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RESEARCH PRODUCT

Improved Mesophyll Protoplast Culture and Plant Regeneration in Arabidopsis thaliana (L.) Heynh., Genotype Landsberg Erecta

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Summary The response of in vitro cultured Arabidopsis thaliana mesophyll protoplasts was investigated with the aim of characterising the crucial factors that affect plating efficiencies and plant regeneration. We selected the genotype Landsberg erecta, which is frequently used for genetic studies, but which is known to respond poorly in protoplast culture. Factors permitting vigorous vegetative growth of the donor plants, i.e. short light period and avoidance of high temperature, were found to be advantageous. Selection of competent leaves and fractionation of protoplast populations indicated that predominantly protoplasts derived from still expanding, yet not highly immature mesophyll cells were competent of dividing. Plating efficiencies between 6 and 10 % were reproducibly obtained by selecting the appropriate type of mesophyll protoplast and by adjusting the culture medium. A medium rich in organic nutrients and low in mineral content, but supplemented with relatively high concentrations of calcium (50 mM CaCl 2 ) and iron (1 mM FeEDTA) was essential during early stages of protoplast culture. Rapid shoot bud formation and plant regeneration could be obtained by adjusting the constituents of the culture media to permit the growth of dense colonies with cells rich in cytoplasm and by correctly timing the transfer to the respective media.