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Ultrasonic-assisted pollen mediated plant genetic transformation method

A pollen-mediated, transgenic technology, applied in biochemical equipment and methods, genetic engineering, and the use of vectors to introduce foreign genetic material, can solve problems such as inability to complete the fertilization process, low seed setting rate, and loss of vitality, and achieve genetic High transformation efficiency, less chance of chimera, and the effect of increasing the number of transformants

Inactive Publication Date: 2012-06-06
AGRI BIOTECH RES CENT OF SHANXI PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the main disadvantages of this method is the low rate of seed setting after pollination, which is apparently due to the fact that most of the ultrasonically treated pollen has lost its viability and cannot complete the fertilization process normally

Method used

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  • Ultrasonic-assisted pollen mediated plant genetic transformation method
  • Ultrasonic-assisted pollen mediated plant genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: The soaking time of corn pollen in the suspension has a significant effect on pollen germination in vitro.

[0012] Table 1 Effect of soaking time of corn pollen in suspension on pollen germination in vitro

[0013]

[0014] Note: The germination rate is about 28°C. The pollen is soaked in 15% sucrose solution for a corresponding period of time, and then a small amount of pollen is absorbed and germinated in the medium for 30 minutes before testing. The formula of corn pollen germination medium in vitro is: 15% sucrose + 50 mg / l boric acid + 300 mg / l calcium chloride + 200 mg / l magnesium chloride + 100 mg / l potassium nitrate + 35 mg / l gibberellin. The letters marked in the table are statistical results at the 5% significant level of multiple comparisons using the DPS data processing system Duncan’s method (the same below).

[0015] It can be seen from Table 1 that with the extension of soaking time, the breakage rate of corn pollen increased and the germi...

Embodiment 2

[0016] Example 2: The sucrose concentration (osmotic pressure) of the suspension has a significant effect on the pollen germination rate.

[0017] Table 2-1 In vitro germination of maize pollen with different sucrose concentrations (2010.5.28—6.10)

[0018]

[0019] Table 2-2 In vitro germination of maize pollen with different sucrose concentrations (2010.7.15—8.5)

[0020]

[0021] The concentration of sucrose in the suspension is mainly reflected in the osmotic pressure of the solution. It can be seen from Table 2-1 and 2-2 that no matter when the samples were taken, the corn pollen had a higher breakage rate in the low concentration (≦5%) sucrose solution. Pollen intact rate increased with the increase of sucrose concentration. But in high concentration (50%) sucrose solution, the pollen germination rate decreased significantly.

[0022] Comparing the in vitro germination of maize pollen in different periods, it can be seen that the responses of different varietie...

Embodiment 3

[0023] Example 3: Effects of storage time and conditions on pollen viability.

[0024]The order of preservation conditions for corn pollen is low-temperature drying > low-temperature humidity > room temperature drying > room temperature humidity, especially the best way to store pollen in a petri dish in a refrigerator at 4°C, which can fully meet the needs of ultrasound-mediated transgenic experiments. It can be seen from Table 3-1 that early sowing corn pollen has a short survival time in vitro and can only be stored in low temperature and dry storage for about 2 hours. After that, the germination ability of the pollen decreases rapidly and it is not suitable for use. Field spring sown corn pollen can still germinate in a petri dish in a refrigerator at 4 ℃ for up to 5 days after dry storage (Table 3-2), and it is found that the pollen just harvested is fragile and has a low germination rate, and the germination rate is obvious when stored for more than 2 hours. The pollen h...

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Abstract

The invention relates to an improved ultrasonic-assisted pollen mediated plant genetic transformation method, and aims to obviously increase the setting percentage of the plant fertilized by pollen subjected to ultrasonic treatment, thereby increasing the number of transformants obtained for each treatment. The method comprises the following steps: by using an agrobacterium Ti plasmid carrying anexogenous gene segment, colibacillus plasmid or any other DNA vector as a genetic donor and using plant pollen as a receptor, mixing the pollen and exogenous DNA in a 5-50% sucrose solution subjectedto aeration and low-temperature treatment, and transferring the exogenous gene into the receptor pollen under the assisting action of ultrasonic; fertilizing the treated pollen onto the stigma of theplant, and harvesting when the grains become ripe; and in the subsequent growth season, sowing the harvested seeds which are obtained after the fertilization of the transformed pollen, screening the germinated seeds and seedlings, carrying out PCR (Polymerase Chain Reaction) amplification and Southern hybridization on the DNA of a seedling sample, and further determining the transformant. The method provided by the invention does not need tissue culture, does not have species or genotype dependency, and can shorten the genetic transformation breeding time and save the manpower and material resources.

Description

technical field [0001] The invention relates to an improved plant pollen-mediated transgene method. Background technique [0002] Transgenic technology can enable cross-species exchange of excellent genes, thereby achieving directional and precise improvement of crop quality and yield traits. Compared with traditional crops, genetically modified crops have significantly improved yield, stress resistance and nutritional quality, and can also greatly reduce production costs and reduce environmental pollution in agricultural production. However, at present, there are only a few types of genetically modified crops that are widely used in agricultural production, such as corn, soybean, rapeseed, cotton, tomato, etc. For most plants, genetically modified technology is still in the laboratory stage. At present, there are two classical methods mainly used in plant transgenic research, one is the Agrobacterium-mediated method; the other is the gene gun bombardment method. These two...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
CPCC12N15/8206C12N15/8205C12N15/82
Inventor 孙毅崔贵梅郝曜山杜建中王亦学杨利艳张红梅张丽君王铭张婷婷
Owner AGRI BIOTECH RES CENT OF SHANXI PROVINCE
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