Page 9 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2 No.2
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treatment, a total of 250 treatment, to remove
contaminated callus, 200 rice explants were used for
callus induction rate statistics analysis, callus
differentiation rate(%)=callus with bud/total number
of callus×100%).
3.5 Starvation and infection treatment of callus
Starvation treatment of callus were done before infection
according to the Lu’s method(Lu et al., 2008), after
precessing, placed the callus in Agrobacterium
suspension (OD600 ≈0.4) to infect for 15 min, then
poure infected callus and absorb the excess bacteria
using the dry sterile paper.
3.6 Dehydration treatment after infection and
co-culture
Callus after infection were treated as following groups:
A group: without drying, directly co-cultured, and
then differentiation culture. B group: dried after
infection. Dried procedure: callus were placed on
absorbent paper within the clean benches and dried 20
minutes. Callus were cultured in media in petriplates
with two filter-papers following the method of Hamid
Rashid et al., 1996 at 28℃ non-light culture for 3
days and using mannitol solution rinse 4-5 times after
co-cultured (mannitol 0.1 mol/L, containing cefotaxime
Cef 300 mg/L).
Calluses were treated as follows after co-cultured: C
group: drying the callus which without drying after
infection. D group: dried the callus with drying after
the infection. Drying methods: air-dried for 20 minutes,
then placed callus on petri-plates with two filter papers,
dried 8 hours under light conditions (50 explants of
rice each treatment, a total of 250 treatment, remove
contaminated callus, 200 rice explants were used in
callus induction rate statistics analysis, callus
differentiation rate(%) = callus with bud / total number
of callus×100%).
3.7 Callus differentiation and acquisition of
resistant plants
The callus after co-culture were transferred on
differentiation medium to culture about 7 days
differentiation with the light culture condition as 2000
lux, 13 h/d and 26 ℃. Then start to differentiate
approximately 14 days, the resistant seedlings were
transferred into the rooting medium. After additional
about 14 days, the regeneration rice plants with
well-developed root refined and transplanted.
3.8 GUS histochemical detection of Transgenic
Rice
GUS histochemical staining was followed with the
procedure of Jefferson’s method (1987).
3.9 PCR Detection
Genomic DNA was extracted from the phosphinothricin-
resistant rice plant following the guide of TIANGEN's
plant genome extraction kit. According UbiI promoter
sequence a pair of primers was designed and synthesized,
that the upstream primer is sequence: 5' -CATCTCTGT
CGCTGCCTCTG-3', and downstream primer
sequence is: 3'-CTGAAGT CCAGCTGCCAGAA -5'.
PCR reaction volume was 25 μL with upstream and
downstream primers each 1.5 μL, 2 μL template DNA,
2.5 μL 10×EX Taq Buffer, 4 μL dNTPs (2.5 mmol/L,
Mg2+ plus), 0.15 μl TaKaRa EX Taq DNA polymerase,
11.35 μL ddH2O. PCR amplification program was
followed as: 94℃ denaturation for 2 minutes, 94℃
30s, 55℃ 40s, 72 ℃ 40s, 35 cycles; 72℃ extension
3 minutes, 4 ℃ preservation. PCR products was
detacted with 1% agarose gel electrophoresis.
3.10 Statistical Analysis
Experimental data were analyzed using statistical
software SPSS 13.0 for significance of difference
analysis based on LSD method for multiple comparisons.
Acknowledgement
This Project was jointly supported by Cooperation Projects of
Ministry of Science and Technology of China (2007DFA31260),
National Science & Technology Pillar Program of Ministry of
Science and Technology of China (2007BAD59B06) and
Transgenic Special Projects of Guizhou Science and Technology
department, China (2004NZO04).
References
Cao M.X., Wei Z.M., and Huang J.Q., 2002, Progress on transformation of
rice mediated by Agrobacterium Tumefaciens, Zhiwu Shenglixue
Tongxun (Plant Physiology Communications), 38(5): 423-427
Clive J, 2010, Global status of commercialized biotech/GM crops: 2009,
Zhongguo Shengwu Gongcheng Zazhi (China Biotechnology), 30(2):
1-22
Duan C.L., Xiao F.H., Carl R., and Lan G., 2001, Establishment of tissue
culture system for the transformation of paddy rice via Agrobacterium,
Xinan Nongye Daxue Xuebao (Journal of Southwest Agricultural
University), 23(6): 528-531
He C.X., and Xia G.M., 1999, Recent advances in gene transformation of
monocotyledons mediated by Agrobacterium Tumefaciens, Zhiwuxue