Method for obtaining embryogenic callus of foxtail millet for genetic transformation and method of genetic transformation

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

Active Publication Date: 2018-09-28
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • 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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  • Method for obtaining embryogenic callus of foxtail millet for genetic transformation and method of genetic transformation
  • Method for obtaining embryogenic callus of foxtail millet for genetic transformation and method of genetic transformation
  • Method for obtaining embryogenic callus of foxtail 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 of foxtail millet for genetic transformation and a method of the genetic transformation. The method for obtaining the embryogenic callus of the foxtail millet for the genetic transformation comprises the following steps: (a1) inoculating an explant of the foxtail millet on an inducing medium for carrying out induction culture to obtain primary callus; (a2) carrying out suspension culture and subculture on the primary callus to obtain an embryogenic callus cell suspension system, wherein the embryogenic callus cell suspension system is the embryogenic callus for the genetic transformation. The invention provides the embryogenic callus derived from mature seeds of the foxtail millet; by suspension culture amplification of embryogenic cells, rapid, efficient and stable genetic transformation of the foxtail millet can be realized. The method disclosed by the invention lays a technical foundation for the study of functional genes with high photosynthetic efficiency (C4), drought and stress resistance and the like of the foxtail millet, and provides a technical support for speeding up genetic breeding research of the foxtail millet and modern agricultural development.

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 Applications(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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