In 1982, Lichter of Germany obtained haploid plants for the first time through free microspore culture of Brassica napus. Since the isolation technology of free microspores of Brassica napus is simple and the efficiency of obtaining embryoids and regenerated seedlings through free microspore culture technology is high, this technology has been widely favored by rapeseed breeders in various countries since its appearance. It is of great significance to apply microspore culture technology on a large scale to the field planting of Brassica napus.
Anther is the male organ of the flower, and anther culture belongs to organ culture. Pollen is a haploid cell. Pollen culture is similar to single cell culture. Anthers and pollen can induce haploid cell lines and haploid plants during the culture process. In 1964, Guha and Maheshwari cultured the mature pollen of Datura stramonium on a suitable culture medium and found that the pollen could transform into an active cell division state, grow embryoids from the chambers, and finally obtain embryoid plants, which verified the theory of cell totipotency at the reproductive cell level. The biggest feature of haploid plants is their high degree of infertility. This is because it has only one set of chromosomes, no homologous chromosomes, and only haploids during meiosis, which cannot undergo synapsis, resulting in irregular chromosome behavior, and the formation of megaspores or microspores with incomplete chromosomes, thus completely losing the ability to reproduce sexually. However, if the number of chromosomes of a haploid plant is doubled, a doubled haploid (DH) plant can be obtained, that is, a homozygous diploid plant or pure line. Rapidly obtaining pure lines has a wide range of application value in breeding. Haploids are of great significance to plant genetic breeding, and their main uses are as follows.
1. Brassica napus seeds were sown in peat/vermiculite nutrient pots.
2. The experimental materials were planted in a greenhouse. The greenhouse conditions were 16 h of light, light intensity of about 200 µmol/(m2·s), and temperature of 25°C/20°C (day/night).
3. Water and fertilize every day after the seedlings emerge. For winter varieties, vernalization was carried out at the 3-5 leaf stage (4°C, 8 weeks).
4. When the first flower of the plant opens, the test material can be used for microspore culture. The material can be continuously collected for 2 weeks, during which the opened flowers and young pods are removed.
5. The size of the flower buds containing embryonic microspores varies with the genotype of the test material and the growth environment conditions. Under normal conditions, the size of the flower buds containing embryonic microspores is no longer than 4.5 mm, and the appearance is green and translucent. Select 3.5-4.5mm flower buds and place them in a stainless steel basket. Sterilize the surface in 6% sodium hypochlorite solution for 10-15 min, then rinse with sterile water 3-5 times, 5 min each time.
6. Move the sterile flower buds into a mortar and add 4°C B5 culture medium to grind. Then filter the ground material with a 50 µm nylon mesh.
7. Pour the filtered microspores into a centrifuge tube and centrifuge at 800-1,000 r/min for 5-8 min. To ensure the purity of the pollen, the centrifugation process can be repeated.
8. Suspend the pure microspores in NLN culture medium with a culture density of 75,000-100,000/mL. The microspore density can be measured with a hemocytometer.
9. Pour 10 mL of NLN medium containing microspores of appropriate density into a 6 cm diameter culture dish, seal it with double sealing film, place it in the dark at 30°C for 14 d, and then move the culture dish to a slow shaker (60 r/min) for another 7 d. The first cell division occurs after 2-3 d of microspore culture, followed by spherical embryos, torpedo-shaped embryos, and cotyledon-shaped embryos.
10. Move the embryoids into B5 solid culture medium for 30-40 d at 27°C, 12 h of light, and a light intensity of 70 µmol/(m2·s).
11. Transplant the well-rooted seedlings into a nutrient pot containing peat/vermiculite. Pay attention to watering and moisturizing the newly transplanted seedlings.
12. Soak the roots of the surviving seedlings in 0.3% colchicine solution for 1.5-3 h for chromosome doubling.
