Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, is applied in the field of fast and efficient Agrobacterium-mediated rice transformation, which can solve the problems of long cultivation period, high requirements for state and genotype, and large economic investment, so as to save manpower and material resources input, reduced cost of genetic transformation, and high transformation efficiency

Inactive Publication Date: 2015-03-25
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the needs in the above-mentioned fields, the present invention provides a genetic transformation method, which avoids the cumbersome technology, strict disinfection and aseptic conditions in plant tissue culture that traditional rice genetic transformation must rely on, long cultivation period, high economic invest

Method used

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  • Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method
  • Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method
  • Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Using this method to obtain Guizhou endemic indica-type red rice Jianhe Honggu transgenic plants

[0038] 1. Preparation of Receptors for Genetic Transformation in Rice

[0039] Take the mature seeds of Jianhe Red Valley (see image 3 Shown), soak the seeds in clear water, change the water once every 12 hours, and after 2 days until the seeds are broken and white, accelerate germination and rooting at 37°C. When the germ grows to 1.5-2.0cm, use a scalpel to cut off the germ and germ from the stem ring. sheath, exposing its shoot apical meristem.

[0040] 2. Agrobacterium culture and activation

[0041] Agrobacterium tumefaciens strain LBA4404, the pCambia-Ubi-CHI vector carrying the bitter melon chitinase gene McCHIT1 and the herbicide resistance gene PAT (this vector was constructed and donated by the laboratory of Professor Pei Yan of Southwest University, and the laboratory of our school has preserved it , can be distributed) see figure 1 As shown, inocu...

Embodiment 2

[0072] Example 2: Using this method to obtain Guizhou endemic japonica type glutinous rice Pingtang black waxy transgenic plants

[0073] 1. Preparation of Receptors for Genetic Transformation in Rice

[0074] Get the mature seeds of Pingtang black glutinous rice (see Figure 4 Shown), soak the seeds in clear water, change the water once every 12 hours, and after 2 days until the seeds are broken and white, accelerate germination and rooting at 37°C. When the germ grows to 1.5-2.0cm, use a scalpel to cut off the germ and germ from the stem ring. sheath, exposing its shoot apical meristem.

[0075] 2. Agrobacterium culture and activation

[0076] Agrobacterium tumefaciens strain LBA4404, pCambia-35s-Pb vector carrying rice pb gene and hygromycin resistance gene HPTII (the vector was constructed by our laboratory, which is stored in the laboratory and can be distributed to the outside world) see figure 2 As shown, inoculate on the YEP solid medium containing 20mg / L rifampici...

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Abstract

The invention discloses a fast and efficient agrobacterium-mediated rice stem tip genetic transformation method. The method comprises the following major steps: directly soaking mature rice seeds in tap water, accelerating germination, then transversely cutting the coleoptiles to expose stem tips as transformation receptors, performing genetic transformation by virtue of agrobacterium mediation without sterilization treatment, putting the transformed materials under an appropriate environment to allow plants to grow, and carrying out transgenic screening and identification to obtain the transgenic rice plants. According to the fast and efficient agrobacterium-mediated rice stem tip genetic transformation method, callus induction and a series of subsequent tissue culture processes are avoided, the defects of materials affected by bacteria, difficult regeneration, vitrification, browning, low survival rate of transplanted tissue cultured seedlings and the like in the tissue culture process are avoided, the rice genetic transformation cost is reduced, the restriction of the rice genotype to the transformation efficiency is effectively overcome, and a large quantity of transgenic rice plants can be obtained in short time; the fast and efficient agrobacterium-mediated rice stem tip genetic transformation method has a significant value in good-quality rice culture and rice genetic transformation research.

Description

technical field [0001] The invention belongs to the fields of agricultural biotechnology and plant genetic engineering, in particular to a fast and efficient Agrobacterium-mediated rice transformation method. Background technique [0002] Rice is one of the most important food crops in the world. More than half of the world's population depends on rice as a staple food, and its stable supply directly affects the global food balance. Breeding a variety of high-quality rice is one of the ways to solve the increasingly prominent food and environmental problems. Conventional rice hybrid breeding has a long cycle and slow results, and has the disadvantages of gene linkage and reproductive isolation. With the help of mutagenesis breeding under some physical and chemical conditions, it is difficult to control the direction of mutagenesis, and the beneficial mutation rate is low. Obtaining hybrid cells by means of cell fusion, cultivating and developing into plant cell engineering...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 赵德刚曾晓芳李岩张笑寒
Owner GUIZHOU UNIV
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