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A protocol for protoplast isolation and culture was developed by Encina (2004) using hyperhydric leaves and etiolated hypocotyl explants of A. cherimola ‘Fino de Jete’. The protoplasts were isolated using CPW salts plus 0.6 M mannitol and 3 mM methyl ethane sulfonate (MES) together with an enzyme solution consisting in 1.2% Cellulase Onozuka RS, 0.2% Macerozyme R10 and 0.25% Pectolyase Y23. Protoplasts were cultured for 5 days in B5 liquid medium (Gamborg et al., 1968), supplemented with 0.6 M mannitol, 0.09 M sucrose, 5 μM 2,4-D, 3 μM BA, 1 μM zeatin, 2 μM NAA and 1% dimethyl sulfoxide (DMSO) until the development of microcolonies. The microcalluses were developed and regenerated at low rate (0.5%) only from protoplasts derived from the hypocotyl.

A reproducible and more efficient protocol for the isolation and culture of both hypocotyl and leaf protoplasts has been described (Padilla et al., 2018). In both cases, protoplast yield was similar and c.4 × 10⁶ protoplast/g FW and >90% of protoplast viability was obtained (Fig. 3.1.3a). A protoplast-to-plant regeneration protocol from both explant types is under development.

Fig. 3.1.3. (a) Hypocotyl protoplast culture with protoplast in division from Annona cherimola. (b) Shoot regeneration from hypocotyl explants of A. cherimola.