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

A pollen-mediated, transgenic technology, applied in the biological field, can solve the problems of failing to give full play to the superiority of anthocyanins, increasing concerns about the safety of genetically modified foods, and paying huge patent royalties, etc., to expand breeding resources, without genotypes Dependence, the effect of speeding up the breeding process

CN103993034AActive Publication Date: 2014-08-20AGRI BIOTECH RES CENT OF SHANXI PROVINCE
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2014-08-20

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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.
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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...

Examples

Embodiment Construction

[0023] Maize was transformed using the vector containing anthocyanin marker gene and pollen-mediated method; the transformation of maize was carried out by using pollen-mediated method and pLtp1CB transformation vector containing exogenous anthocyanin gene. The specific operation steps are as follows:

[0024] Genetic transformation using ultrasound-assisted pollen-mediated method:

[0025] (1) Test material: The plant material is the maize inbred line Zheng 58, and the gene donor plasmid is the vector pLtp1CB (attached figure 1 )

[0026] (2) Transformation treatment method: Corn seeds were sown in the experimental field of our center in Taiyuan in early May, and entered the full flowering period in mid-July. Before flowering and silking, the male and female ears to be transformed were bagged separately, and fresh pollen was collected at around 10 am the next day, suspended in 0.45mol sucrose solution, and subjected to initial ultrasonic treatment, and plasmid DNA (mixing r...