Regeneration and interspecific somatic hybridization in Allium for transfer of cytoplasmic male sterility to leek

The vast majority of the present day leek cultivars is of poor quality. The genetic constitution of leek makes it a difficult crop to breed and consequently mass, or family selection methods, both of which have a low efficiency, are mainly used. F ₁ hybrid breeding seems the appropriate strategy for improvement of leek. With such a breeding system a higher uniformity of the crop, better fixation of desirable traits, such as pest and disease resistance and a better exploitation of heterosis effects can be achieved. Large-scale seed production of hybrid cultivars requires a hybridization system based on cytoplasmic male sterility (CMS), because of the considerable advantage of this system in maintaining the male sterile parent line. Since no source of CMS has yet been found in leek or related forms of A. ampeloprasum , several researchers are focused on introducing cytoplasmic male sterility into leek. In the present thesis the possibility of introducing CMS into leek via somatic hybridization with A. cepa as CMS donor was investigated. Successful application of such a technique requires an efficient system for regeneration of plants from protoplasts. At the beginning of the research project in 1990 such a protoplast-to-plant system for leek was not available. Therefore, substantial effort was directed towards the development of efficient plant regeneration methods from embryogenic callus cultures, suspension cultures and protoplasts.

Initially, much attention was focused on development of regenerative callus cultures from different explants and cultivars. The highest compact callus response was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Significant differences were found between the cultivars and accessions for shoot formation frequency. In addition, a genotype-dependent response of leek embryo explants for the formation of shoots was evident.

In contrast to plant regeneration from compact embryogenic callus of leek, which is efficient and easy to achieve, the establishment of suspension cultures from this type of callus was less successful. Although compact callus cultures in liquid medium retain their ability to form somatic embryos and shoots for a long period, they do not become a finely-dispersed suspension culture. For this purpose, a new friable embryogenic type of callus was induced on immature embryos instead of mature embryos. This friable callus comprised numerous globular embryoids, embedded in mucilage and is highly regenerative when plated on a cytokinin containing medium. It was found that the developmental stage of the immature embryo and the genotype of the donor plant significantly influenced the callus response. Characterization of the two callus types by a histological examination revealed striking differences and supported the suggestion that friable callus is more suitable for initiating suspension cultures than compact callus. A stringent selection within these friable callus cultures was necessary to obtain highly-embryogenic suspension cultures.

A procedure was described for the isolation, culture and regeneration of plants from protoplasts derived from these embryogenic suspension cells. Imbedding in alginate was an important factor in increasing the plating efficiency. The regeneration frequency of the protoplast-derived calli was primarily affected by the type of callus that developed. The protoplast-to-plant system described was reproducible for at least three genotypes. Plants were regenerated within 6 months after protoplast isolation.

Utilising this regeneration procedure for protoplasts, a method for symmetric hybridization between leek and onion as a CMS donor was set up. The fusion experiments yielded large numbers of hybrid calli and plants. The aneuploid status of the hybrid plants could be explained by the use of leek protoplasts, derived from an aneuploid suspension culture. This also implied that using cell suspensions as a protoplast source for fusion remains a restrictive factor in the establishment of a successful hybridization system for leek. The leaf morphology of the hybrids was intermediate between the two parents. It appeared that most of the hybrids possessed leek chloroplasts and a rearranged mitochondrial genome of both parents, but with a predominance of mtDNA fragments from leek.

With the methods described in this thesis the first steps to transfer CMS to leek have been realized. The results described here show that a cytoplasm of onion can be successfully transferred to leek via somatic hybridization. Future research should focus on further improvement of the system. For this, optimization of some aspects of the regeneration process and developing an efficient selection system for the desired hybrids, containing the onion specific CMS mtDNA sequences should be accomplished. The research has shown that somatic hybridization has a high potential to obtain CMS leek plants.