2011 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 seven interspecific amphiploids were used as donor materials for anther culture. The anthers with late uninucleate microspores were dissected and cultured on the callus and embryoid inducing media. The method of soaking heads in 30% bleach solution for 15 min has been successfully used for surface sterilization of explants. Storing heads at 4 C in the dark for 7 days was better for inhibiting anther somatic tissue growth than anthers directly sampled and inoculated. An induction medium with high sucrose content is better for inhibiting anther somatic tissue growth. 2-HNA (2-hydroxynicotinic acid) was used as a chemical inducer for anther culture in eight inbred lines. Six combinations of 2-HNA concentration and pretreatment time were evaluated for anther culture. The anther calli induction rates did not increase when pretreated with 2-HNA before inoculation. RHA274 and Peredovik had the two highest rates of calli induction among the eight inbred lines. Calli induction rates of the interspecific amphiploids were higher than that of the inbred lines. There were also obvious differences among the hybrids and the media used. Some shoots have been successfully regenerated from the calli and are currently in the process of root induction. The shoot regeneration process of calli tends to suggest that the embryoids were produced by secondary growth of calli, followed by shoot regeneration from the embryoids. We will also try to use foreign pollen serving as haploid inducer. Hybrid F1 plants of four cross combinations of cultivars have been planted and some foreign pollen sources collected. A third approach is to attempt to identify induced mutations capable of inducing haploids, and the initial step of pollen irradiation using X-ray is underway.