Characterization and molecular analysis of transgenic plants obtained by microprotoplast fusion in sunflower

Asymmetric somatic hybrid (ASH) plants were obtained by PEG-mediated mass f usion of microprotoplasts from perennial Helianthus species and hypocotyl p rotoplasts of Helianthus annuus. The formation of micronuclei in perennial sunflower cell cultures was induced, at early log phase, by addition of the herbicides amiprophosmethyl or oryzalin. Sub-diploid microprotoplasts were isolated by high-speed centrifugation and the smallest enriched by sequent ial filtration through nylon sieves of decreasing pore size. Fusion product s were cultured and the regenerated plants phenotypically, genetically and cytologically characterized. DNA analysis using RAPD markers revealed that 28 out of 53 regenerated plants were asymmetric hybrids. Subsequent nuclear -DNA flow cytometric analysis showed that these plants had a higher DNA con tent than the receptor H. annuus, suggesting that they represented addition lines. Cytological investigation of the metaphase cells of 16 hybrids reve aled an addition of 2-8 extra chromosomes in these plants. The phenotype of most ASH plants resembled H. annuus. These results indicate that micronucl ear induction and asymmetric somatic hybridization represent a potent tool for partial genome transfer aimed at the specific transfer of economically important traits in breeding programs.