In Vitro Induction of Female Haploids in Sugar Beet

The method of obtaining haploids through pollen (anther) culture is the most common, but in those genotypes with poor male gamete induction response, unpollinated ovary or ovule culture can supplement or replace the pollen (anther) culture method. The phenomenon of unfertilized embryo sac cells producing haploid plants under in vitro conditions is called in vitro gynogenesis. Plant in vitro gynogenesis was initially mainly the culture of unpollinated ovules or ovaries. In recent years, more and more researchers have obtained female haploids by culturing flower buds or even inflorescences. At present, the most successful plant for in vitro gynogenesis is sugar beet, followed by onion, gerbera, tobacco, etc.

Principle

Gynogenesis refers to the process of producing haploid embryos and plants from unpollinated embryo sacs under in vitro conditions. When culturing unpollinated ovaries or ovules, the genetic effect of embryo sac cells developing into embryos or plants without fertilization is similar. The significance of the success of this research is that it has opened up another avenue for haploid breeding, which is particularly important for species where pollen breeding has not yet been successful and for male sterility.

Medium

Procedures

1. Plant the beet plants in a 15 cm diameter nutrient pot and move them into a growth greenhouse. The greenhouse environment temperature is (17±3)°C, the light intensity is about 200 μmol/(m²·s) with 16 h of light. Water and fertilize every day.

2. Harvest the inflorescences that have not yet bloomed and use them as test materials for ovary culture. The inflorescences can be placed in a beaker filled with clean water and stored in a refrigerator at 4-8°C.

3. Select the unopened buds and place them in a stainless steel basket. Disinfect the surface in a 3% sodium hypochlorite solution for 5 min, then rinse with sterile water 3-5 times, 5 min each time.

4. Peel off the ovary under a stereomicroscope in the clean bench and inoculate the peeled ovary into B1 culture medium. 20 ovaries can be inoculated in a 5 cm diameter culture dish. Seal with a double layer of sealing film and culture at 30°C in the dark.

5. After 30-60 days of culture, the embryoids were transferred to a test tube containing B2 medium at 25°C, 16 h of light, and a light intensity of 50 μmol/(m²·s).

6. Every 3-4 weeks, the newly generated embryoids and buds were transferred to fresh B2 medium.

7. The actively growing buds were transferred to a culture bottle containing pre-rooting medium B3, and after 2-3 weeks, they were transferred to a culture bottle containing rooting medium B4 for rooting. Thereafter, subculture was performed every 6-8 weeks until the root system was fully developed. The well-rooted seedlings were transplanted into a nutrient pot containing peat/vermiculite. Newly transplanted seedlings were watered and kept moist. The roots of the surviving seedlings were soaked in a solution of 0.2% colchicine and 0.25% DMSO for 5 h to double the chromosomes.

8. After the plants have doubled and resumed growth, they are subjected to vernalization for 14 weeks at 5°C, 16 h of light, and a light intensity of 60 μmol/(m²·s). After 2 weeks of adaptive growth, the plants can be moved into a growth greenhouse at 12°C, 16 h of light, and a light intensity of 60 μmol/(m²·s).

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