Method for establishing ziziphus jujuba genetic transformation system for receptor by leaves

A genetic transformation system and technology of genetic transformation, applied in the direction of using vectors to introduce foreign genetic material, biochemical equipment and methods, botanical equipment and methods, etc., to achieve the effects of high repeatability, avoidance of variation, and high transformation frequency

Inactive Publication Date: 2016-08-10
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

Compared with the genetic transformation of other fruit trees, the transgenic work of jujube is still in the exploratory stage, and there is no report on the use of jujube leaves as receptors to transfer genes related to anti-aging

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  • Method for establishing ziziphus jujuba genetic transformation system for receptor by leaves
  • Method for establishing ziziphus jujuba genetic transformation system for receptor by leaves
  • Method for establishing ziziphus jujuba genetic transformation system for receptor by leaves

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preparation example Construction

[0075] In the present invention, the preparation method of described Agrobacterium bacterial liquid comprises the following steps:

[0076] A single colony of Agrobacterium strains was inoculated in YEB liquid medium and cultured in dark to OD 600 0.5~0.7, the YEB liquid medium comprises 40~55mg / L rifampicin, 40~55mg / L cephalosporin and 40~55mg / L kanamycin;

[0077] Inoculate the bacterium solution obtained by the dark culture into the YEB liquid medium without antibiotics with an inoculum size of 0.02-0.05%, and continue to cultivate until OD 600 0.4~1.0;

[0078] the OD 600 The bacteria solution with a concentration of 0.4-1.0 is mixed with acetosyringone with a molar concentration of 150-300 μmol / L, and the culture is continued for 0.8-1.5 hours to obtain the Agrobacterium bacteria solution.

[0079] The Agrobacterium strain of the present invention is preferably LBA4404 Agrobacterium tumefaciens, which is purchased from Beijing Huayueyang Biology.

[0080] In the prese...

Embodiment 1

[0142] Select well-growing jujube tissue culture seedlings, cut off the 3rd to 5th leaves from top to bottom in morphology as transformation materials, cut 2 wounds vertically along the midrib of the leaves, cut off the main veins but not the leaves, and touch them with the proximal end culture medium (such as figure 1 , a) Inoculate in WPM adventitious bud initiation medium (TDZ 1.0mg / L+IBA 0.3mg / L+30g / L sucrose+6g / L agar). Agrobacterium infection was carried out after dark culture induction on WPM adventitious bud initiation medium for 3 days.

[0143] The pH value of WPM adventitious bud initiation medium is 5.8. WPM basic medium formula is:

[0144] (1) Major elements: NH 4 NO 3 (400mg / L), Ca(NO 3 ) 2 4H 2 O(556mg / L)K 2 SO 4 (990mg / L), CaCl 2 2H 2 O (96mg / L), KH 2 PO 4 (170mg / L), MgSO 4 ·7H 2 O (370mg / L);

[0145] (2) Trace elements: Na 2 MoO 4 2H 2 O(0.25mg / L), MnSO 4 ·H 2 O (22.4mg / L), ZnSO 4 ·7H 2 O (8.6mg / L), CuSO 4 ·5H 2 O(0.25mg / L), FeSO 4 ·...

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Abstract

The invention belongs to the technical field of plant cell engineering and molecular biology and relates to a method for establishing a ziziphus jujuba genetic transformation system for a receptor by leaves. The method includes: inoculating leaves of ziziphus jujuba into a WPM adventitious bud initial medium to perform dark culture induction for 2-4d to obtain explants; using agrobacterium liquor containing a plant expression vector to infect the explants to obtain infected explants; putting the infected explants on the WPM adventitious bud initial medium to perform dark culture for 2-4d; transferring the leaves subjected to dark culture into a WPM solid selection medium to perform dark culture for 14-16d, transferring to a position under light, performing elongation culture for leaf regeneration in a WPM medium containing 0.1mg/L of heteroauxin and 0.5mg/L of gibberellins to obtain adventitious buds, transferring the adventitious buds to an MS medium to perform propagation to obtain genetically transformed ziziphus jujuba plants. By the method, a proper regeneration system can be provided for ziziphus jujuba, high transformation frequency and high repeatability are realized, and genetic improvement of ziziphus jujuba is benefited.

Description

technical field [0001] The invention relates to the technical fields of plant cell engineering and molecular biology, in particular to a method for establishing a wild jujube genetic transformation system using leaves as receptors. Background technique [0002] Ziziphus jujuba Mill.var.Spinosa is a plant of the genus Jujube in the family Rhamnaceae. It is a native species of Chinese jujube and is widely distributed in my country. Zizyphus jujube has a long flowering period and is a good nectar source plant. The seeds can be used as medicine; jujube fruit has high nutritional value and contains potassium, sodium, iron, zinc, phosphorus, selenium and other trace elements; more importantly, jujube contains a lot of vitamin C , its content is higher than that of ordinary red dates, citrus, apples, grapes and other fruits, so it is favored by consumers and has important value in the production and sales of fruit trees. However, the jujube has a concentrated maturity period and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205
Inventor 李颖岳王欢庞晓明黄飞逸崔艳红侯璐
Owner BEIJING FORESTRY UNIVERSITY
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