Heritable Genomic Variation Induced By Tissue Culture And Associated Phenotypic Changes Under Abiotic Conditions In Rice

Plant tissue culture technology, which was invented in the beginning of 20 th century, is widely-used in plant sciences such as micropropagation, conservation of valuable and rare species, improvement and innovation of germplasm. It has created great social benefits and commercial value. Somaclonal variation generally occurs in plants regenerated from tissue culture. Nevertheless, fundamental issues regarding molecular characteristics, mutation rates and mutation spectra of plant somatic variation as well as their phenotypic relevance have been addressed only recently.Moreover, these previous studies often found highly discrepant results in different plant species, and even in the same plant genotype.In this thesis, whole genome sequencing was performed in selfed somaclonal line TC-reg-2008 and its wild type donor WT(Oryza sativa ssp. japonica cv. Hitomebore)by next-generation sequencing technology. The rate of overall tissue culture induced mutation, single nucleotide polymorphisms(SNPs), small scale insertion and deletions(In Dels), and mobilization of transposable elements(TEs) were analyzed. In addition, we analyzed chromosomal distribution of the various types of genomic mutation, tested the correlations between SNPs and In Dels, and examined the concomitancy between TE activity and its cytosine methylation states as well as the effects of the genomic variations on phenotypes in detail.We found that tissue culture induced massive heritable somaclonal genomic variation within rice, which affected a range of fuctional genes. First, these genomic variations distributed non-randomly across each of the 12 rice chromosomes. Second,the hyper-mutagenic genomic regions overlapped little with hyper-mutable regions resulted from domestication under natural conditions. Third, based on the annotation of these affected genes by Geno Ontology(GO), the genes containing nonsynonymous SNPs were enriched in Biological process and Molecular function,“response to stress’ ’ for example. Fourth, although numerious genomic variations were detected, the phenotype of plants regenerated from tissue culture showed no difference from its WT donor under normal condition but at least one of shoot length,root length or biomass showed significant difference in certain abiotic stress conditions. Lastly, we identified seven mobilizations of transposable elements and observed higher cytosine methylation in the upstream of original copy of activatedtransposable elements.This thesis has a range of implications. First, the distribution of these mutations induced by tissue culture in space(chromosomal distribution) and its underlying mechanism may be different from those occurred in natural settings, Second, a common mutational mechanism might underly SNPs and InDels based on the significant correlation of these two genomic variations. Third, rice can maintain and stabilize the phenotype under normal growing condition via canalization despite a large number of genomic variations. Yet the phenotypic variation can be released due to perturbation of canalization by certain abiotic stresses.In conclusion, our study provided comprehensive information about the rate of tissue-culture-induced genomic mutation and molecular spectrum, which can be useful for better understanding the types, characteristics and mechanisms of somaclonal variations. Our study also has implications for how to apply plant tissue culture technology to improve the development of agriculture, horticulture and forestry in a fast and healthy manner.