Method for large-scale inducing plant gene mutation

A large-scale and large-scale technology, applied in the field of plant genetic engineering, can solve problems such as complicated procedures, inevitable chromosome aberrations, and easy pollution of the environment by mutagens

Inactive Publication Date: 2003-08-06
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The selection of mutants from each generation can only be selected from the offspring of heterozygotes of the previous generation, so the process is complicated
The second method of inducing mutants is chemical mutagenesis and physical mutagenesis. Chromosomal aberrations are inevitable in mutants, and mutagens are easy to pollute the environment.

Method used

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  • Method for large-scale inducing plant gene mutation
  • Method for large-scale inducing plant gene mutation
  • Method for large-scale inducing plant gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] From more than 1,500 transgenic cotton test-tube seedlings, 37 faster-growing test-tube cotton plants transformed by transposition activation tags were selected, and each plant was divided into 6-7 segments to continue selection, and isolated from 42 subcloned plants A plant that grows particularly rapidly on the MSB5 medium prepared by gellan gum can completely hydrolyze the agar and gellan gum in the medium within 10 days. However, neither the other mutants nor the control could hydrolyze the agar and gellan gum in the solid medium.

Embodiment 2

[0070] In 2001, a total of 562 offspring of the 3 transgenic plants (cotton F1) obtained in the previous year transformed by the transposition activation tag structure were planted, and a plant with shriveled anthers and male sterile was obtained by observing the variation of morphological characteristics with the naked eye . The flowers on the upper part of the plant pollinate the pollen of the normal plant, and seeds can be obtained, indicating that the plant is female fertile and male sterile. Observed by fluorescence microscope, all organs of the plant had no green fluorescence, indicating that the transposon tag in the mutant had been fixed.

Embodiment 3

[0072] Using the method of Rueb et al. using Agrobacterium-mediated transformation of rice (S. Rueb et al. 1994, Plant celltissue organ cult. 36: 259-264), the Agrobacterium containing the enhancer trap and transposition activation tag structure was healed with the rice embryo. The wounded tissue was co-cultured for 3 days, and then the callus was transferred to the hygromycin-containing medium for selective culture, and then the expression of GFP gene was observed under a fluorescent stereomicroscope, that is, the occurrence of green fluorescence was observed to determine the transformant. Then the transformed callus was transferred to the differentiation medium to induce the transgenic plantlets, and then transferred to the tray for cultivation after three weeks. According to the expression of the GFP gene and the GUS gene in the leaves, it is proved that the method of the present invention can efficiently induce rice gene mutations. The self-bred progeny of the transgenic p...

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Abstract

The present invention belongs to the field of plant gene engineering technology. It is characterized by that it utilzies agrobacterium mediated conversion method to introduce the artificially-constructed transposon label, i.e. enhancer net and transposition activatino label into plant, specially into the cultivated terrestrial cotton genome, and utilizes the random transposition of transposon andrandom induced gene mutation mechanism to find functional gene on a large scale and create a plant transposon label mutation gene bank containing lots of mutant genes including fixed and non-fixed transposons. Said mutation bank material can further find new functional gene of said crop by means of continuously planting, and the any mutant gene of the mutation bank can be cloned by adopting transposon label method.

Description

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for inducing plant gene mutation on a large scale, in particular to a method for establishing a cotton gene mutation library by using a transposon tagging method. technical background [0002] The use of transposon tagging to clone important functional genes of higher plants or eukaryotes is a very effective and broadly applicable gene cloning strategy. To clone a functional gene that has important utility value in production, the mutant of the relevant gene must be obtained first. Using the principle that transposons randomly induce gene mutations in the plant genome, various functional genes can be identified and isolated according to the phenotypic and biochemical characteristics of the plant. Corresponding transposon tag mutant gene banks have been established in major crops such as rice, tomato, potato and the model plant Arabidopsis, serving as importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00C12N15/09C12Q1/68
Inventor 杨业华王学奎吴征彬
Owner HUAZHONG AGRI UNIV
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