Haploid plants are important materials for genetic research and haploid breeding. At present, tobacco anther culture is the most effective method to produce haploid plants.

Principle

Anthers are the male organs of flowers. Anther culture is an organ culture. Pollen is a haploid cell and pollen culture is similar to unicellular culture in that both anthers and pollen can induce haploid cell lines and haploid plants in culture. In 1964, Guha and Maheshwari cultured mature pollen of Datura innoxia on a suitable medium and found that pollen could be transformed into an active cell division state, growing embryonic bodies from the anthers, and eventually giving embryonic plants.

The most important characteristic of haploid plants is the high degree of infertility, which is due to the fact that it has only one set of chromosomes, no homologous chromosomes, and only haploids are unable to associate during meiosis, resulting in irregular chromosome behavior, formation of macrospore or microspore chromosomes are incomplete, and therefore completely lose the ability to reproduce sexually. However, by doubling the chromosome number of a haploid plant, a doubled haploid plant, i.e., a pure diploid plant or a pure line, can be obtained. The rapid acquisition of pure lines is widely used in breeding. Haploids are important for plant genetic breeding, and their main uses are as follows.

• Through haploid culture and chromosome doubling, we can quickly obtain self-inherited lines and asexual lines of heterogamous pollinated crops.
• Create new germplasm resources through mutations in haploid culture.
• Mutagenesis of haploids can rapidly discover recessive mutations.
• Theoretical research. The aneuploidy obtained by crossing haploids with diploids helps to solve some problems such as determination of linkage groups, chromosome composition of diploids, and the role of gene dosage.

Procedures

1. Tobacco plants were planted in a greenhouse under conditions of 16 h of light, light intensity of about 200 μmol/(m²· s), and temperature of 25°C/20°C (day/night).

2. Take tobacco buds whose anthers have developed to the early stage of two nuclei, and place the buds with sepals and petals of equal length in a culture dish covered with wet filter paper. After being treated in a refrigerator at 5°C for 2-3 d, remove the sepals, stay in 70% alcohol for 0.5 min, and then sterilize in 0.1% HgCl₂ solution for 8 min. After washing with sterile water 3-4 times, peel the petals with pointed tweezers and take out the anthers (be careful not to damage the anthers and remove all the filaments).

3. The macroelements, trace elements, and organic components in the basic culture medium adopt the formula of Nitsch (1967), 30 g/L sucrose, 0.8% agar, pH 5.8. Anther culture was performed in a 6 cm diameter glass culture dish, with 16 anthers inoculated in each dish, arranged in a 4×4 square array. After inoculation, the anthers were cultured in an artificial lighting culture room with a light intensity of 40 μmol/(m²·s) for 12 h and a temperature of (26±1)°C.

4. After the anthers emerged, they were transplanted into nutrient pots. The greenhouse conditions were 16 h of light, a light intensity of about 200 μmol/(m²·s), and a temperature of 25°C/20°C (day/night).

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