阅读说明：本技术 一种农杆菌介导水稻遗传转化方法 (Agrobacterium tumefaciens-mediated rice genetic transformation method ) 是由 顾勇 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种农杆菌介导水稻遗传转化方法,包括如下步骤：制备MS培养基,选取水稻种子,除菌后将种子均匀播种到MS培养基上；进行22-24℃的无光照培养,培养时间8-10天至种子发芽,然后剪取新鲜的子叶备用；活化农杆菌；侵染；转入共培养基进行共培养,同时在光照下培养2-3d；将共培养后的愈伤组织收入培养瓶中,加入羧苄水进行清洗,同时不断摇晃培养瓶,清洗不再浑浊后倒去羧苄水,将愈伤通过无菌滤纸干燥；进行筛选培养；进行分化培养,直至愈伤组织出现绿芽。通过用无光照培养来替代现有技术愈伤组织的诱导培养、继代培养,免去预培养过程,提高了愈伤组织分化能力,进而增加了出苗率。(The invention discloses an agrobacterium-mediated rice genetic transformation method, which comprises the following steps: preparing an MS culture medium, selecting rice seeds, and uniformly sowing the seeds on the MS culture medium after degerming; culturing at 22-24 deg.C for 8-10 days until seed germinates, and shearing fresh cotyledon; activating agrobacterium; infection; transferring into co-culture medium for co-culture, and culturing under illumination for 2-3 d; putting the co-cultured callus into a culture bottle, adding the carboxybenzyl water for cleaning, continuously shaking the culture bottle, pouring the carboxybenzyl water after the cleaning is not turbid, and drying the callus through sterile filter paper; carrying out screening culture; carrying out differentiation culture until the callus shows green buds. By replacing the induction culture and the subculture of the callus in the prior art with the non-illumination culture, the preculture process is omitted, the differentiation capability of the callus is improved, and the emergence rate is increased.)

1. An agrobacterium-mediated rice genetic transformation method is characterized in that:

the method comprises the following steps:

s1, preparing an MS culture medium, adding sucrose, mannitol, agar and a 1% sodium hippurate solution after selecting a basic culture medium, adjusting the pH value of the culture medium to 6, and preparing the MS culture medium for later use after sterilization;

s2, selecting rice seeds, and removing glume shells;

s3, placing the seeds without glumes on a superclean workbench, soaking the seeds for 1min by using alcohol, then disinfecting the seeds by using sodium hypochlorite, then washing the seeds for 10 times by using sterilized water, placing the seeds on sterile filter paper for drying, and finally uniformly sowing the seeds on an MS culture medium in S1;

s4, performing non-illumination culture on the MS culture medium in the S3 at the temperature of 22-24 ℃, culturing for 8-10 days until seeds germinate, and then shearing fresh cotyledons for later use;

s5, activating agrobacterium;

s6, infection, namely inoculating the activated single colony of the agrobacterium in S5 into a natural liquid culture medium, performing shake culture for 24h, and soaking the cotyledon cultured in S4 into the natural liquid culture medium for infection when the logarithmic phase is reached;

s7, after removing the agrobacterium liquid, placing the infected tissue on sterile filter paper to absorb excessive bacterial liquid, then transferring the tissue to a co-culture medium for co-culture, and simultaneously culturing the tissue for 2-3d under illumination;

s8, putting the co-cultured callus into a culture bottle, adding the carboxymethyl water for cleaning, continuously shaking the culture bottle, pouring the carboxymethyl water after the culture bottle is cleaned and is not turbid, and drying the callus through sterile filter paper;

s9, inoculating the callus dried in the S8 on a screening culture medium for screening culture;

and S10, transferring the screened and cultured resistant callus into a differentiation culture medium for differentiation culture until the callus has green buds.

2. The method of claim 1, wherein the genetic transformation of rice is mediated by Agrobacterium, which comprises: in the S1, the basic culture medium comprises 1L of distilled water, 1g/L of ammonium nitrate, 0.5g/L of potassium dihydrogen phosphate, 1.5g/L of disodium hydrogen phosphate, 1g/L of sodium chloride and 0.2g/L of magnesium sulfate heptahydrate, and the pH value of the basic culture medium is adjusted to 7.2.

3. The method of claim 1, wherein the genetic transformation of rice is mediated by Agrobacterium, which comprises: in said S4, the fresh cotyledons to be used are placed at 22-24 ℃ in the absence of light.

4. The method of claim 1, wherein the genetic transformation of rice is mediated by Agrobacterium, which comprises: in S5, the activated agrobacterium contains a target gene.

5. The method of claim 1, wherein the genetic transformation of rice is mediated by Agrobacterium, which comprises: in S10, the differentiation medium was changed once for 9-10d until callus appeared green.

Technical Field

The invention relates to the field of biological genetic transformation, in particular to an agrobacterium-mediated rice genetic transformation method.

Background

With the increasing research on the transformation methods, the agrobacterium-mediated transformation methods are more researched and are most widely applied. The current agrobacterium-mediated transformation method comprises induction culture of callus, subculture of callus, infection, co-culture, removal of agrobacterium, screening culture and differentiation culture of resistant callus. Has the technical problems of long period, weak callus differentiation capability and low emergence rate.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an agrobacterium-mediated rice genetic transformation method so as to at least achieve the aims of reducing the pre-culture process, improving the callus differentiation capacity and further increasing the emergence rate.

The purpose of the invention is realized by the following technical scheme: an agrobacterium-mediated rice genetic transformation method comprises the following steps:

s1, preparing an MS culture medium, adding sucrose, mannitol, agar and a 1% sodium hippurate solution after selecting a basic culture medium, adjusting the pH value of the culture medium to 6, and preparing the MS culture medium for later use after sterilization;

s2, selecting rice seeds, and removing glume shells;

s3, placing the seeds without glumes on a superclean workbench, soaking the seeds for 1min by using alcohol, then disinfecting the seeds by using sodium hypochlorite, then washing the seeds for 10 times by using sterilized water, placing the seeds on sterile filter paper for drying, and finally uniformly sowing the seeds on an MS culture medium in S1;

s4, performing non-illumination culture on the MS culture medium in the S3 at the temperature of 22-24 ℃, culturing for 8-10 days until seeds germinate, and then shearing fresh cotyledons for later use;

s5, activating agrobacterium;

s6, infection, namely inoculating the activated single colony of the agrobacterium in S5 into a natural liquid culture medium, performing shake culture for 24h, and soaking the cotyledon cultured in S4 into the natural liquid culture medium for infection when the logarithmic phase is reached;

s7, after removing the agrobacterium liquid, placing the infected tissue on sterile filter paper to absorb excessive bacterial liquid, then transferring the tissue to a co-culture medium for co-culture, and simultaneously culturing the tissue for 2-3d under illumination;

s8, putting the co-cultured callus into a culture bottle, adding the carboxymethyl water for cleaning, continuously shaking the culture bottle, pouring the carboxymethyl water after the culture bottle is cleaned and is not turbid, and drying the callus through sterile filter paper;

s9, inoculating the callus dried in the S8 on a screening culture medium for screening culture;

and S10, transferring the screened and cultured resistant callus into a differentiation culture medium for differentiation culture until the callus has green buds.

Preferably, in the S1, the basic culture medium comprises 1L of distilled water, 1g/L of ammonium nitrate, 0.5g/L of potassium dihydrogen phosphate, 1.5g/L of disodium hydrogen phosphate, 1g/L of sodium chloride and 0.2g/L of magnesium sulfate heptahydrate, and the pH of the basic culture medium is adjusted to be 7.2.

Preferably, in said S4, the fresh cotyledons to be used are placed at 22-24 deg.C under no light.

Preferably, in the S5, the activated agrobacterium contains a target gene.

Preferably, in S10, the differentiation medium is changed once for 9-10d until callus shows green bud

The invention has the beneficial effects that:

the invention replaces the induction culture and the subculture of the callus tissue in the prior art by the non-illumination culture, thereby avoiding the preculture process, improving the differentiation capability of the callus tissue and further increasing the emergence rate.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to examples, but the scope of the present invention is not limited to the following.