Explant-dependent receptivity to isolation and a cell-wall resynthesis in protoplast culture of recalcitrant yellow lupin

Cell-wall resynthesis was studied in protoplast culture of yellow lupin (Lupinus luteus L.). We optimized protoplast isolation and found that explants excised from young seedling were more suitable sources of protoplasts, in contrast to callus tissue. Incubation in 2% cellulase R-10, 1% pectinase and 0.5% macerozyme solution for 3h effectively released protoplasts from majority of tested explants. Furthermore, we determined the optimal developmental age of explants which was 4, 21, 25 and 35 days for hypocotyls, cotyledons, in-vitro leaf mesophyll and ex-vitro leaf mesophyll, respectively. Explant type, culture medium and genotype influenced both a rate and a pattern of the cell wall regeneration. After 10 days of culture, the number of regenerated cells reached 44%-59% in hypocotyl, 84%-91% in cotyledonary, and 31%-42% in mesophyll protoplasts. Our results show that the earlier wall regeneration begins, the wall surface will be more incomplete. We suggest that unbalanced and inefficient cell-wall resynthesis likely contributes to recalcitrance of yellow lupin to manipulations in protoplast technology.