Evaluation of parameters affecting the yield, viability and cell division of <i>Pinus pinaster</i> protoplasts

Abstract

Various factors affecting the yield and viability of Pinus pinaster Ait. cotyledon protoplasts and the mitotic activity of or regenerated cells are described. A study of the effect of sterilization procedures of the plant material showed that whereas the organs collected from disinfested seedlings allow for good yield and viability of isolated protoplasts, germination under non‐sterile conditions favours a greater germinating capacity and stronger mitotic activity. Numerous clusters of from 10 to 15 cells were formed after 20 days of culture when a 5% aqueous solution of calcium hypochlorite was used as a sterilizing agent. The effects of an additional purification of the enzymes showed that although yield and viability of the protoplasts are only slightly improved, the more highly purified enzymes on the other hand enhanced the mitotic activity markedly. Between the two total enzyme concentrations used (0.2 and 0.4%, and in which the relative ratio of each element was unchanged), only the lowest level supplied a debris‐free protoplast suspension; mitotic activity occurred only in that case. Comparison of the populations of cotyledon protoplasts collected from seedlings at two different growth stages (not fully‐developed or fully‐expanded cotyledons) did not reveal any appreciable difference in their size distribution. Neither was the extent of cellular viability affected by the degree of cell differentiation at the time of collecting. On the other hand, the yield of protoplasts and the mitotic activity of the regenerated cells were greater when partially‐developed organs were used. Moreover a pretreatment of the elongating cotyledons with a mineral (half‐strength MS macronutrients and full‐strength micronutrients) and hormonal (15 μ M BAP, 0.5 μ M NAA) solution improved cell division frequency.