Obtaining embryogenic callus of millet for genetic transformation and method of genetic transformation

An embryogenic callus and callus technology, applied in the field of genetic transformation, can solve problems such as difficulty in genetic transformation of millet

Active Publication Date: 2021-02-19
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to use the mature embryo (seed) to obtain the embryogenic callus of millet, and use this embryogenic callus to carry out efficient and stable genetic transformation mediated by Agrobacterium, thereby solving the key bottleneck problem that the genetic transformation of millet is difficult , to establish a large-scale genetic transformation system for millet, so that millet can truly develop into a model plant for functional gene research

Method used

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  • Obtaining embryogenic callus of millet for genetic transformation and method of genetic transformation
  • Obtaining embryogenic callus of millet for genetic transformation and method of genetic transformation
  • Obtaining embryogenic callus of millet for genetic transformation and method of genetic transformation

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Embodiment 1

[0090] Embodiment 1, the genetic transformation of millet

[0091] 1. Transformation of Agrobacterium with recombinant expression vector

[0092] The vector pCAMBIA1305-GFP was transformed into the Agrobacterium EHA105 strain by heat shock method. The transformation conditions were: 100uL of EHA105 Agrobacterium competent cells, 2μL of the recombinant plasmid, quick freezing in liquid nitrogen for 10 minutes, and warm water bath at 37°C for 10 minutes ; Place on ice for 10 minutes; Inoculate transformed Agrobacterium EHA105 in LB test tubes and culture at 28°C at 200 rpm for 2 hours, then apply to LB solids containing 50 mg / L rifampicin and 50 mg / L kanamycin On the plate, until the correct positive clone grows, pick the positive clone to culture and extract the plasmid, and check whether the recombinant plasmid has been transformed into Agrobacterium by enzyme digestion; in order to reconfirm that the Agrobacterium plasmid has been transformed into Escherichia coli, carry out ...

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Abstract

The invention discloses a method for obtaining embryogenic callus tissue of millet for genetic transformation and genetic transformation. The present invention provides a method for obtaining embryogenic callus of millet for genetic transformation, comprising: a1) inoculating explants of millet on an induction medium for induction culture to obtain primary callus; a2) inoculating said The primary callus is subjected to suspension culture and subculture to obtain an embryogenic callus cell suspension line; the embryogenic callus cell suspension line is the embryogenic callus used for genetic transformation. The present invention provides for the first time an embryogenic callus derived from mature millet seeds, through the suspension culture expansion of the embryogenic cells; rapid, efficient and stable genetic transformation of millet can be realized. The present invention is to develop millet high light efficiency (C 4 ) and drought and stress resistance and other functional genes have laid a technical foundation and provided technical support for accelerating the research on genetic breeding of millet and the development of modern agriculture.

Description

technical field [0001] The invention relates to the fields of plant tissue culture and plant genetic engineering, in particular to a method for obtaining embryogenic callus tissue of millet for genetic transformation and the genetic transformation mediated by Agrobacterium. Background technique [0002] Millet (Setaria italica (L.) P.Beauv.), originated in my country, and its domestication and cultivation history can be traced back to 11,500 years ago. It is one of the oldest crops in the world. Millet belongs to Gramineae, Panicoideae, Paniceae, Setatuubae, Setaria, and corn (Zea mays.), sorghum (Sorghum bicolor), pearl millet (Pennisetum glaucum), millet (Panicummiliaceum), sugarcane (Saccharum officinarum) and other important crops are closely related. Millet has the characteristics of drought tolerance, water saving, barrenness tolerance, high nutritional efficiency and high light efficiency (C 4 Plants) and other characteristics, these characteristics are the developm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04C12N15/82A01H5/00A01H6/46A01H4/00
CPCA01H4/001A01H4/005A01H4/008C12N5/0025C12N5/04C12N15/8201C12N15/8205
Inventor 隋毅张皓珊吴传银刁现民
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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