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Summary This study explores the potential of using haploid embryos of Brassica napus cv. Topas as a model system for study of seed degreening and further, as a means of selecting for rapid degreening and freezing tolerant degreening. Microspore-derived embryos of T opas showed similar pigment composition and content as the developing (75% moisture) zygotic seed of Westar. Canola seed degreening could be mimicked in these haploid embryos in the light by the application of 50 IlM abscisic acid or by dark exposure, providing the embryos were under 5 weeks old and had not been greened for over 1 week. These treatments had little effect on the regenerative ability of the embryos. Exposure of seed, in planta , to -5 °C could not be mimicked directly in the haploid embryos as nucleated freezing was lethal, while supercooling had no effect on degreening. Thus we attempted to desiccate embryos during degreening. This proved to be lethal to dark-degreened embryos, but those embryos that were degreened in the light with 50 µM ABA showed only a slight decrease in viability. The pigment changes after controlled desiccation were similar, but not identical, to freezing-induced changes in the seed. We conclude that microspore-derived haploid embryos constitute a valid system to study seed degreening.

Summary Transport of methionine was determined for Brassica napus suspension culture cells during the induction of freezing tolerance by ABA at room temperature. Methionine uptake was linear over one hour and methionine was concentrated 100-fold above the medium concentration. Alteration of the medium by adding or deleting ABA or BA and altering sucrose concentrations elicited marked differences in transport rates, but these were not correlated with the induction of freezing tolerance. Transport was characterized in both cells with respect to kinetics, pH optima and Ki 's of amino acids. Apparent Km in hardened cells was twice that for nonhardened cells, while Vmax was the same. Both cells exhibited a pH optimum of 6.5. Hardened cells had a greater specificity for D-methionine compared to the L-isomer. Hardened cells continued to accumulate methionine in the cytoplasm and vacuole below 5°C, whereas transport was negligible at this temperature in non-hardened cells. These results suggest that methionine transport may be altered by the hardening process.

Summary ABA was effective in circumventing the requirements for both low temperature and lengthy acclimation periods in the induction of freezing tolerance in an embryogenic microspore-derived cell suspension culture of winter rape (Brassica napus cv. Jet neuf). A higher level of freezing tolerance was induced when cells were cultured for 7 – 8 days in 5 × 10–5 M ABA, and 13 sucrose at 25 °C (60 – 70 % survival at −20 °C) then when cells were cultured in the same medium for 4 weeks at 2 °C (40 % survival at −20 °C). Acclimated cells which survived freezing at −20 °C retained their ability to undergo somatic embryogenesis.