Production of triploid plants of Mooseer (Allium. hirtifolium) through endosperm culture

The endosperm (3n) is the food source for the embryo, which has received two polar nuclei from the maternal parent 2n and one sperm cell from the paternal parent 1n. Traditionally, triploid plants are created by crossing between tetraploid plants and diploid plants. To carry out this method, it is necessary to create tetraploid lines, and it is possible to double the chromosomes of diploid species by using substances that inhibit the formation of the division spindle. The advantage of endosperm culture over the traditional method is that the production of triploid plants in endosperm culture is done in a single step, and the time required to generate a triploid plant is less than in the traditional method. Increasing the level of ploidy of the nucleus can affect the plant's physiological and biochemical processes as well as altering the rate of photosynthesis, respiration, activity of genes, enzymes, and expression of isoenzymes compared with diploid types. They will have a new phenotype and can surpass the range of traits of their diploid ancestors such as increasing biomass and changing the quality and concentration of plant active ingredients, increasing resistance to drought stress, and resistance to pests. Therefore, the chance of their selection will increase.

Immature seeds were collected in late May, about 35 to 45 days after pollination, and mature seeds were harvested after fully ripening approximately in late July, and disinfection using a stereomicroscope with 60 times magnification under a laminar hood. The endosperm tissue was separated from the seed coat and embryo. In this study, mature and immature endosperm tissues of the mooseer plant were cultured onto hormonal compounds for callus induction in dark conditions for three months. In further experiments, the effects of sucrose were investigated at two levels of 30 and 50 gl-1 and the combination of BAP and NAA treatments on mooseer production. The flow cytometry method and chromosome counting were used to determine the ploidy levels of regenerated plants.

In this study, the treatment of 1 mg L-1 of NAA and 1 mg L-1 of BAP indicated the highest percentage of callus formation in both mature and immature tissues of mooseer endosperm. Seedlings produced in the treatment of 0.5 mg L-1 of NAA and 3 mg L-1 of BAP in culture medium with 50 g of sucrose caused the production of bulblet with greater length and diameter. The present study identified the cytogenetic properties, chromosome counts, and stomatal sizes of triploid plants regenerated from both mature and immature endosperm tissue of mooseer, using flow cytometry. A total of 12 triploid seedlings were obtained from mature tissue, while 8 seedlings were obtained from immature tissue.

Traditionally, triploids are produced by hybridization between induced superior tetraploids and
diploids, but endosperm culture is a one-step protocol. In this study, according to the sexual and asexual propagation (propagation through daughter bulbs) of mooseer, the modification and production of the triploid plants in mooseer allow the propagation of this plant through daughter bulbs. Generally, triploid plants were successfully produced through the endosperm culture method.