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Agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance

An Agrobacterium-mediated, vacuum infiltration technology, applied in the field of plant biology, can solve the problems of reduced transgenic plant quantity, callus growth damage, low genetic efficiency of sugarcane engineering strains, etc., and achieve the effect of improving the genetic transformation rate

Inactive Publication Date: 2013-07-17
GUANGZHOU SUGARCANE IND RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some shortcomings in the application of Agrobacterium-mediated genetic transformation of sugarcane. The transformation efficiency is generally below 3%, and the transformation cost is high and the time is long.
One of the main reasons for the low efficiency of Agrobacterium-mediated sugarcane genetics is that the engineered strains and callus cells cannot be fully contacted during the infection process of Agrobacterium strains and sugarcane callus cells. The Agrobacterium carried on the tissue is removed, and the callus needs to be washed and dried. During the treatment, on the one hand, a part of the successfully transformed callus cells are lost, and on the other hand, the growth of the callus cells is also damaged, resulting in the acquisition of transgenic cells in the later stage. Reduced plant volume

Method used

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  • Agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance

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Experimental program
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Effect test

Embodiment 1

[0047] Induction and proliferation of sugarcane heart leaf embryogenic callus in step (1):

[0048] (1.0) The sugarcane variety Yuetang 00-236 was used as the receptor material for transformation. On sunny days, the tip of healthy sugarcane was stripped off the outer leaf sheath, sterilized by 0.1% mercuric chloride solution for 15-20 minutes, and rinsed with sterile water. 3 to 4 times, the young heart leaves of 3 to 5 cm from the top growth point of the sugarcane stem tip were taken as explants for virus-free tissue culture. Under sterile conditions, the explants were cut into disks with a thickness of about 1-2 mm.

[0049] (1.1) Inoculate the explants from step (1.0) on callus induction medium (MS + 2,4-D (2~3) mg / L + 30 g / L sucrose + 8 g / L agar ), and cultured in the dark at 26-28°C for 15 days to obtain embryogenic callus, select light yellow, well-dispersed embryogenic callus for subculture once,

[0050] (1.2) Select light yellow, well-dispersed subcultured embryogen...

Embodiment 2

[0073] Each step of this embodiment is identical with embodiment 1, and the difference between this embodiment and embodiment 1 is:

[0074] Vacuum infiltration in step (3) assisted Agrobacterium infecting sugarcane embryogenic callus:

[0075] (3.1) Take the embryogenic callus that was activated and cultured in step (1.2) for 3 days, place it on the sterile dry filter paper in the ultra-clean workbench, and blow it for about 30 minutes until the surface is dry. Transfer the callus into the Erlenmeyer flask containing the prepared transformation engineering bacteria solution in step (2), co-cultivate for 5 to 15 minutes at 80 rpm, 22~28°C culture conditions; In the container, open the sealing film of the Erlenmeyer flask, give 0.04 Mpa vacuum pressure to assist the Agrobacterium infection for 2 minutes, repeat 3 times;

[0076] (3.2) Transfer the Agrobacterium-infected callus in step (3.1) onto the co-cultivation solid medium (MS + AS 150 μmol / L) covered with filter paper, an...

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Abstract

The invention relates to the technical field of plant genetic engineering, and discloses an agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance. The agrobacterium-mediated sugarcane genetic transformation method includes: (1) induction and multiplication of embryonic callus of sugarcane core leaves, (2) activation and cultivation on agrobacterium tumefaciens with target genes, (3) agrobacterium infestation with vacuum infiltration assistance on embryonic callus of sugarcane core leaves, (4) resistant material screening and germination, and (5) resistant material verification. With the agrobacterium-mediated sugarcane genetic transformation method, the agrobacterium can pass through the gaps among callus cells and enter deep cells on vacuum, and tiny wounds can be generated on the surfaces of the cells, so that phenolic substances are secreted by plants to activate shear and transfer of T-DNA (transferred deoxyribonucleic acid) in Ti plasmids. The agrobacterium-mediated sugarcane genetic transformation system is perfected. Genetic transformation rate of sugarcane is remarkably improved as compared with that of the prior art.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, in particular to a method for genetic transformation of sugarcane callus. Background technique [0002] Sugarcane is the most important sugar crop in my country. In the 2011 / 2012 crushing season, my country’s sucrose production was 11.51 million tons, accounting for 91% of the country’s total sugar production. It can be seen that the sugarcane industry plays a vital role in ensuring the national sugar supply system status. However, in the process of sugarcane planting, frequent occurrence of diseases and insect pests, seasonal drought, and low temperature and chilling damage often occur, resulting in serious production losses, loss of income of sugarcane farmers and sugar enterprises, and a decline in the international competitiveness of my country's sugar industry. Therefore, there is an urgent need to improve the yield and stress resistance of varieties in production, and to cultivate excell...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 樊丽娜齐永文李奇伟邓海华劳方业李昱罗青文周文灵何慧怡陈月桂
Owner GUANGZHOU SUGARCANE IND RES INST
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