Characteristics of genetic variation in the progenies of protoplast-derived plants of rice, Oryza sativa cv Nipponbare.

Genetic variation in protoplast-derived rice (Oryza sativa L.) plants was characterized using first and second generation selfed progenies. A total of 133 regenerated plants were obtained from ten protoplasts of the japonica rice cultivar Nipponbare. Sixty two regenerated plants which set enough seeds for the subsequent field tests at the next generation and were derived from five protoplasts were selected, and their selfed seeds were used as the first selfed-seed progeny (Pt(1) generation). Fifteen plants were selected from each of the 15 Pt(1) lines, and their selfed seeds were used for tests at the Pt(2) generation. Thirty seven Pt(1) lines (60%) segregated plants with detrimental mutant characters of yellowgreen phenotype, dwarf stature, dense and short panicle, or low seed fertility. According to the segregation patterns in the lines having mutated plants among those originated from the same protoplasts, the stages of mutation induction were estimated. Additionally, five quantitative traits were changed in almost all Pt(1) and Pt(2) lines. Varied quantitative traits of heading date, number of spikelets per panicle, and seed fertility, were in a heterozygous state. However, culm and panicle lengths showed high uniformity, whereas reduced culm and panicle lengths were caused by mutational changes in polygenes and/or multiple genes.

Molecular changes in protoplast-derived rice plants.

To determine whether regeneration of rice plants from protoplast culture induces DNA polymorphisms, progeny plants from direct regenerants of such cultures were examined for restriction fragment length polymorphisms (RFLP analysis). Significantly increased levels of DNA polymorphism were found compared with those in non-tissue culture control plants. Analysis with gene sequences representative of different functional domains, revealed that such polymorphisms are apparently widespread and not associated with any particular region. Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.