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Method for acquiring transgene cotton

A transgenic and cotton technology, which is applied in the field of obtaining transgenic cotton by using the cotton stem tip as the receptor, and obtaining transgenic cotton plants through the mediation of Agrobacterium. , strong genotype restriction and high genetic variation rate

Inactive Publication Date: 2010-09-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But usually the conversion rate is low, and because the adventitious buds originate from multi-cells, the restriction of factors such as more chimeras affects the acquisition of regenerated shoots

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, apply the method of the present invention to obtain transgenic cotton

[0030] 1. Obtaining recombinant Agrobacterium

[0031] Recombinant plasmid pTiBO542: China Agricultural University promises to provide it to the public; References: For the detailed construction process of the recombinant plasmid, see Chen H et al. (2005) paper "Transgenic indica rice plants harboring a synthetic cry2A * Gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran ricepests." Page 1131 to 1132; the recombinant plasmid carries the insect-resistant gene Cry2A shown in Sequence 1 of the Sequence Listing * .

[0032] Agrobacterium EHA105: China Agricultural University guarantees public availability; reference: Torregrosa L., et.al. Influence of Agrobacterium Strain, Culture Medium, and Cultivar on the TransformationEfficiency of Vitis vinifera L.Am.J.Enol. , 3: 183-190.

[0033] Agrobacterium EHA105 was transformed with recombinant plasmid pTiBO542 t...

Embodiment 2

[0057] Embodiment 2, apply the method of the present invention to obtain transgenic cotton

[0058] 1. Obtaining recombinant Agrobacterium

[0059] 1. Synthesize the seed-specific expression promoter AGP shown in sequence 2 of the sequence listing.

[0060] 2. Acquisition of recombinant plasmid (pBIαGUS)

[0061] ① Digest pBI121 (Beijing Baierdi Biotechnology Co., Ltd., product number: MP-091) with restriction endonucleases HindIII and Xba I, and recover the large fragment;

[0062] ② Use restriction endonucleases HindIII and Xba I to double digest the seed-specific expression promoter AGP in step 1, and recover the digested product;

[0063] ③ Ligate the large fragment recovered in step ① with the digested product recovered in step ② to obtain the recombinant plasmid pBIαGUS (the constitutive expression promoter CaMV35S in pBI121 is replaced by the seed-specific expression promoter AGP shown in sequence 2). In the recombinant plasmid pBIαGUS, the GUS gene is specifically e...

Embodiment 3

[0088] Embodiment 3, apply the method of the present invention to obtain transgenic cotton

[0089] 1. Obtaining recombinant Agrobacterium

[0090] 1. Synthesize the GFP gene shown in Sequence 3.

[0091] 2. Acquisition of recombinant plasmid (pBIαGFP)

[0092]① Use restriction endonucleases Xba I and Sac I to double digest the pBIαGUS obtained in step 2 of Example 2, and recover the large fragment;

[0093] ② Digest the GFP gene in step 1 with restriction endonucleases Xba I and Sac I, and recover the digested product;

[0094] ③ Ligate the large fragment recovered in step ① with the digested product recovered in step ② to obtain the recombinant plasmid pBIαGFP (the GFP gene shown in sequence 3 is used to replace the GUS gene in pBIαGUS).

[0095] 3. Obtaining recombinant Agrobacterium

[0096] Agrobacterium LBA4404: China Agricultural University guarantees public availability; reference: Torregrosa L., et.al. Influence of Agrobacterium Strain, Culture Medium, and Cultiva...

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Abstract

The invention discloses a method for acquiring transgene cottons. The method comprises the following steps of: (1) cutting cotyledons, leaf stalks and a part of hypocotyl of cotton cotyledon seedlings and keeping r 1-3 cm of the hypocoty to obtain tissues to be infected at 25-30 DEG C and with the relative humidity 40-60%, and culturing for 2-3 days under dark environment; (2) scratching double surfaces from the top of a stem tip to the base part of the hypocotyl with the cut of 0.8-1.2 mm in depth; (3) dip-dyeing for 15-45 min in solution A which contains a recombination agrobacterium carrying exogenous genes; (4) co-culturing for 2-4 days and then carrying out resistance screening to obtain the resistant cotton seedling; and (5) grafting the resistant cotton seedling on a cotton rootstock to obtain the transgene cotton. The invention improves the conversion efficiency through adjusting the growth states of the stem tip, determining the appropriate depth of the agrobacterium-converted stem tip infection, adding acetosyringone and adjusting the proper pH value. Transgene plants can be quickly obtained by using the method, and the higher transgene efficiency is higher.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, and relates to a method for obtaining transgenic cotton, in particular to a new method for obtaining transgenic cotton plants through the mediation of Agrobacterium by using the cotton stem tip as a receptor. Background technique [0002] Cotton is an important economic crop and occupies an important position in the national economy. Cotton transgenic technology has made significant progress in genetic improvement, and has become an example of the successful application of biotechnology in crop genetic improvement. Currently, Agrobacterium-mediated transformation is the most widely used method for genetic transformation of transgenic plants. Agrobacterium-mediated gene transformation technology is not limited by seasons, most of the target genes are single copy or low copy, most of the insertion borders are left and right borders of T-DNA, low rearrangement rate, easy to exclude redun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 华金平刘正杰王彦霞熊敏王玉美
Owner CHINA AGRI UNIV
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