Brassica napus suspension-cultured cells could be hardened in 6 days at 250C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LTso of -17.50C or -180C, respectively, while the LT50 for the comparable nonhardened control (sucrose) was -10°C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4-to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells.Freezing tolerance is the culmination of developmental and physiological changes during growth at low temperature in overwintering plants and other plants capable of acclimating (9). The requirement for low temperature is not absolute, as the same or similar physiological changes conferring hardiness can be induced by ABA in the absence of low temperature. For example, desiccation induces freezing tolerance (3, 4) and this stress is known to increase endogenous ABA levels (15); exogenous ABA circumvents the requirement for low temperature during hardening of stem cultured potato plants (1) and suspension-cultured cells (2, 12). Mefluidide, a synthetic growth regulator which has been shown to increase endogenous ABA levels (13, 18), leads to increased freezing tolerance in stem cultured potato plants (13) and haploid embryos of winter Brassica (1 1). Despite these observations, a direct link between endogenous ABA levels and the attainment of freezing tolerance has not been established.Further insight into the relationship between ABA and freezing tolerance can be gained through the use of growth regulators known to promote or inhibit ABA biosynthesis. Mefluidide enhances endogenous ABA levels (13, 18), whereas fluridone, an inhibitor of carotenoid biosynthesis at ' Supported by Natural Sciences and Engineering Council of Canada operating grants to A. M. J.-F. and H. S. S. the site of phytoene desaturation (6, 14), could block the mefluidide-induced increase in ABA accumulation.The present study utilized a microspore-derived embryogenic culture of Brassica napus cv Jet Neuf which can be hardened at room temperature with ABA to an LT50 of-20°C in 8 d (12). The role of ABA in the induction of freezing tolerance was studied by examining the effect of mefluidide, fluridone, and ABA on freezing tolerance and endogenous ABA levels. MATERIALS AND METHODS Plant MaterialWinter rape (Brassica napus cv Jet Neuf) microsporederived suspension cultures were prepared and cultured in NLN medium as previously described (1 1, 12): Cell suspensions were agitated at 130...

Seed development in canola (Brassica napus) following a mild nonlethal freeze was examined with respect to abscisic acid (ABA) levels, desiccation, and expression of LEA.76 and isocitrate lyase (ICL) transcripts. Plants with seed of 70 and 55% moisture contents were frozen to -50C for 3 hours, and seed development followed after thawing. In addition, similar processes were compared during induction of extreme desiccation tolerance by application of ABA in Brassica microspore-derived haploid embryos in culture. A mild freeze/thaw caused a premature switch in seed developmental direction from predesiccation to desiccation as indicated by an immediate and accelerated loss of seed moisture to levels similar to the mature seed in 7 instead of 35 days, and by elevated ABA levels and induction of low levels of LEA.76 and ICL transcripts. Similarly, addition of ABA to haploid embryos in culture resulted in the induction of desiccation tolerance and low levels of late embryogenesis-abundant (LEA) but not ICL transcripts. In contrast, normal seed development and desiccation of ABA-treated (desiccation-tolerant) embryos resulted in the induction of ICL and very high levels of LEA.76 transcripts. Similarly, desiccation of control (desiccation-sensitive) embryos resulted in very high levels of LEA.76 transcripts. These results indicate that although LEA-type proteins have been implicated in the development of desiccation tolerance, high transcript levels of LEA.76 were not observed in the induction of desiccation tolerance either by a hastening of the maturation process in the developing Brassica seed, or by the exogenous application of ABA to Brassica haploid embryos in culture.