Abstract
Chinese cabbage buds were soaked with Ethyl methanesulfonate (EMS) to induce mutagenesis. The influence of different EMS concentrations and treatment durations on microspore development, embryo production rate and seedling rate were evaluated in five Chinese cabbage genotypes. Mutations in four color-related genes were identified using high resolution melting (HRM) curves of their PCR products. The greatest embryo production and seedling rates were observed when buds were treated with 0.03 to 0.1% EMS for 5 to 10 min, while EMS concentrations greater than 0.1% were lethal to the microspores. In total, 142 mutants with distinct variations in leaf shape, leaf color, corolla size, flower color, bolting time and downy mildew resistance were identified from 475 microspore culture derived Doubled Haploids. Our results demonstrate that microspore derived Doubled Haploids from EMS soaked buds represents an efficient approach to rapidly generate homozygous Chinese cabbage mutants.
Highlights
Occurring mutations represent a major force driving evolution; they occur at an extremely slow rate
The results reveal an efficient method for the rapid production of homozygous mutants by combining chemical mutagenesis and microspore culture in Chinese cabbage
Our results demonstrate that highthroughput identification of mutations in specific genes using high resolution melting (HRM) is suitable for reverse genetic studies of Chinese cabbage
Summary
Occurring mutations represent a major force driving evolution; they occur at an extremely slow rate. Induced genetic variations represent important supplementary variation in plant breeding programs complementary to sources from natural origins (Konzak et al, 1976). Treating the seed or pollen of plants with the chemical mutagen EMS (Ethyl methanesulfonate) is one of the most commonly used approaches to generating large numbers of mutants. Favorable mutations can be identified by screening a mutagenesis library using high-throughput screening methods. EMS-induced mutagenesis is capable of establishing allelic mutations in genes via random point mutations that occur at a very high density, including nonsense, missense, splicing and cis-regulatory mutations (McCallum et al, 2000; Henikoff et al, 2004).
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