Page 4 - BM 2011 Vol.2 No.5

Bioscience Methods
BM 2011, Vo.2, No.5 http://bm.sophiapublisher.com
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Research Article Open Access
Studies on Construction of Regeneration System and Genetic Transformation of
Puccinellia chinampoensis
Tao Wang 1 , Xue Han 1 , Mengqing Zhao 1 , Xinxin Zhang 1 , Takano Tetsuo 2 , Shenkui Liu 1
1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education,; Alkali Soil Natural Environmental Science
Center (ASNESC), Northeast Forestry University, Harbin Hexing Road, 150040
2. Asian Natural Environmental Science Center(ANESC), University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 188-0002, Japan
Corresponding author email: shenkuiliu@nefu.edu.cn; Author
Bioscience Methods 2011, Vol.2 No.5 DOI:10.5376/bm.2011.02.0005
Received: 02 Jun., 2011
Accepted: 20 Jun., 2011
Published: 23 Jun., 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 as:
Wang et al., 2011, Studies on Construction of Regeneration System and Genetic Transformation of Puccinellia chinampoensis, Bioscience Methods,
doi:10.5376/bm.2011.02.0005
Abstract The regeneration system was established and the genetic transformation was processed, using the mature seeds of
Puccinellia chinampoensis as explants. The results showed that the callus induction rate could reach to 39.75% after 19days of
induction with the altered mediumⅠ (adding 4 mg/L 2,4- D, 500 mg/L proline and 500 mg/L glutamine) (pH5.8~6.0). When using
the modified medium S (adding 2 mg/L 2,4- D, 1 mg/L ABA, 500 mg/L proline and 600 mg/L caseinhydrolysate) (pH5.8~6.0) as the
subculture medium, we can get much more embryogenic callus. The differentiation rate could reach to 53.73% using the
differentiation medium (MS as the basic medium, adding 0.4 mg/L ABA, 0.04 mg/L IAA, 500 mg/L proline and 600 mg/L
caseinhydrolysate) (pH5.8~6.0). On the genetic transformation, the transformation efficiency was best when the infect time lasted
30 min, making using of the Agrobacterium tumefaciens mediated pBI121- GUS transformation when OD600 reached to 0.8~1.0.
Keywords Puccinellia chinampoensis; Regeneration system; Genetic transformation
Background
With the expansion of soil salinization globally,
discovering the salt and alkali tolerance mechanisms
of plants is the important evidence for improving the
development and utilization of salty and alkali soil
(Flowers et al, 2004). Puccinellia chinampoensis is a
kind of pioneer plant growing well in the salt and
alkali soil, and it has the other advantages such as low
temperature tolerance and good palatability, so it is the
excellent grass for pasturage. It has been screened out
to manage the salinization since 1980s and have
achieved good results in China (Wang et al., 2004). At
the present time, researches of Puccinellia chinampo-
ensis are focused on the structural anatomy,
physiology ecology and molecular biology and so on.
Separating functional genes related to salt and alkali
stresses from salt plants is the key to discuss the
molecular mechanism of plant salt tolerance and to
breed the new vatieties of plants. Puccinellia chinam-
poensis is not only an excellent grass, but also a
precious resource providing a wealth of salinity
tolerance genes.
In this study, we discussed a series of culture
conditions of callus induction, plant regeneration and
genetic transformation of Puccinellia chinampoensis,
which provided the technical support and theoretical
basis for the further exploring the genetic improvement
and cellular breeding engineer.
1 Results and Analysis
1.1 Impact on the callus induction of hormone 2,4-D
After 7~9 days’ culture on the induction medium,
callus would be induced out at the base of embryo. It
can reduce the induction time at the light cultivate
condition, but at this time, the embryo will grow into a
seedling shoot at low concentrations of 2,4-D, which
can inhibit the induction of callus. To prevent the
formation of seedling shoot, more 2,4-D was
supplemented, and when it reached to 4 mg/L, the
induction rate could be reached to 39.7% after 14
days’ cultivation (Table 1; Figure 1). Additionally, the