TISSUE-CULTURE AND PROTOPLAST ISOLATION IN ASPARAGUS DENSIFLORUS L.

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  • 1 Horticultural Research Center, Departement de phytologie, F.S.A.A., Université Laval, Québec, Qc., Canada, G1K 7P4

We have developed tissue culture and protoplasts isolation protocols for Asparagus densiflorus in order to use this genetic material in the breeding of Asparagus officinalis. For tissue-culture of A. densiflorus, the conditions which optimize the induction and the production of callus are a full MS medium with 1 mg/L of both pCPA and BAP and 0.5 mg/L of thiamine. HCL in the dark. on this medium, we obtained a friable white callus. Indirect organogenesis was obtained if pCPA was omitted from the medium. Replacement of the plant growth regulators by 2,4-D and Kinetin produced a hard and compact callus which did not differentiate. Protoplast have been isolated from 10 days old friable callus. cell wall was digested with 0.3% macerase, 1% cellulase and 0.8% rhozyme for a period of 16h at a temperature of 27°C in a CPW medium. Protoplast yield was 2 ×106 protoplasts/g callus. osmolarity of the digestion solution was 0.8 M provided with a mixture of glucose (0.6 M) and mannitol (0.2 M). cells were then plated at a density of 1 × 105 cells per ml. Microcolonies formed on a 1/2 MS medium with 0,5 mg/L NAA and ZEA and 1 g/L glutamine in the dark.

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