**1. Introduction**

Okra (*Abelmoschus esculentus* [L.] Moench), belonging to the Malvaceae family, is an important crop mainly cultivated as fruits, vegetables and seed oil. It is extensively grown in tropic and subtropic regions [1] and arid and semi-arid regions with limited and erratic

**Citation:** Mkhabela, S.S.; Shimelis, H.; Gerrano, A.S.; Mashilo, J. Drought Tolerance Assessment of Okra (*Abelmoschus esculentus* [L.] Moench) Accessions Based on Leaf Gas Exchange and Chlorophyll Fluorescence. *Life* **2023**, *13*, 682. https://doi.org/10.3390/ life13030682

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 17 January 2023 Revised: 27 February 2023 Accepted: 28 February 2023 Published: 2 March 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/).

rainfall conditions [2]. The tender and immature pods of okra are consumed as cooked vegetables [3]. The pods are rich in protein content (25 %) and amino acids, notably lysine and tryptophan [4], fat, fibre, vitamins (A, C and K), vital mineral elements such as calcium, potassium, sodium, magnesium, iron, zinc and manganese [5], and soluble sugars such as sucrose (110.4 g/100 g FW), fructose (34.8 g/100 g FW and glucose (30.9 g/100 g FW [6]. In addition, minor quantities of organic acids, including citric, oxalic and malic acid, are present in the succulent pods [6]. The mature and dry seeds are a vital source of edible oils. The seed oil content ranges from 20–40%, consisting of the following major fatty acids: linoleic, palmitic, oleic, diacylglycerols and triacylglycerols acids [7].

Continental Asia accounts for a total annual okra production of 6 million tons from 592,375 million hectares of cultivated land, whereas Africa is the second major producer, with 3 million tons per annum from approximately 1.9 million ha of cultivated land [8]. Commercial and small-scale farmers produce okra. In sub-Saharan Africa (SSA), the crop is mainly grown in marginal conditions characterised by low and erratic rainfall, with minimal agricultural inputs and production technologies. In SSA, okra is mainly cultivated under rainfed conditions, and these agro-ecologies face moderate to severe droughts during the growing season [9]. Drought stress significantly reduces growth, biomass and yield [10]. Drought alone accounts for yield losses ranging between 30 and 100% in okra, primarily when the stress occurs during the flowering and pod-filling stages [3]. Breeding okra cultivars with drought adaptation is the major objective in improvement programs. Physiological and complementary phenotypic traits are critical in the selection of drought-adapted crop genotypes.

Phenotyping of plants using gas exchange and chlorophyll fluorescence traits has been reported as a preferred approach for selecting drought-tolerant okra accessions [11]. Some gaseous exchange traits used to assess drought tolerance include photosynthesis rate, stomatal conductance, chlorophyll content and transpiration rate. Further, chlorophyll fluorescence parameters (e.g., minimum fluorescence, maximum fluorescence, effective quantum efficiency of *PSII* photochemistry, photochemical quenching and non-photochemical quenching) have been used in phenotyping for drought tolerance [11–13]. Drought stress affects okra growth and productivity, disrupting physiological functions and the photosynthetic rate, resulting in yield losses [11,13,14]. Mkhabela et al. [3] reported that okra yield loss under drought stress could be significantly minimised by breeding drought-tolerant ideotypes with intrinsic water use efficiency. Hence, understanding the physiological response of diverse okra genotypes under drought stress conditions is essential for the selection of drought-tolerant accessions for production or breeding.

There has been limited progress in the breeding of okra for drought tolerance. This could be due to limited accessions identified as good drought and heat tolerance sources and insect pests and disease resistance [15]. Some unique accessions, including Sabz Pari [16], NHAe 47-4 [17], Pusa Sawari, Iraq P, Hala [1] and Xianzhi [18], were identified as useful sources of genes for enhancing drought tolerance under water-limited conditions. Compared to the highest genetic diversity reported in the cultivated okra [19], the identified accessions with tolerance to drought are relatively few. Therefore, there is a need for concerted research and development in okra to develop market-led and improved varieties for water-limited conditions.

In South Africa, okra is an important but under-researched and under-utilised crop. It is grown under rainfed conditions using local and unimproved accessions with poor adaptation and low yield potential. Genetically unique okra accessions could be sourced from different geographical regions to enhance okra pre-breeding programs [3]. Morphological traits associated with drought tolerance in okra include the number of pods per plant, fresh pod length, number of seeds per pod, hundred seed weight, number of branches per plant, plant height and total pod production [1,19]. Reportedly, a higher number of branches, pod length and number of pods per plant, plant height between 150 and 170 cm and pod weight have a direct influence on pod yield [19]. Drought tolerance assessment of okra accessions using the combination of morphological and physiological

traits could increase the efficiency of identifying and selecting drought-tolerant accessions for cultivar development under dry environments. Therefore, this study aimed to assess the levels of drought tolerance in preliminarily selected okra accessions based on leaf gas exchange and chlorophyll fluorescence to determine best-performing genotypes for drought-tolerance breeding.

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

#### *2.1. Plant Materials and Study Site*

Twenty-five genetically distinct okra accessions were used for the study. The accessions were sourced from the Agricultural Research Council, Vegetable, Industrial and Medicinal Plants (ARC-VIMP) gene bank, and one local variety was included. The accessions were previously studied for their morphological responses to drought stress under field and glasshouse environments [3]. Detailed information on their geographical origin and drought resistance index are presented in Table 1. The experiment was conducted under glasshouse conditions at the Controlled Environment Facility (CEF) of the University of KwaZulu-Natal during the 2020/2021 growing seasons. The first experiment was conducted from September 2020 to December 2020, and the second from February 2021 to May 2021. The accessions were evaluated under non-stressed (NS) and drought-stressed (DS) conditions in the glasshouse environment. Drought tolerance index was calculated as DTI = (Ys/Yn)/(Ms/Mn), where Ys and Yn are the genotype yields under stress and non-stress, and Ms and Mn are the mean yields of the accessions under stressed and non-stressed conditions, respectively [20].

**Table 1.** Accession code, accession number, database, geographical origin, drought tolerance index and stem colour of the okra accessions evaluated in the study.



**Table 1.** *Cont.*

ARC = Agricultural Research Council, DTI = drought tolerance index.

#### *2.2. Experimental Design and Crop Establishment*

Five seeds were initially planted in 5 L capacity plastic pots filled with composted pine bark growing media. Later, two plants were established per pot for each genotype. The day and night temperatures in the greenhouse (GH) were 30 ◦C and 20 ◦C, respectively, and the relative humidity ranged between 45 and 55% during the study. Inorganic fertilizers consisting of nitrogen (N), phosphorus (P) and potassium (K) were applied at a rate of 120, 30 and 30 kg ha<sup>−</sup>1, based on soil fertility recommendations using urea (46-0-0), phosphorus pentoxide (P2O5) and potassium oxide (P2O), respectively.

The trials were established using a 13 × 2 alpha lattice design under drought-stressed and non-stressed conditions with three replications. Drought stress was imposed at 50% flowering until physiological maturity by withholding irrigation until the soil water content reached 30% field capacity for plants under DS. The duration of stress was seven days before sampling. Plants under NS conditions were irrigated regularly to maintain soil moisture content at field capacity until physiological maturity. To determine pod yield, plants reached maturity, and pods were harvested sequentially at the soft, most digestible and immature stage. Tensiometers, moisture monitors (Spectrum Technologies, Inc, Chicago, IL, USA), were used to detect soil moisture levels at the root zone. Agronomic performance of the test genotypes was reported in Mkhabela et al. [19].

#### *2.3. Data Collection*

Gas exchange and chlorophyll fluorescence parameters were measured using an LI-6400 XT Portable Photosynthesis system (Licor Bioscience, Inc. Lincoln, NE, USA) integrated with an infrared gas analyser (IGRA) attached to a leaf chamber fluorometer (LCF) (640040B, 2 cm<sup>2</sup> leaf area, Licor Bioscience, Inc, Lincoln, NE, USA). External leaf CO2 concentration (C*a*) and artificial saturating photosynthetic active radiation (PAR) were set at 400 μmol mol−<sup>1</sup> and 1000 μmol m−<sup>2</sup> s−1, respectively. Water flow rate and relative humidity were maintained at 500 μmol and 43%, respectively. The leaf-to-air vapour pressure deficit in the cuvette was maintained at 1.7 kPa to avoid stomatal closure due to low air humidity. Gas exchange and chlorophyll fluorescence measurements were taken on the third half fully formed leaf inside the sensor head. Under both NS and DS conditions, measurements were taken from five plants of each accession.

The following gas exchange parameters were determined: stomatal conductance (gs), net CO2 assimilation rate (A), transpiration rate (T), intercellular CO2 concentration (C*i*) and the ratio of intercellular and ambient CO2 (C*i*/C*a*) concentrations. The ratio of net CO2 assimilation rate to intercellular CO2 concentration (A/C*i*) was computed according to Kitao et al. [21]. The ratio of A and gs was used to compute intrinsic water use efficiency [22] and the ratio of A and T was used to calculate instantaneous water use efficiency) [23].

To estimate chlorophyll fluorescence variables, a saturation flashintensity of 1300 μmol m−<sup>2</sup> s−<sup>1</sup> was applied. The following parameters were recorded. The minimum (*Fo*- ) and maximum fluorescence (*Fm*- ) of light-adapted leaves under natural glasshouse conditions. The steady-state fluorescence (*Fs*) was also determined in light-adapted photosynthesis. Equation (1) was used to determine the variable fluorescence in light-adapted leaves, while Equation (2) calculated fluorescence changes [24].

$$Fv' = Fm'F\_0'\tag{1}$$

$$
\Delta F = Fm' - Fs \tag{2}
$$

Additional chlorophyll fluorescence parameters were estimated according to Evans [25], *Fv*- /*Fm*- , the maximum quantum efficiency of photosystem II photochemistry, the effective quantum efficiency of photosystem II photochemistry (*φPSII)*, photochemical quenching (*qP*), non-photochemical quenching (*qN*) and electron transport rate (ETR). The ratio of ETR and A was used to calculate a relative measure of electron transport to oxygen molecules. The alternative electron sink (AES) was calculated as the ratio of photosystem II effective quantum efficiency to net CO2 assimilation (A) [26]. Chlorophyll fluorescence was measured using a pulse-amplitude modulated (PAM) fluorometer, which applies a short pulse of light to the sample and measures the resulting fluorescence emitted by the chlorophyll. This measurement provided information on the photosynthetic efficiency and health of the crop. Gas exchange and chlorophyll fluorescence parameters were measured on fully expanded leaves. At the end of the second experiment, yield per plant (YPP) was determined by harvesting fresh pods when 50% of the pods were 3–5 cm long by hand every third day.
