ASEPTIC ISOLATION OF LEAF PROTOPLASTS FROM CITRUS, PONCIRUS, CITRUSXPONCIRUS HYBRIDS AND SEVERINIA FOR USE IN SOMATIC HYBRIDIZATION EXPERIMENTS

An efficient protocol has been developed for the aseptic isolation of leaf protoplasts from Citrus and citrus relatives. Consistently high yields (2–6 × 107 protoplasts/g fresh weight tissue) of leaf protoplasts were isolated from new leaves taken from vigorously growing plants maintained in a growth chamber. Critical parameters included leaf decontamination and reconditioning, the composition of the enzyme cocktail, vacuum infiltration, incubation and clean-up of the resulting protoplasts. The use of leaf protoplasts in fusions with protoplasts isolated from embryogenic callus allowed for the visual identification of heterokaryons and suggests the possible utilization of additional physiological parameters in somatic hybridization selection schemes. Moreover, the number of genotypes which can be utilized in citrus fusion experiments has been greatly increased.