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Method for improving sugarcane genetic transformation efficiency by taking mannose as screening agent

A technology of genetic transformation efficiency and mannose, applied in the field of transgenic plants in the field of plant biotechnology, can solve the problems of low transformation efficiency, low transformation efficiency of genetic transformation system, unsuitable for genetic transformation of different genotypes of sugarcane, etc., and achieves strong regeneration ability. Effect

Inactive Publication Date: 2012-11-07
广西作物遗传改良生物技术重点开放实验室
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Problems solved by technology

However, the current Agrobacterium-mediated genetic transformation of sugarcane still has low transformation efficiency, and individual transformation events have high efficiency but poor reproducibility.
At the same time, because sugarcane genotype, explant type and physiological state have a great influence on the transformation efficiency, the same genetic transformation system is not suitable for the genetic transformation of different genotypes of sugarcane
In addition, the expression efficiency is not high, and the silencing of transgenes in sugarcane is very serious; antibiotics, herbicides and other negative selection agents are mainly used for sugarcane transgenesis, the transformation efficiency of genetic transformation systems is low, the embryogenic callus induction and differentiation time is long and sugarcane Serious defects such as easy browning of callus

Method used

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  • Method for improving sugarcane genetic transformation efficiency by taking mannose as screening agent
  • Method for improving sugarcane genetic transformation efficiency by taking mannose as screening agent
  • Method for improving sugarcane genetic transformation efficiency by taking mannose as screening agent

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Experimental program
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Effect test

Embodiment 1

[0041] 1. Cloning of 6-phosphate mannose isomerase gene manA

[0042] Design primers and carry out PCR cloning according to the 6-phosphate mannose isomerase gene manA (M15380) in GenBank.

[0043] 2. Construction of plant binary expression vector pPMI3301 containing manA.

[0044] The plant binary expression vector used in the present invention is pCAMBIA3301. The cloned 6-phosphate mannose isomerase gene manA replaces the htp gene on the plasmid pCAMBIA3301 with a binary expression vector named pPMI3301. The T-DNA region schematic diagram is as follows figure 1 Shown (LB: left border of T-DNA region; RB: right border of T-DNA region; Ubi: maize ubiquitin promoter; NOS: nopaline synthase termination sequence; manA: mannose-6-phosphate isomerase gene) .

[0045] 3. Obtaining genetically modified sugarcane

[0046] 1. Preparation of transforming receptors

[0047] Take sugarcane ROC22 stem apex 10cm long heart leaf segment as explants, cut into thin slices with a thickness...

Embodiment 2

[0068] 1. Preparation of transforming receptors

[0069]The young ears of sugarcane GT28 were used as explants, cut into sections with a length of about 1-2 mm, inoculated in callus induction medium, and cultured at 28°C in the dark. After 30 days, the callus was transferred to a new subculture medium, subcultured once every 25 days, subcultured twice, and pale yellow, granular sugarcane II embryogenic callus was obtained. The callus induction medium is based on MS medium, with 2mg / L 2,4-D, 10mg / L citric acid, 30g / L sucrose and 0.6% agar, pH 5.8. The callus subculture medium is based on MS medium, supplemented with 1.5mg / L 2,4-D, 10mg / L citric acid, 30g / L sucrose and 0.6% agar, pH 5.8. Before transformation, vigorously growing sugarcane type II embryogenic callus was transferred to fresh subculture medium for culture for 4 days, and the activated callus was used as transformation material. Use tweezers to crush the activated callus into small particles with a size of about 1...

Embodiment 3

[0085] 1. Preparation of transforming receptors

[0086] Take sugarcane GT21 1-2mm stem tips as explants, inoculate them in callus induction medium, and culture them in the dark at 28°C. After 25 days, the callus was transferred to a new subculture medium, subcultured once every 25 days, and subcultured 3 times to obtain pale yellow, granular sugarcane II embryogenic callus. The callus induction medium is based on MS medium, with 2mg / L 2,4-D, 30mg / L citric acid, 30g / L sucrose and 0.6% agar, pH 5.8. The callus subculture medium is based on MS medium, and added with 1 mg / L 2,4-D, 30 mg / L citric acid, 30 g / L sucrose and 0.6% agar, pH 5.8. Before transformation, vigorously growing sugarcane type II embryogenic callus was transferred to fresh subculture medium for 5 days, and the activated callus was used as transformation material. Use tweezers to crush the activated callus into small particles with a size of about 1-2 mm, and place it on an ultra-clean workbench and blow it for...

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Abstract

The present invention belongs to the field of plant transgenic technology, and discloses a method for improving sugarcane genetic transformation efficiency by taking mannose as a screening agent. The method is based on sugarcane young heart leaves-induced yellowish and granular type II embryogenic callus as a transgenic acceptor material, employs a negative pressure assistant and improved agrobacterium tumefaciens mediated method to lead a 6-phosphate mannose isomerase gene into a sugarcane genome, and enables getting transgenic sugarcane plants after screening and differentiating on mannose-containing medium. The method uses a PMI positive selection marker system, requires a short cycle from sugarcane callusing to differentiating into seedlings, is stable in transformation effects, and helps to overcome the defects that a genetic transformation system taking antibiotics and herbicides as negative selection markers is low in transformation efficiency, long time in embryogenic callus induced differentiation, easy in sugarcane callus browning, etc. The transformed callus maintains a high regenerative capacity, and has a transformation rate of 58.97% showed by molecular detection results.

Description

technical field [0001] The invention relates to a method for transgenic plants in the field of plant biotechnology, in particular to a method for improving the efficiency of sugarcane genetic transformation by using mannose as a screening agent mediated by Agrobacterium. Background technique [0002] Sugarcane (Saccharum officinarum L.) is an important sugar crop in China and the world. Today, the cultivation of new varieties of sugarcane mainly relies on conventional cross-breeding. However, due to the allopolyploidy and high heterozygosity of sugarcane genetic background, sexual crosses of sugarcane often have problems such as difficulty in flowering, failure to meet flowering, hybrid infertility and infertility, which makes the conventional breeding cycle of sugarcane long, and resistant varieties Due to limited quality resources, it is difficult to breed new sugarcane varieties with high yield, high sugar resistance and strong stress resistance through conventional cros...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 魏源文邓智年胡春锦李楠秦翠鲜何海波黄诚梅潘有强吕维莉郭文锋曹辉庆李杨瑞
Owner 广西作物遗传改良生物技术重点开放实验室
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