Temperature Effect on Morphobiochemical Characters in Some Black Gram (Vigna mungo) Genotypes

Lack of suitable varieties and genotypes of black gram with adaptation to local conditions is among the factors affecting its production. Efforts to genetically improve the crop mostly involve identifying important morphological descriptors followed by development of advanced breeding lines for locale-specific cultivars. The present day available black gram varieties have not been properly characterized for their thermo sensitiveness with respect to morphological and biochemical characters. Hence efforts were taken in the present research to study the effect of the temperature on these characters in seven black gram varieties over different development stages. We aimed at studying the effect of 3 temperature regimes for identifying suitable stress tolerant genotypes. High percent germination (87.2%), root length (3.68 cm), carbohydrate content (3.72 mg g−1 fresh tissue) among the genotypes was highest at 10°C–20°C temperature. High shoot length (13.39 cm), free amino acid content (3.73 mg g−1 fresh tissue), and protein content (9.54 mg g−1 fresh tissue) was found to be present when the genotypes were exposed to 20°C–30°C temperature. The black gram varieties J.L and PDU-1 performed best in all the temperature regimes over characters. Thus suitable varieties for all temperature regimes were identified using biochemical analysis.


Introduction
Black gram (Vigna mungo) is a tropical leguminous plant, which belongs to the asiatic Vigna species along with V. radiata, V. trilobata, V. aconitifolia, and V. glaberecence. It has high nutritive value and consists of high content of proteins, vitamins, and minerals. V. mungo forms one of the important constituents in the dietary practices of the local communities. It is cultivated as fallow crop aer rice cultivation in India. It is grown in various agroecological conditions and cropping systems with diverse agricultural practices. In recent years, there has been signi�cant decline in its production in India. Lack of suitable varieties and genotypes with adaptation to local conditions is among the factors affecting its production. Efforts to genetically improve the crop are slow with only few efforts to identify important morphological descriptors and develop advanced breeding lines for locale-speci�c cultivars of this crop [1].
Stress is de�ned as an in�uence that is outside the normal range of homeostatic control in a given genotype [2]. Where a stress tolerance is exceeded, response mechanisms are activated, and where the stress is controlled, a new physiological state is established and homeostasis is reestablished. When the stress is removed, the plant may return to the original state or a new physiological state may be established [3]. Each plant species has its unique set of temperature requirements, which are optimum for proper growth and development. Low temperature is one of the abiotic stresses that cause crop failure. Many plants especially those which are native to warm habitat exhibit symptoms of injury when exposed to low nonfreezing temperatures. Low temperature stress induces considerable changes in biochemistry and physiology of plants. It generally causes a decrease in the entire metabolism and also in the photochemical steps of photosynthesis, which are interdependent on the biochemical phase and, as expected, more directly in�uenced by low temperatures [4,5]. Studies of hardiness are essential in evaluating new cultivars of black gram. e general trend of research is to replace or to complete �eld testing by laboratory procedures. Identi�cation of biochemical markers or some physiological indicators is advantageous for breeding and can be used as simple indicators of abiotic stress tolerance.
Here we aim at screening available genotypes of black gram by studying the effect of temperature regimes on various morphological and biochemical parameters at different time periods.

Plant Material and Growth
Conditions. Healthy black gram seeds of 7 genotypes were collected from the Pulse Research Station, Berhampur, Odisha, India. ese varieties are PU 30, SARALA, T9, OBG 17, B.3.3.8, PDU 1, and JAJPUR LOCAL. ey were preserved in an incubator at 19 ∘ C. e seeds were selected for uniformity in size and surface sterilized with 0.1% w/v mercuric chloride (HgCl 2 ) for 5 min followed by thorough washing with sterile doubledistilled water for 4-5 times. e sterilized seeds were soaked overnight. ese soaked seeds were kept in sterilized petriplates containing soaked �lter paper in such a way that each petriplate contained 15 seeds. e effect of different temperature duration on various morphological and biochemical characters was studied. For the present research three temperature regimes A (0 ∘ C to 10 ∘ C), B (10 ∘ C to 20 ∘ C), and C (20 ∘ C to 30 ∘ C) were chosen. To maintain 0 ∘ C to 10 ∘ C temperature the petriplates containing the seeds were kept in an ice basket for germination. For 10 ∘ C to 20 ∘ C, the seeds were allowed to germinate in the culture room where the average room temperature was maintained.
To study the effect of 20 ∘ C to 30 ∘ C temperature, the seeds were germinated at room temperature.

Growth Study.
To study the effect of temperature on morphological characters a number of parameters were studied on 3rd day and 10th day of sowing the seeds. e different parameters studied were percent germination, shoot length, root length, fresh weight, and dry weight of seedlings. e percent germination was calculated by counting the number of seeds that have sprouted in each replication. e fresh plant weight was recorded by taking average of 5 plants from each replication. ese were then kept in hot air oven at 60 ∘ C for 15 min, and their dry weight was recorded.

Biochemical Analysis.
For sugar estimation 500 mg of plant sample was weighted and ground with 50 mL of 80% ethanol (v/v). e homogenate was centrifuged twice each time with 5 mL of 80% ethanol. Supernatant was collected in a beaker and evaporated in water bath. Aer evaporation of all the supernatant, 10 mL of double-distilled water was added to dissolve the sugar le in the beaker. e dissolved sugar was kept in 5 mL bottle at 20 ∘ C. e DNS (dinitrosalicylic acid reagent) method described by Sadasivam and Manickam [6] was followed for reducing sugar estimation.
For extraction of protein, 1 gm of sample was ground in 5 mL phosphate buffer (50 mM, pH 7.5). e homogenate was centrifuged at 8000 rpm for 20 min for �ve times. All supernatant was collected and made up to 50 mL volume with phosphate buffer. 1 mL of this was mixed with 1 mL of TCA (20%) and incubated for 30 min at 4 ∘ C. It was then centrifuged at 8000 rpm for 20 min. e supernatant was discarded, and the pellet was washed with cold acetone and again centrifuged at 8000 rpm for 20 min at 4 ∘ C. e resultant pellet was dissolved in 5 mL NaOH (0.1N) and supernatant was discarded. is was used for determining the protein content.
e total soluble protein of the seedlings was estimated as described by Lowry et al. [7] using BSA as standard. To estimate the amino acid content 0.5 gm of plant sample was ground with small quantity of acid-washed sand. To this homogenate 5-10 mL of 80% ethanol (v/v) was added and centrifuged. e supernatant was collected, and 80% ethanol (v/v) was added and centrifuged. e procedure was repeated �ve times, and supernatant was collected and pooled together. Total volume was made up to 50 mL with 80% ethanol. 5-10 mL of the supernatant was taken and evaporated in water bath. e residue was �nally dissolved in 5 mL citrate buffer (0.2 M, pH 5.0). Amino acid content was estimated by the method of Moore and Stein [8] using L-glycine as standard.

Data Analysis.
All the experiments were repeated three times. Each replication consisted of average of 15 seeds per genotype per character per temperature regime, and observations were recorded. Analysis of variance (ANOVA) for each character among the genotypes was carried out as per the Student's t-test.  (Figure 7) on 3rd day. e genotypes recorded high mean carbohydrate content (3.72 mg g −1 fresh weight), amino acid content (0.87 mg g −1 fresh weight), and protein content (9.28 mg g −1 fresh weight) on 3rd day.

3.2.
Effect of 10 ∘ C-20 ∘ C Temperature. e genotypes recorded high germination % on 3rd day (87.2%) and only 67.74% on 10th day. e mean shoot length (11.48 cm) and root length of the genotypes were higher (3.68 cm) on 10th day. e black gram genotype P.D.U.1 showed the highest fresh weight of 0.90 gm on 3rd day and T-9 the highest (1.23 gm) on 10th day (Figure 2). e genotypes exhibited higher mean fresh weight (0.98 gm) on 10th day. e mean dry weight of the varieties of black gram (Figure 3) was high on 10th day of germination (0.05 gm). O.B.G-17 recorded the highest carbohydrate content (4 mg g −1 fresh tissue) on 3rd day while on 10th day the black gram variety T9 recorded the highest amount of carbohydrate, that is, 3 mg g −1 fresh tissue ( Figure  6). e mean amino acid content of the black gram genotypes (Figure 7)  tissue). e mean protein content (Figure 8) of the black gram genotypes recorded highest on 3rd day (6.28 mg g −1 fresh tissue).

3.3.
Effect of 20 ∘ C-30 ∘ C Temperature. e mean germination was only 66.09% on 3rd day which increased to 79.54% on 10th day of sowing the black gram seeds at temperature 20 ∘ C-30 ∘ C (Figure 1). e mean shoot length of the genotypes ( Figure 4) was higher (13.39 cm) on 10th day than on 3rd day. e mean root length of the genotypes was higher (3.06 cm) on 10th day than on 3rd day (1.40 cm). On 3rd day black gram variety Sarala showed the highest carbohydrate content of 1.3 mg g −1 fresh tissue. e mean carbohydrate content of the black gram genotypes recorded highest on 3rd day (0.98 mg g −1 fresh tissue) which decreased on 10th day to 0.67 mg g −1 fresh tissue ( Figure 6). e mean amino acid content of the black gram genotypes recorded highest on 3rd day (0.53 mg g −1 fresh tissue). e mean protein content of the black gram genotypes (Figure 8) recorded highest on 3rd day (9.54 mg g −1 fresh tissue). On 3rd day PU-30 showed the highest protein content with 12.4 mg g −1 fresh tissue and Sarala highest with 7.2 mg g −1 fresh tissue on 10th day.

Discussion
Black gram is a thermosensitive crop. e temperature during germination, seedling, and reproductive stage is very critical. A number of varieties are available which have high yield potential. But the potential is not realized to the maximum due to its thermosensitiveness. e present day available varieties have not been properly characterized for their thermosensitiveness with respect to morphological and biochemical characters. e differential response of genotypes to temperature stress is considered as the basis for the observed genetic variability. e data revealed signi�cant difference among the genotypes for various morphological and biochemical characters. Any morphological or biochemical adaptation of a genotype is a consequence of gene expression, be due to variation in stress adaptive mechanism among the genotypes. It has been observed that the prolonged exposure of leaves to low temperature resulted in selective inactivation of the oxygen evolving-system in cucumber [9][10][11][12][13], bean [14], and tomato [15]. According to Weis [16], the acclimation of the photosynthetic apparatus to high temperature could be perceived as a long-term response of the leaves to changes in the temperature regime for several days or weeks. However, signi�cant changes for some hours, in diurnal temperature, can oen be observed as well. Acclimation involves both short-term chemical, molecular, and physiological responses and long-term physiological, structural, and morphological modi�cations [17]. In most cases the adaptive mechanisms to different thermal regimes could be considered as compensatory, because they enable plants to buffer the effect of the temperature shi on their metabolic systems [18]. As a result, the amount of speci�c components compensating the temperature effect on the rate of a given reaction changes. e protein synthesizing system plays a crucial role in plant acclimation processes. It has been hypothesized that particular proteins whose synthesis is induced by stress conditions are critical for the survival in that stress [19,20]. Pretreatments which lead to acquisition of thermotolerance are conditions under which heat shock proteins (HSPs) are synthesized [21][22][23]. During HS, these HSPs could interact with other proteins to prevent their aggregation and facilitate reassembly of functional structure [24]. is may explain the occurrence of very high protein content under low temperature conditions, that is, 0 ∘ C−10 ∘ C. e present research indicates sufficient genetic variability existing within the black gram genotypes from tolerance to temperature stress. e black gram varieties J.L and PDU-1 performed best at all the temperature durations over characters.