CN101773039B

CN101773039B - Transgenic cotton regenerated plant transplantation method

Info

Publication number: CN101773039B
Application number: CN 201010103941
Authority: CN
Inventors: 李燕娥; 上官小霞; 吴霞; 张林水; 范小平; 杨淑巧; 翟晓菊
Original assignee: Cotton Research Institute of Shanxi Academy of Agricultural Sciences
Current assignee: Cotton Research Institute of Shanxi Academy of Agricultural Sciences
Priority date: 2010-01-28
Filing date: 2010-01-28
Publication date: 2011-05-25
Anticipated expiration: 2030-01-28
Also published as: CN101773039A

Abstract

The invention discloses a transgenic cotton regenerated plant transplantation method, which solves the problems that the survival rate of the transgenic cotton regenerated plants transplanted by the traditional method is low and the steps are trivial. The method comprises the following steps that: 1) the cover of a bottle containing transgenic cotton regenerated plant seedlings with roots is opened for indoor seedling hardening in the bottle for 2-3 days; 2) culture medium on the roots of the transgenic cotton regenerated plants is washed, the transgenic cotton regenerated plants are planted in a container containing sterilized roseite and are watered thoroughly at one step, and the transgenic cotton regenerated plants are cultured in an incubator with humidity of 88-90 percent and temperature of 20-22 DEG C for 10-15 days; and 3) the transgenic cotton regenerated plants are transplanted into greenhouse with normal conditions and grow to be mature to open bolls. The invention adopts the low-temperature and high-humidity environments for culture, so the survival rate reaches 90-95 percent.

Description

A kind of transgenic cotton regenerated plant transplantation method

Technical field

The present invention relates to the transgene cotton technical field, be specially a kind of transgenic cotton regenerated plant transplantation method.

Background technology

Cotton transforms the transgenosis regeneration strain that foreign gene obtains through agrobacterium-mediated transformation, through Molecular Detection, the proof foreign gene has changed in the acceptor cotton, and the regenerated plant transplantation method of conversion adopts grafting or conventional method for transplanting normally to survive, and is grown to needed transgene cotton then.

The conventional method for transplanting of transgenic cotton regenerated plant is: the seedling of earlier the triangular flask transgenic cotton regenerated plant being taken root is opened bottle cap and was placed between cultivation hardening 1-2 days; Again with transgene cotton flush away root medium, plant in the container that the sterilization vermiculite is housed, disposable water permeable; Put into humidity 70%, 27 ℃～29 ℃ incubators of temperature then, the strain of earlier whole strain lid was cultivated 8～10 days, removed the container that covers cotton plant then, cultivated about 5～7 days at incubator again; Move at last in the greenhouse of normal condition and survive the cotton boll blowing maturation.

The purpose of cotton transgenic is that the external source genes of interest is changed in the conventional cotton, in the hope of obtaining transgene cotton, has been the primary study project of country " 863 " since " 95 " always.The transgenic cotton plant that obtains costs an arm and a leg, but, because existing transgenic cotton regenerated plant transplantation method, need be at high temperature, cultivate in the incubator of low humidity, cause butt rot easily, cause the low problem of motility rate, general survival rate has only 20%～60%, and in incubation, need earlier whole strain to add a cover, and then move lid and cultivate, operating procedure is more loaded down with trivial details, so do not reach ideal results all the time.

Seek a kind of simple to operately, the transgenic cotton regenerated plant transplantation method that survival rate is high becomes the focus of present research.

Summary of the invention

The present invention adopts high temperature, low humidity in order to solve existing transgenic cotton regenerated plant transplantation method, and causes survival rate low, the problem of complex steps, and a kind of transgenic cotton regenerated plant transplantation method is provided.

Transgenic cotton regenerated plant transplantation method of the present invention may further comprise the steps:

1) transgene cotton triangular flask regeneration strain is taken root seedling bottle cap is opened and was carried out indoor in-bottle seeding 2 days～3 days;

2) with transgenic cotton regenerated plant root medium flush away, plant and be equipped with in the container of sterilization vermiculite, disposablely water permeablely, be 88%～90% in humidity, temperature is to cultivate 10～15 days in 20 ℃～22 ℃ the incubator;

3) survive the cotton boll blowing maturation in the greenhouse of immigration normal condition.

Conventional trasplanting method is to carry out under the environment of high temperature low humidity, the strain of earlier whole strain lid was cultivated 8～10 days, remove the container that covers cotton plant then, cultivate again about 5～7 days, the regeneration strain root rots easily in the environment of high temperature low humidity like this, cause whole strain death, and owing to obtain costing an arm and a leg of transgenosis regeneration strain, thereby make cost improve.The present invention is through a large amount of experiments, adopts finally that humidity is 88%～90%, temperature is 20 ℃～22 ℃ low temperature, high humidity culture environment, makes survival rate reach 90%～95%, and save the incubation step with the container cover strain in incubation.

With per 10 strains of transgenic cotton regenerated plant is one group, totally 12 groups, under different temperature and humidity environment, transplant cultivation, and observe survival rate.Concrete experimental data is as described in the following table table 1:

Table 1

As known from Table 1, under the environment of low temperature and high relative humidity, the survival rate of transgenic cotton regenerated plant reaches 90%.On March 15th, 2009, in the transplantation experiments process of transgenic cotton regenerated plant, select for use the environment of low temperature and high relative humidity to carry out, be one group with per 40 strains of regeneration strain, totally 9 groups, observe survival rate, concrete experimental data is as described in the following table table 2:

Table 2

Transgenic cotton regenerated plant transplantation method of the present invention, adopt the culture environment of low temperature and high relative humidity, improved survival rate greatly, and the whole strain of having simplified in the existing transgenic cotton regenerated plant transplantation method is added a cover this step with glass or plastic foil, is a kind of comparatively desirable transgenic cotton regenerated plant transplantation method.

Implement embodiment

Embodiment 1

The transgenic cotton regenerated plant seedling bottle cap of taking root is opened and carried out indoor in-bottle seeding 2 days; Again with the regeneration strain root medium flush away after the hardening, plant in the container that the sterilization vermiculite is housed, disposablely water permeablely, be 90% in humidity, temperature is to cultivate 15 days in 22 ℃ the incubator; Move into to cultivate in the greenhouse and survive the cotton boll blowing maturation.

Embodiment 2

The transgenic cotton regenerated plant seedling bottle cap of taking root is opened and carried out indoor in-bottle seeding 3 days; Again with the regeneration strain root medium flush away after the hardening, plant in the container that the sterilization vermiculite is housed, disposablely water permeablely, be 88% in humidity, temperature is to cultivate 10 days in 20 ℃ the incubator; Move into to cultivate in the greenhouse and survive the cotton boll blowing maturation.

Embodiment 3

The transgenic cotton regenerated plant seedling bottle cap of taking root is opened and carried out indoor in-bottle seeding 3 days; Again with the regeneration strain root medium flush away after the hardening, plant in the container that the sterilization vermiculite is housed, disposablely water permeablely, be 89% in humidity, temperature is to cultivate 13 days in 21 ℃ the incubator; Move into to cultivate in the greenhouse and survive the cotton boll blowing maturation.

Claims

1. transgenic cotton regenerated plant transplantation method is characterized in that may further comprise the steps:

1) transgenic cotton regenerated plant is taken root seedling bottle cap is opened and was carried out indoor in-bottle seeding 2 days～3 days;

2) with transgenic cotton regenerated plant root medium flush away, plant and be equipped with in the container of sterilization vermiculite, disposablely water permeablely, be 88%～90% in humidity, temperature is to cultivate 10 days～15 days in 20 ℃～22 ℃ the incubator;