EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Acanda, Yosvanis; Martı́nez, Óscar; Prado, María Jesús; González, María Victoria; Rey, Manuel

doi:10.1007/s00299-013-1547-6

Cited by 11 publications

(6 citation statements)

References 37 publications

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“…2). This appears to be unique to Indel mutations because this phenomenon was never reported in HRM-TILLING studies characterizing single base-pair substitutions (Gady et al, 2009;Botticella et al, 2011;Lochlainn et al, 2011;Acanda et al, 2014;Bovina et al, 2014). However, in HRM analysis of nine Indel mutations using mixed samples, Cousins et al (2013) also observed one mutation (1-bp deletion) that performed similarly to GZ63m1 and Z9B-lpa, i.e.…”

Section: Detection Of Indel Mutations In Pooled Samplesmentioning

confidence: 66%

“…HRM has already been widely used in plant research for high-throughput genotyping, mapping genes, and testing food products and seeds (Simko, 2016). It has also been used in combination with TILLING (HRM-TILLING) for screening EMSmutagenized populations of tomato (Gady et al, 2009), wheat (Dong et al, 2009;Lochlainn et al, 2011), and grapevine (Acanda et al, 2014). As with most other TILLING systems, HRM-TILLING has been used only for the screening of EMS-induced mutations.…”

Section: Introductionmentioning

confidence: 99%

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High-resolution melting-based TILLING of γ ray-induced mutations in rice

Liu

Fu³

et al. 2018

J. Zhejiang Univ. Sci. B

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Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

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Section: Detection Of Indel Mutations In Pooled Samplesmentioning

confidence: 66%

Section: Introductionmentioning

confidence: 99%

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Liu

Fu³

et al. 2018

J. Zhejiang Univ. Sci. B

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“…Suspension cultures can be synchronized [44] to obtain a homologous population of cells, thereby allowing experimentation on cell physiology, biochemistry, and metabolic events at the cellular level. A cell suspension culture can also aid in mutagenesis studies using CRISPR/Cas9 [45] or chemicals such as ethyl methanesulfonate [46]. The only downside to the system proposed here is that plant regeneration cannot occur directly from suspension cultures, which requires an extra solidified tissue culture step prior to plant regeneration.…”

Section: Resultsmentioning

confidence: 99%

Improved tissue culture conditions for the emerging C4 model Panicum hallii

et al. 2017

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Background Panicum hallii Vasey (Hall’s panicgrass) is a compact, perennial C4 grass in the family Poaceae, which has potential to enable bioenergy research for switchgrass (Panicum virgatum L.). Unlike P. hallii, switchgrass has a large genome, allopolyploidy, self-incompatibility, a long life cycle, and large stature—all suboptimal traits for rapid genetics research. Herein we improved tissue culture methodologies for two inbred P. hallii populations: FIL2 and HAL2, to enable further development of P. hallii as a model C4 plant.ResultsThe optimal seed-derived callus induction medium was determined to be Murashige and Skoog (MS) medium supplemented with 40 mg L−1 L-cysteine, 300 mg L−1 L-proline, 3% sucrose, 1 g L−1 casein hydrolysate, 3 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), and 45 μg L−1 6-benzylaminopurine (BAP), which resulted in callus induction of 51 ± 29% for FIL2 and 81 ± 19% for HAL2. The optimal inflorescence-derived callus induction was observed on MP medium (MS medium supplemented with 2 g L−1 L-proline, 3% maltose, 5 mg L−1 2,4-D, and 500 μg L−1 BAP), resulting in callus induction of 100 ± 0.0% for FIL2 and 84 ± 2.4% for HAL2. Shoot regeneration rates of 11.5 ± 0.8 shoots/gram for FIL2 and 11.3 ± 0.6 shoots/gram for HAL2 were achieved using seed-induced callus, whereas shoot regeneration rates of 26.2 ± 2.6 shoots/gram for FIL2 and 29.3 ± 3.6 shoots/gram for HAL2 were achieved from inflorescence-induced callus. Further, cell suspension cultures of P. hallii were established from seed-derived callus, providing faster generation of callus tissue compared with culture using solidified media (1.41-fold increase for FIL2 and 3.00-fold increase for HAL2).ConclusionsAside from abbreviated tissue culture times from callus induction to plant regeneration for HAL2, we noted no apparent differences between FIL2 and HAL2 populations in tissue culture performance. For both populations, the cell suspension cultures outperformed tissue cultures on solidified media. Using the methods developed in this work, P. hallii callus was induced from seeds immediately after harvest in a shorter time and with higher frequencies than switchgrass. For clonal propagation, P. hallii callus was established from R1 inflorescences, similar to switchgrass, which further strengthens the potential of this plant as a C4 model for genetic studies. The rapid cycling (seed-to-seed time) and ease of culture, further demonstrate the potential utility of P. hallii as a C4 model plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0359-0) contains supplementary material, which is available to authorized users.

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“…This finding was observed in more than 15 plant species [4]. Moreover, Acanda et al [18] found that these transitions caused multiple alleles within a gene, leading to multiple phenotypes. Point mutations occur in the exonic regions [19], with higher methylation rates in the 5' regions [20].…”

Section: Mechanisms Of Action Of Emsmentioning

confidence: 89%

Ethyl Methanesulfonate as Inductor of Somaclonal Variants in Different Crops

Joya-Dávila¹,

Gutiérrez-Miceli²

2020

Phyton

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Ethyl methanesulfonate is a chemical mutagen, which is currently being used in plant breeding, to increase genetic variability in genes of agronomic interest, of species useful in agriculture. It primarily causes single base point mutations by inducing guanine alkylation, resulting in GC to AT transitions. Its effect is different between clones of a genotype and between genotypes of the same species. This review presents the results obtained in recent research, where its effect on plant tissues, callus, and cells in suspension has been evaluated. Changes in the phenotypic expression of somaclonal variants were reported, involving morphology, production of secondary metabolites, changes in metabolic routes of resistance, tolerance to stress, increased seed yield, among others. In addition, this review compiles the doses and guidelines to consider before using this mutagen, which can serve as a guide for future trials in deciding the response variables, the type of plant explants and the selection of the study model. Mutant lines have allowed plant breeders to have a collection of plants with different characteristics, in places where the cultivar does not have its center of origin. It is important to note that it is still necessary to continue evaluating the heritability of mutations and their behaviour in the environment where they will be established, in order to obtain new varieties of plants that can be cultivated with uniformity in their genetic response.

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EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Cited by 11 publications

References 37 publications

High-resolution melting-based TILLING of γ ray-induced mutations in rice

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Improved tissue culture conditions for the emerging C4 model Panicum hallii

Ethyl Methanesulfonate as Inductor of Somaclonal Variants in Different Crops

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