................................. ............... 33
6. TI LIU THAM KHO ........................................................................... ............... 34
PH LC ........................................................................................................ ............... 35
PH LC 1 ................................................................................................. ............... 36
PH LC 2 ................................................................................................. ............... 38
8
DANH SCH CC BNG
Bng Trang
4.1: Kt qu kh mu ht ging hng sau 4 tun nui cy trn mi trng
MS. .............................................................................................................................. 26
4.2: nh hng ca nng BA v NAA ln kh nng to chi ht ging hng sau 6
tun nui cy. ................................................................................................................... 28
4.3: nh hng ca mi trng nui cy n kh nng to chi ca ging hng in
vitro sau 6 tun nui cy. ................................................................................................ 30
4.4: nh hng ca IBA v NAA n s hnh thnh r ca ging hng in vitro sau 4
tun nui cy. .................................................................................................................. 31
9
DANH SCH CC HNH
Hnh Trang
2.1:Cy ging hng ........................................................................................................ 3
2.2:Mu g ging hng ................................................................................................. 4
4.1: Ht ging hng in vitro ny mm .......................................................................... 27
4.2 Cy con ging hng in vitro ................................................................................... 27
4.3: Chi cy ging hng in vitro c to thnh sau 6 tun nui cy trn cc mi
trng khc nhau ............................................................................................................ 29
4.4: Chi cy ging hng in vitro c to thnh sau 6 tun nui cy trn cc mi
trng khc nhau ............................................................................................................ 30
4.5: Cy ging hng in vitro hon chnh sau 4 tun nui cy ....................................... 32
10
DANH SCH CC CH VIT TT
MS : Murashige v Skoog
WPM : Llooyd v Mc Cown
HSTTV : iu ha sinh trng thc vt
PVP : Polyvinyl pyrrolidone
ABA : Acid abxixic
IBA : Indol butyric acid
NAA : Napthlacetic acid
2,4-D : 2,4-Dichlorophenol acetic aicd
IAA : Indol acetic acid
BAP : 6-Benzylaminopurin
Ki : Kinetin
Z : Zeatin
TDZ : Thidiazuron
BA : Benzyl adenin
11
Chng 1.M U
1.1 t vn
Rng l h sinh thi c ngha to ln trong bo v mi trng sng con ngi.
Rng cng l ngun ti nguyn di do p ng c s pht trin ca nn kinh t t
nc. Bn thn rng l h sinh thi ln phc tp v t iu chnh (Siscop, 1978).
Khng ch cung cp ti nguyn phc v i sng con ngi, rng cn gp phn
quan trng trong vic ti to tiu kh hu, lm trong sch mi trng, chng xi mn
t, chng l lt, c bit trong chin tranh, tn ca nhiu khu rng i vo lch
s, tm thc ca ngi Vit Nam nh: Cai Kinh, Tr Lnh, Trng Sn, Ngc
dng lch s ta thy rng gn b lu i vi ngi Vit, nhng tc ngi Vit c
sng trong hang Con Moong t thi c xa sng nh rng m pht trin. Rng cn
i vo thi ca truyn thuyt, cc cu truyn c, C th ni rng l mt phn trong i
sng vn ho ca dn tc ta.
Th nhng qua nhiu thp k, trn quy m ton cu, rng nhit i ang ngy
cng b tn ph, suy kit do nhiu nguyn nhn. Nhiu nh khoa hc nh gi h sinh
thi rng nhit i l phc tp nht nhng cng rt d suy tn, kh nng phc hi km
sau khi b nhng tc ng nghim trng.
Do nhn thc r vai tr c bit quan trng ca rng cng nh ca cy xanh i
vi mi hot ng ca con ngi cho nn trong nhng nm gn y cng tc trng
rng rt c ch trng. Cng nh ho vo nhp pht trin cng ngh sinh hc
trn th gii. Ngnh lm nghip v nh nc u t xy dng nhng trung tm ging
cy trng lm nghip vi quy m hin i nh trung tm nui cy m thuc X nghip
ging v phc v trng rng TP.HCM, Trung tm nghin cu lm nghip Ph Ninh
(Vnh Ph).
Phn ln cy g sinh sn bng ht. Cc nh chuyn mn thng ni: Mun
nn sn xut nng-lm nghip n nh, c nng sut cao, cng tc ging phi i trc
mt bc, ring i vi cy rng th thi gian i trc t nht phi mi nm (L
nh Kh, 1992).
12
Rng nc ta ang b mt dn nhiu loi thc vt c gi tr kinh t cao. Trong
ging hng l loi cy g qu. Hin nay do tnh trng ph rng lm cho tr lng
ca loi ny b gim st nng v nm trong danh sch cc loi cn c bo v.
Vi cc iu kin v c l cng khng cn l qu sm i vi cng cuc
trng rng trong tng lai ca t nc, c s chp thun ca B mn Cng ngh
sinh hc v s hng dn tn tnh ca c Trn Th Dung chng ti thc hin ti:
Nhn ging in vitro cy ging hng (Pterocarpus macrocarpus).
1.2 Mc ch yu cu
1.2.1 Mc ch
- Kho st nh hng ca nng v thi gian kh trng n t l sng ca ht
ging hng in vitro.
- Kho st nh hng ca mi trng nui cy n s nhn chi v kh nng
to r ca cy ging hng in vitro.
1.2.2 Yu cu
- Xc nh c nng v thi gian kh trng thch hp i vi ht ging
hng in vitro.
- Xc nh nng cht iu ho sinh trng thc vt thch hp n kh nng
to chi ca cy ging hng in vitro bng phng php nui cy chi nch.
- Xc nh nng cht iu ho sinh trng thc vt thch hp cho s to r
cy ging hng in vitro trc khi em ra vn m.
1.3 Gii hn ti
Do thi gian c hn nn cha thc hin c th nghim a cy con ging
hng in vitro ra vn m.
13
Chng 2. TNG QUAN TI LIU
2.1 Gii thiu khi qut v cy ging hng
Tn thng gi: Cy Hng
Tn khoa hc: Pterocarpus macrocarpus Kurz
H: Fabaceae
H ph: Faboideae
B:Fabales
2.1.1 M t cy
Ging hng l loi cy g ln, thuc loi cy g thn thng, trn to c tn rng,
c chiu cao khong t 25 n 40 mt, ng knh thn 0,9 mt hay ln hn, thay l
vo ma kh, gc c bnh v, v mu nu sm, nt dc. Khi b thng s c nha c
mu ti chy ra. Cnh non mnh v c lng, cnh gi nhn. L kp lng chim l
mt ln, di t 15-25cm; mang 9-11 l cht. L cht c hnh bu dc thun, gc trn,
u c mi nhn cng. Hoa c mu vng v c mi thm, lm thnh chm nch l,
c cung di v nhiu lng mu nu. i hnh chung cong gc, c 5 rng ngn, gn
bng nhau hay khng bng nhau. Qu hnh trn dp, c mi cong v hng cung,
mu vng nu, gia qu c t 1 n 2 ht, xung quanh c cnh mng v c lng mn
nhung.
2.1.2 Sinh hc
L loi c lng qu c sinh ra hng nm rt nhiu, nhng kh nng ti sinh
ht rt km c th do la rng. Tuy nhin, v kh nng ti sinh chi th rt kho mnh.
Cy con c to t ht mang trng s pht trin nhanh trong thi gian rng non, tng
trng chiu cao mnh nht lc 16-20 nm tui, n giai on trung nin s chm dn.
2.1.3 Phn b
Ging hng phn b ch yu trong rng rm nhit i na rng l, mc
cao di 700-800m, thng mc hn giao vi mt s loi cy l rng khc. Chn hn
nh ging hng thng mc ranh gii vi rng rng l cy h du
(Dierocapaceae), mc hn giao vi mt s loi cy l rng khc nh g (Afzelia
xylocalpa), mung en (Cassia siamea), bng lng (Lagerstromia sp.), bnh linh (Vitex
Hnh 2.1:Cy ging hng
14
sp.), a t c thnh phn t tht nh n trung bnh, phong ha t cc trm tch
v macma acid, c khi c trn t bazan.
2.1.4 c im g v cng dng:
G p, c mi thm, mu nu hng, mn, c vn
p do vng nm kh r rng, tia rt nh, mt cao;
mch to , t trng 0,84 0,90.
Thuc nhm g qu him, c mi thm, hoa vn
rt p, c nhiu ngi a chung, dng lm
g cao cp v mt hng m ngh, t b nt n v khng b mi mt. Ngoi ra nha cn
c th lm thuc nhum mu .
2.1.5 Tnh trng:
L loi cy rng c gi tr kinh t cao. nc ta rt t c din tch rng cy ging
hng mc tp trung tui thnh thc, ch yu l mc ri rc, an xen vi nhng
loi khc, hoc ti sinh t nhin sau nng ry, do ngi dn bo v, nui dng trong
t vn ry, ang tui cn non hoc tui trung nin. Cy c ng knh ln th rt
him. L loi cy qu him ca tnh ang b ngi dn khai thc s dng khng hp
l, lm gim dn v s lng vn khan him li cng khan him hn. Mc e
da: bc V.
2.1.6 Gii php bo v:
Ging hng l i tng c bo v ca cc khu rng cm Mom Ry, Ch.
n,
ngh Kim lm a bn v Chnh quyn a phng qun l cht ch, bng
cch khng cp giy php cht h i vi nhng loi cy trung nin, cha n tui
thnh thc, nhm bo v ngun gen qu him. ng thi, ngh trng xen vo din
tch trng keo l trm v vn ng cng ng trng theo ranh t.
2.2 Nhn ging cy trng in vitro
2.2.1 Khi nim nui cy m t bo thc vt
Nui cy m t bo thc vt l mt cng c cn thit trong nhiu lnh vc
nghin cu c bn v ng dng ca ngnh sinh hc. Nh p dng k thut nui cy
m, con ngi thc y thc vt sinh sn nhanh hn gp nhiu ln tc vn c
Hnh 2.2:Mu g ging hng
15
trong t nhin. Do , to ra hng lot c th mi gi nguyn tnh trng di truyn ca
c th m, lm rt ngn thi gian a mt ging mi vo sn xut.
Hn na, da vo k thut nui cy m c th duy tr v bo qun cy trng qu
him.
Nhn ging v tnh bng k thut nui cy m bt u bng mt mnh nh thc
vt v trng t vo mi trng dinh dng thch hp. Chi mi hay m so m mu
cy ny to ra bng s tng sinh c phn chia v cy chuyn nhn ging.
Nui cy m t bo thc vt cho n nay c chng minh l phng php
nghin cu qu trnh hnh thnh c quan hiu qu nht. Nm 1939, nghin cu qu
trnh hnh thnh c quan trn s hnh thnh chi (White, 1939) v r (Nobercourt,
1939). V cc kt qu nghin cu v s tc ng ca cc nhn t bn trong v bn
ngoi nh hng n s hnh thnh c quan (Thorpe, 1980, 1988). Qua kt qu nghin
cu qu trnh hnh thnh c quan in vitro, cho thy c 3 nhn t nh hng trc tip:
Mi trng nui cy, iu kin nui cy v mu c s dng trong nui cy.
Vn dng qu trnh hnh thnh c quan in vitro qua s tc ng tng h ca
cc nhn t ni trn, c hng ngn loi thc vt c nghin cu qu trnh hnh
thnh chi v r (Brown & Thorpe, 1986).
2.2.2 C s khoa hc chung v nui cy m t bo thc vt
Nui cy m t bo thc vt l mt ngnh khoa hc tr nm trong sinh l thc
vt. nc ta ngnh ny mi c ch v pht trin khong 15 20 nm tr li y.
Trong cng tc ging cy trng, k thut nui cy m t bo thc vt c pht
trin nhng c s l thuyt v t bo hc v c s sinh l thc vt hc nh Nguyn
Vn Uyn (1993) v mt s nh nui cy m nc ngoi nhn nh:
- l tnh ton th ca m v t bo thc vt, cho php ti sinh c cy hon
chnh t m, thm ch t mt t bo nui cy tch ri. y l mt im rt quan trng,
bi v trn c s n v m, t bo, cc nh sinh vt hc thc hin c nhng k
thut tin tin cho vic chn, ci thin v c lai to ging cy trng.
- Kh nng loi tr virus bng nui cy nh sinh trng, to cc dng v tnh
sch bnh cc cy nhn ging v tnh. Vn ny c cc nh khoa hc khai thc
phc trng cc ging khoai ty, cy n tri (cam, qut).
16
- Kh nng dng chi nch, cc th chi protocorm vo nhn ging v tnh vi
tc cc nhanh cy trng phc v sn xut: cy lng thc (khoai ty), cy cnh
(phong lan), cy lm nghip (bch n, tch,...).
- Kh nng bo qun cc ngun gen bng nui cy trong ng nghim, kh nng
trao i Quc t cc ngun gen sch bnh di dng cy nui trong ng nghim.
- Kh nng to cc cy n bi qua nui cy ti phn v ht phn, t to ra
cc dng ng hp t tuyt i v nh rt ngn c chu trnh lai to.
- Kh nng hp thu DNA ngoi lai vo t bo nh cng ngh gen.
- Kh nng nui cy t bo thc vt nh nui cy vi sinh vt v qua kh
nng ng dng di truyn phn t vo thc vt bc cao phc v cng tc to ging.
- K thut nui cy protoplast v kh nng dung hp protoplast ti sinh cy
hon chnh t cc protoplast lai.
- Kh nng s dng nui cy phi khc phc hin tng bt th khi lai xa.
- Kh nng tn tr cc t bo thc vt sng trong thi gian di v nhit
thp khng mt tnh ton th ca t bo.
ng thi nui cy m t bo cng to nhng c s cho qu trnh nghin cu di
truyn thc vt, vai tr cht iu ho sinh trng thc vt.
Ngy nay cng vi cng ngh gen, nui cy m t bo l mt phn quan trng
khng th thiu, thc y vic ng dng cng ngh sinh hc trong ngnh kinh t. Hai
nhim v ln ca cng ngh sinh hc thc vt nc ta t nay ti nm 2010 l: To ra
cc ging cy trng mi bng phng php cng ngh sinh hc thc vt, c bit l
cng ngh gen v nhn nhanh cc ging, dng u vit bng k thut nui cy m t
bo thc vt (Nguyn Vn Uyn, 1995).
2.2.3 Li ch ca nhn ging bng nui cy m t bo thc vt
Theo Bi B Bng (1995), nhn ging bng nui cy m c nhng li im
sau:
To ra cy con ng nht v ging nh cy m. Phn ny ging nh nhn ging
v tnh. i vi cc cy trng thuc nhm th phn cho nh phn ln cc loi cy n
tri, cc cy con sinh ra t ht khng hon ton ng nht, v c th khng ging nh
cy m, trong trng hp ny nhn ging v tnh c li im hn nhn ging qua ht.
17
So vi kiu nhn ging v tnh thng thng (chit cnh, hom), nhn ging bng nui
cy m c u im l c th nhn mt s lng cy con ln t mt c th ban u
trong thi gian ngn.
C th to ra cy con sch bnh nh p dng vic chn lc vt liu ban u mt
cch cht ch hoc lm cho vt liu ban u tr nn sch bnh. Khng chim nhiu
din tch, khng b nh hng bi thi tit, iu kin ngoi cnh. Mt ging cy qu
c th c nhn ra nhanh chng a vo sn xut. Vic trao i ging c d
dng.
2.2.4 Cc phng php nui cy m t bo thc vt
Theo Dng Cng Kin (2002), c mt s phng php nui cy m t bo
thc vt nh sau:
2.2.4.1 Nui cy nh sinh trng
Mt trong nhng phng thc sinh trng t c mc tiu trong nui cy
t bo v m thc vt l nui cy nh sinh trng (bao gm nui cy chi nh v
chi bn).
Sau khi v trng, mu s c nui cy trn mi trng thch hp cha y
cht dinh dng khong v c v hu c hoc mi trng khong c b sung cht
kch thch sinh trng thch hp,
T nh sinh trng, sau mt khong thi gian nui cy nht nh mu s pht
trin thnh mt chi hay nhiu chi. Chi tip tc pht trin vn thn, ra l v r
tr thnh cy hon chnh. Cy con c chuyn ra t dn dn thch nghi v pht trin
bnh thng.
2.2.4.2 Nui cy m so
M so l mt khi t bo pht trin v t chc, hnh thnh do s phn phn
ho ca t bo phn ho. M so s pht trin nhanh khi mi trng c s hin din
ca auxin. Khi m so c kh nng ti sinh thnh cy hon chnh trong iu kin mi
trng khng c cht kch thch to m so.
18
2.2.4.3 Nui cy t bo n
Khi m so c nui cy trong mi trng lng v c t trn my lc c
tc iu chnh thch hp s tch ra thnh nhiu t bo ring l gi l t bo n. T
bo n c lc v nui cy trn mi trng c bit tng sinh khi.
Sau mt thi gian nui cy ko di trong mi trng lng t bo n c tch
ra v tri trn mi trng thch. Khi mi trng thch c b sung auxin, t bo n
pht trin thnh cm t bo m so. Khi trn mi trng thch c t l cytokinin
auxin thch hp, t bo n c kh nng ti sinh thnh cy hon chnh.
2.2.4.4 Nui cy protoplast - chuyn gen
Protoplast (t bo trn) l t bo n tch lp v cellulose, trong iu kin nui
cy thch hp, protoplast c kh nng ti sinh mng t bo, tip tc phn chia v ti
sinh thnh cy hon chnh.
Khi t bo mt vch v tin hnh dung hp, hai protoplast c kh nng dung
hp vi nhau to ra t bo lai, c tnh ny cho php ci thin ging cy trng. Qu
trnh dung hp protoplast c th c thc hin trn hai i tng cng loi hay khc
loi.
2.2.4.5 Nui cy ht phn n bi
Ht phn thc vt c nui cy trn nhng mi trng thch hp to thnh
m so. M so ny c ti sinh thnh cy hon chnh l cy n bi.
2.2.5 Cc giai on nhn ging in vitro
Theo Nguyn Xun Linh (1998), s thnh cng ca vic nhn ging in vitro ch
t c khi tri qua cc giai on:
2.2.5.1 Giai on 1: Kh trng m nui cy
y l giai on ti quan trng quyt nh ton b qu trnh nhn ging in
vitro. Mc ch ca giai on ny l phi to ra c nguyn liu v trng a vo
nui cy in vitro.
Theo ti liu ca Street (1974) cc cht dit nm khun x l m nui cy
nh sau:
19
Tc nhn v trng Nng (%) Thi gian x l Hiu qu
(pht)
Hypochlorit Calcium 9-10 5-30 Rt tt
Natri hypochlorit 2 5-3 Rt tt
Hydroperoxid 10-12 5-15 Tt
Nc brom 1 -2 2 10 Rt tt
HgCl2 0,1 -1 2 10 TB
Cht khng sinh 4 50mg/l 30 60 Kh tt
V trng m cy l mt thao tc kh, t khi thnh cng ngay ln u tin. Tuy
vy, nu kin tr tm c nng v thi gian v trng thch hp th sau vi ln th
chc chn s t kt qu.
2.2.5.2 Giai on 2: Ti sinh mu nui cy
Mc ch ca cc giai on ny l s ti sinh mt cch nh hng cc m nui
cy. Qu trnh ny c iu khin ch yu da vo t l ca cc hp cht auxin,
cytokynin ngoi sinh a vo mi trng nui cy. Tuy nhin, bn cnh iu kin
cng cn quan tm ti tui sinh l ca mu cy. Thng m non, cha phn ho c
kh nng ti sinh cao hn cc m trng thnh chuyn ho su. Ngi ta cng cn
nhn thy rng mu cy trong thi gian sinh trng nhanh ca cy trong ma sinh
trng cho kt qu rt kh quan trong ti sinh chi.
2.2.5.3 Giai on 3: Nhn nhanh
Giai on ny c coi l giai on then cht ca qu trnh. tng h s nhn,
ta thng a thm vo mi trng dinh dng nhn to cc cht iu ho sinh trng
(Auxin, Cytokynin, Gibberellin,), cc cht b sung khc nh nc da, dch chit
nm men, kt hp vi cc yu t nhit , nh sng thch hp. Tu thuc vo tng
i tng nui cy, ngi ta c th nhn nhanh bng kch thch s hnh thnh qua cc
cm chi (nhn cm chi) hay kch thch s pht trin ca cc chi nch (vi gim
cnh) hoc thng qua vic to cy t phi v tnh.
2.2.5.4 Giai on 4: To cy hon chnh
Khi t c kch thc nht nh, cc chi c chuyn t mi trng giai
on 3 sang mi trng to r. Thng 2 3 tun, t nhng chi ring l ny s xut
20
hin r v tr thnh cy hon chnh. giai on ny ngi ta thng b sung vo mi
trng nui cy cc auxin l nhm hormon thc vt quan trng c chc nng to r
ph t m nui cy.
2.2.5.5 Giai on 5: a cy ra t
Giai on a cy hon chnh t ng nghim ra t l bc cui cng ca qu
trnh nhn ging in vitro v l bc quyt nh kh nng ng dng qu trnh ny trong
thc tin sn xut.
y l giai on chuyn cy con in vitro t trng thi sng d dng sang sng
hon ton t dng, do phi m bo cc iu kin ngoi cnh (nhit d, nh sng,
m , gi th,) ph hp cy con t t l sng cao trong vn m cng nh
rung sn xut.
2.2.6 Cc yu t nh hng n nhn ging in vitro
2.2.6.1 Mu nui cy
Murashige (1974) ghi nhn s quan trng ca chn la mu cy thch hp v
ch cho thy hu ht nhng c quan c th dng nui cy m. iu quan trng cho
thy mt s nhn t khi chn lc mu bao gm kiu gen, c quan c chn lc, tui
sinh l, ma v, giai on sinh trng, kho ca mu v ngun mu.
- Kiu gen
Kiu gen nh hng su sc n qu trnh nui cy. Vi loi thuc l c s
dng nh cy kiu mu, Cheng v Smith (1973) ghi nhn s khc nhau gia cc
genom qua nui cy sinh trng m li. Hn na, Jaramillo v Summers (1990) ghi
nhn kiu di truyn nh hng n s lng v ng knh m so qua nui cy ht
phn c chua Lycopersycon esculentum Mill.
- Chn c quan
Murashige (1974) cho rng hu ht cc loi c quan v m u c kh nng s
dng nui cy in vitro. ng cho rng mu nui cy khc nhau cc loi khc nhau,
nh Petunia dng chi nh nui cy, theo Doerschung v Miller (1976) cho rng
chi mm thch hp lm mu nui cy cc cy ny mm t ht.
- Tui v sinh l
Tui thc ca mu nui cy v tui theo ma trong nm ca mu nui cy cho
thy c nh hng quan trng n s bit ho t bo v tui sinh l. C nhiu nghin
21
cu khc nhau v nh hng ca tui sinh l mu nui cy, theo Pierik (1970) ghi
nhn r pht sinh trn l non v khng pht sinh trn l gi.
- Mu in vitro
Trong nhng nm gn y, nhiu kt qu nghin cu cho thy mu in vitro c
kh nng ti sinh cao hn mu ly t cy m trn ng rung hay trong vn m nh
cy Azalea (Economou v Read, 1986). Tuy nhin, Lu et al. (1991) ghi nhn nui
cy ti phn t t l thnh cng cao khi nui cy ti phn trn cy ng rung.
- Sc sng ca mu
iu cn thy rng mu cy m c nh hng rt quan trng n nui cy in
vitro. Morel (1952, 1955) nui cy nh sinh trng loi virus sn xut nhng cy
sch bnh v iu ny ni ln rng cn phi cn thn chn mu nui cy nht l i
vi nhng cy bnh, nu nui cy cy b bnh th s c mt s lng ln nhng cy
bnh c nhn ln.
2.2.6.2 iu kin nui cy
Nhit
Nhit thch hp cho nui cy m l 20 27oC. Theo Murashige (1974), nhit
nh hng su sc n sinh trng v pht trin cy in vitro qua nhng tin trnh
sinh l nh h hp hay hnh thnh t bo hay c quan.
Cng nh sng
Cng nh sng l mt nhn t quan trng trong quang hp, nh hng n
kh nng nui cy in vitro cy c l xanh. nh hng ca nh sng hnh nh c lin h
vi cc loi, c loi chu nh sng cao, nh sng trung bnh v nh sng thp hay ti
(Papachatzi et al., 1981; Miller v Murashige, 1976; Thorpe v Murashige, 1970).
Vic nui cy in vitro tt nht trong iu kin nh sng 1000 lux (Dng Cng Kin,
2002).
Quang k v cht lng nh sng
+ Thi gian chiu sng
nh hng su sc n nhng p ng sinh l cy trng.
+ Cht lng nh sng
22
nh hng trc tip n cy in vitro, v nh sng cao hn nh sng hay nh
sng c nh hng n nhng bin i sinh l trn cy nh ra hoa, ch dinh
dng v nhng hin tng khc nh tng sinh chi in vitro.
+ Cc cht kh
Thnh phn cht kh trong bnh nui cy c nh hng n sinh trng cy in
vitro. O2, CO2 v ethylen l nhng thnh phn cht kh c kho st nhiu trong mi
trng nui cy. m cng c quan tm n, do nh hng n qu trnh lm kh
mu nui cy.
2.2.6.3 nh hng ca mi trng nui cy
La chn mi trng nui cy thch hp trong nui cy m l rt cn thit. V
mi loi cy trng khc nhau u yu cu mt hm lng dinh dng khc nhau. Mt
khc, mi trng cn thay i tu thuc vo s phn ho ca m cy, tu theo trng
hp duy tr m trng thi m so, to r, to mm hay ti sinh cy hon chnh.
Vic la chn mi trng cn da vo ti liu cho cng i tng nui cy
hoc thm d qua mt s mi trng cho xc nh mi trng thch hp cho
mu nui cy.
Cc mi trng u c thnh lp t mt s thnh phn chnh vi nguyn tc
c s cn bng cc yu t trong mi trng.
Cc thnh phn chnh:
- ng lm ngun carbon.
- Cc mui khong a lng.
- Cc vitamin.
- Cc cht sinh trng.
Ngoi ra cc tc gi cn cho thm mt s cht hu c nh: Nc da, nc
chit nm men.
2.2.7 Nhng vn trong nhn ging in vitro
2.2.7.1 Tnh bt nh v mt di truyn
Mc d k thut nhn ging v tnh c s dng nhm mc ch to ra
qun th cy trng ng nht (true-to-type) vi s lng ln, nhng cng to ra nhng
bin d soma qua nui cy m so v nui cy t bo n. Nhng bin d ny c
nghin cu vn dng vo ci thin ging cy trng (Evans v Sharp,1986, 1988;
23
Larkin, 1987). Tn s bin d th hon ton khc nhau v khng lp li (Sreissen v
Karp, 1985; Fish v Karp, 1986).
Nhng nhn t gy ra bin d t bo soma nh:
- Kiu di truyn
- Th bi
- S ln cy chuyn
- Loi m
2.2.7.2 S hoi mu
C hai tc nhn lm h mu nui cy in vitro:
- B vi sinh vt hu hoi, c th kh trng mu trc khi a vo mi trng.
- B virus hay th ging nh virus xm nhim, khng hi mu nhng c nh
hng v sau.
Tuy nhin c s xm nhim ca vi sinh vt nh Agrobacterrium, Bacillus v
Pseudomanas vo nhu m dn truyn s hoi mu khi t bo bt u phn chia.
C th lm gim kh nng hoi mu bng cch:
- Kh trng mu trc khi cy vo mi trng.
- S dng mu nui cy l m phn sinh nh.
2.2.7.3 Vic sn xut cht gy c t mu cy
Thng chng ta hay thy hin tng ho nu hay ho en mu, sinh trng
ca mu b ngn chn hay h mu. Hin tng ny l do mu nui cy c cha nhiu
cht tannin hay hydroxyphenol, c nhiu trong m gi hn m non.
Khi mi trng v m cy b i mu qu mc th absorbent c s dng. Hai
loi absorbent thng thng l polyvinylpyrrolidone (PVP) v than hot tnh. Nhng
mt s nghin cu cho rng nn s dng than hot tnh (Mohamed Yassen et al.,
1995 v Wann et al., 1997)
Nhiu phng php lm gim s ho nu c ngh v c nhiu nh khoa
hc ng nh:
S dng mu cy nh t m non.
Gy t vt thng trn mu khi kh trng.
Ngm mu vo dung dch ascorbic acid v citric acid vi gi trc khi cy.
24
Nui cy trong mi trng lng, oxy thp, khng c n 1-2 tun.
Chuyn mu t mi trng c cht kch thch sinh trng thp qua mi trng
c nng cao hn.
2.2.7.4 S dng thuc khng sinh
C nhiu loi thuc khng sinh s dng trong nui cy m, nhm hn ch s
hoi mu ca vi sinh vt nh kanamycin, penicillin, nystatin, amphotericin B, Nng
s dng 5 -100 g/l ph thuc vo vt liu nui cy nh t bo hay t bo trn. S
hu hoi ca cht khng sinh ln m thc vt xy ra plastid hay mitochondria, x l
cng lu hay nng cng cao d dng dn n s thay i kiu gen ca t bo cht
hay DNA.
2.2.7.5 Hin tng thu tinh th
Thn l phng to cha nhiu nc, cy c dng trong. y l mt dng bnh l
thng thy khi cy c nui trong mi trng m vic trao i kh gia cy v mi
trng bn ngoi b dng li, qu trnh thot hi nc tp trung trong cy.
Mt s phng php hn ch qu trnh ho thu tinh th:
Gim s ht nc ca cy trong in vitro bng cch tng nng ng trong
mi trng cy hoc dng cc cht c p sut thm thu cao.
Trnh gy thng tn trn mu cy v tip xc vi mu cy t nht.
mt s loi c th s dng cht ABA.
Gim nng m trong mi trng cy.
Gim C2H2 trong bnh nui cy bng cch thng gi tt, tng cng nh sng
v gim nhit phng cy.
2.2.8 Cht iu ho sinh trng thc vt (HSTTV)
Cht HSTTV hay hormones sinh trng l cc hp cht hu c (gm cc sn
phm thin nhin ca thc vt v cc hp cht tng hp nhn to). Chng c tc dng
iu tit cc qu trnh sinh trng v pht trin ca thc vt. Tuy nhin, cc cht
HSTTV ch lm tng cng qu trnh trao i cht m khng tham gia trc tip vo
qu trnh trao i cht. N khng th dng thay th cht dinh dng. Cht
HSTTV gy nn tc dng mnh m vi mt lng v cng b ln trao i cht ca t
25
bo, nng cao chng c th hot ng nh cht km hm. Trong thnh phn mi
trng nui cy, cc cht HSTTV lm vic nh chic cha kho ng m s hot
ng ca gen, iu khin s pht sinh hnh thi v tng hp hot cht. Tc dng ca
cht HSTTV lin quan n hin tng km hm v cm ng tng hp enzyme trong
c th thc vt, hot ho cc b phn ca phn t DNA. Mi mt cht HSTTV u
mang mt chc nng ring, nhng trong c th ca thc vt, iu khin nhng hot
ng ca thc vt, chng tham gia vo thng khng phi l mt m l vi cht. Tu
mi giai on nui cy, giai on pht trin ca thc vt, s kt hp cc cht ny c
khc nhau. Trong phm vi nghin cu ca ti chng ti s dng cc cht thuc
nhm auxin v cytokinin.
2.2.8.1 Auxin
Tc dng sinh l ca auxin ch yu lm tng th tch ca t bo, kch thch s
hnh thnh r, km hm s sinh trng ca chi bn, km hm s rng hoa, rng qu.
Auxin hot ho cc hp cht cao phn t (protein, cellulose, pectin) v ngn cn s
phn gii chng. Auxin c xem l hormone thc vt quan trng nht v chng c vai
tr rt c bn trong qu trnh phi hp sinh trng v bit ho t bo cn thit cho s
pht trin bnh thng ca thc vt. Auxin cng vi mt s cht iu chnh khc m
bo cho s to thnh khi cc t bo ang phn chia thnh c th thc vt hon chnh.
Trong nui cy m thng s dng cc cht nh:
- Indol acetic acid (IAA)
- Naphthyl acetic acid (NAA).
- 2,4-D Dichlorophenol acetic acid (2,4-D).
- Indol butyric acid (IBA).
2.2.8.2 Cytokinin
Bao gm cc nhm cht:
- 6-Benzylaaminopurin (BAP).
- Kinetin (Ki).
- Zeatin (Z).
- Thidiazuron (TDZ).
Cytokinin c tc dng kch thch s sinh trng ca t bo cy m v lm tng
tc phn bo. Khi nng cao, n c tc dng kch thch s to chi, ng thi
c ch s phn ho r ca m cy.
26
Cytokinin c hiu qu rt r trn s phn chia ca t bo, trong qu trnh ny
cytokinin cn thit nhng chng khng c hiu qu nu vng mt auxin. Trong mt t
l gia cytokinin v auxin th c kch thch to chi hay to r, thng thng
cytokynin cao hn auxin th kch thch to chi. V ngc li, auxin cao hn cytokinin
th kch thch s to r.
Trong c th thc vt cytokinin c tc dng rt ln l tng cng s tng hp
DNA v protein, kch thch qu trnh trao i cht.
2.2.9 Nhng thnh tu v nui cy m cy rng trn th gii v Vit Nam
2.2.9.1 Trn th gii
Ngnh nui cy m thu c nhiu thnh tu cc lnh vc cy trng cng
nghip (c ph, thuc l, c du, cao su,), cy nng nghip, thc phm (khoai ty,
la, bp ci,), cy cnh (phong lan, cm chng, hu,) (Albert Sassons, 1988;
Nguyn Vn Uyn v cc tc gi, 1993; Bezborogov v cc tc gi, 1994). c bit
trn lnh vc cy cnh th phong lan nui cy m c pht trin rt rng ri c trong
nc v ngoi nc (Nguyn Thin Tch v cc tc gi, 1988; V Th Bch Mai,
1996). Cy n tri lu nm: cy kh, mng cu xim, mng ct, (Nguyn Vn Uyn
v cc tc gi, 1993). Trong tp ch Plant physiology (1988) v nhiu ti liu khc,
cc nh khoa hc Nga cng cho bit kt qu nui cy m nhiu loi cy khc nhau nh
thng, bch dng,
Ngy nay cy trng t cy m khng ch quen thuc vi cc nh nghin cu,
cc nh kinh doanh, sn xut chuyn nghip m c nhng ngi nng dn, ngi lm
vn th cng cng bit v quan tm ti nh cy khoai ty, cy chui,
Tuy nhin trn lnh vc cy trng rng, nht l nhng cy qu, him, lu nm
do nhng c th ring cn t c nghin cu k c v chng loi v quy m nghin
cu. Cc kt qu c u ch yu nghin cu cho cc loi cy mc nhanh, cy c gi
tr kinh t trong thi hn kinh doanh ngn, c kh nng mau chng p ng nhu cu
ph xanh t trng v cung cp c khi lng nguyn liu ng k cho nn kinh t
v i sng ngi dn (nha m, g, ci,).
Reilly v Washev (1977) tch chi mm cy thng (Pinus sp.) c tch t
ht gieo trong ng nghim. T nhng nm 1970 ngi ta nui cy thnh cng mnh
l, cung l, on thn, r bch n (Albert Sassons, 1988). Cc nh nghin cu M
27
to c cy con t nui cy on thn loi Eucalyptus grandis, E. gunni, E.
danrympleama , vo cc nm 1977, 1979.
Nm 1973, Afocel khi s nghin cu nhn ging v tnh cy bch n
nhm mc ch sn xut ln cc dng v tnh chu lnh, nng sut g cao. Ngi ta
to cy t ht ny mm trong ng nghim, hoc ct cc chi non t cc cy chn lc,
t cnh ghp. T nm 1975, cy cy m c bt u trng ra ngoi t vi s lng
20.000 cy / thng.
Mt s ging bch n c nng sut cao, hay gi tr kinh t v tinh du cng
c th nghim nhiu nc nhit i: n , Senegal, hng nm t mt on cnh
c th cho 50.000 cy con hay hn na (Albert Sassons, 1988).
Ti hi ngh Kaset Sart (Thi Lan, 1994) cng bo co kt qu nhn ging
thnh cng 55 loi tre trc v d nh phc v d n trng rng ca Thi Lan, vi sn
lng 1 triu cy con / nm (Pranon Prutgongse, 1994).
Malaysia cng c kt qu vi nhn ging cc loi cy g nh Acacia
mangium; Gmelia arborea (Marziah Mahmood, 1995).
2.2.9.2 Ti Vit Nam
Nc ta c mt s c s ging cy rng, Vin lm nghip, nhiu Trng i
hc Nng Nghip v khoa hc (sinh hc) cng xy dng cc phng nui cy m.
in hnh l X nghip ging v phc v trng rng TP.HCM c chuyn giao
mt Trung tm nui cy m ln t Qung ng (Trung Quc). Hin nay Trung tm c
kh nng cung cp 1 triu cy con / nm vi khong 10 dng bch n v cc loi
tch, keo l trm (bo co ti hi ngh CNSH ln III, 1995).
Trung tm nghin cu lm nghip Ph Ninh-Vnh Ph cng xy dng phng
nui cy m 1994, n thng 5 nm 1995 sn xut 50000 cy con bch n, v
trng th mt s tnh Vnh Ph, Qung Ninh, Gia Lai (Mai nh Hng, 1995). Cng
nh kt qu thng bo nui cy m bch n li thnh cng v trng ra ngoi t t
nhin ca Nguyn Ngc Tn (1995).
Khoa lm nghip trng i hc Nng Lm TP. HCM, cng c nhiu thnh
tu v nui cy m cy rng nh: nui cy m mt loi cy bch n (E.
camaldulensis), cy thng carib (Pinus caribaea), cy gi t (Tectona grandis Linn
28
F.), cy thng ba l (Pinus kharya Royle), cy thng , cy trm hng, cy my
np,... song kt qu cn ch yu dng li giai on ng nghim.
Qua cc kt qu cn rt hn hp nh vy, chng t nhng nm qua nhu cu th
trng cy cy m cho trng rng trong nc cha cao nht l i vi cc loi cy g
qu, him, lu nm. Mc d vy nhng thnh tu c cng l nhng c s, kinh
nghim v hy vng t vn nui cy cy ging hng, l mt loi cy qu ca
rng nhit i ang c nguy c b tiu dit.
Hy vng khng ch t vo kh nng thnh cng t cc th nghim m c trin
vng ng dng trong thc t trng rng nc ta. V ngoi nhng thnh tu khng
nh nu trn, cn phi ni n mt thnh cng rt ln trong nh hng pht trin
ngnh nui cy m cy rng ca nh nc v cc nh chuyn mn lm nghip. l
s u t thch ng chuyn giao nhng cng ngh hn i t Trung Quc, c bit
l X nghip ging v phc v trng rng TP.HCM c nh gi l trung tm nui
cy m ln v hin i nht trong nc hin nay. Trn thc t x nghip trin khai
hiu qu, trng th v cung cp ging cy m cho mt s c s trng rng. Kt qu
cy con (bch n) sau mt nm c th t chiu cao 6-7 m, ng knh gc t 10cm.
Ch sau 3 thng mt s ging bch n o c ng knh gc bnh qun 3,8 cm.
Chiu cao vt ngn 2,5-2,8 m (o thng 10.1996).
Trong thi gian ti, cc trung tm v c s nui cy m khc s cn a ra
nhiu thnh cng nui cy cy rng vi nhiu chng loi hn, phc v c cc k
hoch trng rng ca nc ta. K hoch CNSH nm 1996 2010 trong quan tm
nhn nhanh cc cy lm nghip, cy cnh, cy thuc qu (Nguyn Vn Uyn, 1995).
Hin nay vn khn thit v cn quan tm hn na ti tim nng ca vic ph
bin ng dng vi nhn ging i vi cc loi cy g. nh hng kinh t ca ng dng
nhn ging v tnh cc loi thc vt ny l rt ln i vi nhng vng nhit i v
nhit i (Gamborg, 1995).
29
Chng 3. VT LIU V PHNG PHP NGHIN CU
3.1 i tng th nghim
Th nghim c tin hnh trn cy ging hng (Pterocarpus macrocarpus).
3.2 Thi gian v a im nghin cu
ti c thc hin t ngy 6/2- 18/6/2006 ti B mn Cng ngh sinh hc
trng i hc Nng Lm.
3.3 Vt liu nghin cu
3.3.1 Trang thit b v dng c
Thit b: t v trng, ni hp, my o pH, cn in t, my lnh, nhit k, m
k, k t bnh, n neon,
Dng c: pince, ko, dao cy, bnh thu tinh 500ml, a, n cn.
3.3.2 Mi trng nui cy
Cc mi trng c s dng gm: Mi trng MS, MS, WPM. Trong
mi trng MS l mi trng MS m thnh phn a lng c gim i mt na.
- Mi trng MS ci tin (Murashige v Skoog, 1962)
Thnh phn Nng (mg/l)
Khong a lng NH4NO3 1650
KNO3 1900
CaCl2.2H2O 440
MgSO4.7H2O 370
KH2PO4 170
Khong vi lng MnSO4.4H2O 23,3
ZnSO4.7H2O 8,6
H3BO3 6,2
KI 0,83
Na2MoO4.2H2O 0,25
CuSO4.5H2O 0,025
CoCl2.6H2O 0,025
30
St EDTA Na2.EDTA 37,3
FeSO4.7H2O 27,8
Vitamin myo-Inositol 100
Thiamin (B1) 0,1
Nicotinic acid 0,5
Pyridoxine HCl 0,5
Glycine 2
- Mi trng WPM ( Llooyd v McCown, 1981)
Thnh phn Nng (mg/l)
Khong a lng NH4NO3 400
CaNO3 556
K2SO4 990
CaCl2 96
KH2PO4 170
MgSO4 370
St EDTA FeNa2EDTA 65,1
Khong vi lng MnSO4.4H2O 23,3
ZnSO4.7H2O 8,6
H3BO3 6,2
KI 0,83
Na2MoO4.2H2O 0,25
CuSO4.5H2O 0,025
CoCl2.6H2O 0,025
Vitamin myo-Inositol 100
B1 1
B6 0,5
Nicotinic acid 0,5
Glycine 2
31
Cc cht iu ho sinh trng thc vt c s dng l BA, NAA, IBA (mg/l).
Cc thnh phn khc:
ng sucrose 30 g/l
Agar 7,5 /l
Nc da 10%
Than hot tnh 1g/l
Mi trng c iu chnh v pH=5,7 0,1 (bng KOH 1N v HCl 1N) trc
khi hp kh trng bng autoclave 1atm (1210C) trong 25 pht.
3.4 iu kin nui cy in vitro
Thi gian chiu sng 16 gi/ngy
Nhit 25 2oC
m 75-80%
Cng nh sng 2000-3000 lux
3.5 Phng php kh trng
3.5.1 Vt liu
Ht ging hng do Khoa Lm Nghip trng i Hc Nng Lm cung cp
3.5.2 Phng php kh trng mu
- Bn ngoi t cy:
Ht c bc v em ra bng x bng
Ra sch x bng bng nc my
- Trong t cy v trng:
Ra ht bng nc ct v trng
Lc cn 700 trong 30 giy
Ra li 3 ln bng nc ct v trng
Ngm trong Javel (nng v thi gian theo th nghim)
Ra li 3 ln bng nc ct v trng
3.5.3 Cy mu
To vt thng cho ht
Cy mu vo bnh thu tinh cha 30ml mi trng 1/2MS khng b sung cht
iu ho sinh trng thc vt
32
3.6 Phng php th nghim
Cc th nghim u c b tr theo kiu th nghim n yu t v hon ton
ngu nhin, vi 3 ln lp li. Trong mi bnh cy mt mu. Mi bnh cha 50ml mi
trng
3.6.1 Th nghim 1: nh hng ca nng v thi gian kh trng n t l
sng ca mu cy cy ging hng in vitro.
Mc ch th nghim: Xc nh nng javel v thi gian thch hp cho
vic v trng mu ging hng nhm to ngun mu sch ban u cho qu trnh
nhn ging tip theo.
Vt liu th nghim: Ht ging hng
Th nghim gm: 9 nghim thc
Nghim thc Nng Javel (%) Thi gian kh trng (pht)
NT1 5 5
NT2 5 10
NT3 5 15
NT4 10 5
NT5 10 10
NT6 10 15
NT7 12 5
NT8 12 10
NT9 12 15
Mi nghim thc cy 3 bnh
Tng s mu: 81
Thi gian th nghim: 4 tun
Ch tiu theo di:
T l ht ny mm (%): (Tng s ht ny mm / tng s mu cy)x100
T l ht khng nhim (%): (Tng s ht khng nhim / tng s mu cy)x100
T l ht nhim (%): (Tng s ht nhim / tng s mu cy)x100
3.6.2 Th nghim 2: Kho st nh hng ca cc mi trng ln kh nng to
chi cy ging hng in vitro.
33
3.6.2.1 Th nghim 2a: nh hng ca nng BA v NAA ln kh
nng to chi ca cy ging hng in vitro.
Mc ch th nghim: Xc nh nng thch hp BA v NAA thch hp
cho qu trnh to chi ca cy ging hng in vitro, lm ngun nguyn liu cho
qu trnh vi nhn ging tip theo.
Vt liu th nghim: Chi con in vitro mc ln t ht ging hng
c v trng v nui cy trong mi trng MS th nghim 1 sau 4 tun nui
cy.
Th nghim gm: 8 nghim thc
Nghim thc Mi trng BA(mg/l) NAA (mg/l)
NT1 (C) WPM 0 0
NT2 WPM 0,5 0,1
NT3 WPM 1 0,1
NT4 WPM 1 0,5
NT5 WPM 1,5 0,1
NT6 WPM 1,5 0,5
NT7 WPM 2 0,1
NT8 WPM 2 0,5
Mi nghim thc cy 3 bnh
Tng s mu: 72 mu
Thi gian th nghim: 6 tun
Ch tiu theo di:
S lng chi (chi): Tng s chi/ tng s mu cy
Chiu cao chi (cm): o t mt thch n nh cao nht ca cm chi
H s nhn chi: Tng s chi / mu cy
3.6.2.2 Th nghim 2b: Kho st nh hng ca mi trng nui cy
c bn n kh nng to chi ca ging hng in vitro.
Mc ch th nghim: Xc nh c loi mi trng c bn thch hp
cho s tng trng v pht sinh cm chi ca cy ging hng in vitro nhm lm di
34
do lng mu v m bo cht lng mu v kch thc cng nh kh nng tng
trng mnh cho ln vi nhn ging sau.
Vt liu th nghim: Chi ging hng in vitro th nghim 2.
Th nghim gm: 3 nghim thc
Nghim thc Mi trng BA (mg/l) NAA (mg/l)
NT1(C) WPM 1,5 0,1
NT2 1/2MS 1,5 0,1
NT3 MS 1,5 0,1
Mi nghim thc cy 3 bnh
Tng s mu: 27
Thi gian th nghim: 6 tun
Ch tiu theo di:
Chiu cao chi (cm): o t mt thch ln n nh cao nht ca cm
chi.
S lng chi (chi): Tng s chi / tng s mu cy.
H s nhn chi: Tng s chi / mu cy.
3.6.3 Th nghim 3: Kho st nh hng ca IBA v NAA n s hnh thnh
r ca ging hng in vitro.
Mc ch th nghim: Xc nh nng IBA v NAA thch hp cho qu
trnh to r ca cy ging hng in vitro, nhm chun b cy con kho mnh a
ra vn m.
Vt liu th nghim: Chi tch ra t th nghim 2 c kch thc khong
2 3 cm.
35
Th nghim gm: 9 nghim thc
Nghim thc Mi trng IBA (mg/l) NAA (mg/l)
NT1 (C) WPM 0 0
NT2 WPM 0,5 0
NT3 WPM 1 0
NT4 WPM 1,5 0
NT5 WPM 2 0
NT6 WPM 0 0,5
NT7 WPM 0 1
NT8 WPM 0 1,5
NT9 WPM 0 2
Mi nghim thc cy 3 bnh
Tng s mu: 81 mu
Thi gian th nghim: 6 tun
Ch tiu theo di:
Thi gian chi to r (ngy sau cy): Tnh t lc cy mi to r.
S r /cy (r): m tt c r mi cy khi 50% s cy ra r
Chiu di r (mm): o chiu di r sau 4 tun nui cy
3.6.4 Phn tch thng k
S liu thu thp c x l trn my vi tnh bng chng trnh thng k
Statgraphic 7.0. c kt qu da vo bng ANOVA, bng trung bnh v bng so snh
khc bit gia cc nghim thc (Bng phng php LSD)
36
Chng 4. KT QU V THO LUN
4.1. nh hng ca nng v thi gian kh trng n t l sng ca mu
cy cy ging hng in vitro.
Kh mu l mt bc lm quan trng m bo cho ngun nguyn liu sau
ny ca qu trnh nhn ging in vitro do ngun mu ban u khng sch, ly t t
nhin cn ln bn, t, khng v trng, d mang mm bnh,. Ging hng l loi
cy h u, ht c v bao bc bn ngoi nn cng rt kh c tnh trng nhim khi kh
mu hn l so vi kh mu t cc b phn khc nh: chi, thn,
Bng 4.1: Kt qu kh mu ht ging hng sau 4 tun nui cy.
NT1 33,33a 66,67
c 0,00
a
NT2 66,67b 33,33
b 0,00
a
NT3 100,00c 0,00
a 0,00
a
NT4 100,00c 0,00
a 0,00
a
NT5 66,67b 33,33
b 0,00
a
NT6 100,00c 0,00
a 100,00
d
NT7 100,00c 0,00
a 66,67
c
NT8 100,00c 0,00
a 33,33
b
NT8 100,00c 0,00
a 0,00
a
CV (%) 8,83 1,42 0,67
*Cc gi tr theo sau bi ch ci trong cng mt ct khng cng k t biu hin s
khc bit rt c ngha v mt thng k mc 0,05.
Tuy t l ht khng nhim th rt cao nhng t l ht ny mm li qu thp.
iu ny l do ht c v bc bn ngoi ng vai tr nh bo v ngn cn s ny mm
ca ht.
Nghim thc T l ht nhim T l ht khng nhim T l ht ny mm
(%) (%) (%)
37
Hnh 4.1: Ht ging hng in vitro ny mm Hnh 4.2 Cy con ging hng in vitro
Qua kt qu trn cho thy:
i vi cy ging hng th nng javel v thi gian kh trng thch hp l
10% javel trong 15 pht.
Cc ht ging hng b nhim hu nh l b nhim nm, thng xy ra i vi
trng hp kh trng nng javel v thi gian kh trng thp.
4.2 Kho st nh hng ca cc mi trng ln kh nng to chi ca cy
ging hng in vitro.
Vic nhn chi in vitro c vai tr quan trng trong vic to ra lng ln cy con
in vitro lm nguyn liu cho cc qu trnh nhn ging tip theo. y l qu trnh quan
trng v cng c th ni y l nhim v ca nhn ging v tnh.
Tht ra, bn thn thc vt c kh nng t tng hp v iu chnh cc cht iu
ha sinh trng thc vt thch hp vi mi thi k sinh trng, pht trin, vi thi tit
kh hu, iu kin sng. Vai tr ca cc cht sinh trng th hin nhiu mt nh iu
khin vn ng, iu khin qu trnh ra hoa, cc hot ng sinh l, sinh ha trong c
th thc vt (Oparin, 1977; Marti Mihaly, 1976; Nguyn Vn Uyn, 1995). i vi
m nui cy trong tnh trng d dng, kh nng t tng hp v iu chnh cc cht
iu ha sinh trng l rt hn ch, cn b sung nhng cht ny vo mi trng nui
cy mt lng ph hp. Ty vo mi loi m cy, loi cy, thi gian pht trin ca m
cy v mc ch nui cy m chu s nh hng ca nng cht iu ha sinh
trng khc nhau.
38
4.2.1 nh hng ca nng BA v NAA ln kh nng to chi ca cy
ging hng in vitro.
Bng 4.2: nh hng ca nng BA v NAA ln kh nng to chi cy ging
hng in vitro sau 6 tun nui cy.
CV(%) 13,9 12,99 12,29
*Cc gi tr theo sau bi ch ci trong cng mt ct khng cng k t biu hin
s khc bit rt c ngha v mt thng k mc 0,05.
nghim thc 5 (nng BA=1,5 mg/l; NAA=0,1 mg/l) s lng chi to
thnh v chiu cao chi m bo lm ngun nguyn liu in vitro cho cc qu trnh
nhan ging tip theo. Do nng BA=1,5 mg/l; NAA=0,1 mg/l rt thch hp cho
s nhn chi cy ging hng in vitro.
Bng kt qu trn cho thy ging hng ti sinh chi tt nng BA=1,5
mg/l v NAA=0,5 mg/l. Tuy nhin nghim thc ny phn ln chi to thnh c kh
nng tng trng chiu cao chi rt thp.
nghim thc 2 (nng BA=0,5 mg/l; NAA= 0,1 mg/l) v nghim thc 4
(nng BA=1mg/l; NAA=0,5mg/l) th chi bn pht trin kh tt nhng s lng li
t.
nghim thc 8 ( BA=2 mg/l; NAA=0,5 mg/l) chi hu nh khng xut
hin, chiu cao chi gim v c hin tng to so, nguyn nhn do hm lng cht
iu ho sinh trng cao gy c ch kh nng pht trin chi ca cy ging hng in
vitro.
Nghim
thc
BA
(mg/l)
NAA
(mg/l)
S
chi
(chi)
Chiu cao
chi (cm)
H s
nhn chi
NT1
(C)
0,0 0,0 1,0a
1,17a
1,00a
NT2 0,5 0,1 1,7a
1,43abc
1,67c
NT3 1,0 0,1 1,3a
1,30ab
1,33b
NT4 1,0 0,5 2,0a
2, 01d 2,00
d
NT5 1,5 0,1 6,3b
2,20d 6,33
f
NT6 1,5 0,5 4,7b
1,90cd
4,67e
NT7 2,0 0,1 1,0a
1,77bcd
1,00a
NT8 2,0 0,5 1,0a
1,17a 1,00
a
39
BA=0mg/l; NAA=0mg/l BA=0,5mg/l; NAA=0,1mg/l
BA=1mg/l; NAA=0,1mg/l BA=1mg/l; NAA=0,5mg/l
BA=1,5mg/l; NAA=0,1mg/l BA=1,5mg/l; NAA=0,5mg/l
BA=2mg/l; NAA=0,1mg/l BA=2mg/l; NAA=0,5mg/l
Hnh 4.3: Chi cy ging hng in vitro c to thnh sau 6 tun nui cy
trn cc mi trng khc nhau
40
4.2.2 Kho st nh hng ca mi trng nui cy c bn n kh nng to chi
ca cy ging hng in vitro.
Bng 4.3: nh hng ca mi trng nui cy n kh nng to chi ca cy ging
hng in vitro sau 6 tun nui cy.
Nghim thc Mi trng S chi Chiu cao chi H s nhn chi
(chi) (cm) (chi)
NT1 WPM 2,0a 1,4
a 2,00
b
NT2 MS 1,33b 1,87
b 1,33
c
NT3 MS 3,67a 3,13
a 3,00
a
CV (%) 10,2 8,09 12,77
*Cc gi tr theo sau bi ch ci trong cng mt ct khng cng k t biu hin
s khc bit rt c ngha v mt thng k mc 0,05.
Kt qu trn cho thy nghim thc 2 (mi trng MS) cho s chi nhiu, chi
pht trin kho mnh hn so vi mi trng MS v mi trng dnh cho cy thn
g WPM.
C th trong mi trng cy thn g thnh phn khong a lng thp, chi tuy
c pht trin nhng kch thc chi khng m bo lm ngun nguyn liu cho qu
trnh nhn ging tip theo so vi mi trng MS.
Mi trng MS Mi trng WPM Mi trng MS
Hnh 4.4: Chi cy ging hng in vitro c to thnh sau 6 tun nui cy trn cc
mi trng khc nhau.
41
4.3 Kho st nh hng ca IBA v NAA n s hnh thnh r ca cy
ging hng in vitro.
R l mt b phn quan trng ca cy. N c tc dng ht nc, mui khong
v cht dinh dng cung cp cho cy sinh trng v pht trin. Do , trong vic to
cy in vitro hon chnh ta phi quan tm n b r ca cy, tm mi trng tht s
thch hp cho s to v pht trin ca r. Cy in vitro c b r khe th mi c kh
nng sng trong iu kin vn m do khi chuyn t iu kin in vitro ra vn m
c s tc ng mnh m n cy con in vitro nh: m , nh sng, nhit , mi
trng thun ha khng thch hp,cy rt d b cht.
Bng 4.4: nh hng ca IBA v NAA n s hnh thnh r ca cy ging hng in
vitro sau 4 tun nui cy.
Nghim thc NAA IBA Thi gian S r Chiu di
(mg/l) (mg/l) ra r (r) r (cm)
(ngy)
NT1 0 0 0,0a 0,0
a 0,00
a
NT2 0,5 0 10,0e 6,0
abc 0,75
ab
NT3 1 0 10,0e 4,0
abc 0,25
ab
NT4 1,5 0 7,5d 4,0
abc 1,25
bc
NT5 2 0 4,0b 10,0
c 0,85
abc
NT6 0 0.5 0,0a 0,0
a 0,00
a
NT7 0 1 10,0e 2,5
ab 0,40
ab
NT8 0 1,5 7,0d 8,5
bc 3,50
b
NT9 0 2 5,0c 6,5
abc 2,00
c
CV(%) 6,07 7,67 12,42
*Cc gi tr theo sau bi ch ci trong cng mt ct khng cng k t biu hin
s khc bit rt c ngha v mt thng k mc 0,05.
Cy ging hng in vitro c kh nng to r rt mnh. Trong nghim thc 8
(nng NAA=0 mg/l; IBA=1,5 mg/l) cho kt qu to r nhanh, di nht. Nh vy
nng ny rt thch hp cho s to r ca cy ging hng in vitro.
42
cc nghim thc khc ging hng in vitro cng c kh nng to r nhanh
nhng kch thc r khng m bo cho giai on vn m sau ny do qu ngn.
nghim thc 5 (NAA=2mg/l; IBA= 0mg/l) cng cho ra r sm nhng r ch
pht trin mc nhng si trng, mnh, ngn d rt nhiu. iu ny khng m
bo cho vic em trng vn m.
nghim thc 4 (NAA=1,5mg/l; IBA=0mg/l) chi con ging hng in vitro
cng c to r nhng c xu hng to so.
NAA=2mg/l NAA=1,5mg/l
IBA=2mg/l IBA=1,5mg/l
Hnh 4.5: Cy ging hng in vitro hon chnh sau 4 tun nui cy.
43
Chng 5. KT LUN V NGH
5.1 Kt lun
Qua nhng th nghim thc hin chng ti rt ra c nhng kt lun sau
v vic nhn ging in vitro cy ging hng:
Ht ging hng in vitro c t l sng cao nht khi chng c x l
nng javel l 10% trong 15 pht.
Mi trng MS vi nng BA=1,5 (mg/l); NAA=0,1 (mg/l) rt thun
li cho kh nng to chi ging hng in vitro.
Nng IBA=2mg/l s gip cy ging hng in vitro ra r nhanh v
khe, rt thch hp cho vic em cy ra vn m.
5.2 ngh
Tip tc nghin cu thm tm ra cht iu ha sinh trng thch hp
cho vic nhn chi c bit l tng trng chi.
Nghin cu giai on vn m tm ra gi th thch hp cho s
pht trin ca cy ging hng in vitro.
Nghin cu lp ra quy trnh k thut nhn ging v trng cy ging
hng in vitro c hiu qu kinh t cao nht.
Nghin cu v kh nng nhn ging cy ging hng bng phng php
gim cnh trong iu kin in vivo to ra lng cy con nhanh, khe.
44
TI LIU THAM KHO
TING VIT
1. Trn Th Dung, 2003, Bi ging nui cy m t bo thc vt, Trng i hc
Nng Lm TP.HCM.
2.Trng Mai Hng, 1997, Nghin cu xy dng qu trnh nhn ging in vitro cy
cm lai B Ra (Dalbergia bariaensis Pierre), Trng i hc Nng Lm
TP.HCM.
3. Trn Vn Minh, 2000, Cng ngh sinh hc thc vt, Vin sinh hc nhit i
TP.HCM.
4. Cao Minh Thu Nguyn, 2005, Nhn ging v tnh in vitro cy Bch n chanh
(Eucalyptus citriodora), Trng i hc Nng Lm TP.HCM.
5. Nguyn Kim Thanh, Nguyn Thun Chu, 2005, Gio trnh sinh l thc vt,
NXB H Ni.
6. Phm Minh Tho, 2005, Rng Vit Nam. Nh xut bn Lao ng.
INTERNET
http://sonongnghiep.angiang.gov.vn
http://www.ldd.go.th
http://www.forest.go.th
http://www.springeslink.com
http://sonongnghiep.angiang.gov.vn/http://www.ldd.go.th/http://www.forest.go.th/http://www.springeslink.com/
45
PH LC
46
PH LC 1
Bng s liu gc t cc th nghim
Bng 1: Kt qu kh mu ht ging hng sau 4 tun nui cy
Nghim thc T l ht
khng
nhim (%)
T l ht
nhim
(%)
T l ht
ny mm
(%)
NT1
NT2
NT3
NT4
NT5
NT6
NT7
NT8
NT9
33,33
66,67
100,00
100,00
66,67
100,00
100,00
100,00
100,00
66,67
33,33
0,00
0,00
33,33
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
100,00
66,67
33,33
0,00
Bng 2: nh hng ca nng BA v NAA ln kh nng to chi cy
ging hng in vitro sau 6 tun nui cy.
Nghim thc S chi (chi) Chiu cao chi (cm) H s nhn chi
NT1 (C) 1,0 1,17 1,00
NT2 1,7 1,43 1,67
NT3 1,3 1,30 1,33
NT4 2,0 2,01 2,00
NT5 6,3 2,20 6,33
NT6 4,7 1,90 4,67
NT7 1,0 1,77 1,00
NT8 1,0 1,17 1,00
47
Bng 3: nh hng ca mi trng nui cy n kh nng to chi ca cy ging
hng in vitro sau 6 tun nui cy
Bng 4: nh hng ca IBA v NAA n s hnh thnh r ca cy ging hng
in vitro sau 4 tun nui cy
Nghim thc S chi (chi) Chiu cao chi (chi) H s nhn chi
NT1 2,00 1,40 2,00
NT2 1,33 1,87 1,33
NT3 3,67 3,13 3,00
Nghim thc Thi gian ra r (ngy) S r (r) Chiu di r (cm)
NT1(C) 0,0 0,0 0,00
NT2 10,0 6,0 0,75
NT3 10,0 4,0 0,25
NT4 7,5 4,0 1,25
NT5 4,0 10,0 0,85
NT6 0,0 0,0 0,00
NT7 10,0 2,5 0,40
NT8 7,0 8,5 3,50
NT9 5,0 6,5 2,00
48
PH L C 2
Bng phn tch s liu cc th nghim
THI NGHIEM 1
TY LE HAT KHONG NHIEM
BANG ANOVA
One-Way Analysis of Variance
----------------------------------------------------------------------
Data: THUY.HATKNHIEM
Level codes: THUY.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
-----------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig. level
-----------------------------------------------------------------------
Between groups 9382.9136 8 1172.8642 999.999 .0000
Within groups .0000 9 .0000
-----------------------------------------------------------------------
Total (corrected) 9382.9136 17
0 missing value(s) have been excluded.
_
BANG TRUNG BINH
Table of means for THUY.HATKNHIEM by THUY.NT
---------------------------------------------------------------------
Stnd. Error Stnd. Error 95 % LSD
Level Count Average (internal) (pooled s) intervals for mean
----------------------------------------------------------------------
NT1 2 33.330000 .00000E0000 .00000E0000 33.330000 33.330000
NT2 2 66.670000 .00000E0000 .00000E0000 66.670000 66.670000
NT3 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT4 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT5 2 66.670000 .00000E0000 .00000E0000 66.670000 66.
NT6 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT7 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT8 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT9 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
-----------------------------------------------------------------------
Tota 18 85.185556 .00000E0000 .00000E0000 85.185556 85.185556
49
BANG KET QUA TRAC NGHIEM PHAN HANG
Multiple range analysis for THUY.HATKNHIEM by THUY.NT
-----------------------------------------------------------------------
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
-----------------------------------------------------------------------
NT1 2 33.330000 X
NT2 2 66.670000 X
NT5 2 66.670000 X
NT3 2 100.000000 X
NT4 2 100.000000 X
NT6 2 100.000000 X
NT7 2 100.000000 X
NT8 2 100.000000 X
NT9 2 100.000000 X
-----------------------------------------------------------------------
contrast difference +/- limits
NT1 - NT2 -33.3400 0.00000 *
NT1 - NT3 -66.6700 0.00000 *
NT1 - NT4 -66.6700 0.00000 *
NT1 - NT5 -33.3400 0.00000 *
* denotes a statistically significant difference.
_
TY LE HAT NHIEM
BANG ANOVA
One-Way Analysis of Variance
---------------------------------------------------------------------------
Data: TN1.HATNHIEM
Level codes: TN1.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
---------------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig. level
---------------------------------------------------------------------------
Between groups 9382.9136 8 1172.8642 999.999 .0000
Within groups .0000 9 .0000
---------------------------------------------------------------------------
Total (corrected) 9382.9136 17
0 missing value(s) have been excluded.
50
BANG TRUNG BINH
Table of means for TN1.HATNHIEM by TN1.NT
---------------------------------------------------------------------------
Stnd. Error Stnd. Error 95 % LSD
Level Count Average (internal) (pooled s) intervals for mean
---------------------------------------------------------------------------
1 2 66.670000 .00000E0000 .00000E0000 66.670000 66.670000
2 2 33.330000 .00000E0000 .00000E0000 33.330000 33.330000
3 2 .000000 .00000E0000 .00000E0000 .000000 .000000
4 2 .000000 .00000E0000 .00000E0000 .000000 .000000
5 2 33.330000 .00000E0000 .00000E0000 33.330000 33.330000
6 2 .000000 .00000E0000 .00000E0000 .000000 .000000
7 2 .000000 .00000E0000 .00000E0000 .000000 .000000
8 2 .000000 .00000E0000 .00000E0000 .000000 .000000
9 2 .000000 .00000E0000 .00000E0000 .000000 .000000
---------------------------------------------------------------------------
Total 18 14.814444 .00000E0000 .00000E0000 14.814444 14.814444
_
BANG KET QUA TRAC NGHIEM PHAN HANG
Multiple range analysis for TN1.HATNHIEM by TN1.NT
---------------------------------------------------------------------------
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
---------------------------------------------------------------------------
3 2 .000000 X
4 2 .000000 X
6 2 .000000 X
7 2 .000000 X
8 2 .000000 X
9 2 .000000 X
2 2 33.330000 X
5 2 33.330000 X
1 2 66.670000 X
---------------------------------------------------------------------------
contrast difference +/- limits
1 - 2 33.3400 0.00000 *
1 - 3 66.6700 0.00000 *
1 - 4 66.6700 0.00000 *
1 - 5 33.3400 0.00000 *
* denotes a statistically significant difference
_
51
TY LE HAT NAY MAM
BANG ANOVA
One-Way Analysis of Variance
---------------------------------------------------------------------------
Data: THI1.NAYMAM
Level codes: THI1.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
---------------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig. level
---------------------------------------------------------------------------
Between groups 22222.667 8 2777.8333 999.999 .0000
Within groups .000 9 .0000
---------------------------------------------------------------------------
Total (corrected) 22222.667 17
0 missing value(s) have been excluded.
BANG TRUNG BINH
Table of means for THI1.NAYMAM by THI1.NT
---------------------------------------------------------------------------
Stnd. Error Stnd. Error 95 % LSD
Level Count Average (internal) (pooled s) intervals for mean
---------------------------------------------------------------------------
NT1 2 .000000 .00000E0000 .00000E0000 .000000 .000000
NT2 2 .000000 .00000E0000 .00000E0000 .000000 .000000
NT3 2 .000000 .00000E0000 .00000E0000 .000000 .000000
NT4 2 .000000 .00000E0000 .00000E0000 .000000 .000000
NT5 2 .000000 .00000E0000 .00000E0000 .000000 .000000
NT6 2 100.000000 .00000E0000 .00000E0000 100.000000 100.000000
NT7 2 66.670000 .00000E0000 .00000E0000 66.670000 66.670000
NT8 2 33.330000 .00000E0000 .00000E0000 33.330000 33.330000
NT9 2 .000000 .00000E0000 .00000E0000 .000000 .000000
---------------------------------------------------------------------------
Total 18 22.222222 .00000E0000 .00000E0000 22.222222 22.222222
_
BANG KET QUA TRAC NGHIEM PHAN HANG
Multiple range analysis for THI1.NAYMAM by THI1.NT
---------------------------------------------------------------------------
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
---------------------------------------------------------------------------
NT1 2 .000000 X
NT2 2 .000000 X
NT3 2 .000000 X
NT4 2 .000000 X
NT5 2 .000000 X
NT9 2 .000000 X
NT8 2 33.330000 X
NT7 2 66.670000 X
NT6 2 100.000000 X
---------------------------------------------------------------------------
contrast difference +/- limits
NT1 - NT2 0.00000 0.00000
NT1 - NT3 0.00000 0.00000
52
NT1 - NT4 0.00000 0.00000
NT1 - NT5 0.00000 0.00000
* denotes a statistically significant difference.
TH NGHIM 2
Th nghim 2a
S CHI
BNG ANOVA
One-Way Analysis of Variance
---------------------------------------------------------------------------
Data: SCHOI.SOCHOI
Level codes: SCHOI.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
---------------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig.level
---------------------------------------------------------------------------
Between groups 84.958333 7 12.136905 10.403 .0001
Within groups 18.666667 16 1.166667
---------------------------------------------------------------------------
Total (corrected) 103.62500 23
1 missing value(s) have been excluded.
_
BNG TRUNG BNH
Table of means for SCHOI.SOCHOI by SCHOI.NT
---------------------------------------------------------------------------
Stnd. Error Stnd. Error 95 % LSD
Level Count Average (internal) (pooled s) intervals for mean
---------------------------------------------------------------------------
NT1 3 1.0000000 .0000000 .6236096 .0649778 1.9350222
NT2 3 1.6666667 .6666667 .6236096 .7316444 2.6016889
NT3 3 1.3333333 .3333333 .6236096 .3983111 2.2683556
NT4 3 2.0000000 .5773503 .6236096 1.0649778 2.9350222
NT5 3 6.3333333 .8819171 .6236096 5.3983111 7.2683556
NT6 3 4.6666667 1.2018504 .6236096 3.7316444 5.6016889
NT7 3 1.0000000 .0000000 .6236096 .0649778 1.9350222
NT8 3 1.0000000 .0000000 .6236096 .0649778 1.9350222
---------------------------------------------------------------------------
Total 24 2.3750000 .2204793 .2204793 2.0444197 2.7055803
53
BNG KT QU TRC NGHIM PHN HNG
Multiple range analysis for SCHOI.SOCHOI by SCHOI.NT
---------------------------------------------------------------------------
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
---------------------------------------------------------------------------
NT1 3 1.0000000 X
NT7 3 1.0000000 X
NT8 3 1.0000000 X
NT3 3 1.3333333 X
NT2 3 1.6666667 X
NT4 3 2.0000000 X
NT6 3 4.6666667 X
NT5 3 6.3333333 X
---------------------------------------------------------------------------
contrast difference +/- limits
NT1 - NT2 -0.66667 1.87004
NT1 - NT3 -0.33333 1.87004
NT1 - NT4 -1.00000 1.87004
NT1 - NT5 -5.33333 1.87004 *
NT1 - NT6 -3.66667 1.87004 *
* denotes a statistically significant difference.
_
CHIU CAO CHI
BANG ANOVA
One-Way Analysis of Variance
-----------------------------------------------------------------------
Data: CCCHOI.CCAOCHOI
Level codes: CCCHOI.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
-----------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig. level
-----------------------------------------------------------------------
Between groups 3.5516667 7 .5073810 4.380 .0069
Within groups 1.8533333 16 .1158333
-----------------------------------------------------------------------
Total (corrected) 5.4050000 23
0 missing value(s) have been excluded.
54
BNG TRUNG BNH
Table of means for CCCHOI.CCAOCHOI by CCCHOI.NT
---------------------------------------------------------------------------
Stnd. Error Stnd. Error 95 % LSD
Level Count Average (internal) (pooled s) intervals for mean
---------------------------------------------------------------------------
NT1 3 1.1666667 .1666667 .1964971 .8720446 1.4612888
NT2 3 1.4333333 .1452966 .1964971 1.1387112 1.7279554
NT3 3 1.3000000 .0577350 .1964971 1.0053779 1.5946221
NT4 3 2.0666667 .0666667 .1964971 1.7720446 2.3612888
NT5 3 2.2000000 .1154701 .1964971 1.9053779 2.4946221
NT6 3 1.9000000 .1000000 .1964971 1.6053779 2.1946221
NT7 3 1.7666667 .4702245 .1964971 1.4720446 2.0612888
NT8 3 1.1666667 .0881917 .1964971 .8720446 1.4612888
---------------------------------------------------------------------------
Total24 1.6250000 .0694722 .0694722 1.5208354 1.7291646
_
BNG KT QU TRC NGHIM PHN HNG
Multiple range analysis for CCCHOI.CCAOCHOI by CCCHOI.NT
-----------------------------------------------------------------------
Method: 95 Percent LSD
Level Count Average Homogeneous Groups
-----------------------------------------------------------------------
NT1 3 1.1666667 X
NT8 3 1.1666667 X
NT3 3 1.3000000 XX
NT2 3 1.4333333 XXX
NT7 3 1.7666667 XXX
NT6 3 1.9000000 XX
NT4 3 2.0666667 X
NT5 3 2.2000000 X
-----------------------------------------------------------------------
contrast difference +/- limits
NT1 - NT2 -0.26667 0.58924
NT1 - NT3 -0.13333 0.58924
NT1 - NT4 -0.90000 0.58924 *
NT1 - NT5 -1.03333 0.58924 *
NT1 - NT6 -0.73333 0.58924 *
* denotes a statistically significant difference.
55
HE SO NHAN CHOI
BNG ANOVA
One-Way Analysis of Variance
---------------------------------------------------------------------------
Data: HSN1.HSNCHOI
Level codes: HSN1.NT
Labels:
Means plot: LSD Confidence level: 95 Range test: LSD
Analysis of variance
---------------------------------------------------------------------------
Source of variation Sum of Squares d.f. Mean square F-ratio Sig.level
---------------------------------------------------------------------------
Betwe