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Breeding of Actinidia spp. is restricted by dioecism and polyploidy, and somatic hybridization could be useful to produce material with desirable traits from two species in this genus (Wang and Gleave, 2012). Methods for protoplast isolation have relied on the use of cell wall-degrading enzymes, i.e. Cellulase R-10 and Macerozyme R-10, etc. (Table 1.1.1). Success in plant regeneration from protoplasts has been very limited (Table 1.1.1).

Table 1.1.1. Summary of results of protoplast isolation of different Actinidia genotypes. Genotypes were corrected from the original papers and named according to the current taxonomy of the genus Actinidia (Ferguson, 2016).

Tsai (1988) isolated protoplasts from callus derived from leaves on MS medium with 4.5 μM zeatin. Protoplasts were isolated following incubation for 4–5 h at 28°C in a solution containing 2% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.5 M mannitol and 3 mM MES. Isolated protoplasts were cultured in a series of differentiation media (Tsai, 1988; Revilla et al., 1992) for cell wall regeneration, callus formation and shoot bud induction. Protoplasts from A. eriantha leaves were also obtained (Zhang et al., 1998) using 1% Cellulase R-10, 0.5% Macerozyme R-10 and 0.05% Pectolyase Y-23. Subsequently, callus was recovered and shoots were regenerated on MS medium with 2.5 μM zeatin and 0.5 μM IAA.

Oliveira and Pais (1991) used thin slices of callus derived from petioles as a source of protoplasts after incubation with cellulase and macerozyme and purification by filtration and centrifugation in a 0.6 M sucrose solution. Protoplasts were cultured using a two-step procedure with a SH-based (Schenk and Hildebrandt, 1972) medium in the presence of a complex mixture of phytohormones (NAA, 2,4-D, kinetin and BA). Culture on an MS-based medium with IAA and zeatin stimulated the recovery of shoot buds (Oliveira and Pais, 1991). Raquel and Oliveira (1996) improved the yield of protoplasts by preculturing the leaves for 5–6 days on a zeatin-containing medium.

Fraser and Harvey (1988) isolated protoplasts from microspores obtained at the tetrad stage of pollen development. Protoplasts from male plants remained viable for several weeks in culture, but those isolated from female plants did not survive more than 2 days, suggesting that those pollen grains were already not viable.

It is likely that interest in protoplast technology could be revived with the advent of new genomic technologies such as CRISPR-Cas9.