Somatic hybridization for transfer of resistance against Asparagus virus 1 (AV-1) from Asparagus amarus into A. officinalis

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.