**1. Introduction**

Oilseed rape has become a major crop in North America, with cropland dedicated to rapeseed production increasing from 4,391,660 ha in 2001 to 7,103,725 ha in 2010 in both U.S.A. and Canada (Canola Connection, 2011; National Agricultural Statistics Service, 2011). Most of these are cultivated in spring in the Canadian Prairie Provinces and the northern Great Plains of the USA.

Canola is cultivated both during winter and spring seasons in the United States and this exposes the crop to winter kill, frost, and high temperatures, during the reproductive period. The temperatures during winter and spring are known to influence all the crucial steps of the reproductive cycle including gametogenesis, pollination, fertilization and embryogenesis (Angadi, 2000). Winter rapeseed has been successfully grown in the Pacific Northwest, southern Great Plains, Midwest, and southeast regions of the USA. The hardiest cultivars will routinely survive winters in the north east of USA but survival is inconsistent further south (Rife *et al.,* 2001). Winter-grown canola (*Brassica napus* L.) production is limited mostly by frost and winter-kill in the southern canola-growing regions of the United States (Singh *et al.,* 2008). For instance, the late freeze in 2007 resulted in significant damage to most of the winter canola cultivars at the National Winter Canola Variety Trials in Alabama, U.S. (Cebert and Rufina, 2007). Winter hardiness and freezing tolerance are a major concern for improving production consistency in many regions of the canola growing countries.

<sup>1</sup>*CESTA, Center for Viticulture and Small Fruit Research, Florida A&M University, Tallahassee, USA* 

*<sup>2</sup>Department of Biological and Environmental Sciences, Alabama A&M University, Normal, USA 3Faculties of Agriculture and Veterinary Medicine, University of Nairobi, Nairobi, Kenya*

*<sup>4</sup>Plant Biotechnology Laboratory, College of Agriculture, Florida A & M University, Tallahassee, USA*

*<sup>5</sup>Department of Food and Animal Sciences, Food Biotechnology Laboratory,* 

*Alabama A&M University, Normal, USA* 

*<sup>6</sup>Department of Natural Resources and Environmental Design, North Carolina A&T State University Greensboro, USA*

Prospects for Transgenic and Molecular

temperatures increases.

**1.3 Cold stress symptoms and its after effects** 

Fig. 1. Purpling of leaves due to low temperature.

Fig. 2. Cupping of leaves due to low temperature.

Fig. 3. Leaves damaged by herbicide carryover.

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 3

Cold stress symptoms can arise only after a cold temperature event; however, mild symptoms of herbicide injury may often be confused with symptoms caused by cold stress temperatures or nutrient-deficient soil (Figure 1 to 3). Recovery from cold stress will be rapid as temperatures increase. Nutrient stress symptoms are unlikely to occur at the

Fig. 1 shows that since the 1st and 2nd leaves are of normal size, the purpling observed is not herbicide injury. The purpling is as a result of anthocynin production caused by cold temperatures. Purpling may be towards the base, on the leaf margins or may cover entire

Fig. 2 exhibits cupping caused by cold temperatures and symptoms quickly diminish as

Fig. 3 indicates that cupping was caused by a low level herbicide residue. Variation in herbicide carryover means uninjured (red arrow) and injured yellow arrow and plants may

cotyledon stage as nutrient demands at this stage are generally low (Boyles. 2011).

young leaves of the plant. This symptom will diminish as temperatures increase.

be found in close proximity. Cold stress generally causes more uniform damage.

Introduction and cultivation of new crops in a given environment require management practices and trait selection that enable optimum performance of the crop. Canola is an important oilseed crop and its cultivation is expanding, particularly in the western world because of its importance as both an oilseed and a bio-diesel crop.

#### **1.1 Cold tolerance**

The ability of rapeseed plants to tolerate very low temperatures depends essentially on their development and the degree of hardening it has achieved. Unhardened plants can survive -4°C, while fully-hardened spring-type rapeseed can survive much lower temperatures (-10° to -12°C). Hardened winter rapeseed can survive short periods of exposure to temperatures between 15° and -20°C. Unhardening happens fairly fast after the plants initiate active growth (Sovero, 1993). The plants are typically best adapted to survive the winter in rosette stage with 6 to 8 leaves. Small plants are usually not as capable of surviving over-wintering, while plants with more leaves often start the stem elongation prematurely, exposing the meristem tissue to cold, making it more susceptible to damage. The hardening requirements of rapeseed have not been fully characterized. Winter type canola tend to harden faster, achieve higher degree of cold tolerance and unharden slower than spring types, but it is likely that variable hardening requirements could also be found within both types. Some differences in cold hardiness have been observed among both winter and spring types, however, it is unclear whether these are due to differences in ultimate achievable cold hardiness or differences in hardening requirements. The absence of snow cover during the coldest period of the winter decreases the plants' chances to survive. Ice formation on the soil surface can damage the crown area of the plants and reduce survival rate (Sovero, 1993).

#### **1.2 Vernalization requirement**

Most winter rapeseed cultivars will require three weeks of near-freezing temperatures in the field to get fully vernalized and start rapid generative growth. In controlled environments, eight weeks at 40C temperature is sufficient for full vernalization. In spring planting, winter rape will typically start slow generative growth after the prolonged rosette stage, and some cultivars may start blooming towards the end of the growing season. Differences in this respect are sometimes useful in distinguishing between morphologically similar cultivars. Different vernalization requirements are apparent among winter rape cultivars. A high vernalization requirement does not necessarily result in good winter hardiness, as many of the winter type cultivars from extreme maritime environments, such as Japan, require a long vernalization period yet have little tolerance for low temperatures (Sovero, 1993).

Some spring type cultivars do not exhibit any vernalization response at all, but in some cases the generative development can be accelerated with brief chilling treatment. In spring planting, only a few cool nights are usually needed. Vernalization response in spring types also tends to disappear in a long day environment. In spite of the variability in vernalization requirements within both types, the differences between the two types i.e. winter and spring canola are fairly clear with no overlap in the initiation of blooming in either spring or fall planting (Sovero, 1993).

#### **1.3 Cold stress symptoms and its after effects**

2 Oilseeds

Introduction and cultivation of new crops in a given environment require management practices and trait selection that enable optimum performance of the crop. Canola is an important oilseed crop and its cultivation is expanding, particularly in the western world

The ability of rapeseed plants to tolerate very low temperatures depends essentially on their development and the degree of hardening it has achieved. Unhardened plants can survive -4°C, while fully-hardened spring-type rapeseed can survive much lower temperatures (-10° to -12°C). Hardened winter rapeseed can survive short periods of exposure to temperatures between 15° and -20°C. Unhardening happens fairly fast after the plants initiate active growth (Sovero, 1993). The plants are typically best adapted to survive the winter in rosette stage with 6 to 8 leaves. Small plants are usually not as capable of surviving over-wintering, while plants with more leaves often start the stem elongation prematurely, exposing the meristem tissue to cold, making it more susceptible to damage. The hardening requirements of rapeseed have not been fully characterized. Winter type canola tend to harden faster, achieve higher degree of cold tolerance and unharden slower than spring types, but it is likely that variable hardening requirements could also be found within both types. Some differences in cold hardiness have been observed among both winter and spring types, however, it is unclear whether these are due to differences in ultimate achievable cold hardiness or differences in hardening requirements. The absence of snow cover during the coldest period of the winter decreases the plants' chances to survive. Ice formation on the soil surface can damage the

Most winter rapeseed cultivars will require three weeks of near-freezing temperatures in the field to get fully vernalized and start rapid generative growth. In controlled environments, eight weeks at 40C temperature is sufficient for full vernalization. In spring planting, winter rape will typically start slow generative growth after the prolonged rosette stage, and some cultivars may start blooming towards the end of the growing season. Differences in this respect are sometimes useful in distinguishing between morphologically similar cultivars. Different vernalization requirements are apparent among winter rape cultivars. A high vernalization requirement does not necessarily result in good winter hardiness, as many of the winter type cultivars from extreme maritime environments, such as Japan, require a long vernalization period yet have little tolerance

Some spring type cultivars do not exhibit any vernalization response at all, but in some cases the generative development can be accelerated with brief chilling treatment. In spring planting, only a few cool nights are usually needed. Vernalization response in spring types also tends to disappear in a long day environment. In spite of the variability in vernalization requirements within both types, the differences between the two types i.e. winter and spring canola are fairly clear with no overlap in the initiation of blooming in either spring or fall

because of its importance as both an oilseed and a bio-diesel crop.

crown area of the plants and reduce survival rate (Sovero, 1993).

**1.1 Cold tolerance** 

**1.2 Vernalization requirement** 

for low temperatures (Sovero, 1993).

planting (Sovero, 1993).

Cold stress symptoms can arise only after a cold temperature event; however, mild symptoms of herbicide injury may often be confused with symptoms caused by cold stress temperatures or nutrient-deficient soil (Figure 1 to 3). Recovery from cold stress will be rapid as temperatures increase. Nutrient stress symptoms are unlikely to occur at the cotyledon stage as nutrient demands at this stage are generally low (Boyles. 2011).

Fig. 1 shows that since the 1st and 2nd leaves are of normal size, the purpling observed is not herbicide injury. The purpling is as a result of anthocynin production caused by cold temperatures. Purpling may be towards the base, on the leaf margins or may cover entire young leaves of the plant. This symptom will diminish as temperatures increase.

Fig. 2 exhibits cupping caused by cold temperatures and symptoms quickly diminish as temperatures increases.

Fig. 3 indicates that cupping was caused by a low level herbicide residue. Variation in herbicide carryover means uninjured (red arrow) and injured yellow arrow and plants may be found in close proximity. Cold stress generally causes more uniform damage.

Fig. 1. Purpling of leaves due to low temperature.

Fig. 2. Cupping of leaves due to low temperature.

Fig. 3. Leaves damaged by herbicide carryover.

Prospects for Transgenic and Molecular

components (Cebert and Rufina, 2007).

2007 in North Alabama, U.S.A.

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 5

during mid to late spring (late March to Mid April). In April, 2007, twenty winter canola genotypes at varying stages of flowering and early pod-filling (Figures 4-9) were exposed to three incidences of naturally occurring severe spring frosts that reduced most of the yield

Fig. 4-9 show the extent of damage resulting from cold stress after freeze from April 5 to 9,

Fig. 4. Canola field at flowering after spring 2007 hard freeze in Northern Alabama.

Fig. 5. Early maturity breeding line (lower right) completely destroyed by late spring frost.

Fig. 6. Several cultivars did not suffer much damage. Cultivar, Kadore produced the highest

yield despite the freeze followed by an exceptional drought period.

#### **1.4 Methods of measuring cold stress**

Field sites often exhibit either complete survival or complete winter-kill. Because of this variability, laboratory procedures to measure freezing tolerance have been developed including plant tissue water content (Brule-Babel and Fowler, 1988), ion leakage from plant cells after a freezing stress (Teutonico *et al.,* 1993), and changes in luminescence (Brzostowicz and Barcikowska, 1987). Further, meristem regrowth after plants are subjected to freezing temperatures is commonly used (Andrews and Morrison, 1992).

Laboratory freezing tolerance procedures have allowed investigators to gather information on cold tolerance that would otherwise be unobtainable in the field. Freezing tolerance of plant tissue is evaluated by measuring whether the tissues are alive or dead after subjecting the tissue to a range of freezing temperatures. The extent of damage caused by the freezing can be evaluated by placing plant tissue in distilled water and measuring the electrical conductivity of the resultant solution (Madakadze *et al*., 2003; Murray *et al.,* 1989). An increased rate of electrolyte loss is interpreted as evidence and the extent corresponds to damage. The electrolyte leakage (EL) method (Oakton CON 510TDS electrical conductivity meter: Eutech Instruments, Singapore) is based on objective measurements, which utilizes small quantities of tissue, and is relatively cheap. However, it takes more time than the chlorophyll fluorescence method (OS1-FL portable pulse-modulated fluorometer: Opti-Sciences, Tynggsboro, MA).

Freezing on the functionality of the photosynthetic apparatus can be used to assess the cold tolerance of plant genotypes (Chengci *et al.,* 2005). The photosynthetic apparatus function can be evaluated by measuring the ratio of chlorophyll variable fluorescence (Fv) over the maximum fluorescence value (Fm) (Fv/Fm), which indicates the efficiency of the excitation capture by open photosystem II reaction centers (Frachebound *et al*., 1999; Rizza *et al.,* 2001). A significant reversible decrease in Fv/Fm was found 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 (Rizza *et al*., 2001). Measurement of Fv/Fm is rapid and noninvasive.

#### **2. Effect of cold stress on plant performance**

#### **2.1 Cold stresses reduce plant productivity**

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. Low temperatures reduce both the final percentage as well as the rate of germination, which leads to delayed and reduced seedling emergence of canola (Zheng et al., 1994). Early spring frosts are more problematic with fall-seeded canola, which emerges earlier than canola seeded in early spring (Willenborg *et al.,* 2004). Fall seeding of canola does present some challenges in the Canadian Prairie Provinces and the northern Great Plains of the USA. Fall seeding frequently entails seeding into hard, cold soil, which ultimately results in poor soilto-seed contact (Kirkland and Johnson, 2000).

Mild winter climate in Northern Alabama, USA is conducive for optimum productivity of winter canola (Cebert and Rufina, 2007). However, the region is also vulnerable to late frost

Field sites often exhibit either complete survival or complete winter-kill. Because of this variability, laboratory procedures to measure freezing tolerance have been developed including plant tissue water content (Brule-Babel and Fowler, 1988), ion leakage from plant cells after a freezing stress (Teutonico *et al.,* 1993), and changes in luminescence (Brzostowicz and Barcikowska, 1987). Further, meristem regrowth after plants are subjected

Laboratory freezing tolerance procedures have allowed investigators to gather information on cold tolerance that would otherwise be unobtainable in the field. Freezing tolerance of plant tissue is evaluated by measuring whether the tissues are alive or dead after subjecting the tissue to a range of freezing temperatures. The extent of damage caused by the freezing can be evaluated by placing plant tissue in distilled water and measuring the electrical conductivity of the resultant solution (Madakadze *et al*., 2003; Murray *et al.,* 1989). An increased rate of electrolyte loss is interpreted as evidence and the extent corresponds to damage. The electrolyte leakage (EL) method (Oakton CON 510TDS electrical conductivity meter: Eutech Instruments, Singapore) is based on objective measurements, which utilizes small quantities of tissue, and is relatively cheap. However, it takes more time than the chlorophyll fluorescence method (OS1-FL portable pulse-modulated fluorometer: Opti-

Freezing on the functionality of the photosynthetic apparatus can be used to assess the cold tolerance of plant genotypes (Chengci *et al.,* 2005). The photosynthetic apparatus function can be evaluated by measuring the ratio of chlorophyll variable fluorescence (Fv) over the maximum fluorescence value (Fm) (Fv/Fm), which indicates the efficiency of the excitation capture by open photosystem II reaction centers (Frachebound *et al*., 1999; Rizza *et al.,* 2001). A significant reversible decrease in Fv/Fm was found 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 (Rizza *et al*., 2001). Measurement of

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. Low temperatures reduce both the final percentage as well as the rate of germination, which leads to delayed and reduced seedling emergence of canola (Zheng et al., 1994). Early spring frosts are more problematic with fall-seeded canola, which emerges earlier than canola seeded in early spring (Willenborg *et al.,* 2004). Fall seeding of canola does present some challenges in the Canadian Prairie Provinces and the northern Great Plains of the USA. Fall seeding frequently entails seeding into hard, cold soil, which ultimately results in poor soil-

Mild winter climate in Northern Alabama, USA is conducive for optimum productivity of winter canola (Cebert and Rufina, 2007). However, the region is also vulnerable to late frost

to freezing temperatures is commonly used (Andrews and Morrison, 1992).

**1.4 Methods of measuring cold stress** 

Sciences, Tynggsboro, MA).

Fv/Fm is rapid and noninvasive.

**2. Effect of cold stress on plant performance** 

**2.1 Cold stresses reduce plant productivity** 

to-seed contact (Kirkland and Johnson, 2000).

during mid to late spring (late March to Mid April). In April, 2007, twenty winter canola genotypes at varying stages of flowering and early pod-filling (Figures 4-9) were exposed to three incidences of naturally occurring severe spring frosts that reduced most of the yield components (Cebert and Rufina, 2007).

Fig. 4-9 show the extent of damage resulting from cold stress after freeze from April 5 to 9, 2007 in North Alabama, U.S.A.

Fig. 4. Canola field at flowering after spring 2007 hard freeze in Northern Alabama.

Fig. 5. Early maturity breeding line (lower right) completely destroyed by late spring frost.

Fig. 6. Several cultivars did not suffer much damage. Cultivar, Kadore produced the highest yield despite the freeze followed by an exceptional drought period.

Prospects for Transgenic and Molecular

(Nykiforuk and Johnson-Flanagan, 1994)

**2.2 Plant responses to cold stress** 

Breeding for Cold Tolerance in Canola (*Brassica napus* L.) 7

germination temperature for canola was reported to be 5°C (Morrison *et al*., 1989) and 0.4 to 1.2°C (Vigil *et al*., 1997). Soil temperature in April in most canola production areas of the northern Great Plains is usually lower than the previously given optimum temperatures (Zheng *et al.*, 1994). Spring canola seeded into suboptimal soil temperatures had lower emergence and stand establishment rates due to the seed rotting in the cold soils (Blackshaw, 1991; Livingston and de Jong, 1990). A significant reduction in canola germination was found at temperatures less than 10oC (Nykiforuk and Johnson-Flanagan, 1994), and it took as long as 18 days for 50% emergence at 5°C (Blackshaw, 1991). Vigil *et al.* (1997) reported that 65 to 81 growing degree days (GDD) are required for spring canola seedlings to emerge. Differences in GDD have also been found among the species and seed lots

One can determine an optimal seeding date in the northern Great Plains or other regions with similar climate and soil conditions as central Montana (Chengci *et al*., 2005). First, days from seeding to emergence can be predicted from long-term weather data based on the base temperature for germination (Tb) and growing degree days for 50% emergence. Second, based on the cold tolerance information, one can decide which cultivar to plant and the risk of frost damage for a given early seeding date. Third, using the information on days to 50 % flowering in combination with long-term weather data, the maximum daily temperatures at flowering stage, optimum seeding date can be forecasted; thus, the potential impact of

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

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

maximum temperature on seed yield can be estimated for a given seeding date.

to determine freezing tolerance and not crown meristem survival.

Fig. 7. Primary yield loss due to dropping off of fertilized flowers after late spring frost.

Fig. 8. Destruction of photosynthetic green tissues due to late spring frost.

Fig. 9. Destruction of developing pods due to late spring frost.

Early seeded canola may encounter suboptimal soil temperatures for seed germination and seedling establishment in the northern Great Plains (Chengci *et al*., 2005). The optimum

Fig. 7. Primary yield loss due to dropping off of fertilized flowers after late spring frost.

Fig. 8. Destruction of photosynthetic green tissues due to late spring frost.

Fig. 9. Destruction of developing pods due to late spring frost.

Early seeded canola may encounter suboptimal soil temperatures for seed germination and seedling establishment in the northern Great Plains (Chengci *et al*., 2005). The optimum

germination temperature for canola was reported to be 5°C (Morrison *et al*., 1989) and 0.4 to 1.2°C (Vigil *et al*., 1997). Soil temperature in April in most canola production areas of the northern Great Plains is usually lower than the previously given optimum temperatures (Zheng *et al.*, 1994). Spring canola seeded into suboptimal soil temperatures had lower emergence and stand establishment rates due to the seed rotting in the cold soils (Blackshaw, 1991; Livingston and de Jong, 1990). A significant reduction in canola germination was found at temperatures less than 10oC (Nykiforuk and Johnson-Flanagan, 1994), and it took as long as 18 days for 50% emergence at 5°C (Blackshaw, 1991). Vigil *et al.* (1997) reported that 65 to 81 growing degree days (GDD) are required for spring canola seedlings to emerge. Differences in GDD have also been found among the species and seed lots (Nykiforuk and Johnson-Flanagan, 1994)

One can determine an optimal seeding date in the northern Great Plains or other regions with similar climate and soil conditions as central Montana (Chengci *et al*., 2005). First, days from seeding to emergence can be predicted from long-term weather data based on the base temperature for germination (Tb) and growing degree days for 50% emergence. Second, based on the cold tolerance information, one can decide which cultivar to plant and the risk of frost damage for a given early seeding date. Third, using the information on days to 50 % flowering in combination with long-term weather data, the maximum daily temperatures at flowering stage, optimum seeding date can be forecasted; thus, the potential impact of maximum temperature on seed yield can be estimated for a given seeding date.
