Method for converting cotton germ by agrobacterium with ultrasonic wave aid

Abstract

The invention relates to a method for using ultrasonic wave with agricillin to transfer the cotton germ to cultivate gene-transferred cotton. The inventive method comprises that planting the disinfected and skin-removed cotton on the germ-free cultivate medium; removing two fresh blades to be arranged on the germ-free filter paper; adding 10ml of 0.5 OD600nm agricillin solution into the plastic eccentric tube or glass triangle bottle, then putting the germ into the agricillin solution, transferring the treated germ into the share cultivate medium to be cultivated; washes with germ-free water which contains cefradine, absorbs the surface water, to be transferred to the return cultivate medium, under sunlight to be returned and cultivated to obtain the germ; then transfers it to the selecting cultivate medium to obtain the antibody germ; cuts off the antibody germ to be transferred to the rooting cultivate medium to induce the root or transplanted to the cotton stock, to obtain the regenerated gene transferred plant. The invention can improve the instant expression rate of GFP (green fluorescence protein) gene at blast to 100 times, realizes the gene transferred plant of island cotton, and obtain anti-sect cotton. The test has proved that the external gene is stably transferred into the cotton gene to be expressed and generated.

Description

technical field [0001] The invention belongs to the field of cotton transgene technology in the field of plant transgene technology. In particular, it relates to a new transgenic method for obtaining transgenic cotton plants by using ultrasound-assisted Agrobacterium to transform cotton germs. The method significantly shortens the cotton transgenic cycle and expands the scope of cotton transformation. Relevant to the field of new plant technologies. Background technique [0002] At present, the genetic transformation of plants mainly has the following three methods: Agrobacterium-mediated gene transfer; direct gene transfer using protoplasts, cells or tissues as receptors (such as electric shock, microinjection, PEG-mediated transformation of protoplasts, etc. , gene gun and ultrasonic method can directly introduce exogenous DNA into plant cells with walls, thereby avoiding the long process of protoplast regeneration of plants and making transgenes faster and easier); gene ...

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Problems solved by technology

[0004] As far as the transgenic technology of cotton is concerned, the Agrobacterium-mediated transformation method is widely used in the world at present, but this method requires cotton tissue culture technology as the basic platform. As we all know, cotton is recognized as a relatively difficult in vitro method. The cultured species are mainly manifested in the harsh conditions of tissue culture, long cycle, serious somaclonal variation, and low regeneration efficiency. The biggest shortcoming of this transformation method is that it depends heavily on genotype

Although the transgenic technology of cotton has been developed for two decades, only a few upland cotton varieties can obtain transgenic regenerated plants through somatic cell culture, such as Kezi cotton series, Jihe 321, Jinmian 7, etc. ( Zhang Xianlong et al., 1993) and these varieties belong to the old varieties that have been eliminated in production, and their agronomic characteristics cannot meet the needs of current production, so the value of direct utilization is not great. The above utilization greatly reduces the utilization value of cotton transgenic breeding. It is precisely because of the existence of the above limiting factors that the wide application of cotton genetic transformation is affected.

At the same time, sea-island cotton (long-staple cotton), another cotton cultivar, has not obtained transgenic plants so far. The main reason is that sea-island cotton is difficult to obtain regenerated plants through tissue culture, so it is impossible to combine conventional Agrobacterium infection with tissue culture techniques. Obtain transgenic plants (Zhang Xianlong et al, 1991; Chi Jina et al, 2005, Sakhanokho et al, 2001; Wu et al, 2004; Jin et al, 2005)

The cotton transgenic technology created by domestic researchers—the pollen tube passage method is currently widely used in provincial and municipal cotton breeding units (Zhou et al, 1983; Liu et al, 2002; Zhang et al, 2000), but this method has been transformed Serious shortcomings such as extremely low efficiency (less than 1 / 1,000), many copies of foreign genes, and poor stability of transformation materials have been gradually abandoned by many research institutions.

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