**5. Nodulation**

were obtained and taken to the laboratory for starch analysis. The leaves were bleached in boiling 90% ethanol and incubated in dilute iodine (0.5 M) solution (2:1) for 3 minutes and then rinsed with distilled water. Rinsing is necessary to remove excess iodine solution

20 Plant, Abiotic Stress and Responses to Climate Change

**Figure 4.** Effect of water deficit stress on photosynthetic pigment (chlorophyll) content of soybean plants expressed to CCI. (a) Chlorophyll content of control plants during early growth stages (V3). (b) Leaf chlorophyll content of the control during early reproductive stages. (c) Amount of chlorophyll content in WT 1 plants. (d) Leaf chlorophyll content in WT

**Figure 5.** Iodine test on ethanol bleached leaves. After bleaching and staining with iodine: (a) Show traces of starch on leaflet taken from water stress plants (WT 1). (b) Absence of or minor starch traces on severely water stressed leaflet (WT 2).

(c) Starch content (blue black colour) on leaflet taken from the control plants.

2. Data represent CCI means and the different letters denote significant differences of the means at p < 0.05.

The formation of cell protuberance containing nitrogen-fixing Gram-negative bacteria in the roots of legumes plays an important role in improving plant growth characteristics, crop productivity and maintaining soil fertility. This establishment of lumps on roots of plants (known as nodulation) guarantees the supply of fixed atmospheric N<sup>2</sup> for use in the synthesis of proteins, nucleic acids and other necessary nitrogen-containing compounds required for plant, animal and human growth and development. However, various reports have indicated that, water stress induces low frequencies of nodulation in many legumes, including soybean. Miao et al. [35] provided evidence that verifies sensitivity of soybean nodulating root cells and Rhizobium to water stress. In 2003, Ramos et al. [36] also indicated that, water stress affect nodulation in other legume species like *Phaseolus vulgaris* L. Failure for soybean roots to produce effective nodulations affect the metabolism of nitrogenous and carbonic compounds in the plant. The changes resulting into decreased nodulation could cause reduction in various aspects of plant growth (stem height, stem wood diameter and root dry weight) due to drought as reported by Shetta [37]. Additionally, Shetta indicated that the initiated nodules can become thickened and more resistant to infection by *Rhizobium* as a result of this stress. Poor nodulation can be induced by poor plant nutrition, seed filling, or abiotic stress factors. In WT 2 plants, where irrigation was withheld for 15 days, nodulation was severely affected (**Figure 6f**). It was found that nodules stopped fixing nitrogen and then started decomposing. Nodulation and nitrogen fixation in the WT 1 also decreased following imposed water deficit stress. The nodules turned green (**Figure 6e**) and this predominant green colour indicated inefficient fixation by Rhizobium strain in contrast to highly efficient red-pinkish nodules in the control (**Figure 6d**). This inefficiency may have been caused by the poor amounts of assimilates that are exchanged from soybeans to the bacteria due to reduced rates of photosynthesis in the leaves. Plants do not get fixed nitrogen from Rhizobium for free. For plants to receive fixed atmospheric nitrogen, in a form that is directly available for growth (nitrates– NO3 ¯ and ammonium–NH4 + ), plants must give bacteria sugars. This symbiotic relationship was reported by Dupont et al. [38], Serraj et al. [39] and Stajkovic et al. [40] as the major stimulant of increased plant biomass, stabilising atmospheric CO2 by stabilising C–N ratio. The symbiosis establishment is playing a very critical role in ecological and agronomic supply of N2 , estimated to account for a total of about 65% of the nitrogen fixed in legumes used for agriculture globally.

**Figure 6.** Soybean plants with nodulated roots. (a) Healthy nodules on soybean control plants. (b) Roots of WT 1 with numerous mature nodule structures. (c) WT 2 stressed plant root showing poor nodulation. (d) Nitrogen (N) fixing nodules with *Rhizobia* as observed in the control. (e) Less effective nodules from WT 1 roots. (f) Decomposing root nodule of WT 2 plants.

### **6. Impact of water deficit on flowering and fruiting**

The soybean genotypes showed great differences in the percentage flowering, pod formation and other yield related components. Water stressed plants produced less than 2% yield, in the two soybean cultivars (LS 677 and Peking) that survived induced water stress. A few WT 1 plants subjected to water stress continued their growth until flowering and pod formation stages. However, flower and fruit pod abortions were simultaneously observed leading to 7.0 and 3.0 mean pod number observed in the few plants that had survived (**Table 3**). These numbers were not comparable with the yield component data recorded for these cultivars in the control. Soriano et al. [41] determined a positive relationship between yield quantity by estimating grain number and weight in early planted sunflower by timing induction of environment stress. In line with this report, positive yield characteristics that include; total percentage of flowering plants, mean number of pods and average pod length, pod weight and seed weight (per 100 seeds) were observed in all of the cultivars in the control. In contrast, as a result of water stress, a significant number of flower abortions (10–15%) were observed in cultivar Dundee, LS 677, TGx 1740-2F and TGx 1835-10E which showed the least survival rate at 0%.

growth conditions (**Table 3**). In the control, a single genotypic setback was observed in cultivar TGx 1740-2F and TGx 1835-10E, which were the only ones producing the lowest number of pods, respectively. The effect of water stress in other oilseed grains such as sunflower, common bean, wheat, barley and maize were reported [41–43]. According to Jaleel et al. [19] the changes in the photosynthetic pigments and the decrease in metabolic functioning of the plant lead to decreased yield productivity. Seed yield and seed's morphological characters can also be affected by drought [44]. In cultivar Peking, the interaction between water deficit stress and seed appearance resulting from the genotype was not severely pronounced. The seeds appeared intensely shrinked and decreased in seed size due to loss of seed moisture, immediately after harvesting. This response was observed in another study assessing seed longevity in soybean seeds (data not published), clearly suggesting this as a dormancy or viability mechanism compared to other genotypes. In general, significant differences were observed during flowering, pod formation and seed maturation/ filling, as well as in the seed phenotypic characteristics among all cultivars in the control. Many water stressed plants (WT 1 and WT 2) did not survive to reach flowering as observed in the normally irrigated

**Table 3.** Vegetative growth and flowering response of soybean plants subjected to normal water conditions.

Plant watering was carried out depending on the moisture availability in the soil. Data on yield components was

The mean number of pods produced was determined 2 weeks after the pods were successfully produced in order to avoid counting fruit pods that will eventually not produce seeds. Data represent the means and values followed by

plants of the control (**Tables 2** and **3**).

**Soybean genotypes Mean plant** 

**height (cm)**

Additional data on yield and yield components of untreated soybean plants

different letters are significantly different (in columns) (at p ≤ 0.05) by ANOVA.

Ave. pod length (cm)

Dundee 4.06a 0.44a 18.53a LS 678 3.38b 0.49b 14.06b LS 677 5.23c 0.50c 14.02b Peking 3.96d 0.38d 9.54c TGx 1740-2F 3.40b 0.51c 12.03d TGx 1835-10E 3.94d 0.49b 12.87e

recorded on the day that the experiment was terminated.

**Mean no. of branches**

Ave. pod weight (g)

Dundee 31.0a 6.0a 80.0a 21.0a 80.0a LS 678 41.0b 5.0b 95.0b 32.0b 95.0b LS 677 49.1c 6.0a 100.0c 36.0c 100.0c Peking 51.0d 6.0a 100.0c 29.0d 100.0c TGx 1740-2F 47.1e 5.0b 95.0b 19.0e 95.0b TGx 1835-10E 49.5c 6.0a 100.0c 21.0a 100.0c

**No. of flowering plants (%)**

Water Stress: Morphological and Anatomical Changes in Soybean (*Glycine max* L.) Plants

Seed weight/100 seeds (g)

**Mean no. of pods produced**

http://dx.doi.org/10.5772/intechopen.72899

**Survival frequency (%)** 23

The variation observed in control plants however, did not seem to affect pod development and maturation, thus, could be attributed to the genotype performance than the environmental Water Stress: Morphological and Anatomical Changes in Soybean (*Glycine max* L.) Plants http://dx.doi.org/10.5772/intechopen.72899 23


Additional data on yield and yield components of untreated soybean plants

**6. Impact of water deficit on flowering and fruiting**

nodule of WT 2 plants.

22 Plant, Abiotic Stress and Responses to Climate Change

The soybean genotypes showed great differences in the percentage flowering, pod formation and other yield related components. Water stressed plants produced less than 2% yield, in the two soybean cultivars (LS 677 and Peking) that survived induced water stress. A few WT 1 plants subjected to water stress continued their growth until flowering and pod formation stages. However, flower and fruit pod abortions were simultaneously observed leading to 7.0 and 3.0 mean pod number observed in the few plants that had survived (**Table 3**). These numbers were not comparable with the yield component data recorded for these cultivars in the control. Soriano et al. [41] determined a positive relationship between yield quantity by estimating grain number and weight in early planted sunflower by timing induction of environment stress. In line with this report, positive yield characteristics that include; total percentage of flowering plants, mean number of pods and average pod length, pod weight and seed weight (per 100 seeds) were observed in all of the cultivars in the control. In contrast, as a result of water stress, a significant number of flower abortions (10–15%) were observed in cultivar Dundee, LS 677, TGx 1740-2F and TGx 1835-10E which showed the least survival rate at 0%.

**Figure 6.** Soybean plants with nodulated roots. (a) Healthy nodules on soybean control plants. (b) Roots of WT 1 with numerous mature nodule structures. (c) WT 2 stressed plant root showing poor nodulation. (d) Nitrogen (N) fixing nodules with *Rhizobia* as observed in the control. (e) Less effective nodules from WT 1 roots. (f) Decomposing root

The variation observed in control plants however, did not seem to affect pod development and maturation, thus, could be attributed to the genotype performance than the environmental


Plant watering was carried out depending on the moisture availability in the soil. Data on yield components was recorded on the day that the experiment was terminated.

The mean number of pods produced was determined 2 weeks after the pods were successfully produced in order to avoid counting fruit pods that will eventually not produce seeds. Data represent the means and values followed by different letters are significantly different (in columns) (at p ≤ 0.05) by ANOVA.

**Table 3.** Vegetative growth and flowering response of soybean plants subjected to normal water conditions.

growth conditions (**Table 3**). In the control, a single genotypic setback was observed in cultivar TGx 1740-2F and TGx 1835-10E, which were the only ones producing the lowest number of pods, respectively. The effect of water stress in other oilseed grains such as sunflower, common bean, wheat, barley and maize were reported [41–43]. According to Jaleel et al. [19] the changes in the photosynthetic pigments and the decrease in metabolic functioning of the plant lead to decreased yield productivity. Seed yield and seed's morphological characters can also be affected by drought [44]. In cultivar Peking, the interaction between water deficit stress and seed appearance resulting from the genotype was not severely pronounced. The seeds appeared intensely shrinked and decreased in seed size due to loss of seed moisture, immediately after harvesting. This response was observed in another study assessing seed longevity in soybean seeds (data not published), clearly suggesting this as a dormancy or viability mechanism compared to other genotypes. In general, significant differences were observed during flowering, pod formation and seed maturation/ filling, as well as in the seed phenotypic characteristics among all cultivars in the control. Many water stressed plants (WT 1 and WT 2) did not survive to reach flowering as observed in the normally irrigated plants of the control (**Tables 2** and **3**).
