Pollen-mediated plant transgenic method using pigment-related gene as visual mark

Abstract

The invention provides a pollen-mediated plant transgenic method using a pigment-related gene as a visual mark. The objectives of the invention are to visually select transformed seeds according to color changes of transformed seeds, simplify screening steps and shorten a period for obtainment of transformed seeds. The method comprises the following steps: with Agrobacterium Ti plasmid, Escherichia coli plasmid or other DNA vector carrying a pigment-related exogenous gene and a promoter thereof as a gene donor and male gametophyte pollen of a plant as a receptor, mixing the pollen with a DNA fragment in 0.15 to 1.5 mol of a cane sugar solution having undergone ventilation and low temperature treatment and allowing the exogenous gene to be transferred into the receptor pollen through ultrasonic-assisted action on a pollen fluid suspension; pollinating the treated pollen to the stigma of the plant and harvesting seeds when the seeds are mature; and preliminarily selecting the harvested seeds having undergone transformation according to color changes of seeds in a following growing season, allowing the seeds to germinate and carrying out PCR amplification and verification through molecular identification like Southern hybridization on DNAs of seeding samples so as to further confirm transformed strains.

Description

technical field [0001] The invention relates to an improved plant pollen-mediated transgene method, which belongs to the field of biotechnology. Background technique [0002] Plant transgenic research is of great significance for improving crop yield, resistance to diseases and insect pests, stress resistance and quality improvement. After more than 30 years of theoretical research and technical development, plant transgenic methods have made great progress, and the technology is becoming more and more mature and applied in breeding. Since the first transgenic plant came out in 1983, plant transgenic research has developed rapidly. In 1996, the first transgenic product was commercially produced. By the end of 2013, the commercial planting area of ​​transgenic crops had reached 175 million hectares. increased by more than 100 times (James2014, http: / / www.isaaa.org / purchasepublications / itemdescription.asp,ItemType=BRIEFS&Control=IB046-2013). Although genetically modified pla...

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Problems solved by technology

These two methods have the following common disadvantages: 1) The plant tissue culture process is cumbersome, time-consuming, and inefficient; 2) In order to facilitate the screening of transgenic progeny plants, most plant expression vectors contain antibiotic-resistant or herbicide-resistant, etc. Select marker genes, which increases the concerns about the safety of genetically modified foods; 3) Since only part of the genotypes of some plant species can be used to obtain regenerated plants through tissue culture, there are great genotype limitations in this method for transgenic sex

4) Due to the large amount of tissue culture equipment that must be used, plant transformation work can only be carried out by laboratories with corresponding conditions; conventional breeders cannot use this technology

5) In addition, the two commonly used genetic modification methods are patented by foreign companies, and Chinese researchers can only use these two methods to conduct research. However, if the genetically modified crop varieties selected by these two methods enter large-scale commercialization Production, will likely have to pay huge patent royalties to foreign companies

All of these transgenic studies were done through the plant tissue culture route and thus failed to take full advantage of the advantages of anthocyanins as intuitive selectable markers

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