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Molecular Plant Breeding 2011, Vol.2 No.2, 8
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13
http://mpb.sophiapublisher.com
8
An Article Open Access
Rapid Acquirement of Transgenic Rice Plants Derived from Callus of Mature
Embryos Transformed by
Agrobacterium
Mediation
Gang Guo
1
, Jing Yu
2
, Degang Zhao
1, 3
1. Guizhou Key Laboratory of Agro-Bioengineering, College of Life Sciences, Guizhou University, Guiyang, 550025, P.R. China
2. Guizhou Tongren flue-cured tobacco limited liability company, Tongren, 554300, P.R. China
3. Guizhou Key Laboratory of Green Pesticide and Agricultural Biological Engineering, Ministry of Education, Guiyang, 550025, P.R. China
Corresponding author email:
degangzhao@yahoo.com;
Authors
Molecular Plant Breeding, 2011, Vol.2, No.2 doi: 10.5376/mpb.2011.02.0002
Received: 26, Nov., 2010
Accepted: 20, Dec., 2010
Published: 22, Jan., 2011
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Guo et al., 2011, Rapid acquirement of transgenic rice plants derived from callus of mature embryos transformed by
Agrobacterium
mediation, Molecular Plant
Breeding Vol.2 No.2 (doi:10.5376/mpb.2011.02.0002)
Abstract
In this research we transformed calli of two elite germplasms, Taipei 309 and Nipponbare, derived from mature embryos
using the construct of pCAMBIA0390 mediated by
Agrobacterium tumefaciens
strain EHA105. The expression plasmid pCAMBIA0390
was constructed with expressing elements of
ubi
:
bar-Gus
and
ACT
:
Bt
. Meanwhile We studied the effects of the factors on the
regeneration rates of callus tissues. The results showed that the regeneration frequencies of Taipei 309 and Nipponbare increased up
to 39% and 44% respectively, under the procedures as following: Callus was cultured 21 days in the conditions of, 28
℃
, 2 000 Lx,
and 13 h/d illumination period. Infected callus air-dried 20 minutes and then co-cultured callus air-dried 20 minutes, finally, the
callus desiccated using sterile filter paper of callus 8 hours. It shows that transformation cycle can shortened from 105 days to 60
days in absences of subculture stage, The
GUS
expression and PCR analysis indicated that the exogenous
Bt
gene has been integrated
into the rice genome.
Keywords
Rice; Mature embryos; Rapid regeneration; Transformation mediated by
Agrobacterium
Background
Rice (
Oryza Sativa
L.) is one of the most important
cereal crops. China is the biggest producer in the
world. On November 27, 2009, Ministry of Agriculture
of China (MOA), aproved and issued bio-safety
certificate for two varieties of transgenic Bt rice,
Restorer line, Hua Hui 1 and hybrid line, ShanYou-63,
that indicated genetically modified rice is going from
the lab to the field in China (Clive, 2010).
The early report on the
Agrobacterium
-mediated
transformation with callus of japonica rice cultivar
was documented in 1994 and done by Hiei et al. In
2006, Seiichi Toki et al reported that transgenic rice
plants were acquired in around 30 days with the
procedures of infection of mature embryso of
Nipponbare using
Agrobacterium
after cultured
mature embryo of Nipponbare one day in induction
medium. And Su Yi et al., (2008) in China was
confirmed that T
0
rice plants were regenerated in 40
days using the same methods as Seiichi Toki. These
researches proved that it is possible and feasible for
rapid regeneration of rice mediated by
Agrobacterium
transformation.
Excellent rice callus was recognized as an important
key factors that affects the efficiency of genetic
transformation in the procedures of the
Agrobacterium
mediated genetic transformation of rice. To improve
callus quality, researchers studied lots of effece of the
factors on callus regeneration rates focusing on
endosperm content, illumination culture period, and
drying methods etc. In this studies we developed the
transformation procedures with combination of some
results of previous studies such as mature embryo
soaking, distilled water and removal of endosperm
(Lin et al., 1996), light culture (Duan et al., 2005),
drying method (Masayoshi et al., 1992), waiving
subculture off (Seiichi Toki, 2006), to improve rates of
induction and differentiation of rice mature embryo
callus transformed by Agrobacterium mediation, and
eventually to acquired the transgenic rice plants within