Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Abstract

An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 μM atrazine. Its chromosome number was 36-38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.

Mitochondrial DNA rearrangements and transcriptional alterations in the male-sterile cytoplasm of Ogura radish.

“…(ii) Somatic hybrids can be made between Brassica species by protoplast fusion and plant regeneration (36). Somatic hybrids between plants with the CMS Ogura cytoplasm and Brassica campestris have been shown to contain recombinant mitochondrial DNAs (mtDNAs) (9,37). (iii) The mtDNAs of Brassica species are the smallest and among the best-characterized flowering plant mitochondrial genomes.…”

Plant regeneration from cotyledon protoplasts of Brassica carinata

“…The development of protoplast technology for B. napus, B. oleracea and B. rapa has permitted the resynthesis of oilseed rape [22,24,25], the production of somatic hybrids between B. napus and wild cruciferous species [6,19], and the formation of novel cybrids [3]. However, application of these approaches to the improvement of B. carinata has been hindered by lack of information on protoplast culture and plant regeneration.…”

Cauliflower and Broccoli

“…Efficiency of mass fusions of Brassica protoplasts for plant regeneration has been obtained by using polyethylene glycol Morgan and Maliga, 1987;Robertson et al, 1987). Several methods have been proposed for selecting the desired heterokaryons but the more interesting are based on flow cytometry to individuate the new genetic combinations cells which will be sorted, or on pretreating one parental with iodoacetate or iodoacetamide and irradiating the other parental (Sidorov et al, 1981;Gerdemann-Knörck et al, 1994;O'Neill et al, 1996).…”