The Beneficial Effects of Insect Pollination and Biochar Seed Coating on Okra (Abelmoschus esculentus) Seed Quality at Varying Temperature Conditions

Abstract

Underutilized crops, such as okra, have the potential to alleviate stress on crop production imposed by climate change and farming conditions, but their production is greatly hindered by poor seed quality. Insect pollination and seed coating with organic substances (biochar) may improve okra’s seed performance, but the beneficial effects of biochar seed coating and pollination on the seed quality of okra grown under stressful conditions is unknown. We examined the impact of pollination and biochar seed coating on okra seed performance under varying temperatures. Seeds were obtained from plants grown under complete insect pollination and exclusion. A factorial experiment was conducted in growth chambers with three factors: seed type, seed coating and temperature conditions. Insect-pollinated seeds with biochar coating had the highest chlorophyll content, seedling vigour index, shoot, and root growth and the heaviest seedling mass, but with a reduced speed of germination and emergence. The insect-pollinated seed without biochar coating had a lighter seedling mass (33% lower) than insect-pollinated, coated seed. Low temperature conditions during germination were ameliorated by biochar seed coating but biochar coating could not alleviate high temperature (35/30 °C) stress. Harnessing the pollinator’s benefits and biochar seed coating are possible sustainable solutions to enhance seed quality.

1. Introduction

A main tool to improve yield stability, crop resilience, food and nutritional security with changing climatic conditions is crop diversification with traditional, indigenous, or underutilized crops [1,2]. To achieve the Sustainable Development Goals (SDGs), which aim to balance the demands on land and food production (SDG 2) as well as biodiversity (SDG 15), maximizing the potential of underutilized crops is crucial, because these crops can be productive under marginal farming conditions with greater returns on ecological services [3]. Opportunities for strengthening the management of underutilized crops and their seed quality are paramount, considering the current climatic variability that frequently has a deleterious effect on seed establishment and performance.

A main underutilized crop is okra (Abelmoschus esculentus L.). It is a multi-use crop due to its edible pods, fresh leaves, stems and seeds. It is a valuable source of micronutrients, such as vitamins, calcium, iron, magnesium, proteins, antioxidants and is a rich source of calories [4]. Okra requires little agricultural input, is resilient to climatic change, and thrives well in degraded soil or marginal lands [5,6]. It is a tropical and subtropical crop and prefers temperatures of 20–30 °C for growth, flowering and fruiting [7]. Okra flowers can be self-pollinated, but cross-pollination by insects can improve effective fruiting, yields and the seed quality of okra [8].

Okra’s floral physiology (bilaterally symmetrical whitish-yellow flowers with sticky pollen) encourages insect pollination rather than wind pollen dispersion [8]. In the absence of insect pollination, the crop still produces seeds through self-pollination. Perera and Karunaratne [9] reported that the deprivation of okra from pollination reduced seed production and may affect seed set and seed quality leading to lower seed viability.

Currently, okra cultivation and production are low in South Africa due to its semi-arid growing environment [10,11]. Van der Walt and Fitchett [12] reported a pronounced seasonal difference in the soil temperature affecting cropping practices in South Africa. A shortage of improved okra seeds, poor establishment, poor germination, and unfavourable soil surface temperature are among the major factors limiting okra productivity in South Africa [13]. A poor and non-synchronous germination is caused by delayed permeability through seed-coat-imposed dormancy, causing imperviousness of seed coats to water and gases, which complicates the management of okra production.

Several studies have aimed to overcome seed dormancy hurdles in okra through seed stratification, or chemical and mechanical scarification [14]. Ebert et al. [15] reported that the percentage of hard seeds in okra increased significantly in all cultivars with an increase in seed maturity. There is limited knowledge about the effects of insect pollination and seed coating with organic growth stimulation substances, such as biochar, on improving seed germinability and vigour at varying temperatures. Seed coating can stimulate germination and the early growth of seedlings under adverse weather conditions, and can reduce the impact of environmental stresses by improving water availability [16].

Biochar is a carbon-based product obtained by slow pyrolysis of organic materials at high temperatures (300–450 °C) under low or no oxygen conditions [17]. Biochar seed coating is a cheap technique that makes use of local materials, while mineral or organic fertilizer could be optionally added to enhance nutrient supply to the seedling [18]. Biochar contains smoke derivatives and growth stimulators compounds called Karrikin, which provide strong chemical cues to stimulate seed germination and seedling growth [19] through improved soil physical properties and soil water-holding capacity [20]. Smallholder farmers save seed from the previous season and apply seed coating with biochar [21]. Seed coating offers non-germinated seeds protection against adverse environmental conditions, such as variation in soil temperature, and it provides nutrients to seedlings [22], stimulates root development, and helps to prevent seed predation by insects and birds. The beneficial role of ecological service providers, such as pollinators, on the seed quality of underutilized crops, such as okra, are rarely reported in the literature [1].

Therefore, this study focuses on ways to improve seed germination and seedling vigour in okra, through the indigenous practice of seed coating. Moreover, we aim to assess the benefits of insect pollination on seed quality. We model the variation in soil temperature experienced during the okra seed establishment stage by using varying controlled temperature conditions. The objective of this study is to examine the beneficial impacts of insect pollination and biochar coating on okra seed germination and vigour performance under varying temperature conditions.

2. Materials and Methods

This study presents the results from two experiments: a seed germination and an emergence experiment.

2.1. Seed Germination Experiment

The germination experiment was conducted in a growth chamber of the Department of Soil Crop and Climate Sciences, University of the Free State, South Africa. For the germination experiment, a 2 × 3 × 3 full factorial experimental design with four replications was used [23] to identify the impact of seed coating, pollination and temperature on germination variables.

2.1.1. Seed Source

The seeds were obtained from a field trial with okra at Kenilworth Farm, 15 km northwest of Bloemfontein, the Free State, South Africa (−29°06′ S; 26°15′ E, 1350 m asl). Part of the original okra seed for planting was saved. To obtain non-pollinated seed, a pollinator exclusion approach was used, which entailed the complete exclusion of the flower from insects. Exclusion was ensured using a tulle cloth bag with a 1mm mesh size covering the pod nodes before flowering, while other plant parts, such as leaves, were excluded. As the pods expanded during flowering, the bags were periodically adjusted to avoid contact with the florets. The flowers were labelled at the time of anthesis and four samples of 10 pods each were selected from the same plant (node) position, and manually harvested at 65 days after anthesis (equivalent to 17 WAP). Pollinated seeds were obtained by allowing insects to freely visit the flowers. The pods from both treatments were spread on glasshouse benches and air-dried. The seeds were manually threshed and air-dried to <12% moisture content. The mean hundred seed weight was 5.98 g for the pollinated seeds and 4.64 g for the non-pollinated seeds.

2.1.2. Preparation of Biochar

Pine biochar was obtained from C Fert™ Johannesburg, South Africa, which has a similar carbon content as other wood-derived biochar [24]. The raw material was pinewood (15–25% moisture content; pyrolyzed at 400 to 450 °C). The biochar pellets were finely ground using a coffee blender (Safeway model SBCS167, South Africa). One gram of dried powdered biochar was dissolved in 10 mL distilled water, which was equivalent to 10% (v/v) concentration. The mixture was shaken for two hours at 120 rev. min⁻¹ in the dark, then filtered and the filtrate was used as aqueous biochar for watering the germination paper and seedlings during seed germination and emergence [20].

2.1.3. Chemical Analysis

Three random samples from the powdered biochar were taken for chemical analyses according to the methods of the Soil Fertility Analytical Services Section, Department of Agriculture and Environmental Affairs, KwaZulu-Natal, South Africa. The pH and electrolytic conductivity (EC) were determined with Combo pH and EC waterproof meters HI 98,130 (Hanna Scientific, Woonsocket, RI, USA) respectively. The mineral nutrients, exchangeable K⁺, Ca^2+,, Mg²⁺, Fe³⁺, Cu²⁺, and Zn²⁺ were analysed using an atomic absorption spectrophotometer (AA-7000 Shimadzu, South Africa) with the specific wavelength set for each mineral element [25,26]. The available phosphorus content was determined via the Lancaster method using a spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan). The automated Dumas dry combustion method was used to determine total C and N, and organic carbon was determined using the Walkley–Black method [25].

2.1.4. Seed Germination Assay

Pollinated seeds, non-pollinated seeds, and the original planting seed from the field trial (saved seed) were used. Saved seed was included to model the smallholder farmers’ practices where saved seeds are used to propagate next season’s cultivation. Seed coating treatments consisted of biochar-coated seed and uncoated seeds. Two hundred seeds from each seed source were primed with 50 mL distilled water (non-coated) or 10% biochar solution (coated seed) (see Section 2.1.2). The seeds were primed for 6 h. Subsequently, the seed coating was strengthened with biochar in the ratio 1:5 (seed weight: biochar). The seed coating was air-dried for 12 hours to reduce the moisture content before use.

Germination assays were carried out according to International Seed Testing Association protocols [27] (ISTA 2020). This was conducted in a growth chamber of the Department of Soil, Crop and Climate Sciences, University of the Free State, South Africa. One hundred seeds from each seed lot were taken, and 25 seeds were arranged in a paper towel and rolled, giving four replications. The rolled paper towels were placed in sealed plastic bags to avoid moisture loss and incubated in Labcon growth chambers (Labcon laboratory Equipment Germany L.T.I.E.) at a temperature regime of 25/16 °C (low temperature), 30/25 °C (optimal temperature), and 35/30 °C (extreme temperature) for 12 light/12 dark hour cycles for 7 days.

A daily count of germinated seeds was conducted with germination defined as radicle protrusion of at least 2 mm. The final germination percentage represented the percentage of normally germinated seeds on the 7th day. In addition, seeding morphological indices, such as shoot length (SL) and root length (RL), were manually obtained with a ruler on the 7th day. Root and shoot dry weights were determined after drying the samples at 80 °C for 24 h.

Mean time to germination (MGT) was calculated according to Bewley and Black (1994) as follows:

MGT = (∑_ij^n▒FX)/(∑_ij^n▒F)

where: MGT = mean germination time, F = the number of seeds completing germination on day X, X = number of days counted from the beginning of the germination test, I = day one to day j and j = final day of germination.

Germination velocity index (GVI) which is a measure of the speed of seed germination was calculated according to Maguire (1962) as follows:

GVI = G1/T1 + G2/T2 + ⋯Gn/Tn

where: GVI = germination velocity index, G1, G2…Gn = number of germinated seeds in first, second… last count, and T1, T2…Tn = number of sowing days at the first, second… last count.

Seedling vigour: At the final count (14 days), all seedlings that had complete morphological parts without lesions or defects were selected as vigorous seedlings, and the average seedling length and weight of seedlings were measured for calculating the seedling vigour index as [28]: VI = germination (%) × average seedling length (mm).

2.2. Seedling Emergence Experiment

The emergence experiment was conducted in a climate-controlled growth cabinet of the Department of Soil Crop and Climate Sciences, University of the Free State, South Africa. The experimental design for the emergence assay was a 2 × 3 full factorial experimental design [23] with two independent variables: seed coating (two levels) and seed source (three levels). Two seeds per pot were sown in air-dried sieved soil pots (10 × 10 × 12 cm). Each treatment consisted of eight pots and was replicated four times. Seed coating (biochar-coated seed and uncoated seed) and seed source (pollinated seed, non- pollinated seed and saved seed) were the factors. The pots were placed in a climate-controlled growth cabinet (Conviron E15; Controlled Environments) with a long light period of 16 h, a photosynthetic photon flux density of 350 µmol·m⁻² s⁻¹, a day/night temperature of 30/25 °C, and relative humidity of 60%. Temperature and relative humidity in the chamber were continuously monitored by Conviron series controllers (CMP3243 Controlled Environments Ltd., Winnipeg, MB, Canada). The pots were rotated three times a week to ensure uniform growing conditions in the growth chamber. Before the experiment, the field capacity of the soil (FC) of each pot was evaluated using the formulae below:

FC = (Saturated pot weight − pot dry weight)/(pot dry weight)

Throughout the experiment, the water application rate was determined by measuring the individual pot weight.

A daily count of emerged seeds was conducted. In addition, 21 days after the start of the experiment, seedling morphological indexes, such as shoot length (SL) and root length (RL), were measured. Root and shoot dry weights were determined. Mean time to emergence (MET) was calculated according to [29] as follows:

MET = (∑_ij^n▒FX)/(∑_ij^n▒F)

where: MET = mean emergence time, F = the number of seeds completing emergence on day X, X = number of days counted from the beginning of the emergence test, I = day one to day j and j = final day of emergence.

Emergence velocity index (EVI) which is a measure of the speed of seed emergence was calculated according to [30] as follows:

EVI = E1/T1 + E2/T2 + ⋯En/Tn

(1)

where: EVI = emergence velocity index, E1, E2…En = number of emerged seeds in first, second… last count, and T1, T2…Tn = number of sowing days at the first, second… last count.

Physiological parameters of the plants were measured at 14 and 21 DAP: leaf chlorophyll content index (CCI), plant height (PHT), leaf number (LN). The CCI was measured using a portable SPAD meter (SPAD-502-PLUS chlorophyll meter, Konica Minolta, Ramsey, NJ, USA) on the adaxial leaf surface.

2.3. Statistical Analysis

Seed germination characteristics were analysed by a three-way analysis of variance, with temperature regime, seed coating and pollination status as factors, using the IBM SPSS Statistics package 25.0 (IBM Corp., Armonk, NY, USA). First, the data were tested for homogeneity of variance by Levene tests. Similarly, seedling emergence variables were analysed by a two-way analysis of variance, with seed coating and seed source as factors. The interaction effects of seed coating and seed source on leaf chlorophyll content index and plant growth were determined through a two-factor analysis of variance. The differences between means were assessed with Tukey’s test using a significance level of 0.05. The P-values highlighted in bold denote significance levels at p < 0.05. Linear regression was performed on XLSTAT. Principal component analysis (PCA) was constructed through SPSS by using a correlation matrix.

3. Results and Discussion

3.1. Seed Germination Indices

3.1.1. Effects of Biochar Seed Coating

The chemical constituents of biochar (

Table 1. The mean for various chemical properties of biochar used for seed germination and emergence experiments. EC = Electrolyte conductivity (mS/cm).

Extracts	Biochar
pH	7.8
EC (mS/cm)	0.24
Density (g/mL)	0.67
OC (%)	30.4
N (%)	3.5
P (%)	0.02
K (%)	0.13
Ca (mg/L)	722
Mg (mg/L)	190
Zn (mg/L)	1.3
Cu (mg/L)	0.6
Fe (mg/L)	89.6

Note: EC = Electrolyte conductivity (mS/cm) and OC = Dissolved organic carbon.

) contained highly dissolved organic carbon, and adequate total nitrogen, magnesium, zinc and iron concentrations. Biochar seed coating supplied organic nutrients to the seedling embryo during germination [20,31].

Biochar seed coating significantly influenced germination indices, such as final germination, root length, shoot length, fresh mass, mean germination time and the vigour index of the seedlings (

Table 2. The main effects of seed coating, pollination and temperature regime on okra seed germination indices. Significant p-values are indicated in bold. Different letters in superscript indicate significant differences between values.

Treatments	Levels	FG (%)	RL (cm)	SL (cm)	R:S	FM (g)	MGT (Days)	GVI	VI
Seed source	Saved seed	48 b	7.9 b	4.7	1.7 b	0.4	4.2	2.9 b	423 b
	Pollinated	49 b	7.7 b	4.9	1.7 b	0.4	4.1	3.4 c	471 b
	Non-pollinated	38 a	7.0 a	4.4	1.3 a	0.4	3.8	2.2 a	346 a
	p values	0.001	0.05	0.47	0.053	0.25	0.27	0.001	0.001
	LSD (0.05)	4.6	0.5	0.5	0.14	0.03	0.3	0.3	61.2
Seed coating	Coated	48 b	7.9 b	5.0 b	1.7	0.4 a	4.3 b	2.9 b	343
	Uncoated	40 a	7.5 a	4.6 a	1.7	0.7 b	3.8 a	2.7 a	483
	p values	0.001	0.052	0.032	0.53	0.025	0.001	0.19	0.001
	LSD (0.05)	3.8	0.4	0.4	0.1	0.02	0.2	0.3	50.0
Temperature	25/20	45 a	7.2 a	3.9 a	1.9 b	0.4 a	2.0 a	1.7 b	350 b
	30/25	65 b	8.7 b	5.7 c	1.6 a	0.5 b	3.3 b	4.6 c	610 c
	35/30	43 a	7.4 a	4.8 b	1.6 a	0.4 a	4.9 c	1.3 a	280 a
	p values	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001
	LSD (0.05)	4.6	0.5	0.4	0.1	0.03	0.3	0.3	61.0

FG = final germination percentage, RL = root length, SL = shoot length, R:S = root:shoot ratio, FM = fresh mass, MGT = mean germination time, GVI = germination velocity index, and VI = vigour index.

). Most germination indices responded to biochar seed coating, where the shoot and root lengths increased by 5%, but uncoated seed had a higher speed of germination compared with coated seed. Biochar seed coating substantially enhanced early seedling development in okra, as indicated by the germination indices. The mechanism is likely related to the priming effects of biochar increasing the substrate pH, thereby enhancing seed germination. In addition, biochar improves soil fertility by providing soil organic carbon, Ca, K, Mn, soil exchangeable-P and other essential nutrients for seedling development. The increase in root and shoot lengths could have influenced the germination time (speed of germination) and had a resultant effect on the seedling vigour index. Hammerschmiedt et al. [32] explained that biochar co-application can additively enhance the soil or any growing substrates since it contains humic substances that are rich in organic carbon. Similarly, Lopes et al. [33] showed that biochar application to the growing substrate ensured the availability of nutrients for growing plants, by altering the activity of some enzymes. Akinnuoye-Adelabu et al. [20] explained that biochar might stimulate hormone-like effects in the embryo similar to auxin and cytokinin, thus improving seed germination and vigour.

3.1.2. Effects of Insect Pollination

Pollination significantly influenced seed germination percentage, seedling root length, germination velocity index, and vigour index (Table 2). The germination indices of the pollinated and saved seed were comparable. Although the pollination status of the saved seed is unknown, it likely resembled that of pollinated seeds. The FG, RL, GVI and VI of pollinated seed were 29%, 10%, 55% and 36% higher than non-pollinated seed, respectively. The weight of pollinated seed (5.98 g) was greater than that of non-pollinated seeds (4.64 g), which was reflected in a higher germination velocity index. The movements of insects were expected to produce more outcrossing, which resulted in heavier seed weights in pollinated seeds, thus providing an increase in seedling vigour and fitness. In addition, pollination might have affected offspring quality through its influence on seed size; Labouche et al. [34] reported that seed size could translate into qualitative differences in germination rate, where pollinated seed experienced higher rate of seed germination, larger seedling mass, and higher seedling growth rates. We found the individual effects of insect pollination and seed coating to be the most important factors in determining the overall germination and vigour characteristics in seedlings (Table 2).

3.1.3. Effects of Temperature

The varying temperature regimes significantly influenced all germination indices (Table 2). Among the varying temperature regimes, seedling grown at 30/25 °C gave the highest germination indices, represented by higher values for FG, RL, SL, GVI and VI. Although, the mean germination time (MGT) for okra seedling was the shortest at 25/20 °C. The favourable temperature range of 25–30 °C sped up the germination process, while temperatures below 20 °C likely slowed down the diffusion process which caused disrupted imbibition and the escape of solutes from the seeds [35]. Temperatures above 30 °C led to a decline in the germination rate due to the desiccation of the seed coat and insufficient seed moisture. Sarma and Gogoi [36] reported that an optimal soil temperature between 25 °C and 30 °C led to the fastest okra seed germination. Our study indicates that beyond this range, germination is delayed and weak seeds may not even germinate, which was the experience at a low temperature of 25/20 °C and a high temperature of 35/30 °C.

3.1.4. Interactions between Seed Coating and Pollination on Seed Germination Indices

The interaction of pollination status, seed coating and the temperature regime all significantly affected the germination percentage, root length, shoot length, root:shoot ratio, germination velocity index and vigour index (

Table 3. Interaction effects of seed coating, pollination and temperature regime on okra seed germination indices. Significant p-values are indicated in bold. Different letters in superscript indicate significant differences between values.

Temperature	Seed coating	Pollination	FG (%)	RL (cm)	SL (cm)	R:S	FM (g)	MGT (Days)	GVI	VI
25/20 °C	coated	Non-pollinated	41 b	7.1 b	6.3 c	1.87 bc	0.4	4.3	1.9 b	399 b
		Pollinated	49 b	7.8 b	7.9 e	2.17 c	0.5	3.9	2.7 c	595 c
		Saved seed	37 a	7.6 b	6.4 c	1.61 b	0.4	4.2	1.3 a	229 a
	uncoated	Non-pollinated	31 a	6.1 a	4.4 b	1.90 bc	0.3	3.2	1.7 a	330 b
		Pollinated	29 a	6.5 a	3.6 a	1.80 bc	0.3	4.3	1.1 a	246 a
		Original	30 a	7.0 b	4.8 b	1.82 bc	0.4	4.3	1.2 a	296 a
30/25 °C	coated	Non-pollinated	66 c	7.3 a	7.0 cd	1.52 a	0.4	3.6	4.0 d	697 d
		Pollinated	77 cd	8.9 b	8.9 e	1.27 a	0.5	3.7	6.6 d	764 e
		Saved seed	70 c	9.4 c	7.8 d	2.01 c	0.5	3.9	5.9 d	742 e
	uncoated	Non-pollinated	50 b	7.3 a	7.4 d	1.41 a	0.4	2.9	7.6 e	554 c
		Pollinated	67 c	7.9 a	7.3 d	1.53 a	0.5	2.8	7.3 e	579 c
		Saved seed	69 c	8.4 b	6.9 c	1.73 b	0.4	3.7	6.2 d	326 b
35/30 °C	coated	Non-pollinated	34 a	7.0 b	7.6 d	1.50 a	0.4	5.4	2.1 b	279 a
		Pollinated	39 ab	7.9 c	8.2 e	1.49 a	0.4	5.1	3.5 d	388 b
		Original	29 a	7.6 bc	8.0 e	1.47 a	0.4	4.7	3.4 d	253 a
	uncoated	Non-pollinated	33 a	6.1 a	7.1 d	1.59 a	0.3	4.8	1.2 a	278 a
		Pollinated	31 a	7.3 b	7.2 d	1.66 b	0.4	4.8	1.1 a	253 a
		Saved seed	31 a	6.9 b	7.8 d	1.81 bc	0.2	4.5	1.2 a	266 a
p values			0.001	0.053	0.001	0.032	0.686	0.400	0.001	0.008
LSD (0.05)			11.3	0.8	1.1	0.4	0.1	0.7	0.8	150
CV (%)			23.3	11.9	15.7	14.8	13.4	12.0	20.3	25.6

FG = final germination percentage, RL = root length, SL = shoot length, R:S = root:shoot ratio, FM = fresh mass, MGT = mean germination time, GVI = germination velocity index, and VI = vigour index.

). Our results show that the complementary effects of pollination and seed coating improved the germination percentage by 20% at low (25/20 °C) and by 15% at high (35/30 °C) temperatures compared to the uncoated and non-pollinated seeds (Table 3). Heavier seed was obtained from insect-pollinated and biochar-coated seeds which provided the capacity to hold more water and minerals within the embryo that promoted germination. The coated, saved seed and the coated, pollinated seeds had a comparable performance with regard to germination indices, especially at optimal temperatures (30/25 °C).

Interestingly, coated and pollinated seeds grown at high temperatures developed a longer shoot length (Table 3), which might be due to the ability of the biochar coating to retain water. This was more evident through the root survival ratio, where the interaction significantly enhanced the root:shoot ratio. This could be attributed to the ability of the biochar to increase seedling root surface area per unit of growing medium, indirectly increasing water-use efficiency, nutrient retention capacity and modifying the biological activity in the seedlings. Our findings observed that the coated seed at low temperatures had the highest root:shoot ratio, which suggests that the biochar coating was able to alleviate the effects of low temperature on the root:shoot ratio. A similar result was reported by Akinnuoye-Adelabu et al. [20] where biochar priming boosted the pea seedling root:shoot ratio, although the varying temperature regimes were not considered in their study. However, the seedling’s fresh mass was not significantly affected by coating, varying temperatures, and the interactions of these factors. Although, saved and pollinated seeds with biochar coating had the heaviest seedling mass under optimal (0.5 g) and low temperatures (0.4 g). Coated saved seed and pollinated seeds gave comparable seedling vigour index values. Remarkably, seed coating was able to increase the vigour index of seedlings grown at low temperatures (Table 3). This could be attributed to the biochar application that helped to overcome low temperatures by maintaining the soil moisture level, thus promoting seedling vigour at low temperatures. In addition, the pollinated seed may possess the fitness-related traits that enhance its seedling vigour. Uncoated and non-pollinated seed had the fastest germination velocity index, while the highest number of days to germination, indicating slow germination, were among the coated and pollinated seeds (Table 3). This speed of germination (germination velocity index) of seedlings from non-pollinated and uncoated seeds was 15% more rapid than coated and pollinated seeds. This could be attributed to a lower mass of uncoated and non-pollinated seed giving the seed less time to initiate development in the seedling and resulting in a less vigorous seedling, while the large seed mass from pollinated and coated seeds resulted in a longer period of initial seedling development and more vigorous seedlings. Non-pollinated and uncoated seed germinated earlier and faster than pollinated and coated seeds but had a lower vigour index. Pandey et al. [37] reported that seed priming improved seed germination percentage and vigour in cucumber, more than 60% over unprimed seed; this high increase might be due to the inorganic chemicals used for the priming, while our study used an organic substance (biochar). Ahmed et al. [38] conducted a similar study under stressful growing conditions; their findings showed a 10% increase in germination percentage in seeds treated with moringa leaf extract compared with untreated seed. Our findings observed a higher germination percentage of 29% in coated and pollinated seed compared with uncoated and non-pollinated seed. This implies that the complementary interactions of pollination and seed coating enhanced the seedling performances more than their individual effects.

3.2. Seedling Emergence Experiment

The emergence test was conducted at optimal temperatures (30/25 °C) only. Coating the seed with biochar significantly enhanced seedling root length, shoot length, root:shoot ratio, fresh mass, mean emergence time and emergence velocity index (

Table 4. The main effects of seed coating and pollination on okra seed emergence indicators. Significant p-values are indicated in bold. Different letters in superscript indicate significant differences between values.

Treatment	Levels	FE (%)	RL	SL	R:S	FM	MET	EVI	VI
Pollination	Original	88 b	11.4 b	8.0 b	1.5 b	1.9 b	5.0 b	1.4 b	1762
	Pollinated	89 b	11.8 b	8.2 b	1.4 b	1.8 b	5.1 b	1.4 b	1734
	Non-pollinated	80 a	9.6 a	7.2 a	1.2 a	1.1 a	4.7 a	1.2 a	1511
	p values	0.049	0.002	0.05	0.002	0.001	0.005	0.002	0.08
	LSD (0.05)	7.89	1.146	0.40	0.2	0.306	0.3	0.104	244
Seed coating	Coated	87	11.9 b	8.3 b	1.5b	1.8 b	5.4b	1.4 b	1745
	Uncoated	84	10.0 a	7.6 a	1.3a	1.4 a	4.5 a	1.2 a	1594
	p values	0.505	0.001	0.005	0.027	0.004	0.001	0.006	0.129
	LSD (0.05)	6.44	0.94	0.4	0.1	0.3	0.2	0.1	199

FE = final emergence percentage, RL = root length, SL = shoot length, R:S = root:shoot ratio, FM = fresh mass, MET = mean emergence time, EVI = emergence velocity index, and VI = vigour index.

).

Pollination significantly influenced all seed emergence indices except for the vigour index (Table 4). The emergence percentage, root length, shoot length, root:shoot ratio, fresh mass, mean emergence time and emergence velocity index of pollinated seed were 11.3%, 23%, 14.3%, 25%, 64%, 8.5%, 16.7%, respectively, higher than non-pollinated seed. Pollinated and saved seeds had comparable emergence indices, implying that the farmer-saved seeds had similar germinability with completely pollinated seeds at optimal temperature conditions (Table 4). Moreover, the improvements in the emergence indices of biochar-coated and pollinated seeds could be attributed to the water- and nutrient-holding ability of biochar and the heavier seed weight obtained from pollinated pods.

The combined effects of pollination and seed coating significantly increased seedling emergence percentage, root length and fresh mass (

Table 5. Interaction effects of seed coating and pollination on okra seed emergence indices. Significant p-values are indicated in bold. Different letters in superscript indicate significant differences between values.

Seed Coating	Pollination	FE (%)	RL (cm)	SL (cm)	R:S	FM (g)	MET (Days)	EVI	VI
Biochar	Non-pollinated	78.1 a	10.4 c	8.1	1.3	1.2 a	5.1	1.3	1489
	Pollinated	87.5 a	12.9 d	8.4	1.6	2.3 d	5.6	1.5	1838
	Saved seed	93.8 b	12.4 d	8.4	1.6	1.9 c	5.4	1.4	1908
Uncoated	Non-pollinated	81.2 a	8.9 a	7.5	1.2	0.9 a	4.2	1.1	1533
	Pollinated	90.6 b	9.6 b	8.0	1.3	1.4 b	4.7	1.4	1631
	Saved seed	81.2 a	9.5 b	7.4	1.5	1.9 c	4.7	1.3	1617
p values		0.052	0.05	0.487	0.209	0.028	0.883	0.932	0.349
LSD (<0.05)		11.2	1.6	0.8	0.2	0.4	0.4	0.2	345
CV (%)		8.8	10.0	6.5	10.4	18.2	5.0	7.5	13.9

FE = final emergence percentage, RL = root length, SL = shoot length, R:S = root:shoot ratio, FM = fresh mass, MET = mean emergence time, EVI = emergence velocity index, and VI = vigour index.

). Biochar-coated saved seeds and pollinated seeds had an elongated root length, increased fresh mass and the highest emergence percentage, while the lowest seedling root length, fresh mass and emergence percentage were among non-pollinated and uncoated seeds (Table 5). The uncoated and non-pollinated seedlings had the fastest emergence (4.2 days), while coated and pollinated seedlings took more time to emerge (5.6 days). Coated and pollinated, as well as coated saved seeds, gave the highest seedling vigour. This observed increase in seedling survival and shoot:root ratio and vigour index could be attributed to their increase in seedling mass, root and shoot length due to their larger cotyledons. The possible reason for the improvement in these parameters could be the supply of nitrogen content from the biochar, an improved water-holding capacity and the availability of moisture from biochar which plays a key role in the physiological activities of the seedlings. Similar findings were reported in the seedling establishment of turnip rape by Elliott et al. [39], where seedlings obtained from large seeds had a higher mass than seedlings developed from smaller seeds. This study agreed with Perera and Karunaratne [9] who highlight the fact that pollination exclusion resulted in lower seed weight, seed quality and germination ability of okra seed, although their study did not focus on seed improvement using biochar. Our study highlights that root length, seedling fresh mass and emergence percentage values increased with biochar application and this is also reported elsewhere [40].

Fijen et al. [41] and Prasad et al. [42] reported that biochar application and insect pollination increase seed germination, emergence and seedling growth of tomato and hybrid leek seed production. However, the findings of Meng et al. [43] showed no influence on germination rate but a significant effect on older seedling fresh weight. Ali et al. [44] reported that insect pollination in okra resulted in higher germination, and the addition of biochar improved germination parameters. We observed the complementary benefits of insect pollination and biochar seed coating was more pronounced among seedling emergence indices, such as emergence percentage, root length and fresh mass.

Pollination and seed coating affected the leaf chlorophyll content index of the emerging seedlings, but not plant height and leaf number which had no distinct trend (

Table 6. The main effects of seed coating and pollination on the plant parameters in Experiment 2. Significant p-values are indicated in bold. Different letters in superscript indicate significant differences between values.

Treatments		PHT (cm)	LN	CCI
Pollination	Saved seed	12.4	2.9	37.6 b
	Pollinated	11.8	3.1	37.5 b
	Non-pollinated	11.6	2.9	30.3 a
	p values	0.108	0.350	0.015
	LSD (0.05)	0.9	0.4	1.8
Seed coating	Coated	11.9	2.9	34.4 a
	Uncoated	11.9	3.0	38.3 b
	p values	1.000	0.391	0.001
	LSD (0.05)	0.7	0.3	1.4

PHT = plant height, LN = leaf number and CCI = chlorophyll content index.

).

Likewise, the interaction of insect pollination and seed coating did not influence plant height and leaf number and only affected the leaf chlorophyll content index (

Table 7. Mean values of growth parameters of seedlings under pollination and seed coating treatments. Different letters in superscript indicate significant differences between values.

Seed Coating	Pollination	PHT (cm)	LN	CCI
Biochar	Non-pollinated	11.4	2.8	38.3 cd
	Pollinated	12.0	3.0	39.3 d
	Saved seed	12.4	3.0	37.4 c
Uncoated	Non-pollinated	11.8	3.0	32.3 a
	Pollinated	11.5	3.3	36.0 b
	Saved seed	12.5	2.9	37.9 c
p values		0.558	0.477	0.004
LSD (<0.05)		1.2	0.5	2.5
CV (%)		6.9	11.7	4.5

PHT = plant height, LN = leaf number and CCI = chlorophyll content index.

). There was a distinct trend in leaf chlorophyll content index, where coated and pollinated seeds gave the highest CCI at two weeks after planting (Table 7). It could be deduced that pollination and biochar coating mostly influenced the physiological trait (chlorophyll content) of the seed. Adelabu et al. [20] characterized the effects of biochar on the physiological status of pea seeds and observed a high photosynthetic rate with seedlings treated with biochar which was attributed to its high carbon content in the biochar. Biochar seed coating and pollination enhanced the chlorophyll content of okra seedlings. Extreme temperatures (25/20 °C and 35/30 °C) decreased the physiological growth of chickpea seedlings [45], and biochar application increased the photosynthetic ability of okra [46].

3.3. Correlation, PCA and Linear Regression

Some of the germination indices measured had significant positive and negative correlations with temperature regimes at different relationship strengths. The root length showed a positive significant correlation with the shoot length (r = 0.63), GVI (r = 0.50) and seedling fresh mass, while mean germination time was slightly and negatively correlated with root length (r = −0.31), FM (r = −0.35) and had a strong negative relationship with germination velocity index (r = −0.69). A negative strong relationship existed between shoot length and the root:shoot ratio as well as the germination velocity index and mean germination time. The correlation matrix indicated that the germination indices were interrelated. Likewise, seedling fresh mass and vigour index were positively correlated with root length. Notably, under temperature regimes, R:S had non-significant correlations with the germination velocity index, but had a strong negative correlation with shoot length and moderate negative correlation with seedling fresh mass (

Table 8. Pearson’s correlation coefficients and the significance of the associations between seedling emergence indices. Values with * and ** represent significance levels alpha = 0.05 and 0.001, respectively.

	RL	SL	FM	MGT	GVI	TSL	R:S	FG	VI
RL	1	0.632 **	0.460 **	−0.308 **	0.502 **	0.904 **	0.020	-0.237	0.035
SL	0.632 **	1	0.517 **	−0.173	0.514 **	0.903 **	−0.737 **	−0.354 **	−0.088
FM	0.460 **	0.517 **	1	−0.347 **	0.627 **	0.541 **	−0.263 *	−0.153	0.024
MGT	−0.308 **	−0.173	−0.347 **	1	−0.687 **	−0.266 *	−0.080	−0.041	−0.080
GVI	0.502 **	0.514 **	0.627 **	−0.687 **	1	0.562 **	−0.178	−0.213	−0.063
TSL	0.904 **	0.903 **	0.541 **	−0.266 *	0.562 **	1	−0.397 **	−0.327 **	−0.029
R:S	0.020	−0.737 **	−0.263 *	−0.080	−0.178	−0.397 **	1	0.306 **	0.187
FG	−0.237 *	−0.354 **	−0.153	−0.041	−0.213	−0.327 **	0.306 **	1	0.945 **
VI	0.035	−0.088	0.024	−0.080	−0.063	−0.029	0.187	0.945 **	1

RL = root length, SL = shoot length, FM = fresh mass, MGT = mean germination time, TSL = total shoot length, R:S = root:shoot ratio, FG = final germination, VI = vigour index.

).

Similarly, all the emergence seedling indices measured had significant positive correlations with one another for seed coating, temperature and pollination treatments. Highly significant correlations were recorded between MET and EVI (r = 0.62) and between FM (r = 0.61) and SL (r = 0.54), final seedling emergence and MET (r = 0.43). Likewise, EVI and SL (r = 0.49), as well as FE and MET (r = 0.43), EVI (r = 0.54) and root length (r = 0.45) showed moderate correlations with treatment. Shoot length had positive correlations with MET, EVI and root length (

Table 9. Pearson’s correlation coefficients and the significance of the associations between seedling emergence indices. Values with * and ** present significance level alpha = 0.05 and 0.001, respectively.

Variables	MET (Days)	EVI	FE (%)	FM	SL	RL
MET (days)	1	0.617 **	0.428 *	0.612 **	0.538 *	0.705 **
EVI	0.617 **	1	0.540 *	0.583 *	0.493 *	0.638 **
FE (%)	0.428 *	0.540 *	1	0.235	0.289	0.449 *
FM	0.612 **	0.583 **	0.235	1	0.354	0.651 **
SL	0.538 *	0.493 *	0.289	0.354	1	0.552 *
RL	0.705 **	0.638 **	0.449 *	0.651 **	0.552 *	1

MET = mean emergence time, EVI = emergence velocity index, Fe = final emergence, FM = fresh mass, SL = shoot length and RL = root length.

).

Principal component analysis was applied to nine germination and six emergence variables to examine the relationship among these variables (Figure 1A,

Table 10. PCA for seedling germination variables.

	Initial Eigenvalues				Rotated Component Matrix Loading
Component	Total	% of Variance	Cumulative%	Variables	Total
1	4.022	44.7	44.7	RL	1.775
2	2.011	22.3	67.0	SL	1.478
3	1.289	14.3	81.4	FM	1.324
4	0.920	10.2	91.6	GVI
5	0.532	5.91	97.5	MGT
6	0.201	2.24	99.7	TSL
7	0.018	0.2	99.9	R:S
8	0.006	0.1	100.0	FG
9	−1.001 × 10−13	−1.012 × 10−13	100.0	VI

). The first two principal components (PCs) explained 44.69% (PC1), 21.34% (PC2) in root length and shoot length for seedling germination variables, while 60.16% (PC1), 13.39% (PC2) in root and shoot length for the seedling emergence variables (Figure 1B). The PC1 in root length for germination variables was robustly weighted in SL, FM, MGT, GVI, R:S, FG and VI, contributing 44.69% to the total variance, followed by the SL contributing 22.34% to the total variance (Table 10). Likewise, the PC1 in root length in seedling emergence was strongly weighted in MET, EVI and FE contributing 60.17%, 13.39% and 10.98% to the total variance, respectively (

Table 11. PCA for seedling emergence variables.

	Initial Eigenvalues				Rotated Component Matrix
Components	Total	% of Variance	Cumulative%	Variables	Loading
1	3.610	60.2	60.2	MET (days)	3.462
2	0.803	13.4	73.6	EVI	1.763
3	0.653	10.9	84.4	FE
4	0.376	6.3	90.7	FM
5	0.302	5.0	95.7	SL
6	0.256	4.3	100.0	RL

). The data set showed components were extracted with a cumulative percent of 67.03% and 73.55% of the total variance in seedlings root and shoot, for both the germination and emergences indices, respectively (Figure 1A,B). The first component in Table 10 demonstrated significantly strong positive rotated component matrix loadings of RL (1.78), SL (1.48) and FM (1.32), for the germination variables. Similarly, in the emergence dataset, the first component showed significantly strong positive rotated component matrix loadings of MET (3.46) and EVI (1.76) (Table 11).

The linear regression was carried out only on the emergence indices. A moderate relationship with the temperature regimes were observed among the selected indices (Figure 2). The linear regression of MET and RL (Figure 2A) indicates that as the speed of emergence (MET) increased there was a slight increase in root length, while an increase in root length moderately enhanced the shoot length (Figure 2B), and emergence velocity index (Figure 2C). The regression slope was moderately higher in seedling fresh mass which led to an increase in the speed of emergence i.e., MET and EVI (Figure 2D,E). The mean emergence time and emergence velocity index which represents the speed of germination shows a linear regression (Figure 2F).

Principal component analysis illustrated the speed of seedling germination, root and shoot lengths to be strong germination and emergence indices of okra seedlings. A positive relationship existed between shoot and root elongation of biochar-coated and pollinated seeds. Thus, seed coating and insect pollination altered the germination ability, vigour, and seedling chlorophyll content, thus improving seed quality and the performance of the okra crop. Harnessing the pollination benefits and demand to optimize seed quality is not always straightforward as it requires a combined knowledge of crop management and seedling establishment [47]. Thus, the quality of ecosystem service received by the seed and other environmental factors, such as temperature, can result in complex interactions that affect the harvested seed germination and vigour [48].

4. Conclusions

This study investigated the beneficial effects of pollination and biochar seed coating on okra seed germination and emergence at varying temperature regimes. Pollinated and coated seeds had a higher root and shoot growth, final germination percentage, vigour index as well as chlorophyll content index compared to uncoated and non-pollinated seeds. Uncoated and non-pollinated seeds showed a faster germination and emergence than coated seeds but with a lower vigour index. The beneficial effects of insect pollination and biochar seed coating on seed germination and vigour indices was more visible at 25/20 °C than at 35/30 °C. This reveals that seed coating with biochar reduced the environmental stress because okra seeds are sensitive to chilling and coating enhances its tolerance ability. This finding highlights the potential of biochar seed coating and insect pollination to improve okra seed establishment under extreme temperature conditions.