Location: Sunflower and Plant Biology Research2011 Annual Report
1a. Objectives (from AD-416)
The objective of the project is 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)
Two approaches will be undertaken simultaneously by the postdoctoral affiliate, both approaches having precedents in other crops. (1) Anther culture: C. C. Jan initiated work on anther culture in the 1980s, but put it aside when other projects became higher priority. Dr. Jan envisions that the postdoctoral affiliate would develop a protocol for extraction of anthers from sunflower florets at an optimum stage of development and determining optimum tissue culture media and environmental conditions for induction of embryos, germination to plantlets, chromosome doubling, confirmation of haploid production, and growth to a mature, homozygous plant. (2) Foreign pollen: Under the direction Lili Qi and Brent Hulke, the postdoctoral affiliate will explore the potential for sunflower haploid production by fertilization of sunflower ovules with foreign pollen (as is done with wheat by fertilization by corn pollen, in which the ovule is fertilized, but the corn chromosomes are eventually eliminated, leaving a haploid wheat embryo that can be doubled). The postdoctoral affiliate will identify potential sources of foreign pollen from the family Compositae, but outside of the genus Helianthus, and test them 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.
3. Progress Report
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 4C 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. The ADODR monitors research progress by semiannual meetings with the Cooperator's personnel and by site visits to field plot locations.