**7. Conclusions**

22 Oilseeds

the expression of cold-responsive genes. This requires the involvement of a constitutively

Four orthologues of the *Arabidopsis* CBF/DREB transcriptional activator genes were identified from the winter *Brassica napus*, cv. Jet neuf (Gao *et al.,* 2002). All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs 5, 7, and 16 are very similar to the *Arabidopsis* CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the *Brassica* seedlings and preceded transcript accumulation of the coldinducible BN28 gene, a *Brassica* orthologue of the cor6.6 or KIN gene from *Arabidopsis*. Coldinduced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter *Brassica* but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in *Brassica*, controlling the expression of cold-induced genes through an ABA-independent pathway. Chen *et al.* (2011) indicated that BnCOR25 protein expression was up-regulated by cold, dehydration, and exogenous ABA treatment, suggesting this gene may be activated via ABA-dependent signal pathway. In this study, the data revealed that BnCOR25 was localized on cell periphery, at which the BnCOR25 protein may bind to cell membrane. In addition, BnCOR25 protein also displays a number of dehyrins' features. Thus, BnCOR25 protein may play a role in cold resistance in a similar manner. It can be concluded, however, that DRE/CRT element, Bn CBFs and Bn COR25 are capable of mediating the cold induced

expressed CBF-transcriptional inducer, which would be activated by cold treatment.

expression of genes in *Brassica napus* via an ABA-independent pathway.

The development of genetically engineered cold tolerant Canola plants by introducing or overexpressing selected genes is a viable option for producing an elite plant. It is also the only option if genes of interest originate from different species or distant relatives. However, the increasing availability of data related to large-scale genome homology between *Arabidopsis* and *Brassica* species means that canola is well-positioned to be among the first major crop species to benefit from continuous progress in plant biotechnology and molecular marker technologies. The sequencing of *B. napus* genome and the emergence of detailed physical maps are of great importance towards engineering cold tolerant canola. The availability of complete gene sequences for *Brassica* can enhance advances in detecting

**6. Future research** 

Cold is an environmental factor that adversely affects the productivity and oil quality and limits the geographical distribution and growing season of canola. Although significant progress has been made to elucidate the genetic mechanisms underlying cold tolerance in Canola, our current understanding is limited to single and shallow temperature stress. In cold regions such as Canada, canola plants are subjected to intense levels of cold stresses, and hence, the response of canola to a combination of different cold temperatures deserves more attention. Newly developed varieties offer an opportunity to test the effects of multiple cold temperatures, and to perform extensive field studies under diverse environments to assess their tolerance. Discoveries in cold stress response in model species such as *Arabidopsis thaliana* can be adapted to canola to identify candidate genes, a key element in molecular plant breeding in canola.
