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Genetic transformation method of tartary buckwheat lamina

A genetic transformation method and tartary buckwheat technology, applied in the fields of biochemical equipment and methods, botanical equipment and methods, angiosperms/flowering plants, etc., can solve problems such as the difficulty of artificial hybridization and the failure of major breakthroughs in tartary buckwheat breeding, etc. achieve the effect of increasing the content

Inactive Publication Date: 2012-02-15
CHENGDU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the self-pollination characteristics of tartary buckwheat plants, it is difficult to succeed in artificial hybridization in production and breeding, so there is still no major breakthrough in the breeding of tartary buckwheat

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] (1) Activation culture of strains: Streak inoculation of Agrobacterium rhizogenes Ri1601 on YEB solid medium and culture for 1 day, pick a well-grown single bacterium into YEB liquid medium with an inoculation loop, shake at 28°C Cultivate for about 1 day to the OD of the bacterial solution 600 = 0.5;

[0014] (2) Acquisition of sterile seedlings: peel off the seed coat of tartary buckwheat seeds, put them on the ultra-clean workbench, disinfect them with 75% alcohol for 40 seconds, then disinfect them with 0.1% mercury liter for 5 minutes, and then rinse them with sterile water After 4 times, inoculate on MS solid medium, cultivate under the conditions of 25°C, light intensity 1200lx, and light 10h every day, to obtain tartary buckwheat sterile seedlings;

[0015] (3) Genetic transformation culture: choose tartary buckwheat aseptic seedling leaves with a leaf age of about 12 days and good growth conditions as explants, and inoculate the explants in the pre-medium MS+2...

Embodiment 2

[0017] Changing the explant selected in the embodiment 1 step (3) into leaf age is 30 days, tartary buckwheat leaves in good growth condition, and other steps are with embodiment 1; After cultivating for 25 days, the statistical hairy root conversion rate is lower than that of implementation example 1. It shows that the explants at different growth stages, in the process of genetic transformation with Agrobacterium, the cells that make up the organs are in different competent states; usually the longer the growth period of the organ, the more differentiated the cells are. The higher the value, the less likely it is for the cells to adjust to the competent state where Agrobacterium T-DNA is easy to enter, so the conversion rate of hairy roots will be lower.

Embodiment 3

[0019] This embodiment omits the precultivation step described in the step (3) of embodiment 1, and the blades selected from the aseptic seedlings of tartary buckwheat are directly dipped into the agrobacterium liquid to induce hairy roots, and other steps are implemented in the same manner. Example 1, after cultivating for 25 days, the statistical hairy root conversion rate was significantly lower than that in Example 1. It shows that in the process of genetic transformation mediated by Agrobacterium, the pre-culture treatment can effectively reduce the damage of Agrobacterium explants during the transformation process, and can adjust the explant cells to a state suitable for Agrobacterium-induced transformation.

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Abstract

The invention discloses a genetic transformation method of tartary buckwheat lamina. The method comprises the following steps; (1) inoculating Agrobacterium rhizogene into a YEB culture medium to prepare a desired bacterial liquid; (2) inoculating tartary buckwheat seeds into an MS medium without hormone so as to obtain aseptic seedling; and (3) based on aseptic seedling lamina as an explant, immersing the explant subjected to culture in advance into the activated Agrobacterium rhizogene bacterial liquid so as to fully contact the explant with the Agrobacterium rhizogene, taking out the explant to be transformed into the MS culture medium with the hormone for coculture, and then transforming and inoculating into a bacteria-removing culture medium containing sodium cefetaxime for bacteria-removing culture until a large amount of hairy roots are generated. By using the method in the invention, the content of secondary metabolites can be greatly improved.

Description

technical field [0001] The invention relates to a plant genetic transformation method mediated by Agrobacterium, in particular to a method for genetic transformation of tartary buckwheat leaves mediated by Agrobacterium. Background technique [0002] Buckwheat is a plant with the same source of medicine and food. There are two kinds of buckwheat, tartary buckwheat and sweet buckwheat. Because tartary buckwheat contains a large amount of flavonoids in its seeds, roots, stems, leaves, flowers and other organs, it is known as a "three-lowering food for lowering blood sugar, blood pressure, and blood fat". With the continuous discovery of its nutritional value and medicinal value, tartary buckwheat plant resources are more and more favored by people. Due to the self-pollination characteristics of tartary buckwheat plants, it is difficult to succeed in artificial hybridization in production and breeding, so there is still no major breakthrough in the breeding of tartary buckwhea...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 赵钢王跃华张红玉陈燕汤有举孙雁霞邬晓勇邹亮彭镰心胡一冰
Owner CHENGDU UNIV