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High-efficiency agrobacterium genetic transformation method for heavy metal super-enriched plant Indian mustard

A technology of hyperaccumulation plant and Indian mustard, applied in the field of efficient Agrobacterium genetic transformation of heavy metal hyperaccumulation plant Indian mustard, can solve the problems of immature means of Indian mustard transformation, limited application, low transformation efficiency, etc., and achieves saving seedlings The effect of occupying land, reducing production costs, and stabilizing hereditary traits

Inactive Publication Date: 2008-02-27
GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the immature transformation methods of Indian mustard, the transformation efficiency is very low, which limits its application in production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Agrobacterium-mediated genetic transformation of Indian mustard

[0046] 1. Establishment of primary culture

[0047] Take the mature seeds of Indian mustard, disinfect the surface with 70% alcohol for 30 seconds, and soak them in 0.1% mercury chloride for 8 minutes. Rinse with sterile water 3-5 times, blot dry with sterilized filter paper; culture on seed germination medium for 7 days. The culture conditions are as follows: the light intensity is 5000 lux, the light time is 16 hours / day, and the temperature is 20°C. The seed germination medium is MS medium;

[0048] 2. Pre-culture

[0049] Using germinated and cultured Indian mustard seedlings as materials, cut hypocotyl segments about 5-7 mm long as explants, spread them on the pre-cultivation medium, and pre-cultivate them for 2 days. The culture conditions are: the light intensity is 3000 lux, the light time is 16 hours / day, and the temperature is 20-25°C.

[0050] The components and proportions cont...

Embodiment 2

[0078] Example 2: High-efficiency Indian mustard genetic transformation mediated by Agrobacterium

[0079] 1. Establishment of primary culture

[0080] Take the mature seeds of Indian mustard, disinfect the surface with 70% alcohol for 30 seconds, and soak them in 0.1% mercury chloride for 8 minutes. Rinse with sterile water 3-5 times, blot dry with sterilized filter paper; culture on seed germination medium for 10 days. The culture conditions are: the light intensity is 4000 lux, the light time is 16 hours / day, and the temperature is 23°C;

[0081] The seed germination medium is MS medium;

[0082] 2 pre-culture

[0083] Using germinated and cultured Indian mustard seedlings as materials, cut the cotyledon petiole segment with a length of about 3-5 mm as explants, spread it on the pre-cultivation medium, and pre-cultivate it for 3 days.

[0084] The culture conditions are: the light intensity is 4000 lux, the light time is 16 hours / day, and the temperature is 20-25°C.

...

Embodiment 3

[0113] Example 3: High-efficiency Indian mustard genetic transformation mediated by Agrobacterium

[0114] 1. Establishment of primary culture

[0115] Take the mature seeds of Indian mustard, disinfect the surface with 70% alcohol for 30 seconds, and soak them in 0.1% mercury chloride for 8 minutes. Rinse with sterile water 3-5 times, blot dry with sterilized filter paper; culture on seed germination medium for 15 days. The culture conditions are as follows: the light intensity is 3000 lux, the light time is 16 hours / day, and the temperature is 25°C.

[0116] The seed germination medium is MS medium;

[0117] 2. Pre-culture

[0118] Using germinated and cultivated Indian mustard seedlings as materials, cut cotyledons with a length of about 3 mm2 as explants, spread them on the pre-cultivation medium, and pre-cultivate them for 2 and a half days;

[0119] The culture conditions are: the light intensity is 5000 lux, the light time is 16 hours / day, and the temperature is 20-...

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Abstract

The invention discloses a high effective agro bacterium genetic conversion method of a heavy metal over enrichment plant Indian leaf mustard, which comprises the following steps: culturing the disinfected mature seed of Indian leaf mustard until a pair of cotyledons expand; inoculating seedling hypocotyls, cotyledon stem or cotyledon in the culture medium and culturing; co-culturing the disseminated agro bacterium in the co-culture medium; transferring to the front culture medium until callus expands; getting the resistant callus by transferring to the screening culture medium; differentiating the resistant overgrow bud; culturing the overgrow bud in the basic culture medium; proceeding with the water and growing-root culture by transferring to HeGELanTe culture liquid; planting by transferring the strong sprout with the root to the nursery base material; getting the transferred gene Indian leaf mustard mature plant. The method can remove the influence of the external condition, which is fit for four seasons, saves the nursery sock growing field, reduces the cost, acquires a great deal of strong transferred gene seedlings in the short time, and meets the research and the production application of molecular biology.

Description

technical field [0001] The invention belongs to the field of plant biotechnology; in particular, it relates to a high-efficiency Agrobacterium genetic transformation method for heavy metal hyperaccumulation plant Indian mustard. Background technique [0002] Indian mustard (Brassica juncea.L) belongs to Brassica juncea, widely distributed in China, India, Pakistan and Southeast Asian countries, and is an important economic oil crop. Compared with cabbage-type rape and Brassica napus, Indian mustard has stronger ability to resist drought and cold, and at the same time has the ability to enrich various heavy metals such as Cd, Cr, Ni, Zn, Cu, Au and Se. It has broad prospects for the application of heavy metals and radioactive substances in water bodies, and is one of the main species currently studying the molecular mechanism of heavy metal enrichment and developing phytoremediation technology. Indian mustard is a typical heavy metal hyperaccumulator plant, which can accumul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/04C12N5/10A01H4/00
Inventor 柴团耀徐进魏嵬官子楸韩璐丛林
Owner GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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