Genotypic Variability in the Frequency of Plant Regeneration from Leaf Protoplasts of Four Brassica spp. and of Raphanus sativus

Abstract

Abstract More than 65 different genotypes, including cultivars and inbred lines, from five cruciferous species (Brassica oleracea L., B. campestris L., B. napus L., B. juncea L., and Raphanus sativus L.) were tested for their in vitro response of leaf protoplasts. Protoplasts were cultured in three liquid media and the resulting colonies were placed on seven test regeneration media. Significant differences among the species were found in plating efficiency in the frequency of shoot regeneration. Two broad response groups were identified: 1) Cultivars from B. oleracea and B. napus—these generally yielded protoplasts that were able to divide, form colonies at high frequencies, and regenerate shoots at variable frequencies; and 2) cultivars of the other species evaluated, which typically exhibited low plating efficiencies and little, if any, shoot regeneration. Evaluation for the effect of the cytoplasmic constitution of a few B. oleracea breeding lines on in vitro performance indicated that protoplasts carrying the Ogura (R1) male-sterile cytoplasm regenerated shoots at slightly lower frequencies than the corresponding alloplasmic-fertile lines. Genotypes exhibiting high frequency of shoot formation in one medium also had efficient shoot regeneration in other media as well, while genotypes with low shoot regeneration responded consistently in the different media used. This consistency in response indicates that genotype plays a critical role in determining the success of leaf protoplast culture in the crucifers.