Screening for drought tolerance in cocoa hybrids (Theobroma cacao L.) and expression analysis of identified gene

Abstract

Cocoa (Theobroma cacao L.) is a high-value, export-oriented beverage crop, empowering small-scale farmers through global value chain participation. Originating from the humid rainforests of South America, cocoa requires annual rainfall between 1500–3000 mm, and summer irrigation of about 24 liters per plant every four days. However, climate change-induced rainfall unpredictability, particularly in non-traditional cocoa growing regions of India like Tamil Nadu, Andhra Pradesh, and North Eastern states, severely limits water availability, increasing the vulnerability of crop to drought. Shallow root system further exacerbates its sensitivity to water stress, restricting its ability to access deeper soil moisture and significantly affecting yield and supply. Despite the identification of drought resistant genotypes in controlled conditions, their field level efficacy remains largely unexplored. Plants exhibit complex morpho-physiological and biochemical mechanisms in response to drought, notably through signaling molecules such as reactive oxygen species (ROS) and osmolytes that mediate stress responses. However, translating these findings into field resilience in cocoa remains a major challenge. This study aims to identify drought resistant cocoa hybrids under field conditions by evaluating key biophysiological parameters along with gene expression analysis, thereby contributing to the sustainable enhancement of cocoa production amidst growing climate variability. Twenty cocoa hybrids planted during 2018 at Cocoa Research Centre, which are at yielding stage served as the base population for the present study. Morphological characterization based on qualitative and quantitative characters were carried out. Thirteen pod characters and ten bean characters were studied among the hybrids, of which all the characters except for number of furrows and ridges showed high variability. The hybrids were evaluated for the biochemical properties with a focus on determining fat and total polyphenol content of beans. A significant difference in biochemical characters was observed among the hybrids with the highest fat and polyphenol content recorded in H102 (55 %) and H103 (8.56 %), respectively. Correlation study between the weight of beans per pod and other quantitative traits showed a significant positive association with pod weight, total beans, single bean dry and fresh weight, bean length, and width. Path analysis further indicated that five of these traits exerted a direct positive influence on bean weight per pod, whereas the contribution of bean length was minimal, with a negligible path coefficient value of 0.063. Based on scoring and ranking in terms of yield parameters, H101 and H153 were selected as top performing hybrids. Field screening for drought resistance was conducted on these twenty hybrids by initially flood irrigating until the relative water content reached 80 per cent, followed by drought induction through the suspension of irrigation until the onset of incipient wilting symptoms, and subsequently alleviating the stress by reapplying flood irrigation. Based on the days taken to wilt, hybrids were classified using the score chart. Hybrids exhibited differential responses to drought resistance screening. H152, H153, H101, and H102 were found to be highly resistant where as H141, H169, and H103 exhibited moderate resistance towards drought. Highly resistant hybrids retained relatively high values for physio-biochemical parameters under drought stress than the susceptible ones. Correlation studies between days taken to wilt and physio-biochemical parameters revealed significant positive associations with all parameters except transpiration rate (r = -0.652**) and stomatal conductance (r = -0.524*). According to path analysis, eight variables including Relative Water Content (RWC), chlorophyll content, catalase, membrane stability, glycine betaine, proline, transpiration rate, and stomatal conductance exerted a direct effect on days taken to wilt, with RWC exhibiting a very high positive direct effect (1.33). Principal component analysis delineated three components (PC1, PC2, and PC3) with eigen values exceeding the Kaiser criterion threshold (>1), accounting for 75.78 per cent of total variation, with PC1 contributing maximum variability (56.94 %). RWC and superoxide dismutase exhibited maximum contribution to PC1 with a strong positive correlation of 0.901 and 0.890, respectively with PC1. Scoring and ranking of hybrids based on significant physio-biochemical characters regarded H103 and H169 as drought resistant. According to the prior transcriptomic study, laccases (LACs) were found to be upregulated in drought tolerant genotypes and were selected for the present gene expression profiling. The expression study was undertaken at different soil moisture regimes (100% and 40%), using two contrasting hybrids identified as drought resistant (H103, H169) and susceptible (H8, H55). H103 exhibited a pronounced upregulation with a 9.81-fold increase of the targeted gene under 40 per cent field capacity (FC) relative to its 100 per cent FC. In contrast, the susceptible hybrid (H65) manifested a comparatively attenuated response, with only a 1.89-fold increase, indicating a limited capacity for stress-inducible gene activation. Although H103 and H169 exhibit strong drought resistance, their yield performance under ambient conditions remains suboptimal. Hence, H101 and H141 were identified as top-performing hybrids due to their excellent yield and strong drought resistance.