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Asparagus is a valued vegetable in people's diet in many countries of the world. The production of asparagus increased remarkably in the last few years. Comprehensive evaluations have shown that there exist no cultivars of A. officinalis with resistance against Asparagus virus 1 (AV-1) causing a high loss of yield and quality. Resistance was found in wild species of the genus Asparagus such as in A. amarus. Somatic hybridization could be a tool to overcome crossing barriers between species. The sophisticated technology requires plant species adapted protocols. Here, a method starting with suspension cultures of A. officinalis and A. amarus was presented as source for protoplasts. Fusion was performed using either electromanipulation or polyethylene glycol solution. Fused protoplasts were cultivated in liquid protoplast cultivation medium PPM1 supplemented with 1 g L-1 L-glutamine. First cell divisions were noticed already after eight to ten days. About five weeks after fusion microcolonies were plated on solid Murashige and Skoog medium (MS) + 1 mg L-1 2,4-dichlorophenoxyacetic acid. After several subcultures, developing plants were transferred to MS + 0.2 mg L-1 α-naphthalene acetic acid for further growth. Regenerated plants were acclimatized to greenhouse conditions. By fluorescence flow cytometry the DNA amount was estimated. RAPD analyses revealed regenerated plants with DNA profiles with fragments from only one protoplast donor but also with fragments from both donors as it is expected for somatic hybrids. Resistance to AV-1 was tested by DAS-ELISA.

Rucola is a leafy vegetable from the Brassicaceae family with continuously increasing economic demand. Under this name rocket salad (Eruca sativa) as well as the perennial wall rocket (Diplotaxis tenuifolia) are commercially distributed. So far, for both species no hybrid system is available. Therefore, breeding lines of both species were used to pollinate cytoplasmic male sterile (CMS) cauliflower (Brassica oleracea) plants. Intergeneric Fi-hybrids were achieved by manual crosses and embryo rescue. Allotetraploid plants obtained after colchicine treatment were recurrently backcrossed with the respective rucola parent. The elimination of Brassica genomic DNA was verified using molecular markers, GISH analysis, flow cytometry and chromosome analysis. All backcross plants from BC2 up to the BC6 generation of the B. oleracea x E. sativa (BE) cross showed male sterility and full seed set compared to the E. sativa parent. No further incompatibilities were noticed between the CMS inducing cytoplasm and the E. sativa genome. Agronomical traits such as plant vigor, biomass production, flowering and seed set were recorded for the different backcross generations. The developed alloplasmic lines are diploid with 2n=2x=22 chromosomes and could be introduced without any problems into the breeding process. Also BC1 plants of the cross B. oleracea x D. tenuifolia (BD) had completely male sterile flowers. The phenotype was similar to that of the D. tenuifolia parent. Unlike the BE combination, for BD combination embryo rescue was necessary to establish BC2 plants. The flowers of all BC2 plants of BD combination showed besides the male sterility a pronounced female sterility, expressed by the abort of ovaries and failed seed set. Hence, this combination seems to be not applicable for the breeding process. Reasons for this strikingly different behaviour are under investigation.

Introgression of genes from distantly related donors within the family Brassicaceae is useful for augmenting the genetic base for achieving specific crop improvement objectives. Hypocotyl-derived protoplasts of B. oleracea var. capitata cv. 'Toskama' were fused with non-irradiated hypocotyl protoplasts as well as X-ray irradiated mesophyll protoplasts of Lepidium meyenii to produce symmetric versus asymmetric intergeneric hybrids. In addition, hypocotyl-derived protoplasts of B. oleracea cv. botrytis 'Korso' were fused with X-ray irradiated mesophyll protoplasts of L. meyenii. Using polyethylene glycol, 76 plants were regenerated, 37 with symmetric fusion and 39 with asymmetric fusion. Regenerated plants were characterized by RAPDs, AFLPs and Southern hybridization. Due to the small amount of template DNA (20 ng) required, the RAPD markers are suitable for detecting true fusion plants in very early stages of in vitro development. Whereas the large number of markers provided with AFLP was useful for detecting a high degree of genetic polymorphisms in the fusion plants and for quantifying the degree of asymmetric fusion. More plants with the morphology of B. oleracea were obtained with asymmetric fusion.