Production of intergeneric hybrid calli from C₃ and C₄ species of Amaranthaceae through protoplast fusion

Intergeneric somatic hybrid calli were obtained through electrofusion between protoplasts of Celosia cristata L. (C₃ species) cell suspension and Amaranthus tricolor L. (C₄ species) cotyledon. The highest binary fusion frequency, 4~5%, was attained when the protoplasts were suspended in fusion solution containing 0.4 M sorbitol and 2.0 mM CaCl₂, aligned by an AC field (200 V/cm, 0.5 MHz) for 30 s and fused by a single DC square pulse at 1.6 kV/cm for 40 μs. Fusion products were identified by characteristics of both parents in the hybrid cells : they possessed green chloroplasts or pink plastids from cotyledonary protoplasts as well as a cytoplasm-rich colorless sector derived from cell suspension protoplasts. The occurrence of two types of calli, compact and friable, obtained after culturing on proliferation medium lead to the isolation of putative hybrid calli without any artificial selection because C. tristate protoplasts regenerated only friable calli and A. tricolor protoplasts did not product calli. A total of 14 lines of putative hybrid compact calli were obtained by screening on the basis of callus morphology. An isozyme analysis of acid phosphatase confirmed the hybridity of 14 lines of compact calli. Callus morphology was also studied by scanning electron microscopy.