2013 Annual Report
1a.Objectives (from AD-416):
To develop an efficient protocol for doubled haploid production in sunflower that can be adopted by seed companies with modest laboratory facilities and personnel with some training in tissue culture techniques.
1b.Approach (from AD-416):
A postdoctoral research associate, hired by North Dakota State University, Department of Plant Sciences, will conduct the research in collaboration with the ARS Principal Investigator who serves as the Research Coordinator. Under the research plan, two approaches will be undertaken simultaneously by the postdoctoral associate, both approaches having precedents in other crops. (1) A protocol will be developed for doubled haploid production by extraction of anthers from sunflower florets at an optimum stage of development and the optimum tissue culture media and environmental conditions will be determined for induction of embryos, germination to plantlets, chromosome doubling, confirmation of haploid production, and growth to a mature, homozygous plant. (2) The potential for sunflower haploid production by fertilization of sunflower ovules with foreign pollen will be explored. Potential sources of foreign pollen will be identified from the family Compositae, but outside of the genus Helianthus, and tested for their ability to induce haploid production in sunflower. Special attention will be given to foreign pollen sources that are easy to grow, maintain, and have prolific pollen production. Optimum tissue culture conditions for growth of fertilized embryos to mature plants will be determined.
Eight inbred lines and 18 wild species were used as donor materials for an anther culture approach. Anthers with light yellow-white color were dissected and cultured on callus and embryoid inducing media, which have proven successful for inducting embryos. Various concentrations and combinations of cytokinin and auxin were tested for shoot regeneration, including BAP (0.5-1.0 mg/l), NAA (0-0.5 mg/l) and kinetin (0-0.5 mg/l). Additionally, activated charcoal was added to the medium for testing. The same inbred lines and wild species were also used for microspore culture. Immature buds or inflorescences were surface sterilized with bleach and rinsed with sterilized dH2O, and then a 0.3 M mannitol solution was used to release the microspores. The ground mannitol slurry was filtered in sequence through a size 60 steel mesh, 80 µm nylon filter, and 30 µm nylon filter, respectively. After centrifugation, the pellets were combined and re-suspended in a 0.3 M mannitol solution. A 1.0 M maltose solution was used for microspore isolation. The isolated microspores were then mixed with a liquid culture medium and kept in an incubator without light at 32 oC for 3 days and then move to another incubator without light at 24 oC for 4 to 6 weeks. Various basic media and plant growth regulators of different concentrations and combinations were evaluated. Embryo inducer, 2-HNA, (2-hydroxynicotinic acid) was also incorporated in the culture medium during the experiment. Meanwhile, 0.5 g/l activated charcoal was also added to the culture medium to examine its effect on microspore culture. As a result, MS medium with 0.5 mg/l 2, 4-D and 0.5 mg/l BAP and 0.5g/l activated charcoal worked better for line RHA274 than other medium and other plant materials. Some embryo-like structures were observed. The approach of irradiated pollen haploid induction was recently added to the project. The plan is to use nmsHA89, nmsP21, and cmsHA89 as female parents, and to use selected USDA maintainer and restorer lines as the pollinators.