Intraspecific somatic hybridization of mango (Mangifera indica L.) through protoplast fusion

Abstract: Somatic hybridization of mango via protoplast fusion was attempted at cultivar level. Enzymatically isolated protoplasts from leaves of greenhouse-grown seedlings of cvs. 'Tommy Atkins', 'Keitt' and 'Haden' and from proembryonic masses (PEMs) of cv. 'Kensington Pride' were used. Protoplasts were fused by polyethylene glycol (PEG), embedded in Ca-alginate beads and cultured in shallow liquid culture on shaker (30 rpm). After 4 weeks, Ca-alginate beads were depolymerized and released microcolonies of PEMs were plated onto the solid culture media. After two consecutive subcultures, fast growing large clumps of PEMs were picked up and cultured as PEMs line for analyses. Flow cytometry analysis of 242 PEMs lines revealed 41 tetraploid lines. DNA fingerprinting of the regenerated embryos from the tetraploid lines showed that only four lines were somatic hybrids, all resulting from 'Haden' + 'Kensington Pride' protoplast fusions. By contrast, the tetraploid lines from 'Keitt' + 'Kensington Pride' and 'Tommy Atkins' + 'Kensington Pride' were autotetraploids. Root-tip chromosome counts on resulting germinated cotyledonary embryos confirmed that somatic hybrid embryo lines had a chromosome number of 2n=4x=80 compared to diploid parents (2n=2x=40). Of 50 deflasked somatic-hybrid, in vitro plantlets with true leaves only 3 plantlets formed the healthy apical bud (meristem) in the soil and grew normally.