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Method for agrobacterium mediated gene conversion of grass sorghum

An Agrobacterium-mediated and gene transformation technology is applied in the field of Agrobacterium-mediated gene transformation of Sudan grass, which can solve the problem of difficulty in high-frequency regeneration of plants and achieve the effect of promoting callus differentiation.

Inactive Publication Date: 2010-12-29
JIANGSU ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, because Sudan grass is a forage grass of the genus Sorghum, it is relatively more difficult to obtain high-frequency regeneration plants through tissue culture compared with other crops. There is no official report on the transgenic breeding of Sudan grass Agrobacterium-mediated method in China.

Method used

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  • Method for agrobacterium mediated gene conversion of grass sorghum
  • Method for agrobacterium mediated gene conversion of grass sorghum
  • Method for agrobacterium mediated gene conversion of grass sorghum

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Embodiment 1

[0044]The Agrobacterium tumefaciens strain was LBA4404. Recombinant plant expression plasmid pCAMATBADH682 (He Xiaolan, Wu Jizhong, Liu Guihua, et al. Cloning of betaine aldehyde dehydrogenase gene of Atrigus trifoliata and construction of plant expression vector [J]. Journal of Jiangsu Agricultural Science, 2003, 19(4): 237-241.) The structure is: RB-Gus-35SP-BADH3-NosT-35SP-NPT II-NosT-LB, and the transformant is Sudangrass 2098 (Zhong Xiaoxian, Gu Hongru, Zhou Weixing, etc. Hybrid seed production technology of leaf spot resistant Sudangrass and its Germplasm innovation [J]. Journal of Jiangsu Agricultural Sciences, 2001, 17(3): 184-187.), Sudan grass 2098 is the male restorer line of Sudan grass hybrid Green ACE bred in Japan (Katsuba Z, Nakagawa H, Maeda M, et al.A (Sorghum sudanense) line "2098-2-4-4" as apollen parent for developing hybrid sorghum cultivars [J]. Bulletin of the Hiroshima Prefectural Agriculture Research Center, 1998, 66: 15~23.), Introduced from Japan i...

Embodiment 2

[0065] The Agrobacterium tumefaciens strain was LBA4404. The recombinant plant expression plasmid pBGO (She Jianming, Zhang Baolong, Liang Liufang, et al. Obtaining plants with glucose oxidase gene from Poa annua [J]. Journal of Jiangsu Agricultural Science, 2006, 22(3): 217-221.) has the following structure: RB-Nos.P-Npt II-Nos.T-35SP-Ω-GO-Nos.T-LB. The transformant was Sudangrass 2098. The specific conversion process is as follows:

[0066] 1. Young panicle in vitro culture induces granular callus (same as embodiment 1)

[0067] 2. Agrobacterium infection and co-cultivation

[0068] Take 1 mL of Agrobacterium LBA4404 carrying pBGO plasmid out of the refrigerator at -70°C, inoculate it into 5 mL of YEB liquid medium, and activate it by shaking at 26°C and 200 rpm for 24 hours, then take 1 mL of the activation solution and inoculate it into 50 mL of YEB liquid culture Medium, continue shaking culture for 6h to OD 600 About 0.6, dilute the bacterial solution to OD with ste...

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Abstract

The invention relates to a method for transforming Sudan grass through an agrobacterium mediating gene, which belongs to the biotechnology field. When the agrobacterium mediating method is used to transforming exogenous gene of Sudan grass, a faint yellow granular callus which is obtained from the in-vitro culture and induction of a Sudan grass young ear is taken as acceptor material; after the infection and co-culturing of the agrobacterium , the acceptor material undergoes 10 to 20mg / L kanamycin resistance screening to obtain a resistance callus in a subculture medium of carbenicillin with attached antibiotics of 500mg / L; the resistance callus is translated into a differentiation culture medium of the carbenicillin with the attached antibiotics of 500mg / L to obtain a regenerated plantlet; the plantlet is cultured to have sound seedling and root and survives after transplantation; and a transgenic plant is obtained after the plant passes a PCR test. The Sudan grass genetic transformation method established by the invention has a transformation rate of more than 3 percent, thereby laying the foundation for carrying out the transgenic breeding of the multi-purpose Sudan grass through the agrobacterium mediating method.

Description

1. Technical field [0001] The invention relates to a method for agrobacterium-mediated gene transformation of sudan grass, belonging to the technical field of genetic engineering utilization. 2. Technical background [0002] Sudan grass (Sorghum sudanense) is an annual gramineous forage grass belonging to sorghum. It can be used as green forage, silage and hay preparation, and is preferred by various herbivorous livestock and herbivorous fish. Sudan grass is planted all over the world. It can be cultivated from Hainan in the south to Inner Mongolia in the north. It is the most important warm-season pasture in the middle and lower reaches of the Yangtze River, accounting for more than 70% of the planting area of ​​warm-season pasture in the region. At the same time, Sudan grass As a biomass energy crop, it has been paid more and more attention in recent years. However, in southern agricultural areas, there are more people and less land, and the conflict between humans and an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H1/00
Inventor 钟小仙佘建明蔡小宁何晓兰顾洪如张建丽倪万潮邹轶
Owner JIANGSU ACAD OF AGRI SCI
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