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Breeding objectives. As polyploidy and dioecy in Actinidia restrict breeding, somatic hybridization could enable recovery of fertile hybrids, by combining genetic backgrounds within the same gender. After initial attempts (Lindsay et al., 1995), somatic hybridization of A. deliciosa with A. chinensis (6x + 2x) and A. chinensis with A. kolomikta (2x + 2x) was achieved (Xiao and Han, 1997; Xiao et al., 2004). Protoplasts of A. deliciosa and A. chinensis were isolated from cotyledon callus, while those of A. kolomikta were from the youngest fully expanded leaves of micropropagated shoots.

PROTOCOL. Purified protoplasts of A. deliciosa and A. chinensis were mixed in equal volumes, with 0.2 ml of this mixture being transferred into a glass tube with an equal volume of a mixture (8:1:1) of a polyethylene glycol (PEG) solution (40% PEG 6000, 60 mM CaCl₂, 0.3 M sucrose), glycine buffer (pH 10.3) and dimethylsulfoxide (DMSO). After a short incubation, 5 ml of a dilution solution (W5 with 50 mM 2-N-morpholine ethane sulfonic acid, pH 5.6) was added. After 1 h, the protoplast mixture was centrifuged and briefly incubated in 2 ml of the dilution solution, and the protoplasts were cultured in fresh medium (Nitsch and Nitsch, 1969) supplemented with 4.5 mM 2,4-D, 1.1 mM zeatin, 200 mg/l casein hydrolysate, 1% sucrose, 0.2 M glucose and 0.4 M mannitol.

Some modifications were made for the hybridization of A. chinensis and A. kolomikta. The purified protoplasts were mixed and diluted in W5 solution (154 mM NaCl, 5 mM glucose, 125 mM CaCl₂·2H₂O and 5 mM KCl, pH 5.6) in a ratio of 1:2 and two drops of the protoplast mixture were plated with a spacing of 3–5 mm. After 5 min, two drops of PEG solution were added and incubated for 5 min before two drops of W10 buffer (nine parts of stock A with one part of stock B, prepared freshly; stock A: 10% DMSO, 0.06 M CaCl₂ and 0.3 M mannitol; stock B: 1 M glycine–NaOH buffer, pH 10.5) were added. The mixture was gradually diluted (5 min) with 2 ml of dilution solution (W5 with 50 mM MES). The solution was replaced 20 min later, and the protoplasts were cultured with 2 ml of fresh protoplast medium.

ACCOMPLISHMENTS. Somatic hybrids were obtained and confirmed by RAPD analysis, flow cytometry and intermediate morphology characters (Xiao and Han, 1997; Xiao et al., 2004). Ploidy levels of A. chinensis + A. deliciosa hybrids were octoploid (8x), and one plant had RAPD banding patterns combining parental banding profiles. Four A. chinensis + A. kolomikta plants also had a combination of parental RAPD banding profiles, and the regenerated plants were triploid (3x), tetraploid (4x) or pentaploid (5x). The somatic hybrids between A. chinensis and A. kolomikta were tetraploid (4x) and also showed a combination of parental RAPD banding profiles. As A. kolomikta can grow in higher latitudes than A. chinensis, traits related to cold tolerance were screened. The somatic hybrids displayed values close to A. kolomikta for these traits, suggesting acquisition of chilling tolerance.