Rapid and high-efficiency transgenic method for indica rice

A transgenic and genetic technology, applied in the field of transgenics, can solve the problems of increased frequency of somatic cell mutations, difficulties in phenotypic identification and biochemical analysis of transformed strains, and achieve the effects of reducing mutation frequency, shortening in vitro culture time, and short transformation cycle

Inactive Publication Date: 2011-11-02
HUNAN UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

More seriously, the frequency of somatic mutations increased significantly due to the prolonged callus culture in vitro, which brought unpredictable difficulties to the subsequent phenotypic identification and biochemical analysis of transformed strains.

Method used

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  • Rapid and high-efficiency transgenic method for indica rice
  • Rapid and high-efficiency transgenic method for indica rice
  • Rapid and high-efficiency transgenic method for indica rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Induction of Callus

[0047] Select the seeds of Xiangling 628S, an indica rice variety that is well-ripe, plump, and mildew-free, and use a brown rice machine to remove the inner and outer shells of the seeds. First sterilize with 70% alcohol for 2 minutes, then soak in 30% NaClO+0.1% Tween 20 solution, and put it on a shaker at 100rpm for 30 minutes. On the ultra-clean workbench, the sterilized deveined seeds were washed 5 times with sterile water, then transferred to a petri dish equipped with sterile absorbent paper to absorb the water, and then inoculated in the induction medium. The culture conditions were 32°C, continuous light (100 μmole m -2 the s -1 ). After 7 days, the callus was induced at the scutellum of the seed embryo, and then the whole seed was used for transformation (see figure 2 A).

[0048] 2. Selection of Vector and Preparation of Agrobacterium

[0049] The Agrobacterium strain used was EHA105, and the expression vector was modified pCA...

Embodiment 2

[0067] 1. Induction of Callus

[0068] The seeds of indica rice variety ZR02 with good maturity, plumpness and no mildew were selected, and the specific steps of callus induction were the same as in Example 1.

[0069] 2. Selection of Vector and Preparation of Agrobacterium

[0070]The expression vector is the modified pCAMBIA1301 containing transcriptional activation domain motif VP64. Its T-DNA region contains the Basta resistance gene bar and the red fluorescent protein gene AsRed as screening marker genes, and the recombination border sequence of Gateway is added. The target gene is the transcription factor gene bZIP17 from rice, which is subcloned into the vector by Gateway method, and constitutes a synthetic transcription factor with the transcriptional activation domain motif VP64. Both the screening marker genes bar and AsRed are driven by the CaMV35S promoter, while the target gene is driven by the Ubiquitin promoter (see image 3 A). In addition, the expression v...

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Abstract

The invention discloses a rapid and high-efficiency transgenic method for indica rice. The method comprises the following steps of: exposing indica rice mature seeds to Agrobacterium tumefaciens 5 to 15 days after callus induction, co-culturing, performing resistant callus screening for 10 to 20 days, performing differentiation culture for 10 to 20 days, performing rooting culture for 5 to 10 days, and performing resistance screening again. The transgenic plants of indica rice can be produced within 40 to 50 days, and the conversion efficiency reaches 20-30%. In the conversion process, the culture conditions are as follows: the temperature ranges from 30 DEG C to 33 DEG C and the light is continuously supplied with the intensity of 80 to 120 mu mole/m<-2>s<-1>, except that the co-culture stage is carried out at 25 DEG C in the dark. The method provided by the invention greatly shortens the in vitro culture time of calli, increases the differentiation efficiency, reduces the mutation frequency of somatic cells, simplifies the operation procedure and saves a large amount of labor and material resources.

Description

technical field [0001] The present invention relates to a transgenic method, in particular to a fast and efficient transgenic method for indica rice, specifically refers to using mature indica rice seeds that induce callus for 5-10 days for Agrobacterium infection, combined with the rapid screening and A differentiated indica rice transgenic method belongs to the field of biotechnology. Background technique [0002] Rice (Oryza sativa L.) is one of the main food crops in the world, and nearly one-half of the world's population uses rice as a staple food. Indica rice, in particular, accounts for 80% of the world's rice production and plays a pivotal role in the global agricultural economy. However, in the production process of rice, due to the influence of various diseases and insect pests, bad climate and environment, the high and stable yield of rice is seriously restricted. With the rise of transgenic technology, research on transgenic rice has become a hot spot in the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 刘选明林建中唐冬英周波杜长青周延彪
Owner HUNAN UNIV
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