Generation of Interspecific Hybrids of Trifolium Using Embryo Rescue Techniques

The genus Trifolium Leguminosae (Fabaceae), commonly called clovers, includes 237–290 annual and perennial species, of which about 20 are important as cultivated and pasture crops. Taxonomic distribution supported by molecular analysis indicates that Mediterranean region is one of the main centers of distribution of the genus and also a center of domestication and breeding. Self-incompatibility is prevalent in the genus, controlled by a single, multiallelic gene expressed gametophytically in the pollen. It was suggested that hybridity did not play a major role in the evolution of the genus due to the poor crossability of the species under natural conditions. Interspecific hybridization in the genus Trifolium by conventional crossing techniques has been largely unsuccessful. Post-zygotic barriers appear to be a primary cause of the reproductive isolation, associated with endosperm disintegration and consequent abnormal differentiation and starvation of the hybrid embryo. As hybridization using conventional techniques has almost failed in Trifolium , embryo culture technique was used by breeders to obtain new combinations of interspecific hybrids. Embryo culture has been effectively used in developing interspecific hybrids in Trifolium ambiguum , T. pratense , T. montanum , T. occidentale , T. isthomocarpum , T. repens , T. nigrescens , T. uniflorum , T. sarosiense , T. alexandrinum , T. apertum , T. resupinatum , T. constantinopolitanum , T. rubens , and T. alpestre in various combinations. The successful embryo �rescue and development of hybrid plantlets requires skilled techniques of tissue culture and field practices. It includes hybridization in field; excision of hybrid embryos at appropriate stage; disinfection and culture in suitable culture media to allow maturation of embryo, multiplication of shoots, and rooting; hardening of the plantlets; inoculation with suitable Rhizobium culture; and transfer to field.