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Genetic transformation method of brachypodium distachyon

A genetic transformation method, the technology of Brachypodium distachyda, applied in the biological field, can solve the problems of difficult differentiation process, easy pollution, low induction rate, etc., and achieve the effect of improving the regeneration rate

Pending Publication Date: 2020-08-07
山西师范大学
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing Brachypodium distachyon (Bd21) regeneration system has problems such as low induction rate, easy pollution, and difficult differentiation process, which seriously hinder people's research on the gene function of Brachypodium distachyon

Method used

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  • Genetic transformation method of brachypodium distachyon
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  • Genetic transformation method of brachypodium distachyon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Establishment of a high-efficiency genetic transformation system for immature embryos of Brachypodium distachyda mediated by Agrobacterium

[0107] 1) Material cultivation:

[0108] The material used in this experiment was wild-type Brachypodium distachyon (Bd21 ecotype). The growth conditions are: long-day conditions (18h light / 6h darkness, temperature 20°C), short-day conditions (10h light / 14h darkness, temperature 20°C). In order to promote the simultaneous germination of Brachypodium distachyon, the seeds were vernalized at 4°C for 1 week before sowing. In order to promote the flowering of Brachypodium distachyon in the greenhouse, after the seeds were grown in pots for 2 weeks, they were placed at 4°C for 3 weeks. No significant difference was found between the two types of plants (see figure 1 ).

[0109] 2) The configuration of various culture media of the present invention and other reagents used:

[0110] a. Induction medium (MSB3) medium: MS sa...

Embodiment 2

[0136] Example 2: Effects of different concentrations of 2,4-D on the embryogenic callus rate of Brachypodium distachyon

[0137] 1) Material: 8-12 immature embryos of Brachypodium tachycarpa plants.

[0138] 2) Training method:

[0139] Put the immature embryos of Brachypodium distachyon on the 2,4-D MSB3 medium containing 2.0mg / L, 2.5mg / L, 3.0mg / L, and count the embryogenic callus induction rate (embryogenic callus Rate=number of embryogenic callus / number of inoculated embryos×100%), the results are shown in Table 1. 2,4-D is a kind of auxin analogue, which has the effect of promoting plant growth. There are obvious differences in the embryogenic callus rate and adventitious bud regeneration rate induced by different concentrations. When 2.5mg / L of 2,4-D was used to induce callus, the rate of embryogenic callus was the highest. When the concentration of 2,4-D was reduced to 2.0mg / L or increased to 3.0mg / L, the rate decreased. According to the results in Table 1, it shows...

Embodiment 3

[0142] Embodiment 3: the influence of different concentrations of KT on the regeneration rate of Brachypodium distachydium adventitious buds

[0143] 1) Material: transgenic Brachypodium distachyon callus after one month of screening.

[0144] 2) Training method:

[0145] The transgenic calli after one month of screening were respectively transferred to the regeneration medium containing 0.1mg / L, 0.2mg / L, and 0.3mg / L concentration of KT for differentiation culture, and the statistical results of the adventitious bud regeneration rate are shown in Table 2 (Regeneration rate of adventitious buds=number of regenerated adventitious buds / number of inoculated calluses×100%). The present invention finds that with the increase of KT concentration, the average regeneration rate of adventitious buds first increases and then decreases. When the concentration of KT was 0.2mg / L, the regeneration rate of adventitious buds was better than that of the other two concentrations. The different...

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Abstract

The invention provides a genetic transformation method of brachypodium distachyon. The genetic transformation method at least comprises the following steps: 1) carrying out callus induction on immature embryos of brachypodium distachyon by utilizing an induction culture medium to obtain calluses, and carrying out subculture; 2) co-culturing the subcultured callus and agrobacterium tumefaciens, andinfecting the callus with the agrobacterium tumefaciens by using an infection solution; 3) screening the co-cultured calluses by using a screening culture medium to obtain calluses which grow well and are not browned; and (4) culturing the calluses obtained in step (3) by utilizing a regeneration culture medium until the calluses are differentiated and germinated so as to obtain brachypodium distachyon seedlings. Compared with an existing brachypodium distachyon transformation system, the brachypodium distachyon transformation system of the invention has the following advantages: the callus induction rate can be increased to 85.4% or above, and the regeneration rate of transgenic plants is increased. The efficient agrobacterium tumefaciens genetic transformation system established by theinvention provides certain technical guidance for exploring the gene function of brachypodium distachyon and provides a basis for gene improvement of gramineous crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a genetic transformation method of Brachypodium distachyon. Background technique [0002] Brachypodium distachyon L. is a new model plant of Poaceae, and it is the only plant in Poaaceae whose complete genome sequence has been completed. Its biological characteristics have a series of advantages, such as small genome, simple growth conditions, self-pollination, short growth cycle, small plant size, diverse ecotypes, easy transformation, high seed setting rate, self-fertilization and diploid Inbred line, etc. The above advantages make it an ideal model material for the study of functional genomics of temperate grasses. In order to fully exert its potential as a model plant, Brachypodium distachyda must be optimized and modified for its genetic transformation efficiency. However, the prerequisite for genetic transformation of Brachypodium distachyon is to establish an efficient plant ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/46A01H4/00
CPCC12N15/8205A01H4/008
Inventor 陈慧泽牛娇杜美婷韩榕刘维仲
Owner 山西师范大学
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