**2.2 Plant responses to cold stress**

Plants acclimatize to survive metabolic lesions because of intracellular ice formation, as well as to survive the dehydrative effects of frost (Kacperska, 1984). Fowler *et al.,* (1996) found that after the vernalization requirement was met in wheat (*Triticum aestivum* L.) and rye (*Secale cereale* L.), cold acclimation declined. Laroche *et al.,* (1992) did not observe this reduction in cold acclimation in rapeseed but they estimated cell survival on excised leaves to determine freezing tolerance and not crown meristem survival.

The relationship between vernalization requirements and cold tolerance is not clear as different observations have been reported. While Markowski and Rapacz (1994) found little relationship between these traits by comparing vernalization requirements and frost resistance of winter rape lines derived from doubled haploid, Rapacz and Markowski (1999) found a significant correlation between vernalization requirement and both frost resistance and field survival when looking at older, high erucic acid cultivars. Long vernalization requirements are expected to delay a plant from entering the reproductive growth phase, a cold sensitive plant growth stage (Fowler et al., 1996). Rife and Zeinali (2003) found that rapeseed plants may withstand cold temperatures under field conditions more effectively prior to vernalization saturation than after the vernalization requirement has been met.

Under field conditions, rapeseed plants often survive cold events in December and January only to be killed by less severe cold events in February and March (Rife and Zeinali, 2003). One theory to explain this is that after vernalization saturation takes place, rapeseed plants do not have the same ability to recover after a warming event as unvernalized plants. This has been documented in winter cereals. Fowler et al., (1996) found reductions in lethal temperature 50's (LT50s) of 5°C or more for many cultivars between Day 49 and 84 of

Prospects for Transgenic and Molecular

potential treatments.

observed in polymer coat treatments.

with field-evaluated frost damage.

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 9

than the better areas by one to two weeks. Plant counts at harvest averaged 43 plants/m2. The thin crop that was questioned in spring eventually yielded 2,128 kg/ha gross with 2 to 3% green. Although this crop probably would have yielded higher if frost had not occurred, the yield was satisfactory and equivalent or better than reseeded canola. Early spring germinated stands can suffer damage due to subsequent heavy spring frosts. Polymercoated seeds show promise to reduce the untimely germination but carry extra costs (Canola Council of Canada, 2011). However, Christian *et al.,* (2004)) observed that by decreasing osmotic potential and temperature, germination significantly reduced on both coated and uncoated canola seeds. Polymer-coated seeds exhibited delayed germination even in the absence of moisture stress, an effect that was magnified at more negative water potentials and at a lower temperature. Median germination time of polymer-coated seed was significantly higher than for uncoated control seed throughout all temperature and osmotic

Several types of polymer seed coats have recently been developed with the intent to extend the fall planting period (Zaychuk and Enders, 2001). The polymers are specifically designed to prevent germination of canola seeds until spring by absorbing water into the polymer coat matrix but preventing the passage of sufficient amounts of water to the seed coat to begin germination. After water entry into the polymer coat matrix, freezing is required to create microfractures in the polymer coat that act as water channels for imbibition. Polymer seed coats have been shown to decrease imbibition (Chachalis and Smith, 2001) and final germination percentages (Valdes and Bradford, 1987). This may also be a problem with the polymer coats developed specifically for fall seeding canola as Gan et al., (2001) observed reduced canola emergence during a dry spring following a dry fall. Lower yields were also

To make early spring seeding feasible, suitable canola cultivars must be selected. The suitable cultivars must have quick germination, emergence, and establishment at low temperatures, and seedlings must be tolerant to early spring freezing and thawing events. Freezing on the functionality of the photosynthetic apparatus can be used to assess the cold tolerance of plant genotypes. The photosynthetic apparatus function can be evaluated by measuring the Fv/Fm, which indicates the efficiency of the excitation capture by open Photosystem II reaction centers (Rizza et al., 2001). Rizza *et al* (2001) observed a significant reversible decrease in Fv/Fm in all genotypes of oat (*Avena sativa* L.) during acclimation to low, nonfreezing temperatures, and Fv/Fm measurement was found to be highly correlated

The late freeze of April 5-9, 2007, in Northern Alabama, resulted in significant economic damage to most of the winter canola cultivars that were evaluated for the National Winter Canola Variety Trials at the Hazel Green, Alabama location (Cebert and Rufina, 2007). Five consecutive nights of low temperatures: 0.6, 1.1, -5.6, -5.6, and 0.6ºC with chilling hours of 8, 16, 18, 24, and 17occurred from April 5-9, 2007, respectively. These dates correspond to days 192, 193, 194, 195, and 196 after planting, when all cultivars were beyond 50% blooming (Figures 4-9). Damage on the primary stem was highest in early blooming cultivars (Figure 5). Cultivars which were in full bloom between days 178-183 (March 22-27) suffered a complete loss. Seeds per pod and days to 50% blooming were the two factors other than the extent of freeze damage which influenced seed yield (Table 1). In Alabama

acclimation at 4°C. This study suggested that this may not be the case in three rapeseed cultivars: A112, Ceres, and Plainsman. However, the spike of increased cold tolerance was more pronounced in unvernalized seedlings. Under the variable temperature conditions present during Great Plains winters, this phenomenon could have a substantial impact on winter survival before vernalization saturation and selecting genotypes with increased vernalization requirements could have a positive effect on winter survival.

Early seeding between late March and mid April has been proposed as the key to achieve a good and stable canola seed yield in central Montana, USA (Chengci *et al.,* 2005). This study indicated that the optimal seeding rate for early spring seeded canola is 32 to 65 seeds m-2. Although early spring seeded canola is expected to encounter cold soil temperatures and frequent frosts, canola can germinate at less than 4°C and requires 42 to 81 growing degree days (GDD) for 50% flowering for emergence. Further studies are needed to test the threshold temperature and duration of canola genotypes to cold stress. Several genotypes were found to have favorable characteristics for the semi-arid region in the northern Great Plains, such as low Tb, fast emergence, relatively cold tolerant, early flowering, and good seed yield and oil content. Further, Chengci *et al*., (2005) observed that a high Fv/Fm and low EL reading after a snowstorm event indicated less effect on Photosynthetic System II and cell membranes by cold stress. A large recovery of Fv/Fm and slow leakage rate (longer time to reach 50% total leakage) also indicate a frost tolerance of the plants. There were variances observed among the cultivars in Fv/Fm, Δ(Fv/Fm), EL, and hours to 50% total leakage (HTL50). However, as the authors noted, there was no evidence of cell membrane damage by the snowstorm, EL readings ranged from 12.7 to 20.4 μS cm−1, and the time to reach 50% of the total leakage (≈600 μS cm−1 after autoclaving) ranged from 212 to 276 h. Several cultivars had greater Fv/Fm readings and less Δ(Fv/Fm) than others. Neither EL nor the Fv/Fm readings were correlated with seed yield. However, the positive correlations between Δ(Fv/Fm) and biomass indicated that cultivars having faster biomass growth may be sensitive to cold stress. Results in this study also indicated that canola did not suffer severe frost damage by the snowstorm on 12 May 2004. Canola seedlings at the early seeding dates encountered several snowstorms in May and early June over 3 yr from 2002 to 2004, but no severe frost damage to the seedlings was observed. Canola was also found to have the ability to withstand subzero temperatures in other studies after reclamation. Kirkland and Johnson (2000) found fall-seeded canola survived eight consecutive nights of frost with the temperature dropping as low as −8°C in 1994. Further, Johnson *et al.* (1995) observed that canola seedlings were able to tolerate temperatures of −6°C without significant reductions in plant stands in North Dakota

#### **2.3 Effect of cold stress during flowering and pod formation**

Farmers in central Alberta experienced a record string of killing frosts for their Canola plants in late May to early June, 2000. Seedling canola was severely injured by these frosts and significant reseeding occurred. The surviving density was on average 32 to 43 plants/m2 and about 11/m2 in the worst areas. Continuing frosts hampered the recovery of the canola seedling but eventually new growth appeared after 10 days. The canola stand had a slow recovery for a few weeks following damage, but initially looked quite poor. By flowering, the stand began to fill in but differences in maturity were evident between areas that suffered different amounts of stand thinning. The stand continued to improve through flowering and pod fill. The crop matured in September with the thin areas maturing later

acclimation at 4°C. This study suggested that this may not be the case in three rapeseed cultivars: A112, Ceres, and Plainsman. However, the spike of increased cold tolerance was more pronounced in unvernalized seedlings. Under the variable temperature conditions present during Great Plains winters, this phenomenon could have a substantial impact on winter survival before vernalization saturation and selecting genotypes with increased

Early seeding between late March and mid April has been proposed as the key to achieve a good and stable canola seed yield in central Montana, USA (Chengci *et al.,* 2005). This study indicated that the optimal seeding rate for early spring seeded canola is 32 to 65 seeds m-2. Although early spring seeded canola is expected to encounter cold soil temperatures and frequent frosts, canola can germinate at less than 4°C and requires 42 to 81 growing degree days (GDD) for 50% flowering for emergence. Further studies are needed to test the threshold temperature and duration of canola genotypes to cold stress. Several genotypes were found to have favorable characteristics for the semi-arid region in the northern Great Plains, such as low Tb, fast emergence, relatively cold tolerant, early flowering, and good seed yield and oil content. Further, Chengci *et al*., (2005) observed that a high Fv/Fm and low EL reading after a snowstorm event indicated less effect on Photosynthetic System II and cell membranes by cold stress. A large recovery of Fv/Fm and slow leakage rate (longer time to reach 50% total leakage) also indicate a frost tolerance of the plants. There were variances observed among the cultivars in Fv/Fm, Δ(Fv/Fm), EL, and hours to 50% total leakage (HTL50). However, as the authors noted, there was no evidence of cell membrane damage by the snowstorm, EL readings ranged from 12.7 to 20.4 μS cm−1, and the time to reach 50% of the total leakage (≈600 μS cm−1 after autoclaving) ranged from 212 to 276 h. Several cultivars had greater Fv/Fm readings and less Δ(Fv/Fm) than others. Neither EL nor the Fv/Fm readings were correlated with seed yield. However, the positive correlations between Δ(Fv/Fm) and biomass indicated that cultivars having faster biomass growth may be sensitive to cold stress. Results in this study also indicated that canola did not suffer severe frost damage by the snowstorm on 12 May 2004. Canola seedlings at the early seeding dates encountered several snowstorms in May and early June over 3 yr from 2002 to 2004, but no severe frost damage to the seedlings was observed. Canola was also found to have the ability to withstand subzero temperatures in other studies after reclamation. Kirkland and Johnson (2000) found fall-seeded canola survived eight consecutive nights of frost with the temperature dropping as low as −8°C in 1994. Further, Johnson *et al.* (1995) observed that canola seedlings were able to tolerate temperatures of −6°C

vernalization requirements could have a positive effect on winter survival.

without significant reductions in plant stands in North Dakota

**2.3 Effect of cold stress during flowering and pod formation** 

Farmers in central Alberta experienced a record string of killing frosts for their Canola plants in late May to early June, 2000. Seedling canola was severely injured by these frosts and significant reseeding occurred. The surviving density was on average 32 to 43 plants/m2 and about 11/m2 in the worst areas. Continuing frosts hampered the recovery of the canola seedling but eventually new growth appeared after 10 days. The canola stand had a slow recovery for a few weeks following damage, but initially looked quite poor. By flowering, the stand began to fill in but differences in maturity were evident between areas that suffered different amounts of stand thinning. The stand continued to improve through flowering and pod fill. The crop matured in September with the thin areas maturing later than the better areas by one to two weeks. Plant counts at harvest averaged 43 plants/m2. The thin crop that was questioned in spring eventually yielded 2,128 kg/ha gross with 2 to 3% green. Although this crop probably would have yielded higher if frost had not occurred, the yield was satisfactory and equivalent or better than reseeded canola. Early spring germinated stands can suffer damage due to subsequent heavy spring frosts. Polymercoated seeds show promise to reduce the untimely germination but carry extra costs (Canola Council of Canada, 2011). However, Christian *et al.,* (2004)) observed that by decreasing osmotic potential and temperature, germination significantly reduced on both coated and uncoated canola seeds. Polymer-coated seeds exhibited delayed germination even in the absence of moisture stress, an effect that was magnified at more negative water potentials and at a lower temperature. Median germination time of polymer-coated seed was significantly higher than for uncoated control seed throughout all temperature and osmotic potential treatments.

Several types of polymer seed coats have recently been developed with the intent to extend the fall planting period (Zaychuk and Enders, 2001). The polymers are specifically designed to prevent germination of canola seeds until spring by absorbing water into the polymer coat matrix but preventing the passage of sufficient amounts of water to the seed coat to begin germination. After water entry into the polymer coat matrix, freezing is required to create microfractures in the polymer coat that act as water channels for imbibition. Polymer seed coats have been shown to decrease imbibition (Chachalis and Smith, 2001) and final germination percentages (Valdes and Bradford, 1987). This may also be a problem with the polymer coats developed specifically for fall seeding canola as Gan et al., (2001) observed reduced canola emergence during a dry spring following a dry fall. Lower yields were also observed in polymer coat treatments.

To make early spring seeding feasible, suitable canola cultivars must be selected. The suitable cultivars must have quick germination, emergence, and establishment at low temperatures, and seedlings must be tolerant to early spring freezing and thawing events. Freezing on the functionality of the photosynthetic apparatus can be used to assess the cold tolerance of plant genotypes. The photosynthetic apparatus function can be evaluated by measuring the Fv/Fm, which indicates the efficiency of the excitation capture by open Photosystem II reaction centers (Rizza et al., 2001). Rizza *et al* (2001) observed a significant reversible decrease in Fv/Fm in all genotypes of oat (*Avena sativa* L.) during acclimation to low, nonfreezing temperatures, and Fv/Fm measurement was found to be highly correlated with field-evaluated frost damage.

The late freeze of April 5-9, 2007, in Northern Alabama, resulted in significant economic damage to most of the winter canola cultivars that were evaluated for the National Winter Canola Variety Trials at the Hazel Green, Alabama location (Cebert and Rufina, 2007). Five consecutive nights of low temperatures: 0.6, 1.1, -5.6, -5.6, and 0.6ºC with chilling hours of 8, 16, 18, 24, and 17occurred from April 5-9, 2007, respectively. These dates correspond to days 192, 193, 194, 195, and 196 after planting, when all cultivars were beyond 50% blooming (Figures 4-9). Damage on the primary stem was highest in early blooming cultivars (Figure 5). Cultivars which were in full bloom between days 178-183 (March 22-27) suffered a complete loss. Seeds per pod and days to 50% blooming were the two factors other than the extent of freeze damage which influenced seed yield (Table 1). In Alabama

Prospects for Transgenic and Molecular

Carvallo *et al.,* 2011).

species (Kott *et al.,* 1988).

Fig. 10. The Triangle of U, by Woo Jang-choon (Nagaharu U, 1935).

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 11

especially with the use of model plant *Arabidopsis thaliana* (Yamaguchi-Shinozaki and Shinozaki, 1994; Jaglo-Ottosen *et al.,* 1998; Lee *et al.,* 2001; Taji *et al.,* 2002; Zhang *et al.,* 2004b;

However, contribution towards the improvement of *Brassica* species, which eventually led to the development of canola, is the creation of the Triangle of U (Figure 10) by Nagaharu, U (1935). The interpretation of three common diploid species (*B. campestris*, *B. nigra* and *B. oleracea*) being the origin of the current tetraploid *Brassica* species continues to be the foundation for contemporary development and enhancement for both vegetables and oilseed crops within the genus, indicative in improvement of *B. rapa* by Ofori *et al.,* (2008). An extensive assortment of loci for various physiological and morphological traits in the different *Brassicica* species have been determined and identified through both classical and molecular methods. Through the use of embryo rescue techniques, interspecific and intergeneric hybridization hindrances such as male-sterility and self-incompatibility were overcome (Nishi *et al.,* 1959; Quazi, 1988; Mohaptra and Bajaj, 1987). As delineated by Harberd (1969), the tedious procedures for embryo rescue in *Brassica* species resulted in the exchange of genes to produce cultivars with improved resistance for pests, variation for seed coat color, changes in fatty-acids composition and tolerance to herbicide. Sacristan and Gerdemann (1986), successfully transferred blackleg resistance to *B. napus* from *B*. *juncea* using embryo culture, while similar techniques were successfully employed to transfer both aphid-resistance and triazine herbicide-resistance among and between the various *Brassica*


†Means with the same letter are not significantly different at a = 0.05

Table 1. Estimated freeze damage and yield (Kg ha-1) of canola cultivars following late spring frost damage and exceptional drought conditions in National Winter Canola Variety Trial, Alabama A&M University, USA 2007.

A&M University, U.S. experimental plots, Cebert and Rufina (2007) reported an inverse response among cultivars between freeze damage and seed yield. Cultivars Kadore, Kalif and Plainsman with the highest seed yields were also the last ones to reach 50% blooming, approximately five days before the freeze.

### **3. Breeding for crop improvement**

#### **3.1 Genetics**

As described by Nakashima and Yamaguchi-Shinozaki (2006), the basic characteristics of plants forced them to survive in environments with variable environmental stresses such as cold stress and osmotic stress, which includes drought and high salinity. Plants, therefore exhibit an increase in freezing tolerance in response to low, non-freezing temperatures. This concept of acclimating to stressful environmental conditions has been widely investigated,

**Cultivar Estimated freeze damage (%) Estimated yield (Kg ha-1)** 

Baros 80.0a† 374.6i Virginia 65.0ab 685.7hi Viking 60.0abc 838.9fghi Hybrista 56.7abc 782.8ghi Abilene 55.0abcd 851fghi Taurus 53.3abcd 902.7fghi Trabant 53.3abcd 893.6fghi Baldur 40.0bcde 1195.6defgh Rasmus 35.0bcdef 972.2fgh Falstaff 28.3cdef 1332.5defg Summer 26.7cdef 924.7fghi Ceres 21.7def 1399.2cdef Wichita 21.7def 1721.2bcd Satori 16.7ef 1392.3cdef Jetton 16.7ef 1061.8efgh Kalif 16.7ef 2094.6ab Ovation 10.0ef 1578bcde Kronos 8.3ef 1624.8bcd Kadore 1.7f 2552.0a Plainsman 1.7f 1900.6bc

†Means with the same letter are not significantly different at a = 0.05

Trial, Alabama A&M University, USA 2007.

approximately five days before the freeze.

**3. Breeding for crop improvement** 

**3.1 Genetics** 

Table 1. Estimated freeze damage and yield (Kg ha-1) of canola cultivars following late spring frost damage and exceptional drought conditions in National Winter Canola Variety

A&M University, U.S. experimental plots, Cebert and Rufina (2007) reported an inverse response among cultivars between freeze damage and seed yield. Cultivars Kadore, Kalif and Plainsman with the highest seed yields were also the last ones to reach 50% blooming,

As described by Nakashima and Yamaguchi-Shinozaki (2006), the basic characteristics of plants forced them to survive in environments with variable environmental stresses such as cold stress and osmotic stress, which includes drought and high salinity. Plants, therefore exhibit an increase in freezing tolerance in response to low, non-freezing temperatures. This concept of acclimating to stressful environmental conditions has been widely investigated, especially with the use of model plant *Arabidopsis thaliana* (Yamaguchi-Shinozaki and Shinozaki, 1994; Jaglo-Ottosen *et al.,* 1998; Lee *et al.,* 2001; Taji *et al.,* 2002; Zhang *et al.,* 2004b; Carvallo *et al.,* 2011).

However, contribution towards the improvement of *Brassica* species, which eventually led to the development of canola, is the creation of the Triangle of U (Figure 10) by Nagaharu, U (1935). The interpretation of three common diploid species (*B. campestris*, *B. nigra* and *B. oleracea*) being the origin of the current tetraploid *Brassica* species continues to be the foundation for contemporary development and enhancement for both vegetables and oilseed crops within the genus, indicative in improvement of *B. rapa* by Ofori *et al.,* (2008). An extensive assortment of loci for various physiological and morphological traits in the different *Brassicica* species have been determined and identified through both classical and molecular methods. Through the use of embryo rescue techniques, interspecific and intergeneric hybridization hindrances such as male-sterility and self-incompatibility were overcome (Nishi *et al.,* 1959; Quazi, 1988; Mohaptra and Bajaj, 1987). As delineated by Harberd (1969), the tedious procedures for embryo rescue in *Brassica* species resulted in the exchange of genes to produce cultivars with improved resistance for pests, variation for seed coat color, changes in fatty-acids composition and tolerance to herbicide. Sacristan and Gerdemann (1986), successfully transferred blackleg resistance to *B. napus* from *B*. *juncea* using embryo culture, while similar techniques were successfully employed to transfer both aphid-resistance and triazine herbicide-resistance among and between the various *Brassica* species (Kott *et al.,* 1988).

Fig. 10. The Triangle of U, by Woo Jang-choon (Nagaharu U, 1935).

Prospects for Transgenic and Molecular

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 13

**DREB TFs Species Accession no. Stress response References**  DREB1A *Arabidopsis thaliana* AB007787 **Cold** Liu *et al.,* 1998 DREB2C *Arabidopsis thaliana* At2g40340 Salt, Mannitol, **Cold** Lee *et al.,* 2010 CBF1 *Arabidopsis thaliana* U77378 **Cold** Gilmour *et al.,*

CBF2 *Arabidopsis thaliana* AF074601 **Cold** Gilmour *et al.,* 

CBF3 *Arabidopsis thaliana* AF074602 **Cold** Gilmour *et al.,* 

OsDREB1B *Oryza sativa* AF300972 **Cold** Dubouzet *et al.,* 

OsDREB2B *Oryza sativa* Heat, **Cold** Matsukura *et* 

WCBF2 *Triticum aestivum* **Cold**, Drought Kume *et al*.,

HvDREB1 *Hordeum vulgare* DQ012941 Drought, Salt, **Cold** Xu *et al.,* 2009

PgDREB2A *Pennisetum glaucum* AAV90624 Drought, Salt, **Cold** Agarwal *et al*.,

GmDREBa *Glycine max* AY542886 **Cold**, Drought, Salt Li *et al*., 2005 GmDREBb *Glycine max* AY296651 **Cold**, Drought, Salt Li *et al.,* 2005

PNDREB1 *Arachis hypogea* FM955398 Drought, **Cold** Mei *et al.,* 2009

DmDREBa *Dendronthema3moriforlium* EF490996 **Cold**, ABA Yang *et al.,* 2009 DmDREBb *Dendronthema3moriforlium* EF487535 **Cold**, ABA Yang *et al.,* 2009 PeDREB2 *Populus euphratica* EF137176 Drought, Salt, **Cold** Chen *et al.,* 2009

Table 2. Transcription factors identified as regulators in the expression abiotic stress

OsDREBL *Oryza sativa* AF494422 **Cold** Chen *et al*., 2003

Wounding

ABA, Wound

Drought, Salt, faintly to **Cold**,

ABA

ABA

OsDREB1A *Oryza sativa* AF300970 **Cold**, Salt,

OsDREB2A *Oryza sativa* AF300971

OsDREB1C *Oryza sativa* AP001168 Drought, Salt, **Cold**,

OsDREB1F *Oryza sativa* Drought, Salt, **Cold**,

TaDREB1 *Triticum aestivum* AAL01124 **Cold**, Dehydration,

WDREB2 *Triticum aestivum* BAD97369 Drought, Salt, **Cold**,

ZmDREB2A *Zea mays* AB218832 Drought, Salt, **Cold**,

PpDBF1 *Physcomitrella patens* ABA43697 Drought, Salt, **Cold**,

DvDREB2A *Dendrathema* EF633987 Drought, Heat,

\*Adapted from: Lata and Prasad (2011).

including cold tolerance\*

1998

1998

1998

2003

2003

2003

2003

*al.*,2010

2005

2007

Egawa *et al.*, 2006

ABA Wang *et al.,* <sup>2008</sup>

ABA Shen *et al*., 2003

Heat Qin *et al.,*<sup>2007</sup>

ABA Liu *et al*., 2007

ABA, **Cold** Liu *et al.,*<sup>2008</sup>

Dubouzet *et al.,* 

Dubouzet *et al.,* 

Dubouzet *et al.,* 

Microspore culture and protoplast fusion are other protocols which have been employed successfully in the development of improved *Brassica* cultivars. According to results obtained by Ryschka *et al.,* (2007), hybrid formation between haploid at the level of protoplasts fusion obtained from different species has the potential to combine divergent genomes which may not be possible otherwise. Traditional non-molecular systems provided the foundation to identify Quantitative Trait Loci (QTLs) for early genomic mapping. These systems of hybridization coupled with current biotechnology have energized the search for novel procedures to create distinctive desirable cultivars.

#### **3.2 Breeding for cold tolerance**

Current molecular tools such as SSRs (Single Sequence Repeats), SNPs (Single Nucleotide Polymorphism) and ESTs (Expressed Sequence Tags) are being used to identify genes of economic significance including cold tolerance in *Brassica* species as reported in studies by Ofori *et al.,* (2008) and Thomashow (1999). Specific genes that respond to cold and osmotic stress in plants have been elucidated by Zhang *et al.,* (2004a), and Shinozaki *et al.,* (2003) and summarized by Lata and Prasad (2011). Results from these findings along with molecular makers have enabled the creation of high density genetic and physical maps of new genes that will allow the enhancement of genetic variation for desired traits such as response to cold stress. The most recent of such results is the release of the *Brassica rapa* genomesequence by the "*Brassica* Genome Sequencing Project Consortium" (2011). Initiatives to identify stress related genes have provided significant results in understanding functional genomics of abiotic stress. The Genome Canada/Genome Prairie project studied a range of genomics and proteomics technologies to determine how plants respond to various environmental stresses at the gene level, particularly to cold. A general insight among the findings is the discernment that cold tolerance genes are induced under conditions other than low temperatures but also due to dehydration, high salt and other abiotic stress.

Many studies (Sangwan *et al.,* 2001; Zhu *et al.,* 2009; Chinnusamy *et al.,* 2010; Chen *et al.,* 2011) have reported significant findings that provide novel sources of cold tolerance genes which can be introgressed into new hybrids and open pollinated cultivars. New data are being generated with genomics tools to exploit the use of genetic maps derive from various *Brassica* species and those from *Arabidopsis*. The accumulation of ESTs and SNPs is producing significant information on genome polymorphism and sequence data for all stress related characteristics in *Brassica* species. Kreps *et al.,* (2002) used transcriptome changes for *Arabidopsis* to identify genes in response to stress treatment including cold. Their findings showed approximately 30% of the transcriptones demonstrated sensitivity to regulation to common stress, with most being unambiguous in response for specific stimuli. Genes identified as circadian controlled were also found to be associated in response to cold stress stimuli. Similar findings were also reported by Trischuk *et al.,* (2006) and Nakashima and Yamaguchi-Shinozaki (2006) who further indicated that cold acclimation, while primarily influenced by temperature, is also moderated by factors such as light intensity, day length, cultural practices, and other abiotic stresses such as drought, dehydration and salinity. Dehydration responsive element binding (DREBs) have been identified as important plant transcription factors (TFs) in regulating expression of many stress-inducible genes. These DREBs elements have been identified in many plant species as indicated in Table 2 in their response to cold stress (Lata & Prasad, 2011).

Microspore culture and protoplast fusion are other protocols which have been employed successfully in the development of improved *Brassica* cultivars. According to results obtained by Ryschka *et al.,* (2007), hybrid formation between haploid at the level of protoplasts fusion obtained from different species has the potential to combine divergent genomes which may not be possible otherwise. Traditional non-molecular systems provided the foundation to identify Quantitative Trait Loci (QTLs) for early genomic mapping. These systems of hybridization coupled with current biotechnology have energized the search for

Current molecular tools such as SSRs (Single Sequence Repeats), SNPs (Single Nucleotide Polymorphism) and ESTs (Expressed Sequence Tags) are being used to identify genes of economic significance including cold tolerance in *Brassica* species as reported in studies by Ofori *et al.,* (2008) and Thomashow (1999). Specific genes that respond to cold and osmotic stress in plants have been elucidated by Zhang *et al.,* (2004a), and Shinozaki *et al.,* (2003) and summarized by Lata and Prasad (2011). Results from these findings along with molecular makers have enabled the creation of high density genetic and physical maps of new genes that will allow the enhancement of genetic variation for desired traits such as response to cold stress. The most recent of such results is the release of the *Brassica rapa* genomesequence by the "*Brassica* Genome Sequencing Project Consortium" (2011). Initiatives to identify stress related genes have provided significant results in understanding functional genomics of abiotic stress. The Genome Canada/Genome Prairie project studied a range of genomics and proteomics technologies to determine how plants respond to various environmental stresses at the gene level, particularly to cold. A general insight among the findings is the discernment that cold tolerance genes are induced under conditions other

than low temperatures but also due to dehydration, high salt and other abiotic stress.

Table 2 in their response to cold stress (Lata & Prasad, 2011).

Many studies (Sangwan *et al.,* 2001; Zhu *et al.,* 2009; Chinnusamy *et al.,* 2010; Chen *et al.,* 2011) have reported significant findings that provide novel sources of cold tolerance genes which can be introgressed into new hybrids and open pollinated cultivars. New data are being generated with genomics tools to exploit the use of genetic maps derive from various *Brassica* species and those from *Arabidopsis*. The accumulation of ESTs and SNPs is producing significant information on genome polymorphism and sequence data for all stress related characteristics in *Brassica* species. Kreps *et al.,* (2002) used transcriptome changes for *Arabidopsis* to identify genes in response to stress treatment including cold. Their findings showed approximately 30% of the transcriptones demonstrated sensitivity to regulation to common stress, with most being unambiguous in response for specific stimuli. Genes identified as circadian controlled were also found to be associated in response to cold stress stimuli. Similar findings were also reported by Trischuk *et al.,* (2006) and Nakashima and Yamaguchi-Shinozaki (2006) who further indicated that cold acclimation, while primarily influenced by temperature, is also moderated by factors such as light intensity, day length, cultural practices, and other abiotic stresses such as drought, dehydration and salinity. Dehydration responsive element binding (DREBs) have been identified as important plant transcription factors (TFs) in regulating expression of many stress-inducible genes. These DREBs elements have been identified in many plant species as indicated in

novel procedures to create distinctive desirable cultivars.

**3.2 Breeding for cold tolerance** 


\*Adapted from: Lata and Prasad (2011).

Table 2. Transcription factors identified as regulators in the expression abiotic stress including cold tolerance\*

Prospects for Transgenic and Molecular

**4.1.1 Molecular markers** 

losses after the divergence of *Arabidopsis* and *Brassica*.

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 15

The data indicated that the *Brassica* genome has undergone triplication and subsequent gene

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.

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),

#### **3.3 Limitations of classical breeding**

*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 cultivars.

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 becomes routine.
