C. C. Jan

Wild Sunflower Species as a Genetic Resource for Resistance to Sunflower Broomrape (Orobanche cumana Wallr.)

Abstract Broomrape (Orobanche cumana Wallr.) is a parasitic weed that causes economic damage in sunflower production in many countries, especially in Central and Eastern Europe, Spain, Turkey, Israel, Iran, Kazakhstan, and China. Genes for resistance to broomrape races A, B, C, D, and E are present in varietal populations of cultivated sunflower. Since broomrape is a highly variable parasitic weed, the breakdown of resistance is a frequent phenomenon, and multiple sources of resistance are needed to control the emerging races. Genes that confer resistance to races F, G, and H and others that have not been assigned a race designation have been identified in wild sunflower species and incorporated into hybrid sunflower through interspecific hybridization. The U.S. Department of Agriculture, Agricultural Research Service, National Plant Germplasm System wild sunflower collection contains 2,239 accessions with 1373 annual accessions represented by 14 species and 866 perennial accessions represented by 39 species. Sunflower germplasm evaluations for resistance to broomrape races have demonstrated that the Helianthus species constitute a substantial reservoir of genes conferring resistance to new virulence broomrape races. The resistance to broomrape, including immunity reported in seven annual and 32 perennial species, provides breeders a broad genetic base from which to search for resistance to existing and newly emerging races.

Broomrape (Orobanche cumana Wallr.) resistance breeding utilizing wild Helianthus species

Abstract Wild Helianthus species possess valuable resistance genes for sunflower broomrape (Orobanche cumana Wallr.), especially the 39 largely underutilized perennial species. Resistance to race F has been transferred into a cultivated background via bridging of interspecific amphiploids. More recently, a single dominant gene resistant to race G was identified in annual H. debilis ssp. tardiflorus and transferred into cultivated HA 89. Interspecific crosses between wild annual Helianthus species and cultivated lines are relatively easy compared to those involving wild perennial species, which were made easier only after the development of embryo rescue techniques. Interspecific amphiploids resulting from colchicine treatment of F1 hybrids provide bridging materials for transferring genes without relying on embryo rescue. Among the diploid, tetraploid, and hexaploid perennial species, the speed of gene utilization follows the ploidy level of diploids, tetraploids, and hexaploids due to the time-consuming backcrosses required to eliminate the extra chromosomes in the latter two groups. In the development of pre-breeding materials, the retention rate of genetic material of the wild species is another concern with each additional backcross. For crosses involving tetraploid and hexaploid wild perennials, the use of 2n=51 chromosome F1 or BC1F1 generation, as pollen source, could accelerate chromosome reduction to 2n=34 in BC1F1 or BC2F1, resulting in useful materials with fewer backcrosses for trait selection.

Somatic Embryogenesis from Corolla Tubes of Interspecific Amphiploids between Cultivated Sunflower (Helianthus annuus L.) and Its Wild Species

Abstract Somatic embryogenesis in vitro provides an efficient means of plant multiplication, facilitating sunflower improvement and germplasm innovation. In the present study, using interspecific amphiploids (2n=4x=68) between cultivated sunflower and wild perennial Helianthus species as explant donors, somatic embryos were induced directly from the surface of corolla tubes at the late uninucleate or binucleate microspore development stage. Primary somatic embryos (PSEs) were obtained in amphiploids G08/2280 (H. pumilus×P21) and G08/2260 (NMSHA89×H. maximiliani). The PSE induction frequency of G08/2280 on synthesized Medium A and B was 30.27 % and 42.42 %, respectively, while that of G08/2260 was 5.89 % and 12.16 %, respectively. The difference of PSE induction frequency was significant between G08/2280 and G08/2260 (P=0.0058), but was non-significant between induction Medium A and B (P=0.1997). Secondary somatic embryos (SSEs) were rapidly produced from PSEs on subculture Medium 1 with the induction frequency of 100 %. The mean number of SSEs produced from each PSE was 19.2 and 12.2 in G08/2280 and G08/2260 within 30 d of subculture, respectively. Mature SSEs were gradually converted into young shoots on hormone-free subculture Medium 2, with the mean number of small green shoots produced from each PSE of 22.0 and 18.7 in G08/2280 and G08/2260, respectively. Through the additional process of rooting for some shoots without roots on half-strength of MS medium adding 0.25–0.5 mg/l NAA, 0.5 mg–1.0/l IBA, SE-derived shoots without roots gained about 40 % rooting frequency. Regenerated plants acclimated successfully and displayed similar morphological and chromosome number to the amphiploid donors.

Chromosome Painting by GISH and Multicolor FISH

Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique for identifying chromosomes and mapping specific genes and DNA sequences on individual chromosomes. Genomic in situ hybridization (GISH) and multicolor FISH (mc-FISH) represent two special types of FISH techniques. Both GISH and mc-FISH experiments have general steps and features of FISH, including chromosome preparation, probe labeling, blocking DNA preparation, target-probe DNA hybridization, post-hybridization washes, and hybridization signal detection. Specifically, GISH uses total genomic DNA from two species as probe and blocking DNA, respectively, and it can differentiate chromosomes from different genomes. The mc-FISH takes advantage of simultaneous hybridization of several DNA probes labeled by different fluorochromes to different targets on the same chromosome sample. Hybridization signals from different probes are detected using different fluorescence filter sets. Multicolor FISH can provide more structural details for target chromosomes than single-color FISH. In this chapter, we present the general experimental procedures for these two techniques with specific details in the critical steps we have modified in our laboratories.