LAI V TUYN CHN GING LA CHNG CHU MNTS Nguyn Trung Tin v ThS Phm
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I. T VN t nhim mn l mt trong nhng yu t chnh lm kh khn trong chin
lc pht trin sn xut nng nghip v l th thch ln trong mc tiu an ton lng
thc. La (Oryza sativa L.) l cy trng nhy cm vi mn. Trong nhiu nm
qua, ngi ta c gng ci tin nhiu ging lua c tnh chng chu mn va a thanh
cng bc u. Tuy nhin, vic tm ra ging la c kh nng chng chu mn, cho nng
sut cao, chng chiu c mt s su bnh chinh va co phm cht gao ngon c hiu
qu cao trong vic ci thin san xut la vng t nhim mn la vn khng n
gian. Hin nay vi s tin b ca cng ngh sinh hc ngi ta c th da vo hin
tng marker phn t lin kt vi gen mc tiu, t o c th xc nh c kiu gen
khng hoc kiu gen nhim ngay t nhng th h phn ly u tin. Da vao thanh
tu nay, chung ti chng ti thc hin ti Tuyn chn ging la chng chu mn da
vao marker phn t. II. VT LIU VA PHNG PHP NGHIN CU Hat ging lua C53,
Pokkali, c Phung c ly t Ngn hng gen ca Vin la BSCL. Hat lai F1 cua
cac t hp lai C53/Pokkali (C.P); C53/c Phung (C.P); C53/D51 (C.D) c
gieo trong nh li thu nhn bng la non v NCTP. Cc dng la ti sinh c
trng v quan st trong nh li. Sau khi thu ht cc dng la ny, mt phn ht
s c gieo trong cc a petri thc hin thanh lc mn giai on m v trch DNA,
v lm phn ng PCR nh gi qun th A2 bng ch th phn t.
Nui cy ti phn: To t bo sma t ht phn ca cy lai F1, cho ti sinh to
cc dng la c kh nng CCM . Ht lai F1 ca cc t hp lai c gieo lm hai t
trong nh li thu ti phn. Mu l bng la non c thu vo khong 7 - 9 gi
sng, khi khong cch t gc l ng n gc l th nht t 5 - 10cm (Hnh 2.1), ra
sch sau gi m trong qu trnh x l lnh 10oC trong 7 - 10 ngy. ng la sau
khi c x l lnh, ly nhng ht gia bng c u trn ca ti phn cha vt qu chiu
di ht la. Ht la non c x l v trng bng cch ngm trong cn 70% trong 1
pht, sau ngm trong dung dch thu ngn clorua 0,1% thi gian 15 pht. Ra
sch mu cy 3 ln bng nc ct v trng trc khi a vo nui cy. Mu sau khi cy
c gi trong t ti nhit 25oC. M so hnh thnh c kch thc 2 - 3mm c cy
chuyn vo mi trng ti sinh to cy (to cm chi). Cc cm chi (cy xanh) c
nhn v tnh trn mi trng nhn chi, iu kin 25oC, 10 gi chiu sng trn ngy
m bng 02 bng n hunh quang 40W t trn b mt bnh nui cy cch 30cm (hnh
2.2). Sau khi c s lng chi cn thit, cy chuyn sang bnh tam gic nui
ring trong mi trng MS khng c cht iu ho sinh trng. Gi cc bnh cy
25oC, 10 gi chiu sng trn ngy m bng 02 bng n hunh quang 40W t trn b
mt bnh nui cy cch 30cm. Mt na s cy trong cng dng c to r trn mi trng
to r, mt na s cy cn li s c gi trong phng. Cy c r pht trin c thch
nghi trong dung dch Yoshida trong iu kin nhit phng 3 - 5 ngy trc
khi chuyn cy ra trng trong nh li (hnh 2.3). Cc mi trng c s dng
trong nui cy ti phn la Mi trng to m so: N6 + 2mg/l 2,4D + 60g/lt
Sucroz Mi trng ti sinh: MS +1mg/l BAP + 1mg/l NAA + 1mg/l Kinetin +
30g/l Sucroz Mi trng nhn chi: MS + 2mg/l BAP + 30g/l Sucroz. Mi
trng nhn nhanh: MS + 30g/l Sucroz Mi trng to r: MS +1mg/l NAA +
30g/l Sucroz. Mi trng thch nghi: dung dch Yoshida.
Trng cc dng ti sinh (cy A1) trong nh li Cc cy la c r pht trin c
thch nghi trong dung dch Yoshida c trng ring theo tng dng, lp li 3
ln, mi ln trng 20 cy. Cc dng cho ht lp (dng v tnh cn gi li trong
phng) s c x l Colchicine nng 0,05% trong 10 pht trc khi trng ra nh
li. Cac chi tiu theo doi: Thi gian sinh trng (ngy): c tnh t khi
thch nghi trong dung dch Yoshida n khi s ht trn bng chn 85%. Chiu
cao cy (cm): c tnh t gc cy n chp bng, o chiu cao ca 10 cy. S bng
/bi (bng): m s bng /10 bi, tnh trung bnh. S ht chc /bng (ht): m ton
b s ht chc/bng ca 5 bi, tnh trung bnh. T l lp (%): m ton b s ht
lp/bng ca 5 bi, tnh trung bnh, sau chia cho tng s ht/bng (chc v lp)
x 100. Trng lng 1000 ht (gram): Cn 3 ln trng lng ca 500 ht, tnh
trung bnh x 2. NS l thuyt (tn/ha) = (S ht chc/bng x s bng/bi x
TL.1000 ht x 33 bi) x 10-5. Nng sut c th (gram/cy): Cn s ht chc ca
10 bi, tnh trung bnh.
Thanh lc mn giai on m qun th A2 vi ging chun khng l Pokkali v
ging chun nhim l IR28 Thanh lc theo quy trnh thanh lc mn ca IRRI
(1997) c ci tin. Cy m c nh gi cp im theo tiu chun SES (Standard
Evaluating Score) phn bit t mn cm n khng. Khi cy m no hon ton vng,
khng cn m xanh na th c coi nh l cht. S ngy sng st ca cy m c ghi nhn
t khi cho mui vo mi trng n lc cht. Qua nh gi cp khng mn theo tiu
chun c cp nht ho theo tiu chun ca IRRI nm 1997. Cu th c thc hin nh
sau: Mi l ca v xp gieo hai ht ging ny mm, mi ging gieo 10 l t trong
khay nha c cha dung dch dinh dng Yoshida , r c a vo li nylon, mi
hng 10 cy m (xem nh mt ln lp li), mi ging ba hng, kt qu c phn tch
phng sai, nu khc bit c ngha thng k th tip tc so snh cp Duncan. Ging
chun khng l Pokkali c dng nh gi cc ging c kh nng CCM v ging chun
nhim l IR29.
Ht ging phi c x l 5 ngy trong t sy 40oC ph v trng thi ng ca ht.
Ngm ht ging trong a petri c lt giy thm v 30oC trong 48 gi. Sau 3
ngy, khi cy thch nghi, thm vo mi trng dinh dng mui NaCl. Th nghim c
b tr hon ton ngu nhin, 3 ln lp li vi 3 nghim thc: 0 dS/m, 6 dS/m v
15 dS/m. Dung dch mui c to ra bng cch thm NaCl vo trong dung dch
dinh dng v iu chnh dn in EC theo mong mun (0g; 3g; 7,5g NaCl/l dung
dch dinh dng t EC l 0dS/m; 6dS/m v 15dS/m), dung dch nui cy cn cho
mi khay l 10 lt, pH: 5.0. Sau 3 ngy thm nc ct vo, iu chnh pH v nng
mui v sau 8 ngy thay mi trng mt ln (hnh 3.7). nh gi mc chng chu mn
7 ngy, 14 ngy v 21 ngy sau khi gieo ht. Tiu chun nh gi (SES) mc
chng chu mn giai on tng trng v pht trin (bng 3.2). Cac chi tiu theo
doi: Thi gian sng st (ngy): Ngy sng st ca ht c ghi nhn t lc cho mui
vo mi trng n lc cht. Chiu di r (cm): Tnh t c r n nh sinh trng ca
chp r di nht. Chiu cao thn (cm): Tnh t c r n nh sinh trng ca chi
ngn cao nht. Trng lng kh r (mg): Sy kh r ca 10 cy m, cn v tnh trung
bnh. Trng lng kh thn (mg): Sy kh thn ca 10 cy m, cn v tnh trung
bnh. nh gi mc chng chu theo thang im quy nh bng 3.2
Bng 3.2: Tiu chun nh gi mc CCM giai on tng trng v pht trin Cp 1
3 Quan st Tng trng bnh thng, khng c triu chng trn l Tng trng gn nh
bnh thng nhng chp l hoc phn na ca l pha trn c vt trng, l hi cun li.
Mc chng chu Chng chu tt Chng chu
5
Tng trng chm li, hu ht l b cun, ch c vi l cn c th mc di ra Chng
chu trung bnh Tng trng b ngng hon ton, hu ht nhng l kh di, mt
7
vi chi b cht
Nhim mn
9
Tt c cy b cht hoc kh
Rt nhim mn
Ngun: IRRI, 1997
nh gi kiu gen qun th A2 bng ch th phn t (marker) Phng php trch
DNA Ly thn, l cy m non (10 - 15) ngy tui, ct nhuyn, t vo ci, cho vo
400ml DNA extraction buffer (PL 1). Nghin cc m l bng a thu tinh n
khi dch nc c mu xanh (t bo b ph v v phng thch ra dip lc).Thm vo
400ml DNA extraction buffer, trn u, sau ht 400ml a vo mt tube c th
tch 1,5 ml c ghi nhn tn ging a vo khay nc .
Khi trch cc ging ri, ta cho vo mi tube thm 800ml chloroform, trn
v lc u, sau t cc tube ny vo my li tm 12.000 vng trong 3 pht Ly cc
tube ra, ht lp dung dch pha trn (lp dung dch DNA) chuyn sang mt
tube khc c cng th tch. Thm 800ml ethanol (100%), trn k bng my lc
rung vortex, sau t cc tube ny vo my li tm 13.000 vng trong 5 pht,
ly ra, b phn nc pha trn, ra phn kt ta bng ethanol (70%) v phi DNA
nhit phng trong thi gian khong 30 pht. Ho DNA vo 50ml TE buffer (PL
2) v gi nht -20oC ch lm phn ng PCR. Lm phn ng PCR: Sn phm PCR c
chun b trong 10mM Tris-HCl (pH= 8), 50mM KCl, 1,5mM MgCl2, 1 n v
Taq polymerase, 4nmol dNTP, 10pmol primer v 50ng DNA. Primer c s
dng thc hin phn ng PCR l RM223. S dng my Thermal cycler 9600
(Perkin Elmer), chu k nhn ADN c thit k nh sau: Tch dy n 95oC trong
5 pht, tip theo chy 35 chu k: 94oC trong 60 giy, 55oC trong 30 giy,
v 72oC trong 60 giy. Ln ko di phn ng cui l 72oC trong 5 pht. Sau
khi thc hin PCR, thm vo 13 l dung dch m (98% formandehide, 10mM
EDTA, 0,025% bromophenol, 0,025% xylene cyanol). in di trn gel
agarose 5% kim tra mc a hnh ca sn phm PCR nh ethidium bromide
(Kangle v ctv., 1995). (i) Kt qu thu nhn: Xc nh c kiu gen ca cc dng
la CCM da vo s xut hin ca cc bng hnh. Cc dng xut hin bng c kch thc
140bp tng ng vi Pokkali mang kiu gen khng. Cc dng
xut hin bng c kch thc 160bp tng ng vi IR28 mang kiu gen nhim.
III. KT QUA VA THAO LUN 3.1. Thanh lc mn cc dng la giai on m Sau
khi to ra c 74 dng la qua NCTP, chng ti tin hnh th nghim trng cc
dng la ny ra nh li quan st v nh gi kiu hnh cy A1. Kt qu ban u cho
thy c 72 dng la tr bng v cho ht chc, cn li 2 dng la thuc t hp lai
C.P l C.P20 v C.P21 khng tr bng. c c s xc nh tnh CCM ca cc dng la
NCTP, nhm chn ra c cc dng la c kh nng CCM tt chng ti b tr th nghim
thanh lc mn cc dng la ny trong phng th nghim giai on m vi ba mc
0dS/m, 6dS/m v 15dS/m trn 72 dng la c to ra qua NCTP ca 3 t hp lai.
Kt qu thanh lc mn giai on m trn 72 dng la c to ra qua NCTP trong iu
kin dn in (EC) = 15 dS/m, chng ti phn tch phng sai cc ch tiu thi
gian sng st, chiu cao thn, chiu di r, trng lng kh thn v trng lng kh
r .
3.1.1. Thi gian sng st Kt qu ghi nhn thi gian sng st ca cc dng
thanh lc mn c to ra qua NCTP trong mi trng thanh lc mn EC = 15 dS/m
cho thy c s khc bit c ngha thng k khi so snh cp Duncan mc ngha 5%.
Thi gian sng st lu nht, c bit l lu hn ging i chng Pokkali (22 ngy)
c 2 dng, l C.P42 sng st 31 ngy v C.P44 sng st 29 ngy, chim 2,7%. C
4 dng c thi gian sng st tng ng ging Pokkali (21-24 ngy) l cc dng
C.P11, C.P27, C.P43, C.D4, chim 6,75%. C 6 dng c thi gian sng st gn
bng ging Pokkali (15 -19 ngy) l cc dng C.P17, C.P19, C.P5, C.D5,
C.D8 v C.D9, chim 8,1%. Cc dng cn li c thi gian sng st thp, gm c 61
dng, chim 82,43% . Ging chun nhim IR28 sng st c 8 ngy trong mi trng
mui 15 dS/m (bng 4.1). Ghi nhn thc t t kt qu th nghim chng ti thy
nng mui cng cao th thi gian sng st ca cc dng cng thp v ngc li.
trong mi trng dinh dng, tt c cc dng u sng st sau 35 ngy, nhng mi
trng dinh dng c NaCl vi EC = 6 dS/m, thi gian sng st trung bnh ca
cc dng l 29,36 ngy v mi trng dinh dng c NaCl vi EC =15 dS/m, thi
gian sng st trung bnh ca cc dng l 12,73 ngy.
Bng 4.1: Kt qu thanh lc mn giai on m cc dng la c to ra qua NCTP
trong iu kin mn EC = 15 dS/m ti Vin la BSCL STT 1 2 Tn dng C.P1
C.P2 Thi gian sng Chiu cao thn st (ngy) (cm) 6,4 y 11,29 w-z 8,5
s-y 12,01 v-z Chiu di r (cm) 5,10 x 8,43 c-m Trng lng thn Trng lng
r (mg) (mg) 47,28 O 18,22 CD 71,58 KL 20,39 z-D
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
48
C.P3 C.P4 C.P5 C.P6 C.P7 C.P8 C.P9 C.P10 C.P11 C.P12 C.P13 C.P14
C.P15 C.P16 C.P17 C.P18 C.P19 C.P1 C.P2 C.P3 C.P4 C.P5 C.P6 C.P7
C.P8 C.P9 C.P10 C.P11 C.P12 C.P13 CP14 C.P15 C.P16 C.P17 C.P18
C.P19 C.P20 C.P21 C.P22 C.P23 C.P24 C.P25 C.P26 C.P27 C.P28
C.P29
10,1 n-x 9,3 q-y 13,7 g-k 11,3 j-u 11,8 h-r 11,1 j-w 10,2 n-x
10,4 l-w 7,1 xy 13,1 g-n 13,0 g-n 13,4 g-l 14,4 f-i 12,6 g-p 17,6
cde 8,4 t-y 18,4 c 10,5 k-w 10,4 l-w 11,5 i-t 13,3 g-m 17,8 cd 9,8
o-x 9,7 p-x 10,0 n-x 9,8 o-x 9,3 q-y 21,3 b 14,8 e-h 9,3 q-y 12,2
h-q 9,6 p-x 11,7 i-s 11,6 i-s 15,4 d-g 8,8 r-y 8,1 v-y 11,7 i-s
10,2 m-x 11,2 j-v 13,8 f-j 9,3 q-y 10,0 n-x 21,3 b 13,4 g-l 12,6
g-p
13,59 s-v 13,38 s-v 14,23 r-u 13,04 t-x 12,60 u-y 14,72 q-t
13,85 s-v 14,20 r-u 13,33 s-v 12,34 u-z 12,92 t-x 13,91 s-v 13,38
s-v 13,82 s-v 12,37 u-z 12,94 t-x 13,02 t-x 14,75 q-t 16,89 j-p
16,67 k-q 16,81 j-p 18,18 g-m 13,22 t-w 14,89 p-t 15,34 o-s 13,79
s-v 16,39 l-q 17,15 h-o 16,11 m-r 17,85 g-n 15,31 o-s 19,03 fgh
16,96 i-o 16,27 l-q 17,14 h-o 19,15 fgh 18,17 g-m 23,70 ab 18,60
f-k 16,41 l-q 22,48 abc 17,54 h-n 18,99 f-i 23,37 ab 17,70 h-n
23,43 ab
8,75 b-j 8,97 a-h 9,13 a-f 8,43 c-l 8,30 c-o 9,57 a-d 8,48 c-k
9,57 a-d 10,54 a 7,53 a-s 9,01 a-h 9,16 a-e 9,57 a-d 10,26 ab 8,92
a-i 9,80 abc 9,50 a-d 7,32 g-u 6,66 m-x 6,12 q-x 5,64 u-x 6,15 q-x
5,82 s-x 6,15 q-x 5,81 s-x 5,34 wx 6,66 l-x 6,82 k-x 6,07 q-x 5,49
vwx 6,00 r-x 7,49 e-t 7,31 h-u 6,71 k-x 6,23 q-x 7,48 e-t 8,39 c-m
6,88 k-w 6,51 p-x 7,00 j-w 7,69 e-r 7,08 j-w 7,29 h-u 5,74 t-x 6,16
q-x 6,28 q-x
83,30 x-F 81,55 z-G 78,16 D-K 86,36 v-C 66,83 LM 92,50 o-v 78,55
D-J 100,00 i-n 81,91 z-G 79,97 C-G 81,22 A-G 86,97 u-B 79,22 D-I
77,44 E-K 62,91 M 72,41 JKL 72,72 I-L 78,88 t-A 91,89 p-v 98,86 j-o
104,05 f-k 104,64 f-j 88,72 s-y 94,94 m-s 90,66 q-w 80,47 B-G 97,83
k-p 108,94 d-g 106,50 e-h 90,28 m-r 97,33 l-q 95,89 m-r 115,80 c
97,88 k-p 99,19 i-o 92,89 o-v 110,19 c-g 95,72 m-r 112,14 cde
100,80 h-m 123,25 b 110,69 c-f 115,64 c 106,58 e-h 90,41 q-w 105,47
f-i
19,11 BCD 24,47 m-y 26,33 g-u 21,33 x-C 20,83 y-D 28,05 c-o
23,61 p-A 26,97 d-q 26,75 e-s 24,89 i-y 21,66 v-C 24,91 i-x 26,05
h-u 28,89 c-i 25,16 h-x 26,61 e-u 30,44 b-e 23,30 q-A 27,58 d-p
22,72 s-B 23,97 p-A 24,19 o-A 22,61 u-B 26,33 g-u 24,41 m-z 23,33
q-A 30,97 bcd 32,00 bc 28,28 c-n 23,77 p-A 28,86 c-j 25,78 h-u
28,41 c-m 25,19 h-x 21,47 w-C 25,69 h-v 25,88 h-u 25,91 h-u 32,86 b
28,36 c-m 27,08 d-q 28,75 c-l 24,94 i-x 26,19 g-u 23,86 p-A 24,72
l-y
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
70 71 72 73 74
C.P30 C.P31 C.P32 C.P33 C.P34 C.P35 C.P36 C.P37 C.P38 C.P39
C.P40 C.P41 C.P42 C.P43 C.P44 C.D1 C.D2 C.D3 C.D4 C.D5 C.D6 C.D7
C.D8 C.D9 Pokkali IR 28
11,8 h-r 9,0 r-y 8,1 v-y 12,9 g-o 14,2 f-j 11,4 i-t 8,1 wxy 9,3
q-y 8,6 s-y 9,6 p-x 9,1 q-y 11,2j- w 31,1 a 23,9 b 29,0 a 14,4 f-i
9,8 o-x 11,9 h-r 22,4 b 15,7 c-g 14,9 e-h 14,0 f-j 16,8 c-f 15,6
c-g 22,0 b 8,2 u-y
21,88 bcd 16,92 j-o 19,19 fgh 19,74 efg 18,76 f-j 21,19 cde
15,99 n-r 18,29 g-l 17,93 g-n 16,41 l-q 17,86 g-n 17,47 h-n 21,95
bcd 17,39 h-n 24,30 a 11,88 v-z 9,12 AB 11,16 xyz 13,94 s-v 10,52
zA 11,91 v-z 10,85 yzA 8,25 B 7,62 B 20,31 def 9,08 AB
6,55 o-x 6,61 n-x 6,29 q-x 6,89 k-w 6,84 k-x 6,24 q-x 8,16 c-p
7,82 d-q 6,76 k-x 6,83 k-x 7,47 e-t 8,40 c-m 10,35 ab 9,11 a-f
10,36 ab 9,08 a-g 7,66 e-r 8,72 b-j 8,37 c-n 7,37 f-u 8,31 c-o 9,17
a-e 7,67 e-r 7,19 i-v 9,00 a-h 7,02 j-w
110,25 c-g 103,89 g-l 88,14 s-z 79,55 C-H 80,66 B-G 95,80 m-r
82,39 y-G 91,55 p-v 92,80 o-v 84,11 w-E 92,52 o-v 77,83 D-K 122,89
b 93,89 n-t 93,30 n-u 76,33 G-K 81,66 z-G 68,69 LM 107,61 efg 84,55
w-D 114,91 cd 89,80 r-x 77,08 F-K 72,94 H-L 130,19 a 56,05 N
28,58 c-l 29,19 b-h 25,55 h-v 18,25 CD 17,25 D 22,63 t-B 23,33
q-A 25,08 i-x 26,36 f-u 22,83 r-B 24,00 o-A 30,25 b-g 21,69 v-C
25,47 h-w 20,27 A-D 25,94 h-u 27,61 d-p 24,78 j-y 27,64 d-p 26,69
e-t 24,75 k-y 24,25 n-A 30,41 b-f 28,80 c-k 37,36 a 26,83 e-r
Ghi ch: Nhng s trung bnh trn theo sau cng mt k t th khc nhau
khng c ngha mc 5% bi php so snh DMRT. C.P: C53/c Phng; C.P1 : t hp
lai C53/c Phng, dong s 1; C.P:C53/Pokkali; C.P1 : t hp lai
C53/Pokkali, dong s 1; C.D:C53/D51; C.D1: t hp lai C53/D51, dong s
1;
3.1.2. Chiu cao thn Cc dng la c to ra t cc t hp lai khc nhau c s
khc nhau v chiu cao thn, trong cng mt t hp lai cc dng c to ra cng c
s khc nhau v chiu cao thn, chiu cao cy gia cc mi trng cng c s khc
nhau r rt. Kt qu phn tch thng k cho thy c s khc bit khi so snh cp
Duncan mc ngha 5%. trong iu kin mi trng c dn in EC=15 dS/m, kt qu
ghi nhn cho thy cc dng la th nghim thuc t hp lai C.P a s c chiu cao
thn trung bnh trong khong 12 14cm, cc dng th nghim thuc t hp lai
C.P a s c chiu cao thn trong khong 15 - 19cm, cc dng thuc t hp
lai C.D a s c chiu cao thn trong khong 9 - 11cm. Chiu cao thn
trung bnh ca cc dng trong th nghim l 15,85cm. Cc dng c chiu cao vt
tri, cao hn ging i chng Pokkali (20,31cm) v khc bit c ngha khi phn
tch thng k l C.P44, C.P21, C.P29, C.P27, v C.P24. Cc dng c chiu cao
thn tng ng ging i chng Pokkali (khng khc nhau v mt thng k) l C.P42,
C.P30, C.P36, C.P33 v C.P32. C rt nhiu dng c chiu cao thn kh, cao
hn so vi chiu cao thn trung bnh u thuc t hp lai C.P (38 dng), (bng
4.1). 3.1.3. Chiu di r Chiu di r trung bnh ca cc dng trong b th
nghim thanh lc mn l 7,62cm. Ging i chng c chiu di r 9,00cm, c rt
nhiu dng c chiu di tng ng ging i chng (khc bit khng c ngha thng k),
c bit cc dng C.P11, C.P44, C.P42, C.P16, C.P18, l cc dng c chiu di
r kh (bng 4.1). 3.1.4. Trng lng kh thn
Kt qu ghi nhn trng lng kh thn ca cc dng c trnh by bng 4.6 cho
thy c s khc bit khi phn tch thng k mc ngha 5%, trng lng kh thn
trung bnh ca cc dng l 91,24mg. Ging chun khng Pokkali l ging c trng
lng kh thn cao nht (130,19mg), khc bit rt c ngha vi ging chun nhim
IR28 c trng lng kh thn l 56,05mg. Cc dng cn li c trng lng kh thn
tng i cao, gn bng ging i chng l C.P16, C.P22, C.P24, C.P42, C.D6.
3.1.5. Trng lng kh r Kt qu ghi nhn trng lng kh r ca cc dng c trnh
by bng 4.6 cho thy c s khc bit khi phn tch thng k mc ngha 5%, trng
lng kh r trung bnh ca cc dng l 25,51mg. Ging chun khng Pokkali l
ging c trng lng kh r cao nht (37,36mg), khc bit rt c ngha vi cc dng
cn li trong th nghim. Cc dng c trng lng kh r tng i cao, gn bng ging
i chng l C.P10, C.P11, C.P22, C.P41, C.D8 v C.P19. Ging chun nhim
IR28 c trng lng kh r l 26,83mg, v l ging c trng lng kh r mc trung
bnh. 3.1.6. nh gi mc chng chu mn
nh gi mc CCM 7 ngy, 14 ngy v 21 ngy sau khi gieo ht theo tiu
chun nh gi mc CCM giai on tng trng v pht trin (IRRI, 1997). Kt qu
nh gi cho thy trong mi trng dinh dng khng c mui NaCl, cy m tng trng
bnh thng, khng c triu chng b nhim mn . Trong mi trng dinh dng c thm
mui NaCl vi nng 6 dS/m, cy m bt u b nh hng do mn v mc b nh hng cng
tng dn theo thi gian. 7 ngy sau khi gieo hu ht cc dng cha b nh hng
mn, ch c dng CP1 v IR28 nh hng cp 3, n 14 ngy sau khi gieo cy m b
nh hng ch yu cp 3 - 5. Sau khi gieo 21 ngy a s cc dng vn sng v b nh
hng ch yu cp 5 - 7. Nhng trong mi trng dinh dng nng NaCl EC = 15
dS/m th sau 7 ngy gieo ht tt c cc dng u b nh hng bi mn, ging chun
nhim IR28 c biu hin cp 7, ging chun nhim Pokkali c biu hin cp 3, a
s cc dng khc b nhim cp 5 - 7. Vo 14 ngy sau khi gieo ht th ging
chun nhim IR28 b nhim cp 9 (coi nh thit hi hon ton), thi im ny ging
Pokkali biu hin cp 5 cng vi 7 dng c biu hin tng ng C.P17, C.P19,
C.P5, C.P11, C.P27, C.P43 v C.D4. Bn cnh c hai dng biu
hin CCM cao nh C.P42 v C.P44, c biu hin cp 3. S dng cn sng sau
khi gieo ht 21 ngy l 7 dng nhng mi dng ch cn li mt vi cy b nhim cp
7, ch c dng C.P42 v C.P44 l b nhim nh hn, cp 5 (bng 4.2). Nh vy sau
khi ging chun nhim IR28 b cht (nhim cp 9) th c 2 dng biu hin CCM
(cp 3), 8 dng biu hin mc CCM trung bnh (cp 5), 17 dng biu hin mc
nhim mn (cp 7), (hnh 4.1).
Bng 4.2: Kt qu nh gi cp im chng chu mn ca cc dng la to ra qua
NCTP (EC = 6 dS/m v EC = 15dS/m) n v: cp im STT Tn dng 1 2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 CP1 CP2 CP3 CP4 CP5 CP6 CP7 CP8 CP9 CP10 CP11 CP12 CP13 CP14
CP15 CP16 CP17 CP18 CP19 CP1 CP2 CP3 CP4 CP5 CP6 CP7 CP8 CP9 CP10
CP11 6 dS/m 7 ngy 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 15 dS/m 7 ngy 9 7 7 7 5 7 7 7 7 7 9 5 5 5 5 7 3 7 3 7 7 7
5 3 7 7 7 7 7 3
14 ngy 7 5 5 5 5 5 5 5 5 5 3 3 3 3 3 5 3 5 5 5 3 5 5 5 5 3 3 5 3
5
21 ngy 9 7 5 5 5 5 7 7 7 7 3 3 3 3 3 5 3 5 5 7 3 5 7 7 7 5 5 5 3
7
14 ngy 9 9 9 7 9 9 9 9 9 7 7 7 7 9 5 9 5 9 9 9 7 5 9 9 9 9 9
5
21 ngy
9
9 9 9 9 9 9
9 9
7
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
74
CP12 CP13 CP14 CP15 CP16 CP17 CP18 CP19 CP20 CP21 CP22 CP23 CP24
CP25 CP26 CP27 CP28 CP29 CP30 CP31 CP32 CP33 CP34 CP35 CP36 CP37
CP38 CP39 CP40 CP41 CP42 CP43 CP44 CD1 CD2 CD3 CD4 CD5 CD6 CD7 CD8
CD9 Pokkali IR28
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 3
3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 3 3 5 5 5 5 5 5 5 5 5 5 5 5 3 3
3 3 3 3 3 3 3 3 5 5 1 5
3 5 5 5 5 5 7 7 7 7 7 7 7 7 7 7 7 5 5 5 5 5 5 5 5 7 9 9 9 9 3 3
3 3 3 3 3 5 5 5 7 5 3 7
5 7 7 7 7 7 5 7 7 7 7 7 5 7 7 3 5 7 7 7 7 7 5 7 7 7 7 7 7 7 1 3
1 5 7 7 3 5 5 5 5 5 3 7
7 9 9 9 9 9 7 9 9 9 9 9 7 9 9 5 7 9 9 9 9 9 7 9 9 9 9 9 9 9 3 5
3 7 9 9 5 7 7 7 7 7 5 9
9
9
9 7 9
9
5 7 5 9 7 9 9 9 9 9 7
Ghi ch: CP: C53/c Phng; CP:C53/Pokkali; CD:C53/D51
Hnh 4.1: th biu din t l % cp CCM ca cc dng la to ra qua NCTP
trong iu kin EC = 6 dS/m v EC = 15dS/m 3.2. nh gi kiu gen bng nh du
phn t
3.2.2. Kim tra cht lng DNA K thut in di trn gel l k thut lm hin
th trc tip cc acid nucleic c tin hnh trn mt m keo (agarose gel hoc
arcrylamide), agarose l mt trong cc dng ca polysaccharide. Theo tnh
cht ca n, cc nh sinh hc phn t khai thc c tnh th gel trong in di phn
loi cc DNA. Agarose s to thnh ht agarose sau khi tan nhit cao, hoc
un si trong vi pht. Khi ngui li, nhng ht agrose s kt t li vi nhau.
Gia nhng ht nh vy, c nhng l rt nh. Kch thc ca nhng l ny c th x dch
cht t tu theo nng ca agarose gel. Trong in trng, DNA di chuyn t in
cc m sang in cc dng, v DNA mang in tch m (do tnh cht ca gc
phosphate). Khi DNA di chuyn qua cc l ca agarose, s c xt gia ht
agarose v phn t DNA to ra lc khng lm ngn cn s chuyn dch ny ca DNA.
DNA c phn t cng ln th lc cn cng mnh. Do DNA c phn t cng nh, di
chuyn cng nhanh. Nh vy ngi ta phn loi c cc DNA fragment trn agarose
gel. 3.2.3. Phn ng PCR
T kt qu theo doi tinh CCM qua hinh thai, chng ti chn ra 22 dng
la c biu hin kh nng CCM tt v nhng dng la c biu hin nhim mn r rt thc
hin phn ng PCR. Hai ging i chng c
a vo kim tra l Pokkali v IR28. Kt qu kim tra kiu gen v kiu hnh
ca cc dng la CCM c trnh by trong bng 4.3. Gen mc tiu c chn thc hin
th nghim ny l gen mn trn nhim sc th s 8 (salto). y l mt gen lin kt
cht trn nhim sc th s 8 vi marker phn t (RM223). Gen ny c lin kt vi
mi thm v vi CCM giai an pht dc (Lang v ctv., 2001a). Marker RM223 c
s dng lm marker nh du, marker ny c kch thc l (140bp - 160bp) v c
dng lm khun DNA thit lp cc cp primer c bit. Cc cp primer ny s khuch
i c cc on DNA nh hn nm trong nhim sc th ca cc dng nh phng php PCR.
Cc on DNA nh ny c gi l SSR. DNA cc dng la c to ra qua NCTP c cho
khuch i bng marker RM223 kim tra tnh a hnh. Primer c khuch i l
primer RM223 F 5-gagtgagcttgggctgaaac-3 v RM223 R
5-gaaggcaagtcttggcactg-3 Sau tin hnh kim tra vic khuch i trn gel
agarose 5% trong dung dch TBE (1X). Kt qu th hin trn hnh 4.2. Kt qu
th nghim cho thy tt c cc ct u c dng n hnh, xut hin vi hai bng c kch
thc 160bp tng ng vi IR28 v 140bp tng ng vi Pokkali cho gen chng
chiu mn. C 11 dng xut hin bng c kch thc 140bp tng ng vi Pokkali
mang kiu gen ng hp t alen khng, dng CD9 xut hin 2 bng mang kiu gen
d hp t. Cc dng cn li xut hin bng c kch thc 160bp tng ng vi IR28
mang kiu gen ng hp t alen nhim.
Bng 4.3: Kt qu kim tra kiu gen v kiu hnh cc dng la mang gen CCM
STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Tn dng,
ging IR28 Pokkali C.P1 C.P2 C.P3 C.P17 C.P19 C.P1 C.P2 C.P3 C.P5
C.P11 C.P25 C.P26 C.P27 C.P42 C.P43 C.P44 C.D3 C.D4 C.D5 Kiu gen N
T N N N T T N N N T T N N T T T T N T T Kiu hnh N T N N N T T N N N
T T N N T T T T N T T Ghi ch Nhim Chng chu Nhim Nhim Nhim Chng chu
Chng chu Nhim Nhim Nhim Chng chu Chng chu Nhim Nhim Chng chu Chng
chu Chng chu Chng chu Nhim Chng chu Chng chu
22 23 24
C.D7 C.D8 C.D9
N T N/T
N T T
Nhim Chng chu D hp t
Ghi ch: CP: C53/c Phng; CP:C53/Pokkali; CD:C53/D51
Hnh 4.2: Sn phm PCR ca cc dng la to ra qua NCTP ti locus RM223
lin kt vi gen mn trn nhim sc th s 8, v tr hai bng 160bp v 140bp,
trn gel agarose 5 %, TBE (1X) Ghi ch: s th t tng ng vi cc dng: 1:
IR28; 2: Pokkali; 3: C.P1; 4: C.P2; 5: C.P3; 6: C.P17; 7: C.P19; 8:
C.P1; 9: C.P2; 10: C.P3; 11: C.P5; 12: C.P11; 13: .CP25; 14: C.P26;
15: C.P27; 16: C.P42; 17: C.P43; 18: C.P44; 19: C.D3; 20: C.D4; 21:
C.D5; 22: C.D7; 23: C.D8; 24: C.D9 C.P: C53/c Phng;
C.P:C53/Pokkali; C.D:C53/D51
Tng hp kiu gen v kiu hnh (da vo cp CCM) chng ti ghi nhn qua kiu
gen c 12 dng, ging c biu hin CCM vi chiu di bng l 140bp trong c
Pokkali (i chng) v 11 dng, ging c biu hin nhim mn trong c IR28 (i
chng). Ly nhng dng, ging c biu hin CCM qua kiu gen i chiu vi kiu
hnh qua cp CCM, chng ti ghi nhn c 2 dng CCM tt, th hin qua kiu gen
v kiu hnh (cp 3), l dng CP42 v CP44. Bn cnh c 8 dng, ging c kh nng
CCM kh, c biu hin CCM qua kiu gen v kiu hnh (cp 5) l C.P17, C.P19,
C.P5, C.P11, C.P27, C.P43, C.D4 v Pokkali (bng 4.4).
Bng 4.4: So snh tnh CCM theo kiu gen v kiu hnh ca mt s dng CCM
to ra t 3 t hp lai (dng CCM c band di 140bp) STT Dng CCM qua kiu
gen Dng CCM qua kiu hnhnh gi mc CCM (EC =15dS/m) K hiu Tn dng, ging
Cp 3 Cp 5 Tt Kh Pokkali Pokkali Pokkali
1
2
2 3 4 5 6 7 8 9 10 11 12
6 7 11 12 15 16 17 18 20 21 23 Tng
C.P17 C.P19 C.P5 C.P11 C.P27 C.P42 C.P43 C.P44 C.D4 C.D5 C.D8
12
CP42 CP44 2
CP17 CP19 CP5 CP11 CP27 CP43 CD4 8
CP42 CP44 2
CP17 CP19 CP5 CP11 CP27 CP43 CD4 8
Ghi ch: CP: C53/c Phng; CP:C53/Pokkali; CD:C53/D51
3.2.4. Kim tra mc chnh xc gia vic nh gi ging theo kiu hnh v da
vo marker phn t Phng php SSR marker vi marker RM223 c kim tra, vi
mc chnh xc n 82% gia kiu gen v kiu hnh trn giai on pht dc v 92%
giai on m ( Lang v ctv., 2001a) . Chng ti tin hnh vic kim tra mc
chnh xc ca phng php SSR vi marker RM223. Trc ht xc nh kiu hnh thanh
lc i vi gen mn chn ra cc dng khng v nhim r rt nh gi kiu hnh v kiu
gen cho nhanh bng marker phn t. Kt qu ghi nhn v s lin h gia kiu hnh
v kiu gen (bng 4.5) cho thy trong cc dng la NCTP c kim tra c: Trong
12 dng mang tnh khng v kiu hnh, c 11 dng mang kiu gen ng hp t alen
khng (T), mc chnh xc 91,66%. Trong 10 dng mang tnh nhim v kiu hnh,
c 10 dng mang kiu gen ng hp t alen nhim (S),
mc chnh xc 100%. y c iu lu rng nu xt v gc di truyn th RM223 tch
c c d hp t trn dng C53/D51-9. Chng t dng ny cn phn ly, cha thun v
cho c hai alen (140bp - 160bp). Nh vy, c s lin quan gia kiu hnh
chng chu v kiu gen (91,66%), gia kiu hnh nhim v kiu gen (100%). iu
ny cho thy rng qu trnh biu hin t kiu gen ra kiu hnh l mt qu trnh
phc tp gm nhiu nhn t quyt nh, trong quan trng nht l s tng tc gia
kiu gen v mi trng. Phng php ny cho thy kh nng d on kiu gen chng chu
v kiu hnh chng chu rt cao, do c th p dng chn lc nhng ging chng chu
cho iu kin mn, lm ngun vt liu lai cho nhng chng trnh lai to ging la
mi hin nay. Bng 4.5: Kim tra li chnh xc gia nh gi kiu gen v kiu hnh
ca mt s dng la to ra qua NCTP vi s CCM Kiu hnh Nhim Chng chu S cy
10 12 Kiu gen RR 0 11 Gi tr chnh xc (%) RS 0 1 SS 10 0 100,00
91,66
IV.
KT LUN V NGH
4.1. Kt lun Trong thi gian nghin cu ti Lai v tuyn chn ging la
chu mn, chng ti c mt s kt lun sau: (ii) C 3 t hp lai a vo nui cy ti
phn u hnh thnh m so v cho cy ti sinh. T l % hnh thnh m so ca cc t
hp lai l : C53/c Phng: 7,20%; C53/Pokkali: 8,73% v C53/D51: 6,73%.
T l % ti sinh cy xanh ca cc t hp lai l: C53/c Phng: 10,5%;
C53/Pokkali: 22,0% v C53/D51: 4,5%. (iii) Dng marker SSR vi primer
RM223 nh gi cho gen khng mn trn 24 dng, ging, cc band hnh cho thy
tch gia dng chng chu mn v dng nhim mn, kch thc phn t 140-160bp. Kim
tra li chnh xc gia nh gi kiu gen v kiu hnh, s lin quan gia kiu hnh
chng chu v kiu gen l 91,66%, gia kiu hnh nhim v kiu gen l 100%.
(iv) Kt qu nghin cu xc nh tnh chng chu mn ca cc dng la to ra qua
nui cy ti phn bng phng php marker phn t vi primer RM223 v cc tnh
trng hnh thi nh thi gian sng st, cp chng chu mn v nng sut: c 2 dng
c kh nng chng chu mn tt l C53/Pokkali-42 v C53/Pokkali44. Ring dng
C53/Pokkali-44, ngoi c tnh chng chu mn tt cn biu hin nng sut cao.
Bn cnh c 7 dng chng chu mn kh l C53/c Phng-17, C53/c Phng-19,
C53/Pokkali-5, C53/Pokkali11, C53/Pokkali-27, C53/Pokkali-43 v
C53/D51-4.
4.2.
ngh
Do thi gian thc hin ti c hn nn chng ti ch thc hin c mt s th
nghim v thu c nhng kt qu nht nh. Nu ti c tip tc nghin cu ti xin
ngh: (v) Tip tc theo di v chn lc cc dng nui cy ti phn ngoi ng rung,
c bit ch cc dng c kh nng chng chu mn, c nng sut cao. (vi) Da vo kt
qu t c chng ta c th s dng phng php SSR marker vo vic nh gi tnh chng
chu mn cy la. (vii) S dng cc dng c kim tra kiu hnh v kiu gen chng
chu mn lm vt liu lai cho cc chng trnh lai to ging la chng chu mn.
PHN PH LC PL 1 Thnh phn Tris (pH 8.0) EDTA (pH 8.0) NaCl SDS H2O PL
2 Thnh phn Tris (pH 8.0) EDTA (pH 8.0) H2O Nng 10 mM 0.5mM 50 ml
0.5 ml 0.1 ml 49.4 ml Nng 50mM 25mM 300mM 1% 5 ml 0.25 ml 0.25 ml
0.3 ml 0.5 ml 3.7 ml
Nghin cu ng dng maker phn t trong chn ging la chu mn to bng k
thut nui cy ti phn
16/04/2009
ti do PGS.TS. Nguyn Th Lang (Vin La BSCL), Phm Th Xim (Trung tm
Ging tnh Kin Giang), GS.TS. Bi Ch Bu (Vin KHKT Nng nghip min Nam)
thc hin nghin cu ng dng marker phn t trong chn to
ging la chu mn bng k thut nui cy ti phn phc v cho sn xut BSCL.
Th nghim nghin cu c thc hin vi ba t hp C53/Pokkali, C53/c Phng,
C53/C51 trn mi trng N6 phi hp N6 + 2 mg/l 2,4 D + 60g/lt Sucroz.
Phng php nghin cu gm trng cy ti sinh (cy A1) trong nh li, thanh lc
mn giai on m qun th A2 vi ging chun khng l Pokkali v ging chun nhim
l IR28, nh gi kiu gen qun th A2 bng ch th (marker) phn t. Qua quan
st v nh gi kiu hnh ca cy A1 ca 72 dng la c to bng nui cy ti phn
trong nh li, khng nhn thy c dng no phn ng vi quang k. Cc dng c to
ra qua nui cy ti phn th tng ng nht, ch c mt dng c s biu hin ca cy
khc dng. C mt s dng thuc t hp lai C53/D51 nh C53/D51 -3, C53/D51
-7, C53/D51 -8 v C53/D51 -9 cho nng sut c th kh cao v cc c tnh nng
hc tng i tt. T kt qu thanh lc mn giai on m thng qua cc d liu marker
SSR vi primer RM 223 s dng trn 72 dng, cc band hnh cho thy tch gia
ging chng chu v ging nhim. Kch thc ny nm trong khong kch thc phn t
140-160bp. Phng php nh gi kiu gen da trn marker phn t th tng i chnh
xc, da vo marker SSR c th nh gi gin tip s hin din hay khng hin din
ca gen chn lc nh marker m khng b nh hng ca mi trng. Cc dng la ti
sinh qua nui cy ti phn: C53/c Phng -17, C53/c Phng -19, C53/Pokkali
-5, C53/Pokkali -11, C53/Pokkali -27, C53/Pokkali -42, C53/Pokkali
-43, C53/Pokkali -44, C53/D51 -4, C53/D51 -5 v C53/D51 -8 l cc dng
c kh nng chng chu tt vi iu kin mn. LV (ngun: TC NN&PTNT,
8/2008) Cc tin khc:
Hiu qu lm sng, huyt hc, sinh ha ca lc mu chu k bnh nhn suy thn
mn giai on cui - (16/04/2009 12:09) Nghin cu c im hnh thi ng ty rng
ca hm di - (16/04/2009 12:07) Nhn xt mt s c im lm sng ca khi u kt
gic mc - (16/04/2009 12:05) Nghin cu bo ch pellet omepprazol -
(13/04/2009 10:41) Th nghim tng ng sinh hc glucofin
