PROTOPLAST ISOLATION AND FUSION BETWEEN BRASSICA CARINATA BRAUN. AND BRASSICA RAPA L.

M. BERÁNEK, M. BECHYNĚ, M. KLÍMA

Abstract
Brassica carinata mesophyll and B. rapa hypocotyl protoplasts were used for fusing experiments using polyethylene glycol (PEG). Treatment with 30% PEG 6000 MW for 10 minutes was more effective, while combinations with 30% PEG 6000 MW/15 min, 30% PEG 4000 MW/15 min and 30% PEG 4000 MW for 10 minutes reduced viability of protoplasts and cell wall regeneration, cell division and production of microcolonies. Fusion frequency was about 25% with the treatment of 30% PEG 6000 MW/10 min. Cell colonies forming callus was observed after 14 days of cultivation on liquid B medium with addition of liquid C medium. Microcalli were subsequently transferred to solid E and F medium. Shoot regeneration from calli was induced via indirect organogenesis and somatic embryogenesis on MS hormon free medium. In total 58 calli were obtained from fusions between B. rapa 31/96 and B. carinata line 1; 14 calli produced shoots. According to the morfological diferencies half of shoots were heterocaryonicals. This observation approved the events from Flow cytometry. Approximately 40% of analyzed samples were only B. carinata plant type established via protoplasts fusion, 60% were of B. carinata and B. rapa.

Key words:
PEG, protoplast, Brassica, fusion Abbreviations: PEG, Polyethylene glycol; MS, Murashige and Skoog (1962)

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