Genetic variations in rice in vitro cultures at the EPSPs–RPS20 region

Abstract

In vitro cultures of plant cells have often been utilized to generate genetic variations, which are designated somaclonal variations. Little is known about the major genetic alterations in the cultured cells and the nature of these genetic changes. Here, we examined different lines of rice Oc cells that have been cultured for more than 20 years on agar media or in liquid media. We surveyed 35 clones obtained from PCR amplification of the 3-kb EPSPs-RPS20 region. The sequence divergence among the Oc cells was even greater than that between Japonica and Indica rice cultivars. The divergent sequences appeared to be maintained as multiple copies in a single cell. Surprisingly, the nucleotide substitutions in the Oc cells were characterized by an extremely high frequency of transition mutations of A/T-to-G/C, a feature which is similar to that of the mutations caused by chemical mutagens such as 5-bromouracil and 2-aminopurine. Although no replacements in the exons of this region were observed among the AA-genome Oryza species, our results revealed that the nucleotide substitutions of the cultured cell lines occurred more frequently at replacement sites in the exons than at synonymous sites. These distinct mutation biases found in rice in vitro cultures might contribute importantly to somaclonal variations.

Age-associated alterations in the somatic mutation level in Arabidopsis thaliana

“…In our analysis, we found that most of the detected single nucleotide substitutions that accumulated during A. thaliana growth and aging were A:T?G:C transitions, which suggests that the transitions did not result from an ultraviolet light-induced mutagenesis. The data correlate with observations that most of the single nucleotide substitutions that accumulated during the long-term cultivation of Panax ginseng and Oryza sativa callus cultures were also A:T?G:C transitions (Noro et al 2007;Kiselev et al 2009Kiselev et al , 2011Kiselev et al , 2013. However, in contrast to the somatic mutagenesis observed for the A. thaliana plants in this study, no insertions or deletions were detected in the endogenous gene sequences analyzed for P. ginseng in cell cultures that had been propagated for more than 20 years (Kiselev et al 2009(Kiselev et al , 2011(Kiselev et al , 2013.…”

“…The authors suggest that even long-lived clonal plants are vulnerable to senescence. It has also been shown that single nucleotide substitutions accumulate in several endogenous genes and transgenes during the long-term cultivation of plant cell cultures (Noro et al 2007;Kiselev et al 2009Kiselev et al , 2011Kiselev et al , 2013. To our knowledge, there is no other experimental data where somatic mutagenesis has been investigated in relation to ageing in plants or plant cell cultures.…”

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

“…Growth of transgenic regenerants often suffers from the effects of environmental factors such as humidity, temperature, macro-and micro-nutrient deficiencies or excesses, and microbes (pathogens), especially in the initial growth stage after transfer to soil. Alternatively, in the course of tissue culture for selection and regeneration of transgenic plants, a variety of both epigenetic (e.g., DNA methylation, gene silencing, activation of retrotransposons and transposable elements) and genetic (e.g., nucleotide substitutions, deletions, insertions, rearrangements) changes could happen in the plant genome (Kaeppler et al 2000;Cheng et al 2006;Noro et al 2007). These factors, i.e., abiotic and biotic factors and tissue-culture-induced variations, may more or less influence the growth conditions and consequently the phenotypes of FOX plants, and may somewhat increase the rate of phenotype alteration in our FOX lines.…”

Development and Identification of Transgenic Rice Lines with Abiotic Stress Tolerance by using a Full-length Overexpressor Gene Hunting System

“…This is consistent with a previous report (Nakamura et al 2007) and underscores the notion that transgenic regenerants often suffer from the effects of environmental factors such as humidity, temperature, macro-and micro-nutrient deficiencies or excesses, and microbes (pathogens), especially in the initial growth stage after transfer to soil. Alternatively, during tissue culture for selection and regeneration of transgenic plants, a variety of both epigenetic (e.g., DNA methylation, gene silencing, activation of retrotransposons and transposable elements) and genetic (e.g., nucleotide substitutions, deletions, insertions, rearrangements) alterations may occur in the plant genome (Kaeppler et al 2000;Cheng et al 2006;Noro et al 2007). These factors, i.e., abiotic and biotic factors and tissue culture-induced variations, may influence the growth conditions to some degree, and consequently, alter the phenotypes of transgenic plants.…”