**5. Using transgenic approaches to develop cold tolerance**

Biotechnology offers new strategies that can be used to develop transgenic canola plants with improved tolerance to cold stress. Rapid advancement in recombinant DNA technology and development of precise and efficient gene transfer protocols have resulted in transformation and regeneration of transgenic lines in canola and other plant species (Wani *et al.,* 2008; Gosal *et al.,* 2009; Wani *et al.,* 2011). Genes that respond to freezing stress have been isolated and characterized, and studies suggest that they contribute to chilling tolerance and cold acclimation (Knight *et al*., 1999; Hsieh *et al.*, 2002). Therefore, the transgenic approach should be pursued actively in canola breeding to improve cold tolerance. Efforts have been made to generate transgenic lines, which have shown improved tolerance to cold stress (Savitch, *et al.,* 2005).

Transgenic technology has the ability to improve cold stress in plants by introducing or down-regulating genes that regulate specific trait (Kumar, 2006). In the last few years, several efforts have been made to identify and characterize cold-responsive (*COR*) genes. Homologous components of the *Arabidopsis CBF* cold response pathway have also been found in many plants (Yamagachi-Shinozaki and Shinozaki, 2006). Some of these putative orthologs have been analyzed, and functionally tested. Most of the studies indicate that the expressions of *CBFs* and *CORs* in response to cold stress are similar in many plant species. Thus, they involve rapid cold induced expression of the *CBFs* followed by expression of *CBF*-targeted genes that increase freezing tolerance. It is important to note that as we aspire

treatment. Sumoylation/desumoylation of proteins has been shown to have a pivotal role in cold acclimation (Miura *et al*., 2007). Sumoylation is a post-translational protein modification where small ubiquitin-related modifier (SUMO) proteins are conjugated to protein substrates in a process dependent on SUMO E3 ligases, whereas desumoylation is the removal of SUMO proteins from their target proteins by SUMO proteases. It might protect target proteins from proteasomal degradation because sumoylation prevents ubiquitination (Ulrich, 2005). SIZ1, an *Arabidopsis* SUMO E3 ligase is shown to be required for the

Transgenic attempts with many structural genes have also been made with a moderate degree of success. The overexpression of genes encoding LEA proteins can improve the stress tolerance of transgenic plants. Expression of the citrus gene encoding a LEA protein, CuCOR19 increased the cold tolerance of transgenic tobacco (Hara *et al.,* 2003). Likewise, the freezing tolerance of *Arabidopsis* was increased by the ectopic expression of the wheat gene WCS19 (Dong *et al.,* 2002), the *Arabidopsis* gene COR15A (Artus *et al.,* 1996), and the coexpression of the genes RAB18 and COR47, and XERO2 and ERD10 (Puhakainen *et al*., 2004). The freezing tolerance of strawberry leaves was enhanced by expression of the wheat dehydrin gene WCOR410 (Houde *et al.,* 2004). On the other hand, the expression of two cold-induced LEA proteins from spinach (Kaye *et al.,* 1998) and three desiccation-induced LEA proteins from *C. plantagineum* (Iturriaga *et al.,* 1992) in tobacco did not induce any significant changes in the freezing or drought tolerance of the respective transgenic plants. This may indicate either that not all LEA proteins make a significant contribution to plant stress tolerance, or that they need a particular background to function in, as suggested for

Biotechnology offers new strategies that can be used to develop transgenic canola plants with improved tolerance to cold stress. Rapid advancement in recombinant DNA technology and development of precise and efficient gene transfer protocols have resulted in transformation and regeneration of transgenic lines in canola and other plant species (Wani *et al.,* 2008; Gosal *et al.,* 2009; Wani *et al.,* 2011). Genes that respond to freezing stress have been isolated and characterized, and studies suggest that they contribute to chilling tolerance and cold acclimation (Knight *et al*., 1999; Hsieh *et al.*, 2002). Therefore, the transgenic approach should be pursued actively in canola breeding to improve cold tolerance. Efforts have been made to generate transgenic lines, which have shown improved

Transgenic technology has the ability to improve cold stress in plants by introducing or down-regulating genes that regulate specific trait (Kumar, 2006). In the last few years, several efforts have been made to identify and characterize cold-responsive (*COR*) genes. Homologous components of the *Arabidopsis CBF* cold response pathway have also been found in many plants (Yamagachi-Shinozaki and Shinozaki, 2006). Some of these putative orthologs have been analyzed, and functionally tested. Most of the studies indicate that the expressions of *CBFs* and *CORs* in response to cold stress are similar in many plant species. Thus, they involve rapid cold induced expression of the *CBFs* followed by expression of *CBF*-targeted genes that increase freezing tolerance. It is important to note that as we aspire

accumulation of SUMO conjugates during cold stress.

transgenic strawberry plants (Houde *et al.,* 2004).

tolerance to cold stress (Savitch, *et al.,* 2005).

**5. Using transgenic approaches to develop cold tolerance** 

to engineer cold stress in Canola, the contitutive expression of *Arabidopsis CBF* genes in other plants resulted in increasing freezing tolerance (Yamagachi-Shinozaki and Shinozaki, 2006). There are also other structural genes that have been used to engineer cold tolerance in some plants with amoderate degree of success. An example is tobacco that was engineered by over-expressing chloroplast glycerol-3-phosphate acyltransferase (*GPAT*) gene from squash and *Arabidopsis* (Murata *et al.,* 1992). The transgenic tobacco showed enhanced cold tolerance and an increase in the number of unsaturated fatty acids present in the plant cell wall. In another study, Pennycooke *et al.,* (2003) down-regulated α-Gal (α –Galactosidase) in petunia, and this resulted in transgenic plants with an increased freezing tolerance. This suggested that transformation with α-Gal is another way in which freezing tolerance of plants can be genetically improved. The genes encoding *LEA* proteins can also improve tolerance to cold stress if they are overexpressed in other plants. Citrus gene encoding a *LEA* protein, *CuCOR19* was over-expressed in tobacco and an increased cold tolerance of transgenic tobacco was achieved (Hara *et al.,* 2003). The expression of the wheat dehydrin gene WCOR410, in strawberry leaves also enhanced freezing tolerance (Houde *et al*., 2004). In a separate study, Kim *et al*., (2007) engineered tobacco with ring zinc finger protein (RDCPt) from hot pepper and their results indicated that the expression of this gene improved cold tolerance in transgenic plants when compared to wild type. In another study Su *et al.,* (2010), determined that *MYBS3* was critical in cold adaptation in rice and it enhanced cold tolerance. Their report indicated that transgenic rice constitutively overexpressing *MYBS3* tolerated 4°C for at least 1 week and there were no interferences with the yield. All these studies demonstrate that the relationships among different pathways regulated by cold acclimation are complex. Therefore, it is important to understand the mechanism regulating cold-regulated genes in order to engineer cold tolerant canola. In developing transgenic canola, one can study the genes aforementioned with the aim of over-expressing or down-regulating them in Canola plants. The advent of molecular genetics and biotechnology offers a possibility to genetically engineer Canola to be more tolerant to cold. The technology has been modified to significantly improve breeding efficiency, thus resulting in rapid and accurate incorporation of cold tolerant genes into Canola plants.

#### **5.1 ABA-independent gene regulation to cold stress**

Environmental stresses induce the expression of many genes that can be classified into two groups. The first group corresponds to proteins involved in transduction pathways, such as transcription factors, whereas the second group includes effector proteins like the enzymes of osmolyte biosynthesis. Many studies have been focused on transcription factors involved in gene expression regulation. For each signal (salt, drought, and cold), several pathways can be distinguished depending on ABA dependent and ABA independent. ABAindependent expression of stress-responsive genes can occur through dehydrationresponsive element (DRE)/C-repeat (CRT) cis-acting elements. The binding factors CBF/DREB1 (CRT-binding factor/DRE-binding factor 1) and DREB2 mediate gene expression in response to cold and drought/salinity, respectively). Interestingly, the CBF4 protein seems to mediate drought response unlike the other CBFs (Haake *et al*., 2002). A particular feature of CBF proteins is their early and transient cold induction, which precedes

Prospects for Transgenic and Molecular

through molecular genetics.

element in molecular plant breeding in canola.

Available from:

**7. Conclusions** 

**8. References** 

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 23

polymorphisms for many agronomically important candidate genes for cold tolerance. It is also possible to elucidate the genetic control of cold tolerance through allele-trait association studies. This can be implemented by combining SNP haplotype data for *Brassica* candidate genes with pedigree and quantitative trait information. DNA-chip and high-throughput SNP genotyping technologies will accelerate our understanding of cold tolerance in canola

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

Andrews, J., & Morrison, J. (1992). Freezing and ice tolerance tests for Winter *Brassica*. *Journal of the American Society of Agronomy,* Vol. 84, No. 6, pp. 960-962. Angadi, V. (2000). Response of three *Brassica* species to high temperature stress during

Aroca, R.; Irigoyen, J. & Sanchezdiaz, M. (2003). Drought enhances maize chilling

Artus, N., Uemura, M., Steponkus, L., Gilmour, J., Lin, C., & Thomashow, F. (1996).

Blackshaw, E. (1991). Soil temperature and moisture effects on downy brome vs. winter

Boyles, M. (2011). Oklahoma State University. Herbicide foliar and residual injury. Cold

Brzostowicz, A., & Barcikowska, V. (1987). Possibility of frost resistance testing of *Brassica napus* with the help of delayed luminescence intensity. *Cruciferae Newsletter*, pp. 12, 27. Canola Connection. (2011). The Canola Council of Canada, markets & statistics. Available

canola, wheat and rye emergence. *Crop Sci*. Vol. 31, pp. 1034-1040.

 http://www.canola.okstate.edu/herbicides/foliarresidualinjury/index.htm Brule-Babel, L., & Fowler, D. (1988). Genetic control of cold hardiness and vernalization

tolerance.II. Photosynthetic traits and protective mechanisms against oxidative

Constitutive expression of the cold regulated *Arabidopsis thaliana* COR15a gene affects both chloroplast and protoplast freezing tolerance. *Proceedings of National* 

temperatures symptoms. Symptoms similar to herbicide carryover injury.

reproductive growth. *Can. J. Plant Sci*., Vol. 80, pp.693-701.

stress. *Physiologia Plantarum*, Vol. 117, pp. 540-549.

requirement in wheat. *Crop Sci*. vol. 28, pp. 879–884.

from: http://www.canolacouncil.org/acreageyields.aspx

*Academy of Sciences,* Vol. 93, pp. 13404–13409.

the expression of cold-responsive genes. This requires the involvement of a constitutively expressed CBF-transcriptional inducer, which would be activated by cold treatment.

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

#### **6. Future research**

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 polymorphisms for many agronomically important candidate genes for cold tolerance. It is also possible to elucidate the genetic control of cold tolerance through allele-trait association studies. This can be implemented by combining SNP haplotype data for *Brassica* candidate genes with pedigree and quantitative trait information. DNA-chip and high-throughput SNP genotyping technologies will accelerate our understanding of cold tolerance in canola through molecular genetics.
