Luận văn Nhân giống In Vitro cây Giáng hương.pdf

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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

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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

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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

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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).

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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

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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

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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

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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

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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

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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

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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).

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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.

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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

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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

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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).

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- 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.

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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.


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


Tng s mu: 72 mu


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.


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


Tng s mu: 27


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


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


Tng s mu: 81 mu


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



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



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


---------------------------------------------------------------------------

Data: TN1.HATNHIEM

Level codes: TN1.NT

Labels:



---------------------------------------------------------------------------


---------------------------------------------------------------------------

Between groups 9382.9136 8 1172.8642 999.999 .0000


---------------------------------------------------------------------------



50

BANG TRUNG BINH

Table of means for TN1.HATNHIEM by TN1.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

_


Multiple range analysis for TN1.HATNHIEM by TN1.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

---------------------------------------------------------------------------


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


---------------------------------------------------------------------------

Data: THI1.NAYMAM

Level codes: THI1.NT

Labels:



---------------------------------------------------------------------------


---------------------------------------------------------------------------

Between groups 22222.667 8 2777.8333 999.999 .0000


---------------------------------------------------------------------------



BANG TRUNG BINH

Table of means for THI1.NAYMAM by THI1.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

_


Multiple range analysis for THI1.NAYMAM by THI1.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

---------------------------------------------------------------------------


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


TH NGHIM 2

Th nghim 2a

S CHI

BNG ANOVA


---------------------------------------------------------------------------

Data: SCHOI.SOCHOI

Level codes: SCHOI.NT

Labels:



---------------------------------------------------------------------------

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

---------------------------------------------------------------------------



_

BNG TRUNG BNH

Table of means for SCHOI.SOCHOI by SCHOI.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

---------------------------------------------------------------------------


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 *


_

CHIU CAO CHI

BANG ANOVA


-----------------------------------------------------------------------

Data: CCCHOI.CCAOCHOI

Level codes: CCCHOI.NT

Labels:



-----------------------------------------------------------------------


-----------------------------------------------------------------------

Between groups 3.5516667 7 .5073810 4.380 .0069

Within groups 1.8533333 16 .1158333

-----------------------------------------------------------------------



54

BNG TRUNG BNH

Table of means for CCCHOI.CCAOCHOI by CCCHOI.NT

---------------------------------------------------------------------------



---------------------------------------------------------------------------

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

-----------------------------------------------------------------------



-----------------------------------------------------------------------

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

-----------------------------------------------------------------------


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 *


55

HE SO NHAN CHOI

BNG ANOVA


---------------------------------------------------------------------------

Data: HSN1.HSNCHOI

Level codes: HSN1.NT

Labels:



---------------------------------------------------------------------------

Source of variation Sum of Squares d.f. Mean square F-ratio Sig.level

---------------------------------------------------------------------------

Betwe

Documents

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