Abstract
BackgroundInsertional mutagenesis represents one of the most effective ways to acquire information about a plant gene’s function. However, it is hindered by the autosomal genome being diploid and therefore, most mutations being recessive. The problem is addressed by inducing the transposition during anther culture so that selected mutations can be transmitted and then regenerated to a homozygous state.ResultsTo this end, we treated transgenic rice floral tissues containing the inducible transposon with an inducer, salicylic acid. Excision events were detected in regenerated calli and subsequent plantlets. DNA blot and PCR assay were used to determine the homogeneity of knockout mutants. About 5% of the mutants containing transposition events were homozygous. Furthermore, the inducible transposon was active during calli regeneration.ConclusionsThis strategy could be applicable to improve transposition efficiency in microspore development stages to create stable di-haploid mutants in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-27) contains supplementary material, which is available to authorized users.
Highlights
Insertional mutagenesis represents one of the most effective ways to acquire information about a plant gene’s function
This system was introduced into rice plants, and 34 transgenic lines containing a single copy of COKC were identified and the excision efficiencies analyzed
Many regenerated plantlets yielded untransposed COKC signals (Figure 3c), so the transposition events occurred during calli regeneration, which resulted in heterogeneity of the transposed COKC
Summary
Insertional mutagenesis represents one of the most effective ways to acquire information about a plant gene’s function. It is hindered by the autosomal genome being diploid and most mutations being recessive. One limitation of tagging experiments is the problem of the autosomal genome being diploid and most mutations are recessive. Loss-of-function screens with mutant cells that lack expression of a particular gene are difficult in diploid cells, because in most cases both alleles of a gene must be knocked out to result in a phenotype. Homozygous lines are required for screening to obtain the desired mutant phenotype. Creation of biallelic TE-insertion mutants is time-consuming and requires more breeding processes
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