Transformation of Cucumber

Cucumber originates from the tropical rainforest areas at the southern foothills of the Himalayas. The botanical classification is Cucurbitaceae. Improving existing cucumber varieties through bioengineering means and cultivating new cucumber varieties with high yield, high resistance, multiple resistance and excellent quality have become the development direction of cucumber breeding. An efficient cucumber regeneration and transformation system is the basis for cucumber transgenic breeding. Therefore, establishing a cucumber transformation system has important theoretical and practical significance.

Principle

Certain pathogenic organisms (Agrobacterium, viruses, etc.) can integrate the pathogenic DNA into the genome of the host cell by infecting the host cell. Therefore, these pathogenic DNAs can be modified and their infection ability can be used to introduce foreign genes into plant or animal cells to produce transgenic plants or animals. Agrobacterium tumefaciens is the most widely used vector system in plant transgenic engineering .

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Procedures

1. Select plump and complete cucumber seeds, sterilize them in 70% alcohol for 30 s, and then sterilize them in 2% sodium hypochlorite solution for 15 min. Rinse with sterile water several times and then sow on MS medium. The next experiment will be carried out after the cotyledons have just broken out.
2. Pick Agrobacterium (a single colony of Agrobacterium carrying the recombinant plasmid) and inoculate it into YEB medium containing 50 mg/L kanamycin and 70 mg/L Rif, shake at 28°C, 200 r/min, and shake overnight. Then add 2 mL of bacterial solution (add a certain amount of bacterial liquid according to the concentration of the preserved bacterial liquid, if too much is added, the OD₆₀₀ may exceed 0.6-0.8 on the second day) to 50 mL YEB medium containing 50 mg/L kanamycin and 70 mg/L rifampicin, and culture with shaking for about 14 h until the OD₆₀₀ is 0.6-0.8 in the late logarithmic growth phase. Centrifuge at 5,000 r/min for 5 min, collect the cells, wash them once with 1/2 MS liquid culture medium, and dilute them into 1/2 MS liquid culture medium with 5 times the volume of the bacterial solution before centrifugation, and measure the OD₆₀₀ to be about 0.2, ready for infection.
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3. Take cucumber seeds that have grown for 2 d, cut off the growth point and hypocotyl, cut off the upper half (1/3-1/2) of both cotyledons, and keep the lower half of the cotyledons. Use the bacterial solution diluted in the second step to infect the cotyledons for 15 min. After the infection is completed, use sterilized filter paper to absorb excess bacterial fluid, and inoculate the cotyledons face up on the differentiation medium. Co-culture for 2 d at 28°C.
4. After 2 d of co-culture, the explants were transferred to MS differentiation medium containing 25 mg/L kanamycin and 500 mg/L carbenicillin, and placed in a tissue culture room for culture.
5. After 15-20 d, when the resistant shoots grow to 1-1.5 cm, cut them off and move them into MS rooting medium (plus kanamycin 100 mg/L) to induce rooting to obtain resistant plants.
6. After the root system of the transgenic cucumber has developed (5-6 leaves), move it into a flowerpot filled with sterile soil and cover it with plastic wrap to maintain moisture. Cultivate in the artificial climate chamber for 1 week, then move to the greenhouse, adapt in the greenhouse for 3-5 d, and finally plant the transgenic seedlings in the greenhouse for routine management.

Note:

• Genetic transformation operations and culture processes are all conducted under sterile conditions.
• Appropriate selection pressure should be selected when selecting for culture.

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