Production of somatic hybrids between Asparagus officinalis and A. amarus to transfer Asparagus virus 1 (AV-i) resistance

摘要

Interspecific somatic hybridization was performed between suspension protoplasts of Asparagus officinalis 'Rapsody' (2n=2x=20) and the wild relative A. amarus (2n=6x=60) by chemical fusion with polyethylene glycol. The A. amarus protoplast donor is resistant to Asparagus virus 1 (AV-1), whereas A. officinalis is highly susceptible. Within 5 weeks, fused protoplasts developed into microcalli in liquid medium PPM1 supplemented with 1 g L 1 L-glufamine. Soon after the transfer to solid medium Murashigeand Skoog (MS) + 1 mg L1 2,4-dichlorophenoxyacetic acid, calli showed embryoid-like structures and caulogenesis. Rooting of 78 plants took place on MS with 0.2 mg L1 ct-naphthalene acetic acid. The plants were then transferred to soil. The hybrid character of the plants was verified by RAPD showing the presence of fragments of both protoplast donors in the somatic hybrids. However, the number of donor fragments varied between the somatic hybrids. The cytoplasmic genome was investigated with CAPS markersspecific for cp and mt genes, respectively. 73 plants revealed a cytoplasmic genome derived from A. officinalis. The five remaining plants showed a contribution of both protoplast donors to the cytoplasmic genome. The DNA content was evaluated by flow cytometry. DNA amount of somatic Asparagus hybrids and the protoplast donors was estimated in relation to the internal DNA reference Pisum sativum 'Ctirad' (9.09 pg). In relation to the internal reference A. amarus suspension cell clusters had 7.24 pg andA. officinalis suspension 3.43 pg. The DNA content of the somatic hybrids was estimated between 11.69 and 13.77 pg. The habitus of the hybrid plants was similar to A. amarus. Growth disorders were not observed. For resistance evaluation 40 primary fusion plants were infected with AV-1 via the aphids Myzus persicae. For resistance assessment DAS-ELISA was performed. All 40 tested somatic hybrids were evaluated as resistant to AV-1.