EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

Abstract

BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin.ResultsHere, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified.ConclusionsThis new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Kill curve analysis and response of first generation Capsicum annuum L. B12 cultivar to ethyl methane sulfonate

“…Thus, mutagenesis can affect the percent germination (Serrat et al, 2014). As shown in Figure 1, a significant reduction in seed germination was observed with increasing EMS concentrations (P ≤ 0.05).…”

“…That may be due to the use of different presoaking treatments before EMS treatment, which may affect the quantity of EMS absorbed by the germinating embryo. Injury is also dependent on the quantity of EMS that reaches the meristematic region where the germ cells are contained (Serrat et al, 2014). In cotton plants, Aslam et al (2013) observed that the percent of injury increased with increasing EMS concentration.…”

“…It produces random mutations in genetic material by nucleotide substitution with base pair transition (G:C to A:T), thereby producing only point mutations without killing the plants (Greene et al, 2003). In seed-propagated plants, such as peppers, chemical mutagenesis protocols use seeds during the germination process, thus causing the mutagen to be absorbed by the germinating embryo and reach the meristematic region, where the germ cell is present (Serrat et al, 2014). The EMS concentration that yields 50% seed lethality (LD 50 ) is used as an indicator of high mutation frequency (Devi and Selvakumar, 2013).…”

Identification of variability for agronomically important traits in rice mutant families

“…Mutation induction assists breeding programs by increasing variability and therefore obtaining genotypes with agronomically important traits, more adapted to different environmental conditions (Serrat et al 2014). The spontaneous mutation rate in higher plants is very low, ranging from about 10 −5 to 10 −8 (Jiang and Ramachandran 2010).…”

Mutagenic effectiveness and efficiency of Gamma rays and EMS in Lablab purpureus (L.) Sweet var. typicus

“…Mutagenesis provides a powerful technique to improve plant breeding and assist functional and genomic analyses of crop plants. This technique was first introduced with the use of x-ray and radium radiations followed by fast neutron and gamma radiation (Serrat et al, 2014). Because such application of physical mutagens required specialized equipment, chemical mutagens were introduced later.…”