A Comparative Study on Root Traits of Spring and Winter Canola (Brassica napus L.) under Controlled and Water Stressed Conditions

Root system in canola (Brassica napus L.) varies largely in different growth habit types. A study was conducted with five winter and five spring types of canola germplasm. The objective was to identify the gradual change of root traits at different growth habits stages under controlled and water stressed conditions. Two experiments, controlled and water stressed, were conducted in a greenhouse. Data on different root traits were collected at 30, 40, 50 and 60 days after planting. In controlled experiment, no significant difference was observed for root traits between winter and spring types at 30 days after planting. However, significant variations were appeared for taproot length (F = 10.17***) and root dry weight (F = 16.96***) between winter and spring types at 40 days after planting. All other root parameters such as basal taproot diameter (F = 22.14***), bottom taproot diameter (F = 4.59*), primary root branches (F = 78.70***) and root vigor (F = 47.18***) were significantly higher in the winter types compared to those of the spring types at 60 days after planting. Growth pattern curves indicated that all the root traits of spring types increased in a steady fashion, where the root traits of winter types increased rapidly after 40 days of planting. In water stressed experiment, the water stress was applied from 20 to 60 days after planting, and data was taken at 60 days after planting. All the root parameters except taproot length were significantly (P < 0.001) lower in the stressed spring and winter plants compared to the control plants. The root growth reduction in stressed winter type germplasms was higher. Basal taproot diameter, bottom taproot diameter, primary root branches, root vigor, and root dry weight were decreased by 43%, 63%, 19%, 31% and 53%, respectively in stressed winter type plants. In contrast, the root growth reduction of the spring type germplasms were relatively lower. This study indicated that winter type canola generates vigorous root system in comparison to spring types under normal growing conditions, but ceases its root growth rate more than the spring types under water stressed conditions.


Introduction
Root system plays the major role that provides great anchorage and support to the plant, and allows of mining water and nutrient from the soil.Deep and vigorous root system can facilitate higher moisture and nutrient acquisition from the soil, which can increase the yield largely (Marschner, 1998).On the other hand, less vigorous and shallow root system can uptake less amount of moisture and nutrients, which might end up with reduced yield and biomass production.Moreover, shallow root system cannot uptake moisture from deeper soil, and therefore, become vulnerable in drought prone soil.Crop plants use the nutrient and water to perform the necessary metabolic processes, which definitely affect the crop growth and yield positively.For example, maize root system exhibits root growth variation under low phosphorus (P) level in soil and the genotypes having higher lateral root growth were able to uptake more P and maintained good crop stand (Zhu & Lynch, 2004).Seed yield is positively correlated with longer root system in rice (Steele et al., 2013), canola (M.Rahman & McClean, 2013), soybean (Brown & Scott, 1984) and maize (Mackay & Barber, 1986;Hochholdinger et al., 2008).
Three canola growth habits, including winter, semi-winter, and spring types, differ greatly in terms of shoot morphology, root growth and flowering time.Winter types canola have vigorous root system with higher root length, root diameter, root mass, root branches over the spring types canola (Rahman & McClean, 2013;Arif Uz Zaman, Mamidi, McClean, & Rahman, 2016).With these superior root characteristics, root system of winter types might be able to cover more area and depth in soil and better access to moisture and nutrient.As the root length of canola is positively correlated with seed yield (Rahman & McClean, 2013), it can be hypothesized that moisture and nutrient uptake capability is higher in winter types canola, which might play a major role in the higher yield over the spring types.These canola growth habits belong to different genetics groups (Kebedi, Thiagarajah, Zimmerli, & Rahman, 2010).Therefore, huge variation in the root system of winter and spring canola can serve as significant source of genetic diversity in breeding for high yielding spring canola.Direct selection for root traits in the traditional breeding programs is not very popular yet due to several constraints, including phenotyping, root plasticity etc. Detecting the root phenotypic variation in a large scale field trial regarded as one of the main constraints.In addition, root plasticity or preferential growth towards the area of higher moisture and nutrient may deceive plant breeders in highly heterogeneous soil (Arif Uz Zaman et al., 2016).Identifying quantitative trait loci (QTL) associated with the genomic region that control root variation in canola and subsequent marker assisted selection (MAS) could be an alternative solution.However, appropriate phenotyping of a trait is always very critical in the process of identifying genomic region associated with that trait.
We observed in a preliminary study that there is no major variation in root traits between winter and spring types at early growth stages, rather variations observed in the matured plants.There is no report available that monitored the variations of root growth pattern of winter and spring types canola.The lack of adequate information in this regard limits the scope of proper phenotyping of root traits in a mapping population, as well as traditional selection for root traits in the breeding program.Considering these factors, the objectives of the current study was to detect the plant growth stages at which the variation of root traits initiate and reach to maximum.Our secondary objective was to study the root growth behavior under simulated water stress conditions in spring and winter types canola.

Plant Materials
A total of 10 canola germplasms, five winter types (Wichita, Lindora-00, KSU 8, KSU 10, Regal) and five spring types (Oro, DH45, Kanada, Regent, Wester), were used for this experiment.The plants were grown in long pots (40 cm × 10 cm) in a greenhouse.A mixture of sand and peat soil in a ratio of 8:2, respectively, were used to grow the plants.The growing media was supplemented with 10g/pot Osmocote® slow-release fertilizer (Scott's Company LLC, Marysville, OH, USA).Before potting, the pots were lined with plastic bags to facilitate root extraction procedure.The plastic bags were perforated at the bottom to allow the excess water to drain out.Plants were watered daily to saturate all pots and fertilized with water-soluble 20-20-20 fertilizer once a week.

Experimental Design
Four sets of experiments with the same canola germplasm panel (5 winter and 5 spring types) were grown under greenhouse conditions.Each set was planted in a randomized complete block design (RCBD) with four replications.Each pot contains a single canola plant and considered as an experimental unit.These four sets of experiments were grown for four different time periods such as 30 days, 40 days, 50 days, and 60 days after planting.

Data Collection
Data were collected from the plants at 30 days after planting (30 d), 40 days after planting (40 d), 50 days after planting (50 d), and 60 days after planting (60 d).Data on number of leaves and stem diameter were taken from the freshly harvested plants.The plants were cut at the base of the root.The pots were taken to root washing zone.The roots with plastic bag were taken out from pots, placed on sink containing a fine plastic net, and the soils covering root masses were washed with running water.This procedure facilitates to avoid root loss during washing.Absorbent papers were used to soak the water from the extracted clean root system and kept them for 1 hour at room temperature.Data on taproot diameter were taken at two points, just at the below of soil level where the first root was initiated (basal taproot diameter) and at 10 cm below from the place of first root diameter (bottom taproot diameter).Data on bottom taproot diameter were not taken at 30 d.In addition, data on tap root length, and number of root branches were taken.Total root system were visually scored on the basis of root vigor and root mass on a scale of 1-5 according to Rahman and McClean (2013), where score 1: weak bottom and surface roots, score 2: more bottom and surface roots, score 3: intermediate bottom and surface roots, score 4: strong bottom and surface roots, and score 5: the strongest bottom and surface roots (Figure 1).The entire root system of each plant were stored in a perforated plastic bags and dried in 60 ºC until constant weight.Data on root dry weight were recorded for each plant.

Data A
The root t SAS 9.3 st separately winter typ

Water
A water st winter can Water stre (approxim moisture le dry at a so 60 days a technologi basal tapro weight we

Stem D
At 30 day (Table 1      With the increasing popularity of molecular breeding methods, high throughput genotyping obtained a substantial improvement in last two decades, however, phenotyping did not receive much attention yet (Zhu, Gore, Buckler, & Yu, 1008).For the complex traits like roots, phenotyping for large scale association study is even more difficult.The pattern of root growth variation observed in this study will be very useful in the future study of large scale association mapping for different root traits in canola.Phenotyping at 50-60 days after planting will be most effective time to capture maximum amount of phenotypic variation for different root traits in canola.This information might also be helpful in selecting or phenotyping individual root trait for special need.

Number o plants and lower in th statistically
Crop growth and yield are significantly affected by drought stress (Martin, Waldren, & Stamp, 2006;Saidi, Ookawa, & Hirasawa, 2010).Crop root system plays a vital role in avoidance or adapting plants under low moisture content in the soil (Loomis & Connor, 1992).Modification or alteration of root system under different abiotic stress including drought is a common adaptive measure of plants.In this process, different root traits comprising the whole root system, may respond differently under low water regime (Franco, Bañón, Vicente, Miralles, & Martínez-Sánchez, 2011;Licht, Wright, & Lenssen, 2013).Basal taproot diameter and bottom taproot diameter were found significantly higher (p < 0.001) in both spring and winter types of control plants comparing to those of stressed plants.This finding is consistent with those observed in decreasing root diameter under low water conditions in pea (Eavis, 1972), soybean (Read & Bartlett, 1972), maize (Sharp, Silk, & Hsiao, 1988;Liang, Sharp, & Baskin, 1997) and Silene vulgaris (Franco, Arreola, Vicente, & Martínez-Sánchez, 2008).Sharp et al. (1988) concluded that, decreasing root diameter is an adaptive measure under low moisture regime so that plant concentrates their resources for root elongation to reach water level.A significant reduction was observed in root vigor and root dry weight of winter and spring types grown in water stressed conditions compared to their normal growing conditions.Similar responses were described by Martin et al. (2006) and Saidi et al. (2010) in water stressed plants.In Arabidopsis, reduction of root dry weight under severe water stress has been identified (van der Weele, Spollen, Sharp, & Baskin, 2000).
We did not find any growth habit difference in terms of root length under control and water stressed conditions.This result is in agreement with Licht et al. (2013) who reported that soybean root elongation was unaffected under water deficit condition.Saidi et al. (2010) observed similar phenomena in maize where no significant difference for total root length was found between different water potential in soil.Franco et al. (2008) studied root and shoot growth in Arabidopsis at very early stage in nutrient-agar media and reported that the root elongation was actually stimulated under a certain limit of water potential deficit.Plants usually improve osmotic adjustment at the root growing zone under low moisture availability which might help plants to maintain their root elongation (Martin et al., 2006).
Upper ground traits such as stem diameter and number of leaves were significantly decreased (p < 0.001) in water stressed plants compared to control plants of spring and winter types.To our knowledge, there is no report available on the effect of water stress on stem diameter and leaf number.These changes are expected as many researchers reported that the effect of water stress on shoot growth is higher than the root growth (Saidi et al., 2010;Licht et al., 2013;Franco et al., 2008;van der Weele et al., 2000).
We have observed a differential root growth of spring and winter types canola starting from 40 days after planting.The root growth of spring types significantly reduced at 40 days after planting when the plants start to initiate buds.Winter types do not flower without vernalization and therefore they continue to grow for roots and shoots.All the root traits of the winter type cultivars are highly affected under water stressed conditions over the control experiment.However, this effect is much lower in stressed spring types cultivars compared to their control study.This might be due higher water requirement by the winter type cultivars as they possess higher root and shoot vigor compared to the spring types.In addition, it could be a fact that spring types had higher relative water use efficiency compared to the winter types.However, future investigation is needed to confirm this hypothesis.
Figure 1 interme Figure 4 water str diameter, d

Table 1
Rahman and McClean (2013)ated at around 40 d.Rahman and McClean (2013)observed a significant difference of root vigor of winter and spring types during flowering time of spring plants.