Phenotyping drought stress tolerance in citrus rootstocks using high-throughput imaging and physio-biochemical techniques.

Background: Drought stress, the most prevalent abiotic stress, has a significant effect on citrus production worldwide. The differential mechanisms to overcome the drought stress has been reported in citrus rootstock genotypes. This study evaluated nine citrus rootstock genotypes, including indigenous rough lemon variants, for drought tolerance. The genotypes were subjected to well-watered, drought stress, and re-watering conditions to assess morphological, physiological, and biochemical responses. High-throughput imaging techniques were employed to non-destructively assess chlorophyll fluorescence, digital leaf area, and plant tissue water content during drought stress.

Results: For rapid and accurate screening of rootstocks, phenomics and physio-biochemical tools were used to know morpho-physiological responses to drought. Citrus rootstock genotype X639 demonstrated superior performance under drought stress conditions. It maintained the highest growth in terms of relative shoot increment (8.09%), number of leaves (79.00), and specific leaf area (62.45 cm g). X639 also excelled in root morphological parameters, including root length, projected area, diameter, surface area, volume, and number of tips, forks, and crossings. Trifoliate hybrids X639 and Troyer citrange exhibited larger stomata (54.73 and 43.82 µm) compared to mono-foliate species, with minimal impact of drought on stomatal pore area. X639 maintained the highest relative water content, membrane and chlorophyll stability indices, leaf gas exchange parameters, and antioxidant enzyme activity. RLC-1 and RLC-4 genotypes showed pronounced accumulation of leaf proline and antioxidant enzymes during drought, contributing to better recovery after re-watering.

Conclusion: In this study, Cleopatra mandarin, Grambhiri, and RLC-2 were identified as drought-susceptible rootstocks based on their responses. Rootstock genotypes X639 and RLC-4 proven a superior drought-tolerant genotypes. Their robust root system enables efficient water uptake and the maintenance of water relations during drought stress. The drought tolerance of X639 was evidenced by its ability to maintain plant tissue moisture, membrane and chlorophyll stability, and higher photosystem II efficiency. High-throughput imaging techniques have proven effective in rapidly assessing and differentiating drought-tolerant and drought-susceptible citrus rootstocks based on their photosystem- II efficiency, leaf area, and tissue water content during induced drought stress. These findings will contribute to the selection and development of drought-tolerant citrus rootstocks to improve citrus production under water-limited conditions.