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

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 LD₅₀ 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 M₁ regenerants. The study highlights the alternative high frequency in vitro mutagenesis protocol for induction of solid mutants in Kinnow mandarin and related citrus species.