In vitro Mutation of Neolamarckia cadamba (Kelampayan) Using Ethyl Methanesulfonate (EMS)

Delvino Disonneo, Dalin (2022) In vitro Mutation of Neolamarckia cadamba (Kelampayan) Using Ethyl Methanesulfonate (EMS). Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Neolamarckia cadamba is a lightweight hardwood timber tree species that is known to be fast-growing. Increasing demand for high quality planting material promotes the need for producing new variations. In vitro mutagenesis of N. cadamba by using ethyl methanesufonate (EMS) could accelerate the natural mutation and breeding programme for superior planting material. Thus, the current study aimed to determine the lethal concentration 50 (LC50) of EMS, evaluate responses of EMS-treated N. cadamba explants, and screen putative mutants using gene-specific markers. Nodal explants of N. cadamba were subjected to 0 (control), 0.1, 0.3, 0.6, and 1% EMS for 1-, 2-, 3-, and 4-hour treatments. The LC50 of EMS treatments 1.2589%, 0.3715%, and 0.3311% for 2-, 3-, and 4-hour treatment, respectively. Variability of responses among the treated explants were observed by the increasing concentrations of EMS and duration of treatments. Percentages of survival, shoot formation, and callus formation were reduced, while no clear trend was observed from plant heights, rooting percentages, and survival after the acclimatization stage. Morphological abnormality among the survived explants was also identified in the current study. The SNPs analysis in cellulose synthase gene from three mutation lines revealed that 166 SNPs were found in C1 (Tajima’s D = -0.5612, π = 0.04235), 138 SNPs in C3 (D = -2.34147, π = 0.00567), and 82 SNPs in C5 (D = -1.83398, π = 0.00480). Meanwhile for sucrose synthase gene, 10 SNPs were screened in C3 (D = -1.50776, π = 0.00093), and 5 SNPs in C5 (D = -0.02839, π = 0.00083). From this molecular DNA marker-assisted screening, 46 putative mutant seedlings were identified (C1 = 12, C3 = 17, and C5 = 17). The current in vitro mutagenesis approach has shown the effectiveness of using EMS to increase genetic variability of N. cadamba trees, mimicking natural mutation process in the wild population. Current molecular DNA marker-assisted screening approach is also considered to be practical in mutant screening.

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