Morpho-physiological Indications of Moisture Stress Tolerance  in Green Gram (Vigna radiata L.)

Green gram (Vigna radiata L.) is a significant legume crop belonging to the Fabaceae family with chromosome number 2n =22. The crop possesses wide adaptability and demands minimal input resources. Its robust root system architecture actively participates in fixing atmospheric nitrogen (30-50 kg/ha) through symbiosis with Rhizobium bacteria, contributing significantly to soil fertility enhancement and sustainable agricultural yields. Additionally, as a rich source of vegetable proteins, micronutrients and antioxidants like flavonoids and phenolics, green gram serves various purposes including food, animal feed, fodder and gre0en manure (Choudhary et al., 2024). 
 

Despite its economic importance, the productivity of green gram remains stagnant due to unpredictable weather patterns and various environmental stresses. Among these stresses, drought poses the greatest challenge to green gram cultivation, hindering its growth and development (Basu et al.,  2019). Drought stress impacts various physiological processes crucial for growth and molecular functioning, resulting in reduced pod yield (51% to 85.5%) in pulses (Islam et al.,  2024). The characters, plant height, seed weight, root architecture and crop yield are significantly reduced under the drought stress conditions in green gram and other legumes. Hence there is an utmost need to develop drought tolerant varieties to improve crop productivity especially under the changing climate. This study was conducted to screen drought tolerant green gram genotypes by assessing various biometrical, biochemical and genetic parameters.

The experiment was conducted at the Department of Genetics and Plant Breeding, College of Agriculture, Vellayani (8.5^oN, 76.9^oE and 29 m above mean sea level) during February to April 2024. Seed material representing 50 accessions of green gram collected from Indian Institute of Pulse Research (IIPR) Kanpur, Tamil Nadu Agricultural University (TNAU) Coimbatore, National Pulse Research Centre (NPRC) Vamban, Sardarkrushinagar Dantiwada Agricultural University (SDAU) Gujarat and National Bureau of Plant Genetic Resources (NBPGR) Vellanikkara, under Kerala Agricultural University (KAU) and various other local accessions were used in the study (Table 1).


       
Seeds were initially treated with 0.1% HgCl₂ for surface sterilization and residual chlorine was eliminated by thoroughly washing of seeds with distilled water. Seeds from each accession were raised by the roll paper towel method and subjected to different osmotic potentials using 5%, 10% and 20% PEG 6000 concentrations in the laboratory. The experiment was carried out during February 2024, using completely randomized design (CRD) with fifty green gram accessions in three replications. At 5% and 20% no significant variations were obtained, so the observations recorded at 10% only were included in further analysis. Observations on various morpho-physiological characters were examined in the seedling stage (10^th DAS) for drought tolerance viz., germination percentage [GP], root length [RL], shoot length [SL], root shoot ratio [RSR], seedling vigour index I [SVI I], vigour index II [SVI II] and seedling dry weight tolerance index [SDTI].
       
These fifty accessions of green gram were further raised in pots in completely randomized design (CRD) with three replications from March to April 2024. The meteorological data of the experimental site is given in Table 2. Moisture stress was imposed in the pot by withdrawing irrigation for 15 days during critical stages of growth viz., flowering and podding stage of the crop (reproductive stage). Soil moisture was also measured during this period by following gravimetric method (Reynolds, 1970). For comparison, a control with all genotypes was maintained under irrigated conditions. Twelve days after induction of droughtthe plants were pulled out and various morphological and biochemical parameters were recorded from the selected observational plants. The observations included root length (cm) [RL], shoot length (cm)[SL], seedling dry weight (g) [SDW], root dry weight (g) [RDW] and root diameter (cm) [RD] along with proline content (μ mol g^-1), total chlorophyll content (mg g^-1) and total phenol content (mg g^-1).


 
Biochemical analysis
 
The various relevant biochemical parameters namely proline content (μ mol g^-1)(Bates  et al., 1973), total chlorophyll content (mg g-¹) (Yoshida et al., 1971) and total phenol content (mg g^-1)  (Sadasivam and Manickam, 1996) were estimated as per standard procedures.

Statistical analysis
 
The data were subjected to analysis of variance (ANOVA) and various genetic parameters were worked out using GRAPES software, version 1.1.0 (Gopinath et al., 2020).

Analysis of variance with respect to the various morpho-physiological parameters and the biochemical constituents revealed significant difference among the genotypes under study, which suggests ample scope for selection for improvement (Table 1). The morpho-physiological parameters in response to drought stress imposed by 10% PEG concentration revealed wide variation as illustrated in Table 2. The highest GP was obtained for Andhra local (100%) which was on par with VBN 3 (90%), ML1415 (80%), Co 8 (80%) and four other genotypes. With the imposition of stress, GP was reduced in all the genotypes. Reduction in GP due to increase in the PEG concentration were reported by Rakavi et al., (2022) in green gram and Eesha et al., (2023). The length and diameter of root of the seedlings were also drastically affected by PEG application. The accessions Co 8 (3.12 cm) was least affected which was on par with ML 1415 (2.64 cm), GM 6 (2.13 cm) andhra local (2.12 cm) and 23 other genotypes. The higher the RL greater will be the capacity of the plants to absorb more water and thus have the ability for resistance against drought. The RD ranged from 0.33 cm (C5-SML 668) to 0.76 cm (PLM 38). The genotype EC 396142 (0.71 cm) was on par with PLM 38. These findings were in accordance with Amarapalli (2022) in green gram and Mishra et al., (2024) in soybean. Reduced water potential imposed by PEG 6000 concentrations suppressed SL growth in all the genotypes. Corresponding observations were reported by Imtiaz et al., (2020) in green gram and Mishra et al., (2024) in soybean. The shoot length ranged from 9.42 cm (VBN 5) to 55.00 cm (EC 165632). Taller genotypes are comparatively more sensitive to drought stress compared to shorter ones.


Under stress, GM 9 (8.53) recorded highest RSR which is on par with GM 9 (8.53) and ten other genotypes. A significant decrease in RSR was observed under moisture stress condition which was in accordance with the findings of Himaja et al., (2023). The RDW ranged from 0.10g (C2 IPM2 14-2) to 2.76 g (Co 8) and TPDW ranges from 0.10 g (IC 548369) to 6.26 g (VBN 3). The SdDW was maximum for IPM 031 (42.65 mg) which is on par with GM 6 (37.50 mg) and lowest was recorded by IC 548369 (0.00 mg). Root and seedling dry weight showed a significant increase during moisture stress conditions (Imtiaz et al., 2020).
       
Estimation of various tolerance indices will enable us to understand the impact of stress on the various genotypes. In the present study, vigour index was suppressed with increasing PEG concentration which was also reported by Bhujel et al., (2024) in green gram. Among the genotypes andhra local (408.00) and GM 6 (2250.00) recorded the highest vigour index I and II respectively under the stress condition (Table 3). These indexes can be used us an important tool to identify genotypes that are superior in drought prone areas.


       
The genotypes also showed significant variations with respect to biochemical parameters like proline content, total chlorophyll content and total phenol content with the imposition of drought stress (Table 4). An increase in proline content was found in all the genotypes (Islam et al., 2023). The increased levels of proline in tissues can be used as an indicator of the degree of water deficit and can also as a criterion for screening drought tolerant varieties (Kabbadj et al., 2017). The genotype ML 1415 (35.59μ molg-1), IC 148530 (34.12 μ molg^-1) and C5 SML 668 (31.84μ molg^-1) was on par with IC 395518.


         
The chlorophyll content decreased in all the genotypes and the reduction may be due to inhibition of chlorophyll biosynthesis and photo-oxidation. Genotypes exhibiting enhanced drought tolerance due to chlorophyll stability was reported by Islam et al., (2023) in green gram. Total phenol content ranged from 0.04 mgg^-1 (VBN 3) to 9.47 mgg^-1 (IC 607183). Drought stress increased the total phenol content in leaves of majority of the genotypes as compared with the well watered control. This was in conformity with the results of Ashraf and Siddiqi (2024) in sunflower.
               
The phenotypic and genotypic coefficient of variation for the characters GP, SdL, SdRL, SdSL, SdDW, RSR, SVI I and II, SDWTI, RL, SL, RD, TPDW, RDW, proline content, total chlorophyll content and phenol content were estimated to be high indicating a high variation among the genotypes (Table 5). Estimates of phenotypic coefficient of variations (PCV) were higher than genotypic coefficient of variations (GCV) for all the characters under study which indicated some environmental influence on these characters. High values of GCV and PCV were also observed for drought tolerance traits such as chlorophyll content (spad chlorophyll meter reading), leaf water potential, proline content, relative water content and specific leaf area by Kanavi  et al. (2020). High heritability and genetic advance estimates were recorded for most of the characters in the studyindicating the preponderance of additive gene action suggesting selection strategies can improve these characters (Kanavi et al., 2020 and Bordoloi et al., 2023).

Extensive variation with respect to the drought tolerant parameters were revealed from the study which suggests ample scope for improvement in green gram. The genetic parameters namely PCV, GCV, heritability and genetic advance also suggested preponderance of additive gene action for most of the parameters evaluated namely, germination percentage, seedling shoot traits, seedling root traits, tolerance indices, proline content, total chlorophyll content and phenol content. Considering both the experiments the genotypes Andhra local, ML 1415, C_o 8 and GM 6 can be considered as source of moisture stress tolerance. The superiors genotypes identified in the study can be recommended for cultivation as well as may be used as donor parents in future breeding programmes.
 

The first author is grateful to Kerala Agricultural University for granting the Junior Research Fellowship for PG programme.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarilyrepresent the views of their affiliated institutions. The authors are responsible for the accuracy andcompleteness of the information provided, but do not accept any liability for any direct or indirect lossesresulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal careand handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. Nofunding or sponsorship influenced the design of the study, data collection, analysis, decision to publish,or preparation of the manuscript.