Plant regeneration from sugar beet leaf protoplasts: analysis of shoots by DNA fingerprinting and restriction fragment length polymorphism

Shoots were regenerated from leaf protoplasts of cytoplasmic male sterile and male fertile diploid sugar beet (Beta vulgaris L.) genotypes. Protoplasts cultured in Murashige and Skoog medium supplemented with 5 µM naphthaleneacetic acid, 2 µM 6-benzylaminopurine, 100 µM n-propyl gallate, and diamine putrescine at concentrations of 50, 100, or 500 µM were able to synthesize a new cell wall and entered successive mitotic divisions leading to the formation of callus colonies. Shoots were obtained via organogenesis by continuous culture of calli on the same basal medium supplemented with either cytokinin alone, or with a combination of cytokinin and auxin. The regenerants of both lines were characterized with regard to ploidy, and the regenerants of the male sterile line were further characterized with regard to possible somaclonal variation and organization of two mitochondrial genes: atpA and atp6. Chromosome counting revealed that tetra-, hexa-, and octa-ploids were present among regenerants. Random amplified polymorphic DNA (RAPD) analysis identified one somaclonal variant among 31 shoots tested, whereas hybridization with both mitochondrial probes showed no notable changes in the organization of mtDNA within these loci.Key words: Beta vulgaris L., protoplasts, regeneration, random amplified polymorphic DNA (RAPD), atpA, atp6.