Plant in-situ regeneration method and application thereof in genetic transformation

A technology of in situ regeneration and genetic transformation, applied in the field of genetic transformation of in situ organ regeneration of plant cells combined with Agrobacterium infection, can solve the problems of difficulty in making breakthroughs and difficulty in tissue culture, and achieve the solution of avoiding cumbersome tissue culture conditions. Effect

Inactive Publication Date: 2012-07-25
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since tissue culture regeneration involves complex conditions such as the type of medium, the type and concentration of plant growth regulators, temperature, light, etc., even for such an important economic crop as soybean, after years of exploration, it is still a world-recognized difficulty in tissue culture. , so it is difficult to make a breakthrough in this way in a short time

Method used

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  • Plant in-situ regeneration method and application thereof in genetic transformation
  • Plant in-situ regeneration method and application thereof in genetic transformation
  • Plant in-situ regeneration method and application thereof in genetic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the method for plant regeneration in situ

[0038] Step 1, the acquisition of scion

[0039] The tomato variety Liaoyuanduoli (Solanum lycopersicum L, purchased from Liaoning Gardening Seedling Co., Ltd.) was selected as the experimental material. Take the well-developed and disease-free Liaoyuan Dolly tomato seeds, soak them in washing powder water for 1 hour, and rinse them; soak them in 75% alcohol for 30 seconds on an ultra-clean workbench; disinfect them with 10% NaClO solution for 20 minutes, and then wash them with sterile water. Rinse with water for 5 times; inoculate in MS medium, the formula of MS medium is the general formula (see Table 1), incubate at 25°C for 16 hours under sunlight, and cultivate for 7 days to obtain sterile seedlings.

[0040] Step 2. Grafting

[0041] Take a robust Liaoyuan Dolly tomato or a potted tomato with 6-10 leaves as a rootstock, cut off the head below the top bud of the rootstock, and cut vertically downwards. T...

Embodiment 2

[0042] Embodiment 2: Application example 1 of the method for in situ regeneration of plants in genetic transformation

[0043] Step 1, the acquisition of scion

[0044] The tomato variety Liaoyuanduoli (Solanum lycopersicum L, purchased from Liaoning Gardening Seedling Co., Ltd.) was selected as the experimental material. Take the well-developed and disease-free Liaoyuan Dolly tomato seeds, soak them in washing powder water for 1 hour, and rinse them; soak them in 75% alcohol for 30 seconds on an ultra-clean workbench; disinfect them with 10% NaClO solution for 20 minutes, and then wash them with sterile water. Rinse with water for 5 times; inoculate in MS medium, the formula of MS medium is the general formula (see Table 1), incubate at 25°C for 16 hours under sunlight, and cultivate for 7 days to obtain sterile seedlings.

[0045] Take the aseptic seedlings whose cotyledons are fully unfolded but the true leaves have not yet germinated, cut the cotyledons and hypocotyls int...

Embodiment 3

[0063] Embodiment 3: Application example 2 of the method for in situ regeneration of plants in genetic transformation

[0064] The acquisition of the scion of step 1 and the preparation of the agrobacterium of step 2 are all the same as embodiment 2.

[0065] Step 3: Agrobacterium infection

[0066] Use robust potted tomatoes with 5-6 leaves as rootstocks. Cut off the head below the top bud of the rootstock, and then cut vertically downward. Apply the Agrobacterium solution prepared in step 2 on the wound with a sterile cotton swab to infect the plant.

[0067] Step 4: Grafting

[0068] The Liaoyuan Duoli fresh callus obtained on the induction medium was cut into wedges as scions under aseptic conditions, and inserted into the infected potted tomato rootstock for grafting ( figure 2 ), so that the two fit closely, cover the grafting site with plastic film and wrap it tightly. Remove all axillary buds, and the axillary buds that germinate in the future should also be remo...

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Abstract

The invention belongs to the biology and biological technical field, and discloses a plant in-situ regeneration and agrobacterium transformation method. Agrobacterium is coated on a wound of a plant root stock, a callus which is generated by inducing a plant in-vitro tissue on a culture medium is used as a scion, and the scion is grafted onto the root stock through a cleft graft method. Root stock wound cells can realize in-situ organ regeneration under the induction of an exogenous callus, so that a transgenic regeneration bud can be obtained. Compared with the traditional plant genetic transformation method combining the tissue culture, a one-by-one genotype fumbling tissue culture condition can be avoided, a novel technological platform can be provided to the genetic transformation of plants, meanwhile, a novel plant regeneration system is established, and a research system is provided to the study of plant development biology.

Description

technical field [0001] The invention belongs to the field of biology and biotechnology, and relates to a genetic transformation method of in situ organ regeneration of plant cells combined with Agrobacterium infection. Background technique [0002] Plant organ regeneration (organ regeneration) is established on the basis of the theory of cell totipotency. It is to cultivate the organs, tissues and even single cells of plants under artificially controlled sterile conditions to make them grow and differentiate into complete plants. the process of. After hundreds of years of improvement and development, this tissue culture technology has achieved the cultivation of thousands of plants. It has been widely used in the industrial production of medicinal plants and secondary substances. [0003] In the field of plant genetic engineering research, plant organ regeneration in vitro is also an important experimental technology. Plant genetic engineering needs to transform the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A01G1/06
Inventor 曹慧颖夏润玺张立军吕淑霞崔振海张宁张少斌
Owner SHENYANG AGRI UNIV
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