Resilience in the Rainfed tropics: Identifying drought-tolerant black pepper germplasm for climate-adaptive spice farming

Introduction

Black pepper (Piper nigrum L.), known as the "King of Spices" or "Black Gold," is a major commercial spice in India. Global production reached 1,103,024 MT in 2021, with India ranking fourth (61,004 MT) (FAO, 2020) [6] . Although India accounts for 42.55% of the global area, its production share is only 16.76%. Kerala contributes 80-90% of national output, with Idukki and Wayanad together covering 64.21% of area and 76.16% of production. Climate variability has significantly affected productivity. A long-term analysis (1840-2014) showed Kerala's average rainfall (2817±406 mm) follows a cyclic trend, with declining annual and southwest monsoon rainfall but increasing post-monsoon rainfall in recent decades (Kandiannan et al., 2014) [10] . These shifts have contributed to yield decline. Low productivity is further linked to poor genetic potential, senile vines, biotic and abiotic stresses, lack of quality planting material, and suboptimal agronomic practices. Climate change remains a key constraint, particularly for smallholders. Developing climateresilient varieties tolerant to heat, drought, flooding, salinity, and cold is essential. Among abiotic stresses, drought is most critical in Kerala's rainfed systems, though recent floods have also caused damage (Maheswari et al., 2012) [12] . Stress tolerance depends on a plant's ability to survive, reproduce, and recover with high productivity after stress.

Proline content (μ mol/g) = μ proline/ml x ml toluene/115.5(5/g sample) 2.5 Chlorophyll (mg g -1 ): Chlorophyll content was determined using the Arnon (1949) method. Leaf samples were extracted in 80% acetone: DMSO (1:1), and absorbance was recorded at 645 and 663 nm.

Total chlorophyll (mg g -1 ) = (20.2 x A645 + 8.02 x A663) x 1 x V/(1000 x W)  Absorbance at specific wavelength  V-Final volume of chlorophyll extract in 80% acetone:

DMSO mixture (25 ml)  W-Fresh weight of tissue extracted 2.6 Estimation of Peroxidase (activity/min/g): Peroxidase activity was estimated following Srivastava (1987). Enzyme extract was prepared in phosphate buffer, and activity was measured using pyrogallol and hydrogen peroxide by recording changes in absorbance at 420 nm.

Peroxidase activity (activity/minute/g) = (difference in absorbance in 60 seconds/50) x 5000

Polyphenol oxidase activity was determined using the method of Mayer et al. (1965). The reaction mixture contained phosphate buffer and enzyme extract, and the reaction was initiated with catechol. Absorbance was recorded at 495 nm.

Polyphenol oxidase activity = (difference in absorbance in 60 seconds/50) x 5000

Glutathione reductase activity was measured by homogenizing leaf tissue in phosphate buffer with EDTA, followed by centrifugation. The reaction mixture included NADPH and oxidized glutathione, and the decrease in absorbance at 340 nm was recorded.

1 unit of enzyme activity = the amount of enzyme that catalyses the reduction of 1 μ mol of NADPH/minute. N moles of NADPH/ml = (difference in absorbance at 340/minute)/6.22x10-3 μ m -1

Results and Discussion

The collected varieties were screened for drought tolerance in pot culture study. Seven characters including physiological and biochemical parameters were recorded. Results are presented in table 1 to 7. Soil moisture was recorded for every observation and is presented in table 8. Drought tolerance is a complex, quantitatively inherited trait governed by multiple genes, making it difficult to analyze and characterize. Plants adopt diverse strategies to survive water deficit, such as enhancing root growth to improve water uptake and reducing transpiration to limit water loss. They may also employ combined adaptive mechanisms, including the production of protective proteins that maintain membrane stability and cellular function. Additionally, osmolytes like proline, sorbitol, and glycine betaine accumulate in vacuoles, helping reduce cellular water loss. A clear understanding of biochemical and molecular responses to drought is essential for elucidating resistance mechanisms. Drought stress affects plant growth, yield, membrane integrity, pigment content, osmotic balance, water relations, and photosynthesis (Benjamin and Nielsen, 2006) [4] . The degree of susceptibility varies depending on stress intensity, associated environmental factors, species, and developmental stage (Demirevska et al., 2009) [5] . Plant acclimation to water deficit involves structural, physiological, and biochemical adjustments, including changes in growth rate, osmotic potential, and antioxidant defense systems. Therefore, improving drought resistance in crops is critical to maintaining productivity under adverse conditions. The development of climate-resilient crop varieties with tolerance to multiple stresses such as drought, heat, salinity, cold, and flooding is essential for sustaining yields under climate change. Among these, drought remains a major constraint to plant growth and productivity (Maheswari et al., 2012) [12] . In Kerala, drought is the primary abiotic stress affecting pepper cultivation, although recent floods have also caused significant damage. Identifying key traits that support plant growth and survival under stress is crucial for developing tolerant cultivars.

Screening of germplasm for drought under artificial conditions

The collected varieties were screened for drought tolerance in pot culture study. Seven characters including physiological and biochemical parameters were recorded. Mean values are presented in table 4. Mean soil moisture was recorded for every observation and is presented in table 5.

The availability of water is the major factor influencing the growth, development and productivity. Relative water content is considered a measure of plant water status, reflecting the metabolic activity in tissues. It is used as an index for dehydration tolerance. RWC of leaves is higher in the initial stages of leaf and reduces as the dry matter accumulates. A decrease in the RWC in response to drought stress has been noted in wide variety of plants as reported by Nayyar and Gupta (2006) [13] . The relative water content is high for all the genotypes under control when compared to the stress induced ones (Table 1). In five days after stress induction the relative water content ranged from 43.35% (Cholakkodi) to 85.74 (Sadanandapuram local). The mean value for the control is 83.97% and for five days after stress induction is 70.81%. For ten days after stress induction the RWC ranged from 35.86% (Cholakkodi) to 85.04% (Sadanandapuram local). The mean value for control is 81.50% and for ten days after stress induction is 61.44% For fifteen days after stress induction the RWC ranged from 30.19% (Cholakkodi) to 68.37% (Sadanandapuram local). The mean value for control is 78.32% and for fifteen days after stress induction is 46.16%. Comparatively, higher RWC and low membrane leakage are associated with drought tolerant genotypes. Thankamani and Ashokan (2004) [17] reported that in all the varieties tested the maximum relative water content was observed in plants without water stress. The daily irrigated plants of the varieties Kalluvally-l, Kalluvally-2, Panniyur-4 recorded RWC above 90 percent. A stable RWC under the stress may be considered as one of the indices for screening water stress tolerant varieties. Earlier reports also showed very good correlation between RWC and water stress tolerance.

The electrolyte leakage technique to evaluate the cell membrane stability as a mechanism of water stress tolerance in plants.Relative water content and membrane integrity were reduced with increasing stress intensity. Relative water content was positively correlated with membrane integrity and negatively correlated with stress intensity. The Angamaly (PRS-64) showed highest relative water content, membrane integrity which was on par with Panniyur-5 (PRS-115), PRS-160, Panniyur-8 (PRS-153) (Table 2). The lowest relative content, membrane integrity observed in Panniyur-1 (PRS-44) which was par on Karimunda (Pallavi, 2015) [14] . In five days after stress induction membrane leakage ranged from 20.13 (Arikkilazhi) to 66.78 (Padappan). The mean value for the control is 30.20% and for five days after stress induction is 46.90%. For ten days after stress induction the membrane leakage ranged from 38.33% (Palakkodi) to (Narayakodi). The mean value for control is 33.57% and for ten days after stress induction is 60.28%. For fifteen days after stress induction the membrane leakage ranged from 52.64% (Pandithitta local) to 88.09% (Kumbanadan). The mean value for control is 37.02% and for fifteen days after stress induction is 72.85%. Drought is one of the major abiotic stresses affecting plants which can result in limited growth and photosynthesis. The RWC and membrane leakage of drought induced black pepper leaves were analyzed, and we have observed that the drought tolerant genotype, Acc 4216, had higher RWC and lower membrane leakage compared to the susceptible genotype, Sreekara at 10 DASI. The higher RWC and lower membrane leakage is an indicator for drought tolerance in black pepper (Krishnamurthy et al., 1998) [11] . As these physiological parameters are multi-gene controlled traits, the variation in the traits between tolerant and susceptible black pepper genotypes may reflect the differential expression of important drought responsive genes at the molecular level. The relative expression study of differentially expressed genes can help in understanding the key genes and mechanism involved in drought tolerance in black pepper (George et al., 2017) [7] . Thankamani and Ashokan (2004) [17] reported that water stress significantly influenced the membrane stability of the varieties tested. The membrane stability decreased significantly when the plants were subjected to water stress for two days; highest membrane leakage was observed in TMB1 (96.3) and lowest in Kottanadan (21.4). Only, six varieties survived• after four days of water stress and the leakage was Maximum in Uthirankotta-2 (47.3) and least in Poonjarmunda (30.8). A stable RWC under the stress may be considered as one of the indices for screening water stress tolerant varieties. Earlier reports also showed very good correlation between RWC and water stress tolerance (Balasimha et al., 1988 and Vasantha, 1996) [2,18] . Varieties which tolerated four days of water stress showed relatively less membrane leakage. Vasantha (1996) [18] imposed water stress on eight months old rooted cuttings of black pepper of fourty cultivars. Among them only six cultivars viz., Kalluvally-4, Kumbakodi, Padarpan, Panniyur-5, Poonjarmunda and Uthirankotta-2 were survived even after the fourth day of water stress. These cultivars showed highest RWC and membrane stability index. Relative water content decreased and membrane leakage increased due to water stress in different black pepper genotypes. The RWC in black pepper accessions were reduced after stress induction which ranges from 4.7 (acc 1466) to12% (acc 1411) after 4 days of stress and from 16% (acc 1466) to 46.3% (acc 1411) after 8 days of stress. The membrane leakage increases from 35% (acc 1368) to 83.4% (ace 1409) after 4 days of stress and from 105% (acc 1368) to 253.5% (acc 1411) after 8 days of stress (Pallavi, 2015) [14] .

In five days after stress induction proline content ranged from 4.24% (Cholakkodi) to 17.95% (Pandithitta local) (Table 3). The mean value for the control is 5.32% and for five days after stress induction is 10.93%. For ten days after stress induction the proline content ranged from 4.89% (Panniyur 1) to 24.57% (Pandithitta local). The mean value for control is 6.88% and for ten days after stress induction is 13.75%. For fifteen days after stress induction the proline content ranged from 5.19% (Panniyur 1) to 28.23% Sadanandapuram local. The mean value for control is 8.53% and for fifteen days after stress induction is 17.60%.

In five days after stress induction total chlorophyll content ranged from 0.502% (Thekkan) to 1.701% (Pandithitta local) (Table 4). The mean value for the control is 1.72% and for five days after stress induction is 0.94%. For ten days after stress induction the total chlorophyll content ranged from 0.313% (Padappan) to 1.261% (Annavariyan). The mean value for control is 1.46% and for ten days after stress induction is 0.67%. For fifteen days after stress induction the total chlorophyll content content ranged from 0.128% (Panniyur 7) to 0.833% (Sadanandapuram local). The mean value for control is 1.28% and for fifteen days after stress induction is 0.45%.

In five days after stress induction peroxidase content ranged from 1.96% (Kumbanadan) to 11.27% (Sakthi) (Table 5).The mean value for the control is 3.09% and for five days after stress induction is 6.50%. For ten days after stress induction the peroxidase content ranged from 2.13% (Panniyur 6) to 14.65% (Sadanandapuram local). The mean value for control is 6.84% and for ten days after stress induction is 8.77%. For fifteen days after stress induction the peroxidase content content ranged from 2.27% (Panniyur 6) to 18.69% (Pandithitta local).The mean value for control is 3.80% and for fifteen days after stress induction is 11.41%.

In five days after stress induction polyphenol oxidase content ranged from 4.37% (Wild 2) to 8.88% (Adukkanazhi) (Table 6). The mean value for the control is 4.02% and for five days after stress induction is 6.56%. For ten days after stress induction the polyphenol oxidase content ranged from 4.54% (Wild 2) to 11.30% (Shubhakara). The mean value for control is 4.14% and for ten days after stress induction is 7.60%. For fifteen days after stress induction the polyphenol oxidase content ranged from 6.61% (Wild 2) to 13.97% (Champakkulam local). The mean value for control is 4.28% and for fifteen days after stress induction is 9.35%.

In five days after stress induction glutathione reductase content ranged from 2.09% (Wild 3) to 8.23% (Panniyur 7) (Table 7). The mean value for the control is 2.70% and for five days after stress induction is 4.82%. For ten days after stress induction the glutathione reductase content ranged from 2.28% (Aimpiriyan) to 11.09% (Pandithitta local). The mean value for control is 2.77% and for ten days after stress induction is 5.96%. For fifteen days after stress induction the glutathione reductase content content ranged from 2.27% (Aimpiriyan) to 12.00% Murithothan.The mean value for control is 2.92% and for fifteen days after stress induction is 7.94%.

For five days after stress induction in control the mean value ranged between 5.41% (Kottinadan) to 20.17% (Sadanandapuram local) (Table 8). For stress induced plants the mean ranged between 3.83% (Kottinadan) to 12.88% (Sadanandapuram local). For ten days after stress induction the mean control value ranged between 7.83% (Murithothan) to 16.15% (Wild 3). The mean treatment value ranged between 3.07% (Meenadan) to 9.83% (Kuthiravali). The mean control value for 15 days after stress induction ranged between 8.12% (Cholakkodi) and 21.20% (Meenadan). Mean treated value ranged from 1.85% (Wild 3) to 6.90% (Meenadan). Analysis of variance of soil moisture in control and different drought intensities revealed non-significant variation among different genotypes at 5 DASI, 10 DASI and control at 15 th day. It suggests that there were no significant differences in soil moisture levels between the genotypes under study at those specific time points. Soil moisture was lower in the stress induced condition compared to control implying that the drought stress treatment did have a significant effect on soil moisture levels (Fig. 1).

Identification of resistant genotypes in the germplasm

The results recorded were interpreted in two-way factorial analysis using GRAPES software version 1.1.0. (Gopinath et al., 2021) [9] to identify resistant genotypes by statistical analysis of data. Analysis of variance for various physiological and biochemical characters are presented in Table 9-15.

The Accumulation of proline under stress in many plant species has been correlated with stress tolerance, and its concentration is usually higher in stress tolerant than in stress sensitive plants (Silvente et al., 2012) [16] . In general, physiological and biochemical markers for biotic and abiotic stress condition are one and the same. Flooding tolerance is evaluated (quantified) in terms of the growth response, the level of injury sustained and survival in relation to specific flooding characteristics, mainly flooding level (depth) and duration. (Glenz et al., 2006) [8] . Plant responses to submersion vary. They include injury, inhibition of seed germination, changes in vegetative and reproductive growth, changes in plant anatomy, and promotion of early senescence and mortality.

Selection Index Scores

The selection index technique can theoretically determine the genotypic worth of individuals or families in an objective manner. In general, selection indices provide a useful method for quantifying selection potential as well as providing a good chance for more efficient selection. Selection index was calculated on the basis of all the physiological and biochemical characters viz., relative water content, membrane leakage, proline content, chlorophyll content, peroxidase content, polyphenol oxidase content and glutathione reductase.

Based on these stress responsive characters recorded, selection indices were calculated for all the 36 genotypes for the selection of tolerant ones (Table 16) (Figure 1). Index score was highest for Sadanadapuram local, followed by Pandithitta local, Kottinadan, Adukanazhi, Palakkodi and Murithothan.

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