Wheat Growth As Affected By Planting Density, Planting Time And Nitrogen Application


 The growth and development of wheat and quality of wheat flour is dependent on the genotype, fertilizer application, sowing date and plant density. Therefore, plant growth and protein content studies were conducted on two wheat genotypes during the winter planting seasons of 2015 and 2016. The experiment was laid out in a Randomised Complete Block Design in a split-split-plot arrangement, with the main plots being two wheat genotypes; sub plots being two plant density treatments, sub-sub plots being five nitrogen doses and sub-sub-sub plots of two planting dates, replicated three times. The application of 125 kg/ha nitrogen increased the number of days to anthesis, grain protein content and plant height. Early planting reduced days to emergence but delayed days to anthesis and physiological maturity, whereas the protein content and plant height were increased by early planting. The leaf dry mass, crop growth rate and net assimilation rate were similar at each crop stage for the planting densities, and they were increased at 125 kg/ha application of nitrogen. The genotypes showed the presence of glutens of both high molecular and low molecular weight which are likely to contain genes that supports good baking quality of flour. The interaction of plant density, planting time and nitrogen at 125 kg/ha contributed more protein bands with low and high molecular weight glutenin’s in wheat genotypes.


Introduction
The world population is increasing at an alarming rate but the land for cultivation of arable crops is decreasing. This calls for cultivation of high value crops such as wheat (Triticum aestivum L.) to increase yield per unit area. Wheat is important as it provides more protein than any other cereal crops [1].
Currently wheat is not grown in Botswana, as such the country import wheat and for it to start producing wheat, agronomic studies are needed to determine varieties and optimal planting conditions. The wheat grains are ground into our, and the greatest portion of the wheat our is used for bread making. The grain protein content and composition of wheat is affected by both genotype and the environment in which the crop is grown. Nitrogen is applied to plants to improve crop quality and to increase yield, and its application also contributes considerably to protein content, especially when fertilizer rates satisfy the requirements of both yield and protein formation [2]. The correct fertilizer application especially nitrogen is important to achieve higher yields and good grain quality of wheat [3].
In addition to grain protein content, the presence of gluten in wheat makes it unique [4]. The glutens are important our quality determinants because they are responsible for dough extensibility and elasticity of wheat our, which determine the processing qualities of a wide range of wheat products [5]. The amount of gluten in wheat is normally in uenced by the environment and the grain protein content, but also the genotype [6]. Wheat grain protein can be divided into two groups: non-gluten proteins (albumin and globulins) and gluten proteins (gliadins and glutenin's). Gluten protein subunits are divided into High Molecular Weight Glutenin (HMW-GS) and Low Molecular Weight Glutenin's (LMW-GS), [7]. The molecular structure of the glutens determines the quality of the bread and controls the interactions of gluten subfractions during processing [8].
Besides regular nutrition of plants for achieving high yields, the genotype, plant density and planting time also play an important role in good grain quality for bread making in wheat [3,9]. Optimum densities vary between areas, climatic conditions, planting time and varieties. Since cultivars genetically differ for yield components, individual cultivars need to be tested for proper density [10]. Management practices play a vital role in determining yield and end-use quality of wheat. Therefore, the aim of this research was to investigate the in uence of plant density, nitrogen rate, and planting time on plant growth and the protein content as well as the molecular pro le of the grain proteins in two wheat genotypes.

Experimental site
The eld studies were conducted at the Botswana University of Agriculture and Natural Resources (BUAN), Sebele, Gaborone, over the winter planting seasons of 2015 and 2016 under irrigation, in sandy loam soil. Botswana is arid to semi-arid region with rainfall in the summer and dry winters.

Experimental design and treatments
The experiment was laid out in a Randomised Complete Block Design (RCBD) under split-split-plot arrangement with three replications. The main plot consisted of two spring wheat genotypes, Baviaans and 14SAWYT308; and sub plots comprised of two plant density treatments, (0.2m × 0.15m) 333,333 plants/ha and (0.2m × 0.2m) 250 000 plants/ha; sub-sub plots were ve nitrogen doses, 0 kg/ha (N 0 ), 50 kg/ha (N 50 ), 75 kg/ha (N 75 ), 125 kg/ha (N 125 ) and 200 kg/ha (N 200 ); and sub-sub-sub plots of two planting dates, 21 st April and 05 th May. The nitrogen fertilizer applied was urea (46.5% N) and half of the nitrogen was applied at the time of planting while the remaining half was applied one week after seedling emergence. Two weeks after sowing, thinning was done to one plant per hill. Phosphorus (single super phosphate, 9%) was applied at sowing at the rate of 55 kg/ha. Irrigation was done two times a week for two hours each time to eld capacity.

Growth parameters
At 28, 56, 84, 112, and 140 days after sowing (DAS), three plants were randomly selected from each plot and measurements on leaf dry mass, crop growth rate, net assimilation rate, and leaf area were taken.
The leaves from the three plants selected at random from each sub-plot were dried in the oven at 70 ± 5 C° until reaching a constant weight after 48 hours and weighed to get the leaf dry mass. The leaves from each plant were placed on an electronic LI-3100 leaf area meter (USA-model), to get the leaf area. Leaf area index (LAI) for each experimental unit was computed using leaf area values as the ratio of total leaf area to land for each experimental unit [11].
Leaf area duration (LAD) was calculated by the formula as proposed by [12]. The LAD was determined starting from 28 DAS up to 112 DAS. LAI 1 = leaf area index at t 1 , LAI 2 = leaf area index at t 2 , T 1 = time for rst observation, T 2 = time for second observation The dry matter accumulation was determined starting from 28 DAS up to 140 DAS by taking three plants at random from each sub-plot. After taking samples they were dried in the oven at 70 ± 5 C° until reaching a constant weight after 48 hours. Then samples dry weight was calculated and used to nd out crop growth rate (CGR) by the formula as given by [12]. W 2 = dry weight per unit land area (gm -2 ) at second harvest, W 1 = dry weight per unit land area (gm -2 ) at rst harvest, T 2 = time taken to second harvest, T 1 = time to taken rst harvest The net assimilation rate (NAR) was determined by the formula as stated by [12]. The NAR was determined starting from 28 DAS up to 112 DAS using the same leaves as for CGR. The days to emergence was calculated as difference between date of emergence and date of sowing; days to anthesis was computed as difference between date of anthesis and date of sowing; days to maturity was computed as difference between the date of physiological maturity and date of sowing, and plant height at maturity was measured using a ruler from the base of the node to the top of the plant.

Grain protein content
The content of nitrogen on grain samples was determined by micro Kjeldahl method described by [13]. Protein percentage was determined by multiplying grain nitrogen content by 5.7, which is the appropriate factor worked out for wheat [14].

Protein pro ling
Five wheat seeds from each treatment were ground with an electric laboratory grinder (model A10 57-Kinematica Switzerland) and 10 mg of the seed our was taken into a 1.5 mL micro tube. A 400 µL of protein extraction buffer (1% β-mecaptoethanol, 5M urea, 0.2% SDS, 0.05 M tris) and 0.0001% bromophenol blue were added and content vortexed for 5 minutes. The crude homogenates were centrifuged using a micro centrifuge set at 13 000 rpm at room temperature for 10 minutes. The separation gel (12.25%) was prepared using 5 mL Solution A (3.0 M Tris-HCL pH 9.0, 0.4 % SDS), 7.5 ml Solution C (30 % Acrylamide, Acrylamide/Bis = (30:0.8), 200 µL of 10% Ammonium per sulphate (APS)), 7.5 mL of distilled water and 15 µL of TEMED. Glass plates were cleaned with 70% ethanol and xed with clips, and separation gel poured to the cell layered with water, and after 30 minutes distilled water was removed and stacking gel added. A comb was inserted into stacking gel, and the supernatant was separated and 15 µL of each sample (supernatant) along with protein molecular weight marker (11 -190 kDa broad range) was loaded into gels. Electrophoresis was done at constant voltage of 100 volts, until the dye front reached the bottom of the gel. The gels were then removed from plates, dyed in staining solution (Coomasie Brilliant Blue -R250, 6% Acetic acid, 44% methanol) and shaken using an electric shaker (model MK 161-Japan) until protein bands appeared (2 hours). Gels were then washed with distilled water for 5 minutes then transferred to de-staining solution (20% methanol, and 5% acetic acid) then shaken in electronic shaker until protein bands appeared (2 hours). The gel was observed under gel documentation system Bio-RAD with white light illuminator and photographed.

Data analysis
Data on agronomic traits were subjected to an Analysis of Variance (ANOVA) using STATISTICA package, version 13.1 after testing for normality. Where there were signi cant differences, mean separation was done using the Least Signi cance Difference (LSD) method. For protein pro ling, data electropherograms for each treatment were scored and the presence (1) or absence (0) of each band recorded and entered into a binary data matrix, and similarity matrix generated was used to construct dendrogram using a statistical package NTSYC-PC, Version 2.0 [15].

Results
The genotype, plant density and the application of nitrogen fertiliser had no effect on the number of days to emergence of wheat in both years, but planting date affected the number of days to emergence, with earlier planted wheat (21 April), emerging quicker ( Table 1). The wheat genotypes and planting density had no effect on number of days to anthesis except for plant density in 2016. Applying nitrogen increased the number of days to anthesis and plants that received 125 kg/ha of nitrogen (N) exhibited the highest number of days to anthesis, and plants grown on 21st April reached the anthesis later than those planted on 5th May in both years. The days to physiological maturity was not affected by genotypes, planting density and nitrogen application. Planting date signi cantly affected days to physiological maturity with the earlier planted plants taking longer to reach physiological maturity in both years.  The highest plant density showed the highest leaf dry mass but did not differ signi cantly to that of the lowest plant density at each measuring time (Figure 1). The leaf dry matter values increased from early development stage (28 DAS) until they reached maximum values at owering stage (84 DAS), and thereafter dropped until plants reached maturity. Also, the crop growth rate showed peak values at owering time, 84 DAS ( Figure 2).
The net assimilation rate (NAR) was the same at each growth stage for both planting densities (Figure 3). At 56 DAS there was a decline in the NAR, and then it increased at owering and slightly decreased towards maturity.
The application of nitrogen enhanced leaf dry matter of wheat genotypes (Figure 4). There was more leaf dry mass accumulated with the application of 125 kg/ha at each sampling time (Figure 4), but the means did not differ signi cantly from each other (p≤0.05) for a sampling time. Leaf dry mass accumulation increased steadily from the vegetative stage until it reached peak at owering stage (84 DAS), thereafter reduced gradually until physiological maturity (140 DAS).
The crop growth rate and net assimilation rate showed the highest values at owering time ( Figure 5 and 6). The application of 125 kg/ha nitrogen increased the crop growth rate and the net assimilation rate at owering and maturity time. A total of 70 protein bands were detected and were identi ed by common characteristics from main cluster 3 (Table 3). The highest number of bands (17)

Discussions
The addition of nitrogen fertiliser increased number of days to anthesis of wheat, and this may be attributed to prolonged vegetative growth stage at tillering stage. However, nitrogen had no effect on number of days to emergence and physiological maturity of wheat. Over the two years, nitrogen application signi cantly affected plant height of wheat genotypes, with the tallest plants recorded at the nitrogen dose of 125 kg/ha. At the rate of 125 kg/ha, nitrogen was enough to increase the protein content of the cells, as a result cell size increased, evident by more crop growth rate, consequently the leaf area enlarged, and photosynthetic activity increased and ultimately plant height increased as well. The results of increased plant height at higher nitrogen level are supported by previous studies [16,17]. The nitrogen application at 125 kg/ha also in uenced leaf dry mass, crop growth rate and net assimilation rate.
The wheat crops planted late took longer to emerge than those planted on early during both seasons. Delayed emergence may be due to slightly lower average minimum temperature compared to those of April. Normally when temperatures go down the process of germination becomes slow and ultimately the crown root initiation stage is prolonged, resulting in delayed emergence. Earlier research has demonstrated that growth of later-planted wheat is generally slower because of lower temperatures [18], and this might have contributed to signi cantly longer days to emergence by seedlings planted late. The results agree with [19] who found that early sowing had signi cantly maximum plant height and grain yield.
The earlier planted plants took more time to reach anthesis and physiological maturity than those planted late. This is probably because of lower temperatures in the month of July coinciding with anthesis period for the 21st April sowing date which probably lowered photosynthetic activity which resulted in delayed anthesis and physiological maturity. The results agree with ndings [20], who found that longer maturity period was taken by early planted crops. In addition, [21] reported similar ndings of longer physiological maturity with earlier planting due to effect of lower temperature and more grain lling time. In this study, planting date did not have a signi cant effect on crop growth rate, although the crop growth rate value was maximum at owering. It is therefore unlikely that vigorous vegetative growth might be the cause for delayed anthesis and physiological maturity, hence the lower temperature effect is probable.
The higher grain protein content (GPC) was recorded during the early planting suggesting that grain lling period was longer at the earlier sowing, and more assimilates, together with vegetative nitrogen, was channelled to grains resulting in improved grain protein content. [22] reported that the highest grain protein content and grain yield can be produced by planting wheat crop at proper sowing time. The results of increased protein content agree with earlier planting studies by [23,24]. The highest grain protein content was observed with the application of 125 kg/ha N compared to the control. This showed that wheat genotypes needed the application of nitrogen to increase protein content, hence the gluten content, which is needed for good break making. [25] observed that nitrogen rate signi cantly improved wheat grain yield, grain protein content and protein yield.
The two wheat genotypes were clustered in a dendrogram showing protein band size similarities based on the 40 treatments of the current study using Un-weighted Pair Group Method with Arithmetic mean cluster analysis (UPGMA). From the four main clusters demarcated at coe cient of ≈ 0.63, the highest number of treatment interactions was recorded in main cluster 3. The protein bands identi ed were of sizes 25, 32, 58, and 75 kDa, and these results suggest the presence of Low Molecular Weight (LMW) and High Molecular Weight (HMW) glutenin in the cluster 3 treatments. The sub-clusters of main cluster 3 were grouped into A to F, on which demarcation of treatments was at coe cient 0.88 or more, implying that they share at least 88% HMW and/or LMW protein bands amongst them. Studies have shown that the HMW glutens have the highest in uence on the rheological properties of dough and bread-making quality [26,27].
The main cluster 2 comprised of only one treatment interaction, and the binary data matrix analysis of this cluster was able to identify protein band sizes of 22, 46, and 58 kDa, thus, classifying them mainly as low molecular weight glutenin. The main cluster 1 showed that the treatment interaction contained protein of band sizes 11 to 58 kDa, suggesting that they are low molecular weight protein glutenin's. The Low Molecular Weight glutenin's have been documented to contain Glu-A3d, Glu-B3b and Glu-B3g subunits, which are associated with good baking quality [7,28] Therefore, the effect of genotype (all Baviaans at sub-cluster F) and nitrogen (dominance of 125 kg/ha of N at both cluster E and F), and 21st April planting date (16 bands from sub-cluster E) seem to have played a bigger role in clustering of protein bands.

Conclusions
The application of 125 kg/ha nitrogen signi cantly increased the number of days to anthesis, grain protein content and plant height. The interaction of plant population, planting time and nitrogen at 125 kg/ha contributed more protein bands with low and high molecular weight glutenin's and that would produce wheat our with good baking qualities in the wheat genotypes. Effect of plant density on net assimilation rate (g m -2 day -1 ) in wheat genotypes from 28 to 112 DAS during 2015.

Figure 4
Effect of nitrogen doses on leaf dry mass (g m -2 ) in wheat during 2015.

Figure 5
Effect of nitrogen on crop growth rate (g m -2 day -1 ) in wheat during 2015 Effect of nitrogen on net assimilation rate (g m -2 day -1 ) in wheat during 2015 One dimensional SDS-PAGE separation of proteins of wheat genotype 14SAWYT308. Molecular weight maker was represented by label M.