**4. Molecular breeding for crop improvement**

#### **4.1 Genomic approach**

Functional genomics strategies to understand a plant's response to abiotic stress and to exploit this knowledge to improve crop yield, quality & quantity under adverse environmental conditions is a priority focus for northern temperate climates. Genomic approaches are likely to have particular value for *Brassica* crop improvement because they have the potential to identify transcriptional, biochemical, and genetic pathways that contribute to agronomic properties. Examples include revealing transcriptional pathways that are correlated with oil quality and disease resistance (e.g. specific resistance genes and downstream transcriptional pathways). The application of such knowledge to *Brassica* crop improvement program is likely to take the form of improved cultural practices and precise molecular breeding. Approaches such as marker-assisted selection and transgenesis will facilitate transfer of genes for desirable traits into elite or classic cultivars of *Brassica*, with the goal of improving agronomic performance while preserving traditional quality traits.

A complete genome sequence provides unlimited information in the sequenced organism as well as in the related taxa (Yang *et al.,* 2005). Korea *Brassica* Genome Project (KBGP) in conjunction with Multinational *Brassica* Genome Project (MBGP) have sequenced chromosome 1 (cytogenetically oriented chromosome #1) of *Brassica rapa*. They selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. They also reported that the comparative genome analyses of the EST sequences and sequenced BAC clones from *Brassica* chromosome 1 revealed homeologous partner regions on the *Arabidopsis*  genome and a syntenic comparative map between *Brassica* chromosome 1 and *Arabidopsis*  chromosomes. In-depth sequence analyses of five homeologous BAC clones and an *Arabidopsis* chromosomal region revealed overall co-linearity, with 82% sequence similarity.

*Brassica* species having characteristics such as sporophytic pollen self-incompatibility, male sterility along with restoration for cytoplasmic male sterility offered overwhelming possibilities for genetic modification. The production of haploids and doubled haploids using microspores has accelerated the production of homozygous lines in the *Brassica* species (Cardoza and Stewart, 2004). Somatic cell fusion has facilitated the development of interspecific and intergeneric hybrids in the sexually incompatible species of *Brassica*. Those characteristics have been further exploited by combining traditional non-molecular protocols with modern biotechnology including molecular markers in marker-assisted selection and breeding, and transformation technology to introduce desired genes into elite

However, classical breeding which relies largely on homologous recombination between chromosomes to generate genetic diversity (Garcia *et al*., 1998), limits the extent of further improvement as it does not allow the precise understanding of genomic composition. The continuation of current technology in identifying selectable markers to aid in the assortment of segregating population is a major contribution that will extend the use of classical breeding. However, the use of plant transformation which allows the isolation and insertion of single genes into elite cultivars could accelerate crop improvement once the technology

Functional genomics strategies to understand a plant's response to abiotic stress and to exploit this knowledge to improve crop yield, quality & quantity under adverse environmental conditions is a priority focus for northern temperate climates. Genomic approaches are likely to have particular value for *Brassica* crop improvement because they have the potential to identify transcriptional, biochemical, and genetic pathways that contribute to agronomic properties. Examples include revealing transcriptional pathways that are correlated with oil quality and disease resistance (e.g. specific resistance genes and downstream transcriptional pathways). The application of such knowledge to *Brassica* crop improvement program is likely to take the form of improved cultural practices and precise molecular breeding. Approaches such as marker-assisted selection and transgenesis will facilitate transfer of genes for desirable traits into elite or classic cultivars of *Brassica*, with the goal of improving agronomic performance while preserving traditional quality traits.

A complete genome sequence provides unlimited information in the sequenced organism as well as in the related taxa (Yang *et al.,* 2005). Korea *Brassica* Genome Project (KBGP) in conjunction with Multinational *Brassica* Genome Project (MBGP) have sequenced chromosome 1 (cytogenetically oriented chromosome #1) of *Brassica rapa*. They selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. They also reported that the comparative genome analyses of the EST sequences and sequenced BAC clones from *Brassica* chromosome 1 revealed homeologous partner regions on the *Arabidopsis*  genome and a syntenic comparative map between *Brassica* chromosome 1 and *Arabidopsis*  chromosomes. In-depth sequence analyses of five homeologous BAC clones and an *Arabidopsis* chromosomal region revealed overall co-linearity, with 82% sequence similarity.

**3.3 Limitations of classical breeding** 

cultivars.

becomes routine.

**4.1 Genomic approach** 

**4. Molecular breeding for crop improvement** 

The data indicated that the *Brassica* genome has undergone triplication and subsequent gene losses after the divergence of *Arabidopsis* and *Brassica*.

Cheung *et al*. (2009) analyzed homoeologous regions of *Brassica* genomes at the sequence level. These represented segments of the *Brassica* A genome as found in *Brassica rapa* and *Brassica napus* and the corresponding segments of the *Brassica* C genome as found in *Brassica oleracea* and *B. napus*. Analysis of synonymous base substitution rates within modeled genes revealed a relatively broad range of times (0.12 to 1.37 million years ago) since the divergence of orthologous genome segments as represented in *B. napus* and the diploid species. Similar and consistent ranges were also identified for single nucleotide polymorphism and insertion-deletion variation. Genes conserved across the *Brassica* genomes and the homoeologous segments of the genome of *Arabidopsis thaliana* showed almost perfect collinearity. Numerous examples of apparent transduplication of gene fragments, as previously reported in *B. oleracea*, were also observed in *B. rapa* and *B. napus*, indicating that this phenomenon is widespread in *Brassica* species. They also concluded that the majority of the regions studied, the C genome segments were expanded in size relative to their A genome counterparts. Further, they observed considerable variation, even between the different versions of the same *Brassica* genome, for gene fragments and annotated putative genes suggesting that the concept of the pangenome might be particularly appropriate when considering *Brassica* genomes. Thus characterization of complete *Brassica* genome and comparative genome analyses with *Arabidopsis* using genomics approach will increase the knowledge of the biological mechanisms of the *Brassica* species that will allow targeted approaches to reduce the number and impact, which could enable a sustainable and environmentally-sound, farming policy.

#### **4.1.1 Molecular markers**

Molecular markers have been used extensively to analyze genetic diversity and to create linkage maps in *Brassica* crops. Most importantly, they have been used widely to map agronomically important genes in *Brassica* genomes and to assist canola breeding and selection procedures. According to Snowdon and Friedt (2004), the major challenges that face *Brassica* geneticists and breeders are (i) the alignment of existing genetic and physical maps in order to agree on a consensus map with a standardized nomenclature, and (ii) to compile and integrate relevant phenological, morphological, and agronomic information with allelic information for *Brassica* oilseed germplasm. Therefore, association mapping can be used to exploit available genetic resources outside the narrow rapeseed gene pool. Molecular markers linked to agronomically important traits have been reported, and most of them are now integrated into oilseed breeding programmes. It is important to note that genome research and marker assisted applications in *Brassica* began to flourish in the late 1980s (Snowdon and Freidt, 2004), when the first restriction fragment length polymorphism (RFLP) linkage maps for *B. oleracea* (Slocum *et al.,* 1990), *B. rapa* (Song *et al*., 1991), and *B. napus* (Landry *et al.,* 1991) were developed. The development of PCR techniques over the last two decades has lead to the rise of new marker technologies, and this has enabled the generation of high-density molecular maps through the amplification of highly polymorphic anonymous PCR fragments. Some of the DNA-based marker systems that have been used in *Brassica* are random amplified polymorphic DNA markers (RAPD; Williams et al., 1990),

Prospects for Transgenic and Molecular

photochemical efficiency and photosynthetic capacity.

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 17

of two *Brassica* CBF/ DREB1-like transcription factors (BNCBF5 and 17) in *Brassica napus* cv. Westar. They reported that in addition to developing constitutive freezing tolerance and constitutively accumulating COR gene mRNAs, BNCBF5 and 17 over expressing plants also accumulate moderate transcript levels of genes involved in photosynthesis and chloroplast development as identified by microarray and Northern analyses. These include GLK1 and GLK2-like transcription factors involved in chloroplast photosynthetic development, chloroplast stroma cyclophilin ROC4 (AtCYP20–3), β-amylase and triose-P/Pi translocator. In parallel with these changes, increases in photosynthetic efficiency and capacity, pigment pool sizes, increased capacities of the Calvin cycle enzymes, and enzymes of starch and sucrose biosynthesis, as well as glycolysis and oxaloacetate/malate exchange were seen, suggesting that BNCBF over expression has partially mimicked cold-induced photosynthetic acclimation constitutively. Taken together, they suggested that BNCBF/DREB1 over expression in *Brassica* not only resulted in increased constitutive freezing tolerance but also partially regulated chloroplast development to increase

The role of hsp90 in adaptation to cold temperature stress has also been studied. Characterization of the expression of hsp90 genes of *Brassica napus* using northern blot analysis and immunoblotting have shown that the hsp90 mRNA and protein are present in all *B. napus* tissues examined, albeit at different levels (Krishan *et al.,* 1995). High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5°C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20°C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of *B. napus*. Further, determining the cellular localization of the above genes and proteins during cold acclimation and identifying the proteins associated with them will provide more clues to the cellular basis of cold tolerance.

**4.1.3 Microarray based monitoring of gene expression during cold stress** 

Genome wide transcription analysis in response to stresses is essential to providing the basis of effective engineering strategies to improve stress tolerance in *Brassica* crop plants (Lee et al., 2008). In order to perform transcriptome analysis in *Brassica rapa*, Lee et al. (2008) constructed a *B. rapa* oligo microarray, KBGP-24K, using sequence information from approximately 24,000 unigenes and analyzed cold (4 degrees C), salt (250 mM NaCl), and drought (air-dry) treated *B. rapa* plants. Among the *B. rapa* unigenes represented on the microarray, 417 (1.7%), 202 (0.8%), and 738 (3.1%) were identified as responsive genes that were differently expressed 5-fold or more at least once during a 48-h treatment with cold, salt, and drought, respectively. These results were confirmed by RT-PCR analysis. In the abiotic stress responsive genes identified, they found 56 transcription factor genes and 60 commonly responsive genes. The authors suggested that various transcriptional regulatory mechanisms and common signaling pathway are working together under the abiotic stresses in *B. rapa*. In conclusion, they reported that their new developed 24K oligo

amplified fragment length polymorphisms (AFLP; Vos *et al.,* 1995), and inter-simple sequence repeats (ISSR; Zietkiewicz *et al*., 1994).

PCR-based markers that meet the requirements and capacity of rapeseed breeders has emerged due to the ability to convert anonymous PCR markers that are closely linked to loci controlling traits of interest into sequence characterized amplified region (SCAR) or sequence tagged site (STS). Progress has also been made with the development of simple sequence repeat markers, also termed microsatellites (SSR; Grist *et al*., 1993). These markers are highly polymorphic, robust, and relatively inexpensive. They are valuable to use for map alignment among different crosses simply because of their co-dominant nature. The number of publicly available *Brassica* SSR primers is increasing as a result of publicly funded international initiatives (www.brassica.info/ssr/SSRinfo.htm). Another important marker system is the single-nucleotide polymorphisms (SNPs), which results from single-base substitutions in the DNA sequence. These are the most abundant form of DNA polymorphism in most organisms, and they offer an opportunity to develop extremely fine genetic maps. This is because they can be used to uncover allelic variation directly within the expressed sequences, and to develop haplotypes based on gametic phase disequilibrium for analysis of quantitative traits.

The application of all these marker techniques towards developing maps for cold tolerance in *Brassica* crops will greatly improve the production of canola in temperate regions. Collaborative research with many research groups to improve stress tolerance in canola by utilizing these marker systems should be encouraged through funding. The results obtained through these collaborative studies can contribute to the sustainable oil and food production in canola.

#### **4.1.2 Gene expression during cold stress in** *Brassica* **species**

Exposure of cold-hardy species to low, non-freezing temperatures induces genetic, morphological and physiological changes in plants, which results in the development of cold hardiness and the acquisition of freezing tolerance (Savitch *et al*., 2005). The ability of plants to acquire freezing tolerance from cold acclimation has been shown to involve the reprogramming of gene expression networks (Seki *et al.,* 2001, Fowler and Thomashow, 2002; Kreps *et al*., 2002; Seki *et al*., 2002). Photosynthetic cold acclimation has been reported to be an essential component of the development of cold hardiness and freezing tolerance and requires the complex interaction of low temperature, light and chloroplast redox poise (Gray *et al*., 1997, Wanner and Junttila, 1999).

The long-term cold acclimation has also been shown to be associated with morphological changes, such as compact dwarf morphology, increased leaf thickness caused by increased mesophyll cell size, increased specific leaf weight, marked decrease of leaf water content and an increase in cytoplasmic volume relative to vacuole volume (Stefanowska *et al.,* 1999; Strand *et al*., 1999; Stefanowska *et al*., 2002). In fact, it has been suggested that such structural changes might be necessary to account for the increase in stromal and cytosolic enzymes and metabolites in cold-acclimated leaves (Strand *et al.* 1999).

Several genes associated with cold hardiness have been studied in various crops. But in *Brassica napus,* limited report exists. Savitch *et al*. (2005) studied the effects of over expression

amplified fragment length polymorphisms (AFLP; Vos *et al.,* 1995), and inter-simple

PCR-based markers that meet the requirements and capacity of rapeseed breeders has emerged due to the ability to convert anonymous PCR markers that are closely linked to loci controlling traits of interest into sequence characterized amplified region (SCAR) or sequence tagged site (STS). Progress has also been made with the development of simple sequence repeat markers, also termed microsatellites (SSR; Grist *et al*., 1993). These markers are highly polymorphic, robust, and relatively inexpensive. They are valuable to use for map alignment among different crosses simply because of their co-dominant nature. The number of publicly available *Brassica* SSR primers is increasing as a result of publicly funded international initiatives (www.brassica.info/ssr/SSRinfo.htm). Another important marker system is the single-nucleotide polymorphisms (SNPs), which results from single-base substitutions in the DNA sequence. These are the most abundant form of DNA polymorphism in most organisms, and they offer an opportunity to develop extremely fine genetic maps. This is because they can be used to uncover allelic variation directly within the expressed sequences, and to develop haplotypes based on gametic phase disequilibrium

The application of all these marker techniques towards developing maps for cold tolerance in *Brassica* crops will greatly improve the production of canola in temperate regions. Collaborative research with many research groups to improve stress tolerance in canola by utilizing these marker systems should be encouraged through funding. The results obtained through these collaborative studies can contribute to the sustainable oil and food production

Exposure of cold-hardy species to low, non-freezing temperatures induces genetic, morphological and physiological changes in plants, which results in the development of cold hardiness and the acquisition of freezing tolerance (Savitch *et al*., 2005). The ability of plants to acquire freezing tolerance from cold acclimation has been shown to involve the reprogramming of gene expression networks (Seki *et al.,* 2001, Fowler and Thomashow, 2002; Kreps *et al*., 2002; Seki *et al*., 2002). Photosynthetic cold acclimation has been reported to be an essential component of the development of cold hardiness and freezing tolerance and requires the complex interaction of low temperature, light and chloroplast redox poise

The long-term cold acclimation has also been shown to be associated with morphological changes, such as compact dwarf morphology, increased leaf thickness caused by increased mesophyll cell size, increased specific leaf weight, marked decrease of leaf water content and an increase in cytoplasmic volume relative to vacuole volume (Stefanowska *et al.,* 1999; Strand *et al*., 1999; Stefanowska *et al*., 2002). In fact, it has been suggested that such structural changes might be necessary to account for the increase in stromal and cytosolic enzymes

Several genes associated with cold hardiness have been studied in various crops. But in *Brassica napus,* limited report exists. Savitch *et al*. (2005) studied the effects of over expression

**4.1.2 Gene expression during cold stress in** *Brassica* **species** 

and metabolites in cold-acclimated leaves (Strand *et al.* 1999).

(Gray *et al*., 1997, Wanner and Junttila, 1999).

sequence repeats (ISSR; Zietkiewicz *et al*., 1994).

for analysis of quantitative traits.

in canola.

of two *Brassica* CBF/ DREB1-like transcription factors (BNCBF5 and 17) in *Brassica napus* cv. Westar. They reported that in addition to developing constitutive freezing tolerance and constitutively accumulating COR gene mRNAs, BNCBF5 and 17 over expressing plants also accumulate moderate transcript levels of genes involved in photosynthesis and chloroplast development as identified by microarray and Northern analyses. These include GLK1 and GLK2-like transcription factors involved in chloroplast photosynthetic development, chloroplast stroma cyclophilin ROC4 (AtCYP20–3), β-amylase and triose-P/Pi translocator. In parallel with these changes, increases in photosynthetic efficiency and capacity, pigment pool sizes, increased capacities of the Calvin cycle enzymes, and enzymes of starch and sucrose biosynthesis, as well as glycolysis and oxaloacetate/malate exchange were seen, suggesting that BNCBF over expression has partially mimicked cold-induced photosynthetic acclimation constitutively. Taken together, they suggested that BNCBF/DREB1 over expression in *Brassica* not only resulted in increased constitutive freezing tolerance but also partially regulated chloroplast development to increase photochemical efficiency and photosynthetic capacity.

The role of hsp90 in adaptation to cold temperature stress has also been studied. Characterization of the expression of hsp90 genes of *Brassica napus* using northern blot analysis and immunoblotting have shown that the hsp90 mRNA and protein are present in all *B. napus* tissues examined, albeit at different levels (Krishan *et al.,* 1995). High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5°C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20°C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of *B. napus*. Further, determining the cellular localization of the above genes and proteins during cold acclimation and identifying the proteins associated with them will provide more clues to the cellular basis of cold tolerance.

#### **4.1.3 Microarray based monitoring of gene expression during cold stress**

Genome wide transcription analysis in response to stresses is essential to providing the basis of effective engineering strategies to improve stress tolerance in *Brassica* crop plants (Lee et al., 2008). In order to perform transcriptome analysis in *Brassica rapa*, Lee et al. (2008) constructed a *B. rapa* oligo microarray, KBGP-24K, using sequence information from approximately 24,000 unigenes and analyzed cold (4 degrees C), salt (250 mM NaCl), and drought (air-dry) treated *B. rapa* plants. Among the *B. rapa* unigenes represented on the microarray, 417 (1.7%), 202 (0.8%), and 738 (3.1%) were identified as responsive genes that were differently expressed 5-fold or more at least once during a 48-h treatment with cold, salt, and drought, respectively. These results were confirmed by RT-PCR analysis. In the abiotic stress responsive genes identified, they found 56 transcription factor genes and 60 commonly responsive genes. The authors suggested that various transcriptional regulatory mechanisms and common signaling pathway are working together under the abiotic stresses in *B. rapa*. In conclusion, they reported that their new developed 24K oligo

Prospects for Transgenic and Molecular

and imparting tolerance to dehydration stress, respectively.

cold-response pathway related to that found in *Arabidopsis*.

chaperones (Guy *et al*., 1998), respectively, could have protective effects.

**4.2.1 Protein expression during cold stress in** *Brassica* **species** 

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 19

and responsive to ABA proteins which suggests a possible role in preventing ice formation

*B. napus*, a member of the Cruciferae family, cold acclimates just like *Arabidopsis thaliana.* Jaglo *et al.* (2001) showed that *B. napus* has a cold-response pathway related to the CBF coldresponse pathway of *Arabidopsis*. cDNA clones encoding two different CBF-like proteins were identified by screening *B. napus* cDNA libraries using PCR-generated probes. The *B. napus* CBF-like proteins were 92% identical in amino acid sequence to each other and approximately 76% identical in sequence to *Arabidopsis* CBF1. An alignment of the *B. napus* proteins with *Arabidopsis* CBF1 indicated that the sequence identity extended throughout the protein, but was greatest in the AP2/EREBP DNA-binding domain includes an alignment of one *B. napus* CBF protein against *Arabidopsis* CBF1). A sequence for a third *B. napus* CBF polypeptide has been deposited by others (accession no. AF084185; N. Zhou, G. Wu, Y.-P. Gao, R.W. Wilen, and L.V. Gusta). Transcripts encoding *B. napus* CBF-like proteins were found to accumulate rapidly (within 30 min) upon exposure of plants to low temperature. They reasoned that if *B. napus* had a similar CBF-like cold-response pathway, then expression of the *Arabidopsis CBF* genes in transgenic *B. napus* might also activate expression of *Bn115* and other cold-regulated genes containing the CRT/DRE-related regulatory elements and increase plant freezing tolerance. Constitutive expression of *Arabidopsis* CBF1, CBF2, and CBF3 in transgenic *B. napus* caused the accumulation of transcripts for *Bn115* and *Bn28* without a low temperature stimulus; *Bn28* encodes an ortholog of the CRT/DREregulated cold-responsive gene *COR6.6* (Hajela *et al.,* 1990). Electrolyte leakage experiments indicated that expression of the *Arabidopsis CBF* genes in *B. napus* resulted in an increase in freezing tolerance. The experiments presented above indicated that *B. napus* encodes a CBF

There is emerging evidence that certain novel hydrophilic and late embryogenesis abundant (LEA) polypeptides participate in the stabilization of membranes against freeze-induced injury. These hydrophilic and late embryogenesis abundant polypeptides are predicted to contain regions capable of forming amphipathic α-helices which are shown to have strong effect on intrinsic curvature of monolayers and their propensity to form hexagonal II phase. They are said to defer their formation at lower temperatures (Epand *et al.,* 1995). An additional hypothesis suggests that the extensive water binding capacity of these hydrophilic proteins might provide a protective environment in the proximity of membranes during freezing and result in membrane stabilization. In addition, there is evidence that protein denaturation occurs in plants at low temperature (Guy *et al.,* 1998) which could potentially result in cellular damage. In these cases, the enhancement of antioxidative mechanisms (Aroca *et al*., 2003), increased levels of sugars in the apoplastic space (Livingston and Henson, 1998), and the induction of genes encoding molecular

Recently (Chen *et al.,* 2011) studied a gene encoding novel cold-regulated protein with molecular mass of 25 KDa that was isolated from *B. napus* cDNA library using microarray analysis, and is consequently designated as BnCOR25. The data presented in this study revealed that BnCOR25 transcripts were significantly accumulated in roots after cold

microarray will be a useful tool for transcriptome profiling and this work will provide valuable insight in the response to abiotic stress in *B. rapa* and other *Brassica* species.

#### **4.1.4 Micro RNA could modify regulator gene expression during cold in** *Brassica* **species**

MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules (on average only 22 nucleotides long) found in all eukaryotic cells except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. Several miRNAs have been identified that regulate complex process. Kutter et al. (2007) demonstrated that the density and development of stomatal complexes on the epidermis of Arabidopisis thaliana leaves depends on the microRNA-mediated regulation of Agamous-like16 (AGL16), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. They reported that expression of a miR824-resistant AGL16 mRNA, but not the wild type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines over expressing miR824 decreased the incidence of stomata in higher order complexes. Non overlapping patters of AGL16 mRNA and miR824 localization led to the proposal that the miR824/ AGL16 pathway functions in the satellite meristemoid linage of stomatal development. Since *Brassica* and *Arabidopsis*, derived from same line and diverged around 12 to 20 million years ago, similar conserved miRNA families can be identified in *Brassica* species which help in plant development. Hence, microRNAs so identified will play an essential function in regulating gene expression both in mutlicellular plants and animals.

#### **4.2 Proteomic approach**

In the past few decades, considerable efforts have been directed at identifying coldregulated protein in plant species. Various biochemical responses of plants to low, freezing temperatures have been widely documented. They involve changes in protein content and enzyme activities, metabolic modifications and changes in lipid composition and membrane structure. It is also recognized that *Brassica* species are of value for investigating important key areas for resistance, especially cold resistance, and the great advances have been made in terms of cold-induced genes and cytological mechanisms in the shape of cold resistance (Jiang *et al.,* 1996; Diaz *et al*., 1997; Mieczyslaw, 1999; Rapacz, 2002). Cold acclimation is a complex adaptive process by which plants increase their tolerance to extracellular freezing. This process is induced by exposure of the plant to low but nonfreezing temperatures and is accompanied by a variety of biochemical and structural changes in plant cells (Thomashow, 2001). Proteins encoded by cold-regulated genes have an interesting feature in common; they are hydrophilic and remain soluble upon boiling (Gilmour *et al.,* 2000). It is postulated that proteins that display these distinctive properties may have roles in cryoprotection (Thomashow, 2001). Other cold-regulated gene products show similarities to antifreeze proteins or to drought-induced late-embryogenesis-abundant proteins (Gilmour *et al.,* 2000)

microarray will be a useful tool for transcriptome profiling and this work will provide

MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules (on average only 22 nucleotides long) found in all eukaryotic cells except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. Several miRNAs have been identified that regulate complex process. Kutter et al. (2007) demonstrated that the density and development of stomatal complexes on the epidermis of Arabidopisis thaliana leaves depends on the microRNA-mediated regulation of Agamous-like16 (AGL16), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. They reported that expression of a miR824-resistant AGL16 mRNA, but not the wild type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines over expressing miR824 decreased the incidence of stomata in higher order complexes. Non overlapping patters of AGL16 mRNA and miR824 localization led to the proposal that the miR824/ AGL16 pathway functions in the satellite meristemoid linage of stomatal development. Since *Brassica* and *Arabidopsis*, derived from same line and diverged around 12 to 20 million years ago, similar conserved miRNA families can be identified in *Brassica* species which help in plant development. Hence, microRNAs so identified will play an essential function in regulating gene expression both

In the past few decades, considerable efforts have been directed at identifying coldregulated protein in plant species. Various biochemical responses of plants to low, freezing temperatures have been widely documented. They involve changes in protein content and enzyme activities, metabolic modifications and changes in lipid composition and membrane structure. It is also recognized that *Brassica* species are of value for investigating important key areas for resistance, especially cold resistance, and the great advances have been made in terms of cold-induced genes and cytological mechanisms in the shape of cold resistance (Jiang *et al.,* 1996; Diaz *et al*., 1997; Mieczyslaw, 1999; Rapacz, 2002). Cold acclimation is a complex adaptive process by which plants increase their tolerance to extracellular freezing. This process is induced by exposure of the plant to low but nonfreezing temperatures and is accompanied by a variety of biochemical and structural changes in plant cells (Thomashow, 2001). Proteins encoded by cold-regulated genes have an interesting feature in common; they are hydrophilic and remain soluble upon boiling (Gilmour *et al.,* 2000). It is postulated that proteins that display these distinctive properties may have roles in cryoprotection (Thomashow, 2001). Other cold-regulated gene products show similarities to antifreeze proteins or to drought-induced late-embryogenesis-abundant proteins (Gilmour *et al.,* 2000)

valuable insight in the response to abiotic stress in *B. rapa* and other *Brassica* species.

**4.1.4 Micro RNA could modify regulator gene expression during cold in** *Brassica*

**species** 

in mutlicellular plants and animals.

**4.2 Proteomic approach** 

and responsive to ABA proteins which suggests a possible role in preventing ice formation and imparting tolerance to dehydration stress, respectively.

#### **4.2.1 Protein expression during cold stress in** *Brassica* **species**

*B. napus*, a member of the Cruciferae family, cold acclimates just like *Arabidopsis thaliana.* Jaglo *et al.* (2001) showed that *B. napus* has a cold-response pathway related to the CBF coldresponse pathway of *Arabidopsis*. cDNA clones encoding two different CBF-like proteins were identified by screening *B. napus* cDNA libraries using PCR-generated probes. The *B. napus* CBF-like proteins were 92% identical in amino acid sequence to each other and approximately 76% identical in sequence to *Arabidopsis* CBF1. An alignment of the *B. napus* proteins with *Arabidopsis* CBF1 indicated that the sequence identity extended throughout the protein, but was greatest in the AP2/EREBP DNA-binding domain includes an alignment of one *B. napus* CBF protein against *Arabidopsis* CBF1). A sequence for a third *B. napus* CBF polypeptide has been deposited by others (accession no. AF084185; N. Zhou, G. Wu, Y.-P. Gao, R.W. Wilen, and L.V. Gusta). Transcripts encoding *B. napus* CBF-like proteins were found to accumulate rapidly (within 30 min) upon exposure of plants to low temperature. They reasoned that if *B. napus* had a similar CBF-like cold-response pathway, then expression of the *Arabidopsis CBF* genes in transgenic *B. napus* might also activate expression of *Bn115* and other cold-regulated genes containing the CRT/DRE-related regulatory elements and increase plant freezing tolerance. Constitutive expression of *Arabidopsis* CBF1, CBF2, and CBF3 in transgenic *B. napus* caused the accumulation of transcripts for *Bn115* and *Bn28* without a low temperature stimulus; *Bn28* encodes an ortholog of the CRT/DREregulated cold-responsive gene *COR6.6* (Hajela *et al.,* 1990). Electrolyte leakage experiments indicated that expression of the *Arabidopsis CBF* genes in *B. napus* resulted in an increase in freezing tolerance. The experiments presented above indicated that *B. napus* encodes a CBF cold-response pathway related to that found in *Arabidopsis*.

There is emerging evidence that certain novel hydrophilic and late embryogenesis abundant (LEA) polypeptides participate in the stabilization of membranes against freeze-induced injury. These hydrophilic and late embryogenesis abundant polypeptides are predicted to contain regions capable of forming amphipathic α-helices which are shown to have strong effect on intrinsic curvature of monolayers and their propensity to form hexagonal II phase. They are said to defer their formation at lower temperatures (Epand *et al.,* 1995). An additional hypothesis suggests that the extensive water binding capacity of these hydrophilic proteins might provide a protective environment in the proximity of membranes during freezing and result in membrane stabilization. In addition, there is evidence that protein denaturation occurs in plants at low temperature (Guy *et al.,* 1998) which could potentially result in cellular damage. In these cases, the enhancement of antioxidative mechanisms (Aroca *et al*., 2003), increased levels of sugars in the apoplastic space (Livingston and Henson, 1998), and the induction of genes encoding molecular chaperones (Guy *et al*., 1998), respectively, could have protective effects.

Recently (Chen *et al.,* 2011) studied a gene encoding novel cold-regulated protein with molecular mass of 25 KDa that was isolated from *B. napus* cDNA library using microarray analysis, and is consequently designated as BnCOR25. The data presented in this study revealed that BnCOR25 transcripts were significantly accumulated in roots after cold

Prospects for Transgenic and Molecular

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

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 21

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

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 accumulation of SUMO conjugates during cold stress.

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