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)