Method for separation of Chinese cabbage microspores at different development stages

Abstract

The invention discloses a method for separation of Chinese cabbage microspores at different development stages. The method includes: preparing a percoll separation liquid, placing a Chinese cabbage microspore crude extracted solution to the top, carrying out 150rpm low speed centrifugation to separate microspore at different development stages. Microspores at different development stages have close relation with the formation of vacuoles and other organelles, i.e. usually showing the difference of relative density. Therefore, separation of microspores at different development stages can be achieved by percoll density gradient centrifugation. The method provided by the invention can efficiently and stably separate microspores at different development stages, overcomes the disadvantages of difficult grasp of petal length / anther length ratio, flower bud length and the like in experiments utilizing traditional morphological indices, solves the problem of in-vitro preparation of microspore cells at similar development stages, and can provide materials for the mechanism study of Chinese cabbage microspore culture and provide receptors for gene gun mediated Chinese cabbage genetic transformation.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for isolating microspores of Chinese cabbage in different development stages. Background technique [0002] As the free microspore culture technology of cruciferous crops is becoming more and more mature, the research on the mechanism of microspore embryogenesis is becoming a hot spot, but the method of isolating microspores at specific developmental stages has not been successfully solved so far; at present, with the development of functional genomics , the construction of a genetic transformation system is becoming more and more important, and it is difficult to construct a Chinese cabbage regeneration system. At present, researchers are beginning to use microspore culture and cell-penetrating peptides combined with a gene gun for transformation. Because microspores are haploid, they are involved in embryogenesis and regeneration. The seedlings will natur...

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Problems solved by technology

[0002] As the free microspore culture technology of cruciferous crops is becoming more and more mature, the research on the mechanism of microspore embryogenesis is becoming a hot spot, but the method of isolating microspores at specific developmental stages has not been successfully solved so far; at present, with the development of functional genomics , the construction of a genetic transformation system is becoming more and more important, and it is difficult to construct a Chinese cabbage regeneration system. At present, researchers are beginning to use microspore culture and cell-penetrating peptides combined with a gene gun for transformation. Because microspores are haploid, they are involved in embryogenesis and regeneration. The seedlings will naturally double during the process, so the pure transgenic plants obtained after transformation have become important transformed recipient cells

However, in the traditional microspore isolation process, the selection of developmental stages depends on morphological indicators such as petal-to-drug ratio, flower bud length, and sepal color. In specific experiments, different researchers have different empirical standards based on morphological indicators. Difficult to be consistent, so repeatability is difficult to guarantee

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