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Culture medium and kit for preparing embryonic callus of cotton and applications of culture medium and kit

A technique for embryogenic callus and callus is applied in the fields of preparing transgenic cotton plants, preparing medium for cotton embryogenic callus, and preparing cotton regeneration plants, and can solve problems to be improved, etc.

Inactive Publication Date: 2013-11-13
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of the regeneration system of cotton, especially upland cotton, and the method of obtaining transgenic plants still need to be improved

Method used

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  • Culture medium and kit for preparing embryonic callus of cotton and applications of culture medium and kit
  • Culture medium and kit for preparing embryonic callus of cotton and applications of culture medium and kit
  • Culture medium and kit for preparing embryonic callus of cotton and applications of culture medium and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Cotton regeneration system is established

[0050] Adopt Xinluzao No. 39 and Xinluzao No. 42 two upland cotton varieties, carry out following experiment, determine the concrete condition of each step respectively, so that establish the cotton regeneration system of the present invention:

[0051] 1. Obtaining sterile cotton seedlings

[0052] Two upland cotton varieties, Xinluzao 39 and Xinluzao 42, were used to obtain sterile cotton seedlings according to the following steps:

[0053] The seeds were first treated with concentrated sulfuric acid (H 2 SO 4 ) After delinting, rinse with water and dry. Then take an appropriate amount of seeds and sterilize them with 75% absolute ethanol solution for 30 seconds, then sterilize them with 30% hydrogen peroxide for 4-5 hours, wash them with sterile water for 5-6 times, and soak the seeds in sterile water for 16-24 hours until they turn white. Then peel off the seed coat under aseptic conditions, and inoculate ...

Embodiment 2

[0093] Example 2 Preparation of Transgenic Cotton Plants

[0094]According to the method of embryogenic callus induction and propagation in Example 1, the embryogenic callus of the upland cotton variety Zhongmiansuo No. 44 was prepared, and then the conventional plasmid pBI121 was used to prepare transgenic cotton plants through the Agrobacterium-mediated method. Among them, the plasmid host Agrobacterium strain is EHA105, the target gene carried by the plasmid is the CarNAC5 gene, and its T-DNA region contains the GUS gene and the kanamycin resistance gene expressed in plants, and the expression of the genes is initiated by rd29A subdrive. Among them, the gene transformation method is as follows:

[0095] 1. Experimental materials:

[0096] Medium: The medium used in the experiment is shown in Table 7 below:

[0097] Table 7

[0098]

[0099]

[0100] 2. Experimental method:

[0101] Preparation of embryogenic callus: The upland cotton variety Zhongmiansuo No. 44 w...

Embodiment 3

[0104] Example 3 PCR molecular detection of transgenic plants

[0105] The transgenic plants obtained in Example 2 were subjected to PCR molecular detection according to the following steps:

[0106] 1. Solution configuration: 1) DNA extraction solution: 50mmol / L tris-HCl (pH=8.0), 100mmol / L EDTA (pH=8.0), 1.5mmol / L NaCl, 1%SDS, 1%PVP40, 30μL β-Mer ;2) DNA lysis buffer: 50mmol / L Tris-HCl (pH=8.0), 100mmol / L EDTA (pH=8.0), 1.5mmol / L NaCl, 3%CTAB, 3%PVP40; 3) Electrophoresis buffer: 10×TBE: 48.4g / L Tris base, 11.42mL glacial acetic acid, 20mL 0.5mol / L EDTA (working buffer is 1×TBE).

[0107] 2. Genomic DNA extraction of transgenic plants: 1) Weigh 0.2g of seedling leaves, grind them thoroughly, put them into a 2.0mL centrifuge tube, add 800μL of extract pre-cooled to 4℃, 30μL of β-hydroxyethanol, shake vigorously, and Centrifuge at 4°C and 1200rpm for 15min, pour off the supernatant; 2) Add 600mL of lysis buffer solution preheated to 65°C, and stir quickly evenly, after mixing...

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Abstract

The invention discloses a culture medium and a kit for preparing embryonic callus of cotton and applications of the culture medium and the kit. The culture medium comprises a callus induction medium, an embryonic callus induction medium and an embryonic callus proliferation medium, wherein the callus induction medium is an MSB medium into which 0.1mg / L of KT, 0.05mg / L of 2,4-D and 1.5g / L of gelrite are added; the embryonic callus induction medium is an MSB medium into which 1.9g / L of KNO3 and 2.0g / L of gelrite are added; the embryonic callus proliferation medium is an MSB medium into which 1.9g / L of KNO3, 0.1g / L of asparaginate, 0.1g / L of glutamine and 1.8g / L of gelrite are added. The culture medium disclosed by the invention is used for preparing embryonic callus of cotton, especially upland cotton and has the advantages that the initiating rate and the embryonic callus occurrence rate can be remarkably increased, the occurrence of abnormal embryos is reduced and the proliferation amount of the embryonic callus is very large.

Description

technical field [0001] The invention relates to the establishment of a cotton regeneration system and the field of cotton transgenic technology. Specifically, the invention relates to a culture medium, a kit and an application thereof for preparing cotton embryogenic callus. More specifically, the present invention relates to a medium for preparing cotton embryogenic callus, a kit and uses thereof, a method for preparing cotton embryogenic callus, a method for preparing cotton regenerated plants and a method for preparing transgenic cotton plants. Background technique [0002] Cotton is an important economic crop in the world, and its genetic improvement has always attracted the attention of the world. However, the establishment of cotton regeneration system is the key to restrict the success of cotton transgenic technology. At present, cotton tissue culture, as well as somatic embryogenesis and plant regeneration are still relatively difficult, mainly due to its strong dep...

Claims

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Application Information

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IPC IPC(8): C12N5/04A01H4/00A01H5/00
Inventor 陈全家曲延英阿依夏木姑丽·司马依力孙国清
Owner XINJIANG AGRI UNIV
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