Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 199

Papaya protoplast technology was developed to combine genomes of species or lines that had hybridization barriers. Litz and Conover (1979) described the isolation of V. cauliflora and V. stipulata leaf mesophyll protoplasts that could be used to fuse with those of papaya to develop PRSV-resistant progeny. Isolation of protoplasts of papaya was also reported by Krishnamurthy et al. (1983). Protoplasts from papaya and the PRSV-resistant V. pubescens were isolated and fused. Leaves of young greenhouse-grown plants of V. pubescens were disinfested and placed into osmoticum (mannitol or sucrose) followed by incubation in medium containing ‘Onosuka’-R 10 and Macerozyme-R, mannitol, 2-(N-morpholino)ethane sulfonic acid (MES) and CaCl₂ (Jordan et al., 1986). Papaya protoplasts were prepared from calluses generated from hypocotyls. Protoplast yields were determined by enzyme concentration, digestion temperatures, nutrient media and tissue types. Protoplasts of the two species were exposed to PEG 20,000 osmoticum prior to plating. Mixtures of the two species that were plated on modified MS (Murashige and Skoog, 1962) resulted in some fused cells. Although fused protoplasts of each species and early cell cluster formation of V. pubescens were observed, further development of fused papaya and V. pubescens protoplasts was not reported.

Using embryogenic suspensions of papaya × V. cauliflora (Chen et al., 1991), protoplasts were isolated and cultured, and plants were regenerated (Chen and Chen, 1992). Immature zygotic embryos of the hybrid were cultured on modified MS medium containing 3.8 μM abscisic acid (ABA) (Chen et al., 1991), and embryogenic suspension cultures were recovered. The suspensions were incubated with Cellulase R-10, Macerozyme R-10, Driselase and mannitol. Protoplasts were cultured in liquid medium for 14 days and plated on semisolid medium for somatic embryo development. Chromosome counts indicated that the regenerants were normal diploids (2n = 18).