**3. Evaluation of shoot growth and yield components of** *Rj* **gene-accumulated soybean**

### **3.1 Experimental site, design, and tested soybean variety**

A 2-year field trial was conducted in 2016 and 2017 in the experimental field of the Agricultural Science Section, Education and Research Center for Biological Resources, Faculty of Life and Environmental Science, Shimane University, Japan (35°30′55″N, 133°06′36″E). The experimental sites were located at 35°30′60″N, 133°06′35″E in 2016 and 35°31′02″N, 133°06′40″E in 2017. Both experimental fields had gray lowland soil (paddy conversion fields). Soil pH (H2O) and electrical conductivity (mS cm−1) were respectively 6.42 and 0.10 in 2016 and respectively 6.72 and 0.06 in 2017. Before sowing, nitrogen, potassium, and phosphorus were applied at doses of 40, 100, and 100 kg ha−1, respectively. To correct soil pH, magnesium lime was applied at the dose of 1000 kg ha−1. The experiment followed the split-plot design with three replicates. Three soybean cultivars, namely "Enrei," "Sachiyutaka," and "Fukuyutaka," as well as F10 or F11 plants of three *Rj* gene-accumulated soybean *Breeding of* Rj *Gene-Accumulated Soybean Genotypes and Their Availability for Improving… DOI: http://dx.doi.org/10.5772/intechopen.102833*

lines with different flowering and ripening periods, namely B × F − E, B × F − M, and B × F − L, were tested. "Enrei" and "Sachiyutaka" were registered in 1971 and 2001, respectively [45]. "Enrei" presents the *rj*4 genotype [37]. "Sachiyutaka" may present the *Rj*4 genotype, because it is bred through backcrossing "Enrei" with F2 plants from a cross "Enrei" and "Fukuyutaka" [45]. All soybean seeds were sown at a depth of 3–4 cm on June 20, 2016, and June 21, 2017, respectively, and the planting density was 11.1 and 10.1 plants m−2 in 2016 and 2017, respectively.

### **3.2 Data collection and analysis**

Soybean growth was evaluated during the flowering and harvest periods. Samples were collected by from 10 consecutive plots per replicate. During the flowering period, plant height, node number, branch number, stem and leaf dry weight, and main culm dry weight (2017 only) were measured. During the harvest period, plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were measured. Plant dry weight was measured after drying at 70°C for over 72 h in a drying apparatus. All statistical analyses were performed using R version 4.0.3 [49]. Soybean growth parameters during the flowering period were analyzed using Tukey's honestly significant difference (HSD) test for multiple comparisons using the R package "multcomp." Soybean yield components were subjected to two-way analysis of variance using anovakun version 4.8.5 [50]. Meteorological data during soybean cultivation were collected from past information provided by the Japan Weather Association (**Table 2**).

### **3.3 Growth and yield of** *Rj* **gene-accumulated soybean**

The results of soybean growth during the flowering period in 2017 are presented in **Table 3**. The measurements during the flowering period were conducted on August 10, 2017, for B × F − E and "Enrei"; August 17, 2017, for B × F − M and "Sachiyutaka"; and August 26, 2017, for B × F − L and "Fukuyutaka." The plant height of *Rj* gene-accumulated soybean lines tended to increase in the order B × F − E < B × F − M < B × F − L, indicating dependence on the lateness of the flowering period. Similarly, the plant height of other soybean cultivars tended to increase in the order of "Enrei" < "Sachiyutaka" < "Fukuyutaka," indicating dependence on the lateness of the flowering period. Branch number was the highest in "Fukuyutaka" and the lowest in "Enrei." In addition, among the *Rj* gene-accumulated soybean lines, plant height tended to be higher in B × F − L than in the other lines, although the difference was not significant. There were no significant differences in shoot dry weight among the cultivars, although "Fukuyutaka," B × F − M, and "Enrei" showed higher values in that order.

The results of yield components of soybean cultivars during the harvest period in 2016 and 2017 are presented in **Table 4**. In ANOVA, all yield components, except 100-seed weight, significantly differed between years and among cultivars. Specifically, pod and seed number and yield were significantly higher in 2016 than in 2017. Conversely, plant height, node number, and shoot dry weight were significantly higher in 2017 than in 2016. Based on the average values of the 2 years, pod and seed number in B × F − M was significantly higher than that in the other cultivars. Moreover, the yield of B × F − M and "Sachiyutaka" was significantly higher than that of B × F − E, B × F − L, and "Enrei." Furthermore, 100-seed weight of "Sachiyutaka" was significantly higher than that of the other cultivars, except "Fukuyutaka." Plant height and shoot dry weight of B × F − L tended to be higher


*Values indicate monthly averages, and each value was calculated based on meteorological data provided by the Japan Weather Association.*

### **Table 2.**

*Meteorological data during soybean cultivation in 2016 and 2017.*


*Values are presented as the means of three replicates. \*p < 0.05, \*\*\*p < 0.001, and ns = not significant. Different letters indicate significant differences (Tukey's HSD test) at p < 0.05 for different soybean cultivars.*

### **Table 3.**

*Growth of soybean cultivars during the flowering period in 2017.*

than those of the other cultivars. The interaction between year and cultivar was detected for all test parameters, except seed number and yield. Therefore, multiple comparison analysis was performed among 12 cohorts for each test item, and the results are shown in **Figure 2**. Briefly, pod and seed number and yield were lower in all soybean cultivars in 2017 than in 2016. Furthermore, pod and seed number of B × F − E, B × F − M, "Enrei," and "Sachiyutaka" decreased significantly. While the yield of "Sachiyutaka" decreased significantly, that of B × F − E, B × F − M, B × F − L, "Enrei," and "Fukuyutaka" tended to decrease, albeit without significant differences.

*Breeding of* Rj *Gene-Accumulated Soybean Genotypes and Their Availability for Improving… DOI: http://dx.doi.org/10.5772/intechopen.102833*


*Values are presented as the means of three replicates. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, and ns = not significant. Different letters indicate significant differences (Tukey's HSD test) at p < 0.05 for different soybean cultivars.*

### **Table 4.**

*Yield components of soybean cultivars in 2016 and 2017.*

"Enrei," "Sachiyutaka," and "Fukuyutaka" are soybean cultivars that are suitable or possible to cultivate in the Chugoku region of Japan, including Shimane prefecture, where the cultivation test was conducted in the present study [51, 52]. Additionally, pod and seed numbers are the most important soybean yield components, which are primarily determined during the period from before and after flowering to pod set, including the beginning of the seed filling period [53]. However, increasing temperature during the growing season can negatively affect soybean leaf photosynthesis, growth, flowering, pod and seed number, and yield [54, 55]. In the present study, the monthly mean maximum temperature in August during the flowering period of soybean was 32.2°C in 2016 and 31.5°C in 2017 (**Table 2**). Specifically, in early August of 2017, when B × F − E and "Enrei" were flowering, the temperature remained above

#### **Figure 2.**

*Yield components for each soybean cultivar in 2016 and 2017 growing seasons. Values are presented as the mean ± SE of three replicates*. *Different letters indicate significant differences (Tukey's HSD test) at p < 0.05. (a) Plant height. (b) Node number. (c) Shoot dry weight. (d) Pod number. (e) Seed number. (f) 100-seed weight. (g) Yield.*

35°C for 3 consecutive days. Furthermore, in late August of 2017, the temperature remained above 32°C for 4 consecutive days. Additionally, in October 2017, nearly 3.5 times the amount of precipitation in 2016 was recorded (**Table 2**). Soybean pod and seed number and yield in 2017 were significantly lower than the values in 2016 due to the effects of these meteorological factors (**Table 4**). Moreover, the 100-seed weight of B × F − M was lower than that of "Sachiyutaka" and "Fukuyutaka" (**Table 4**). Therefore, backcrossing with these cultivars is expected to produce soybean cultivars with larger seeds and higher yield.
