Mutant selection in the self-incompatible plant radish ( L. ) using two-step TILLING.

Radish ( L. var. ), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BCM line, that is, progenies of M plants crossed with wild-type, and the second step is performed to identify BCM individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BCM lines using our newly developed TILLING strategy and obtained six mutant lines for the six genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.