Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant

Abstract

Traditionally, mutagenesis has been used to introduce novel genetic variability in ornamental crops. More recently, it has become a powerful tool in gene discovery and functional analyses in reverse genetics approaches. The present work aimed to compare the efficiency of physical and chemical agents in generating mutant populations of petunia. We have indirectly evaluated the genomic damage by analyzing developmental characteristics of the plantlets derived from treated seeds employing gamma radiation at 0, 20, 40, 60, 80 and 100 Gy and the alkylating agent ethyl-methanesulfonate (EMS) at 0, 0.05, 0.1, 0.15, 0.2 and 0.25% (v/v). Gamma rays and EMS caused developmental defects and decreased seedling viability in plants obtained from the mutagenized seeds. High mutagen doses reduced in approximately 44% the number of plants with primary leaves at 15 days after sowing (DAS) and decreased seedling survival rates to 55% (gamma) and 28% (EMS), in comparison to untreated controls. Seedling height decrease was proportional to increasing EMS dosage, whereas 40 and 60 Gy of gamma irradiation caused the most significant reduction in height. Moderate DNA damage allowing a high saturation of mutant alleles in the genome and the generation of viable plants for reverse genetics studies was correlated to the biological parameter LD 50 , the dose required to kill half of the tested population. It corresponded to 100 Gy for gamma radiation and 0.1% for EMS treatment. The optimized mutagen treatments were used to develop petunia mutant populations (M 1 and M 2 ) and novel morphological mutants were identified. Key words: EMS, gamma irradiation, genomic damage, mutagenesis, reverse genetics Mutagênese em Petunia x hybrida Vilm. e isolamento de um novo mutante morfológico. A mutagênese tem sido tradicionalmente usada para gerar variabilidade genética em plantas ornamentais. Recentemente, tornou-se uma ferramenta poderosa na descoberta e análise da função gênica em genética reversa. Este trabalho objetivou comparar a eficiência da mutagênese física e química na geração de populações mutantes de petúnia. O dano genômico foi avaliado indiretamente por características de desenvolvimento de plântulas após o tratamento com doses de radiação gama de 0, 20, 40, 60, 80 e 100 Gy e do agente alquilante etil-metanossulfonato (EMS) de 0; 0,05; 0,1; 0,15; 0,2 e 0,25% (v/v). Radiação gama e EMS causaram danos ao desenvolvimento e reduziram a viabilidade das plântulas derivadas das sementes tratadas. As maiores doses de mutagênico diminuíram o número de plantas com folhas primárias aos 15 dias após a semeadura (DAS) em aproximadamente 44% e reduziram as taxas de sobrevivência a 55% (gama) e 28% (EMS) em relação aos controles. A redução na altura das plântulas foi proporcional ao aumento das dosagens de EMS, enquanto 40 e 60 Gy de radiação gama provocaram a redução mais significativa na altura de plantas. Abordagens de genética reversa requerem danos genômicos moderados, que permitam alta saturação de alelos mutantes com pequena redução no número de...

A reverse genetics approach identifies novel mutants in light responses and anthocyanin metabolism in petunia

“…The identification of photoreceptor mutants by forward genetic approaches requires monochromatic light conditions and intensive experimental care (Fankhauser and Casal 2004). The identified sequences may provide important reverse genetic tools for further indepth investigation of available petunia mutant populations (Berenschot et al 2008;Vandenbussche et al 2008).…”

“…'Mitchell Diploid' flowers are white and were deliberately chosen to investigate the efficiency of mutant identification in the absence of visual color phenotypes; thus, proving the concept of reverse genetics screening. Two mutant populations were independently generated by gamma irradiation and EMS treatment and evaluated for moderate DNA damage allowing a high saturation of mutant alleles (Berenschot et al 2008). Seeds representing 100 progenies (M2 populations) from selfpollinations were sown and 5,000 plants per population (M3) were grown in trays under standard greenhouse conditions.…”

“…In a previous study, two saturated mutant populations of petunia were generated by chemical (EMS, ethyl-methane sulfonate) and physical (gamma irradiation) mutagenesis and crossed to generate M2 families (Berenschot et al 2008). The sequences of anthocyanin metabolism and light signaling identified in this study were used to design gene-specific primers to screen the populations for mutations in the genes of interest by a high-throughput PCR-based method, in the absence of direct phenotypic screenings.…”

“…The use of bioinformatic and comparative genomic tools allowed the establishment of the genetic framework for photoperception, light signaling and anthocyanin metabolism in petunia. The compiled sequences were used in a PCR-based reverse genetics approach to identify mutant alleles of the genes of interest in two M3 populations of Petunia x hybrida Mitchell Diploid, generated by gamma irradiation and treatment with the alkylating agent, ethyl methanesulfonate (EMS) (Berenschot et al 2008). Novel mutant alleles were identified for genes involved in anthocyanin biosynthesis (CHALCONE SYNTHASE ) and regulation (PH4), and light signaling (CONSTANS ) in the absence of phenotypic screenings.…”

Optimization of EMS Mutagenesis on Petunia for TILLING

“…The TILLING of petunia for mutant alleles in light signaling and anthocyanin metabolism genes was recently reported [14]. There are published protocols for the mutagenesis of petunia with ethyl methanesulphonate (EMS), but they vary in terms of concentration (0.1% to 0.5%), exposure time (10 to 24 hours), and seed pre-treatment [15][16][17]. In this paper, we describe the optimization of mutation parameters, the development of an EMSmutagenized population that could facilitate TILLING.…”