In vitro mutagenesis using habituation and PBR autotrophy based indirect somatic embryogenesis (ISE) system in Kinnow mandarin.

Solid mutant induction using specialized habituation and PBR (Plant bio-regulator) autotrophy-mediated suspension-based ISE system was the prime aim of present investigation. Based on survival of cell clumps after mutagen treatment, the probit analysis was calculated. The result revealed LD at 54.31 Gy in gamma, while for EMS (ethyl methanesulfonate), it was 0.1% for 3 h and 0.5% for 1 h. Based on embryogenesis efficiency, a dose rate of 100 Gy and 0.1% EMS for a 3-h exposure were selected for regeneration. As compared to control, significant decrease in the embryogenesis efficiency was recorded at 100 Gy (85.92%) with similar reduction trends in embryo production (79.49%), germination (13.43%), conversion (2.48%), establishment (15.78%) and acclimatization (60.92%). The growth-related parameters such as root and shoot length and number of leaves/regenerant were also significantly reduced to 67.29%, 30.19% and 5.03%, respectively, in the regenerated plants after gamma irradiation as compared to control. In the EMS treatment, at the dose rate of 0.1% for 3-h, the embryogenesis efficiency was reduced to 43.67% with similar diminution trends in embryo production (59.49%), germination (8.95%), conversion (1.94%), establishment (4.37%) and acclimatization (29.9%). The growth-related parameters in the EMS treatment, decreased to 91.00% (root length), 71.34% (shoot length) and 35.03% (no. of leaves). The molecular marker based varied amplifications confirmed the occurrence of mutations in both gamma and EMS induced M regenerants. The study highlights the alternative high frequency in vitro mutagenesis protocol for induction of solid mutants in Kinnow mandarin and related citrus species.