2012 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.
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 10 min has been successfully used for surface sterilization of explants. Storing heads at 4oC in the dark for 7 days was better for inhibiting anther somatic tissue growth than anthers directly sampled and inoculated. An experiment of 3 genotypes, 3 levels of sucrose, 3 phytohormones and 2 organic additions resulted in significant differences in embryonic callus formation. The effectiveness of the best combination was confirmed using different inbred lines and interspecific amphiploids. Buds treated with three chemical haploid inducers including 2-HNA, PCIB, and EBR used at various concentrations did not increase the induction rate of calli or embryo-like structure formation. 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. Results from shoot regeneration using ten media suggested varying effectiveness of media and subculturing with changing ratios of cytokinin and auxin. Eight rooting media were evaluated and significant differences among them were detected, and the addition of activated charcoal was beneficial. 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. For the approach of using foreign pollen serving as haploid inducer, hybrid F1 plants of four cross combinations pollinated with H. tuberosus did not produce haploid, but progenies having 2n=68 chromosomes. For the approach of identifying induced mutations capable of inducing haploid, X-ray irradiated pollen were used to pollinate a specific cultivated line and progenies evaluated.