**1. Introduction**

Legumes play a significant role in human diet because they not only complement the nutrients in a cereal diet but also improve the taste and texture of staple dishes [1,2]. Chickpea is a nutrition-rich grain legume and serves as an inexpensive source of high-quality daily protein as compared to animal protein, so is vital for nutritional security in developing countries, especially the vegetarian people of India [3,4]. It also serves as an enhancer of soil fertility through biological nitrogen fixation and fits in various crop rotation systems for the improvement of soil fertility [5,6]. It is also known as Bengal Gram or Garbanzo, and originated from Turkey [7]. It is ranked third after dry beans and peas worldwide [8,9]. Globally, chickpea occupies 14.8 Mha area, spanning over 59 countries, with an annual production of 15.1 million tons [10]. The major global production of chickpea comes from Asian countries; India shares 70% of the global chickpea area and 67% global chickpea production as the largest chickpea-producing country, followed by Pakistan, Turkey, Australia and Myanmar [11]. Based on seed morphological traits, chickpea is separated into two groups, i.e., desi type with microsperma and Kabuli type with macrosperma [12,13].

**Citation:** Tiwari, P.N.; Tiwari, S.; Sapre, S.; Babbar, A.; Tripathi, N.; Tiwari, S.; Tripathi, M.K. Screening and Selection of Drought-Tolerant High-Yielding Chickpea Genotypes Based on Physio-Biochemical Selection Indices and Yield Trials. *Life* **2023**, *13*, 1405. https://doi.org/ 10.3390/life13061405

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 24 April 2023 Revised: 14 June 2023 Accepted: 15 June 2023 Published: 17 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Desi type is more important than Kabuli type, as it covers approximately 80–85% of global chickpea production [14]. Desi chickpea is a potential source of nutritional components, i.e., high-quality proteins composed of albumins and globulins in large quantities, amino acids, essential fatty acids, trace elements and minerals [15].

Chickpea is frequently grown as rainfed crop in arid and semiarid regions, where water requirement is mainly received with either seasonal rainfall or stored moisture under soil [16,17]. In the last few years, unpredicted climatic changes resulting in high temperature (heat stress) and unusual rainfall (floods) and drought stresses are becoming major threats for crop production [18–22]. Among climatic changes, low moisture and high temperature stresses are the most important yield-limiting stresses in chickpea [23]. Chickpea is most sensitive to water stress at pre-flowering and early pod filling stages [1,4]. It is estimated that terminal drought alone can cause up to 50% of yield losses in chickpea [4,24].

Genetic improvement could be a less expensive and more long-lasting solution for better drought adaptation in chickpea than agronomic options. However, an understanding of yield maintenance under low water supplies becomes increasingly difficult because of several mechanisms employed by plants for maintaining growth and development [25]. To experience better stability of grain yield under drought, trait-based breeding strategies are being increasingly emphasized above yield-based breeding because grain yield is greatly affected by genotype × environment interactions and depicts low heritability [26]. Traitbased breeding also enhances the probability of crosses, which result in additive gene action under drought conditions.

For chickpea breeders, the breeding of drought-tolerant cultivars has been a tough task because of the unavailability of good selection indices. The lack of genetic divergence and a good source of resistance/tolerance to different abiotic stresses has been a major obstacle in the development of high-yielding drought-tolerant chickpea cultivars [27]. The screening and selection of chickpea germplasm line (s) based on diverse morpho-physiological and biochemical traits becomes a pre-requisite for crop improvement under drought stress [26]. Although similar efforts have been made with a major focus on morpho-physiological and biochemical traits contributing to drought tolerance in chickpea [4,16,26,28], limited detail about the terminal stage drought tolerance of the same genetic material are available. Thus, to fill this gap, the present investigation was conducted to assess the effect of terminal drought stress in chickpea genotypes by evaluating key drought-tolerant indicator traits and to select high-yielding drought-tolerant chickpea genotypes, especially those cultivated in India.

#### **2. Materials and Methods**

The experiment was performed in a randomized completely block design (RCBD) with three replications during the post-rainy seasons of 2020–2021 and 2021–2022 under a rainout shelter at Biotechnology Centre, Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur (23◦10- N 79◦59- E). To study the effect of normal irrigated and terminal drought-stressed conditions on the morpho-phenology, physiology, biochemistry, yield and other traits of desi chickpea at reproductive stage, forty chickpea genotypes, including drought-resistant types, released varieties and advanced breeding lines, were obtained from Lead Centre, All India Coordinated Research Project (AICRP) and the Department of Plant Breeding Genetics, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhaya Pradesh, India (Supplementary Material Table S1). The field was prepared with 1 m wide bed flanked by 0.45 m furrows and fertilized with di-ammonium phosphate (DAP) containing nitrogen (18.0 kg/ha) and phosphorus (20.0 kg/ha). Seeds were treated with Bavistin (2.0 g per kg seed weight), Chlorpyriphos 20EC (10.0 mL per kg seed) and Rhizobium (5.0 g per kg seed). Seeds were sown at a depth of 2–3 cm manually, maintaining a row-to-row distance of 45 cm. For the uniform emergence of seedlings, 20 mm irrigation was applied immediately after sowing. Thinning was performed after two weeks of seed germination to maintain a plant-to-plant distance of 10 cm within rows. Subsequently, drought stress was imposed by

withholding the water supply to the stressed set of plots before the onset of pod initiation up to the harvesting [29].

#### *2.1. Physiological Traits*

Relative water content (RWC) and canopy temperature depression (CTD) were estimated according to Gontia-Mishra et al. [30] and Purushothaman et al. [26], respectively. The leaf gas exchange parameters, viz., photosynthesis rate (Pn), stomatal conductance (gs), transpiration rate (Tr) and internal CO2 concentration (Ci), were recorded using a portable infra-red gas analyzer (IRGA) LiCor-6400 (LiCor Instruments, Lincol, NE, USA).

## *2.2. Biochemical Traits*

Chlorophyll content was estimated according to Gontia-Mishra et al. [30], while protein content was determined using an extraction buffer, as mentioned in the Bradford assay [31]. To determine the oxidative stress of a cell, hydrogen peroxide (H2O2) content, lipid peroxidation content (malondialdehyde; MDA) and electrolyte leakage (EL) were measured as described by Velikova et al. [32], Naservafaeito et al. [33] and Sachdeva et al. [28], respectively. To estimate the osmolytes accumulation of a cell, free proline content of leaf using ninhydrin [30] and total soluble sugar content using an anthrone reagent methodology [34] were determined.

To determine the enhanced activity of antioxidant enzymes, crude enzyme was extracted using an enzyme extraction buffer. Superoxide dismutase (SOD) activity was determined according to Sharma et al. [35], and one unit of enzyme activity was defined as the amount of enzyme that decreased the absorbance by 50%. The estimation of peroxidase (POD) activity was performed following Rao et al. [36], and enzyme activity was calculated as per extinction coefficient of tetra-guaiacol ∈ = 26.6 mM−<sup>1</sup> cm−1. Catalase (CAT) activity was estimated according to Aebi et al. [37], and enzyme activity was calculated as the amount of H2O2 decomposed per min. Ascorbate peroxidase (APX) activity was determined as described by Nakano et al. [38], and enzyme activity was calculated as per extinction coefficient of ascorbate ∈ = 2.8 mM<sup>−</sup>1cm−1.

#### *2.3. Morpho-Phenological Traits, Yield and Yield Attributing Traits*

Plant height was recorded from the ground level to the shoot tip. The date when half of the plants in a replication had at least one flower opened and the date when more than 75% of the pods of a plant turned brownish yellow from the days after sowing (DAS) were recorded as days to 50% flowering (DTF) and days to maturity (DTM), respectively. At the time of harvesting, all the seed-filled pods of a plant were counted as numbers of pods (NOP), and the weight of the plant including the pods was recorded as biological yield per plant (BYPP). The harvested seeds of a plant were weighed to obtain seed yield per plant (SYPP). Harvest index (%) was calculated as the ratio between seed yield per plant and biological yield per plant multiplied by 100.

#### *2.4. Statistical Analysis*

From each treatment, three plants were randomly selected to record the various drought-related morpho-phenological, physio-biochemical, and yield traits in two successive Rabi seasons (2020–2021 and 2021–2022). The data of both seasons were pooled for all 40 chickpea genotypes under both water conditions. The significance was established by analysis of variance (ANOVA) and Duncan Multiple Range Test (DMRT) at *p* < 0.05 using STAR V2.0.1 and SPSS V20 software, respectively. Principle component analysis (PCA) and PCA-based biplots were constructed to select reliable chickpea genotype (s) under drought-stressed conditions using XLSTAT software. Cluster analysis was also constructed, employing algometric hierarchical clustering for all chickpea genotypes under drought stress by applying STAR V2.0.1.

## **3. Results**

#### *3.1. Effect of Terminal Drought Stress on Physiological Traits*

Under terminal drought-stressed condition, all studied physiological traits were significantly decreased in comparison to normal irrigated conditions in all chickpea genotypes (Supplementary Materials Tables S2 and S3). A higher RWC was maintained by genotype JG63 (77.66%), whereas lower RWC was noted in the genotype JG2016-36 (57.03%) (Figure 1). Higher CTD was obtained by genotype ICCV19616 (2.18 ◦C), whereas the lowest CTD was reported in genotype JG6 (1.08 ◦C). In terms of *C*i, the highest value was achieved in genotype PG205 (195.9 μmol CO2 m−2s−1), whereas the lowest *C*i was recorded in genotype JG2022-75 (123.78 μmol CO2 m−2s−1) (Figure 2). Figures 2 and 3 show that genotype JG2016-44 exhibited the maximum *P*n (18.31 μmol CO2 m−2s−1) and gs (0.31 mol H2O m−2s−1), while the minimum *P*n (10.31 μmol CO2 m−2s−1) and gs (0.17 mol H2O m−2s−1) were found in genotype JG2022-75. Higher *T*r was maintained in genotype JG2016-44 (15.4 mmol H2O m−2s−1),and the lowest was seen in genotype JG2022-75 (8.62 mmol H2O m<sup>−</sup>2s−1).

**Figure 1.** Effect of terminal drought stress on (**A**) RWC and (**B**) CTD of studied chickpea genotypes, where RWC and CTD indicate relative water content and canopy temperature depression, respectively.
