Impact of CO₂ Enrichment on Growth, Yield and Fruit Quality of F1 Hybrid Strawberry Grown under Controlled Greenhouse Condition

Abstract

Carbon dioxide enrichment inside a greenhouse is a sustainable approach to increasing crop production worldwide. Recently, the F1 hybrid strawberry became an alternative to runner-propagated cultivation as an innovative method to shorten the production period and increase strawberry production. This work aims to present CO₂ enrichment as a sustainable tool that improves the yield in a controlled greenhouse and addresses the efficiency of three F1 hybrid strawberry varieties grown under Saudi Arabian conditions. A greenhouse experiment was conducted at the National Research and Development Center for Sustainable Agriculture (Estidamah), KSA, to study the impact of two CO₂ levels (400 ppm (“ambient”) and 600 ppm (“enrichment”)) on the growth, photosynthesis traits, fruit yield and fruit quality of three F1 hybrid strawberry varieties grown under soilless culture conditions. The results show that CO₂ enrichment significantly improved the phenotyping of strawberry growth traits at 60 days post-transplanting. The physiological response of the varieties to CO₂ enrichment reveals a significant increase in the photosynthetic rate (129.7%) and intercellular CO₂ (43.7%) in the leaves of strawberry exposed to CO₂ enrichment rather than in ambient conditions, combined with a significant increase in the number of fruits per plant (27.5%) and total fruit yield (42.2%). A similar pattern was observed with varieties D and S in terms of fruit number, length and diameter. However, CO₂ at 600 ppm promoted total soluble solid accumulation and vitamin C for the tested varieties. In contrast, CO₂ enrichment significantly decreased nitrogen, phosphorus, potassium and magnesium accumulation in the leaves of the exposed plants in comparison to 400 ppm of CO₂. These results suggest that increasing CO₂ enrichment could contribute to an increase in strawberry yield and nutritional value and demonstrate that understanding the response of each variety to CO₂ enrichment is important to support selecting suitable greenhouse strawberry varieties to improve crop yield.

1. Introduction

Sustainable agriculture leads to enhanced agricultural productivity by strengthening resilience to the unfavorable impacts of climate change [1], in order for the planet to feed 10 billion people by 2050 [2]. One of the challenges for global food security is the limitation of agricultural land due to soil erosion and nutrient depletion, mainly in arid zones [3]. Numerous reports have shown that protected culture is a key answer to provide markets with adequate quantities of vegetables to cope with global demand because of its capability for optimal plant development [4]. Saudi Arabia has ~124 thousand greenhouses producing ~584 thousand tons of different crops, with USD ~335 million of annual sales [5]. However, improving greenhouse production in arid zones requires a new generation of technology and supporting systems to achieve the maximum production with a sustainable cost [6]. In this respect, advanced greenhouse technologies are becoming more essential for maximizing crop production per cultivation unit area in hot climates [7]. The performance of crop growth and productivity in the greenhouse is basically controlled by several factors, like water, fertilizers and greenhouse climate conditions [8]. Therefore, adapting the microclimate conditions inside the greenhouse is considered a suitable approach to increase crop yield and quality [9]. One of the factors that limits crop growth is frequent insufficient carbon dioxide (CO₂) inside the greenhouses during the daytime [10]. Atmospheric CO₂ has risen from 280 to 390 mmol mol⁻¹, and it is expected to reach 500–1000 µmol mol⁻¹ by the next century [11], which will potentially increase the global temperature by 1.8–5.8 °C. This is contributing a major share to global warming [12], which subsequently influences plant growth performance and crop yields [13]. Carbon dioxide significantly affects crop photosynthesis and productivity when its concentration is within the appropriate range [14,15,16,17]. Under low-ventilation circumstances within the greenhouse, levels of CO₂ decrease to less than 250 ppm, which is below the optimal required concentration of 400~450 ppm [18,19]. CO₂ enrichment has demonstrated an increase in lettuce yield and growth rate [20,21], cucumber fruit biomass and leaf area index [22] and total yield of tomato [23]. Furthermore, the authors of ref. [24] reported that CO₂ fertilization improved bell pepper growth and yield associated with changes in stomatal traits, leaf photosynthesis and foliar nitrogen. Lettuce fresh and dry weight increased with increasing CO₂ concentrations, with the greatest increases observed between 400 and 800 ppm [25]. In strawberry, a 25% increase in the marketable fruit yield and 1.2% rise in fruit brix were determined for the cultivar Sagahonoka [26], while the cultivar Koiminori presented a 21–28% increase in average fruit weight and 29–35% increase in fruit numbers under CO₂ enrichment [27]. However, another study [28] confirmed that elevated CO₂ increased the yield of vegetables by 34%, the harvest index by 23% and the root to shoot biomass ratio by 8%. Several studies have shown that different technologies can be used for CO₂ enrichment inside the greenhouse, since CO₂ is dissipated to the outside if the greenhouse is ventilated. For instance, these include natural ventilation by using windows or fans, the direct supply of compressed CO₂ [29], carbonaceous fuel burning [30,31] and decomposition of agricultural wastes by microbial fermentation to release CO₂ for crop production [32]. Strawberry (Fragaria X ananassa) is produced worldwide, because of its great importance to producers and consumers [33,34]. According to the authors of ref. [35], the global production of strawberry has exceeded 8.3 million tons produced on ~372 thousand hectares. The fruit of strawberry has high-quality nutritional properties, like polyphenols, ellagic acid, anthocyanins and vitamin C [36]. In Saudi Arabia, the domestic market depends on imported strawberries to fulfill the needs of consumers. In 2020, the country imported a total of ~21,969 tons of strawberries (fresh and frozen), with a value exceeding USD ~71 million. This high import volume is attributed to the limited supply of locally produced strawberries, which are currently priced in a higher range. Local farmers keep using runner-propagated plants to cultivate a new crop cycle, which has led to concern about the quality of the seedlings and a subsequent decrease in yield each year. However, in several countries, a new approach to strawberry cultivation has been adopted, which involves the use of hybrid strawberry seeds [37]. This innovative method is anticipated to shorten the production period of seedlings, with no transmission of pests and diseases. A small quantity of seed gives logistical advantages in storing and transportation, and enhances overall productivity compared to the traditional cultivation methods. This work investigates the effect of CO₂ enrichment on crop production in a controlled greenhouse and addresses the efficiency of F1 hybrid strawberry varieties to increase fruit yield under Saudi Arabian condition. The morphological and physiological characteristics of strawberry plants will be evaluated and compared in terms of growth, photosynthesis traits, fruit yield and fruit quality under soilless culture.

2. Materials and Methods

2.1. Plant Materials and Greenhouse Setup

A greenhouse experiment was carried out at the National Research and Development Center for Sustainable Agriculture (Estidamah, Riyadh, Kingdom of Saudi Arabia). A Hi-Tech greenhouse with 400 m² (16 m × 25 m) and 6 m height was used; the compartments were covered with a diffuse tempered single glass, with 91% light transmission, 80% hemispherical light transmission and 75% haze. Both compartments are supported by air conditioning system, to provide sufficient cooling and avoid ventilation with outside air during the growth season. Conditioned air is distributed by air ducts placed on the growing gutter. The greenhouse climate was monitored by three separated measuring boxes while the nutrient recipe was applied through drip irrigation system [38], whereas the setpoint of EC and pH of 1.8 and 5.8, respectively, was adjusted. Seeds of the three F1 hybrid varieties of strawberry Fragraria X anansa Duch “Delizzimo”, “Estavana”, “Soraya” ABZ seeds, Netherlands, were sown in rockwool cubes on 15 September 2023. Two months later, seedlings were transferred to the compartments under controlled conditions of 20 ± 1 °C/day and 16 ± 1 °C/night, with 2.5 plants per m² plant density.

2.2. Carbon Dioxide Injection System

One-week post-transplanting, plants were treated with two carbon dioxide concentrations. Therefore, two separate greenhouses were used in this study: carbon-rich (600 ppm) and ambient CO₂ (400 ppm). Each CO₂ concentration was adjusted by an automatic greenhouse CO₂ control system (Figure 1). CO₂ was supplied through injection system to supplement the natural levels of CO₂ in the greenhouse atmosphere. The system consists of a carbon dioxide tank, evaporator and pressure regulator outside the greenhouse (Figure 1). The tank stores the CO₂ gas in a compressed form. Hoses are connected to the tank, allowing a controlled release and distribution of CO₂ into the greenhouse environment. The CO₂ outlets are strategically positioned below the planting gutter-lines, ensuring that the released CO₂ is evenly distributed throughout the growing area (Figure 2).

2.3. Vegetative Growth

Plant height, total number of leaves per plant, number of crowns per plant, crown diameter, fresh and dry weight of plant leaves were recorded at two months post-transplanting. Leaf area was measured using the LI-COR leaf area meter, model 3000A (LI-COR, Inc., Lincoln, NE, USA). The crown diameter was determined using a Vernier caliper.

2.4. Yield and Its Component

The total yield per plant was determined by continuous fruit harvesting of free-decay fruit, with 90% red color. The number of fruits per plant was counted via dividing the total harvesting fruit number by the number of planted plants. Representative fruit samples were dried at 70 °C to determine fruit dry weight. Ten fully colored fruits were randomly selected to measure fruit length and diameter with digital caliper at 90 days posttransplant. Finally, the fresh fruit weight was measured to determine the average fruit weight.

2.5. Vitamin C

Strawberry fruit juice was used to determine vitamin C by the titration method, as illustrated by [39]. Vitamin C in fruit juice was calculated as:

$$\mathrm{Ascorbic} \mathrm{acid}={\displaystyle \frac{\mathit{y}}{\mathit{x}}}\times 100$$

(1)

where y = volume of dye used milliliters in titrating 2 milliliters fresh juice. x = volume of dye used (mL) in titrating 2 milliliters standard stock solution.

2.6. Titratable Acidity (TA)

The titratable acidity (TA) of strawberry fruit juice was determined by titration with NaOH (0.1 N). Five milliliters of filtered juice was added into 50 mL flask, and two drops of phenolphthalein (PhPh) indicator were added to the juice. The exact amount of NaOH solution was used to calculate the acidity of fruit juice according to [39].

2.7. Total Soluble Solids (TSS)

Total soluble solids percentages (Brix %) were measured in sweet pepper juice samples via a hand digital portable refractometer (PR.101 model, ATAGO Co., Tokyo, Japan).

2.8. Nutrient Analysis

Leaves of strawberry plants were collected at 60 days post-transplant and cleaned with double-distilled water, then oven dried at 105 °C for 24 h. Thereafter, plants were grounded and sieved with a 0.5 mm sieve and preserved until digestion; then, 0.1 g from each sample was weighted in a 100 mL conical flask. The samples were digested with acids (hydrogen peroxide and concentrated sulfuric acid) using a hotplate. After digestion, the sample volume was completed to a 100 mL by adding distilled water and filtrated using filter paper. Total macro- and micronutrients were determined using (Kjeldahl method for nitrogen, spectrophotometer for phosphorus and flame-photometer for potassium and calcium, and inductively coupled plasma–optical emission spectrometry (ICP-OES) for micro-nutrients) [40,41].

2.9. Leaf Gas Exchange Properties

Photosynthesis rate (Pr), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr) were determined by a portable photosynthesis system (LI-COR 6400, Lincoln, NE, USA) using a chamber head with a natural light window under ambient air temperature and relative humidity conditions. Two months post-transplanting, five plants were selected from each treatment for photosynthetic measurement, and the photosynthetic rates were measured for four different strawberry leaves per plant with a minimum of three repeated measurements for each leaf of the four expanded leaves. Measurements were performed from 8:00 to 10:00; the chamber relative humidity was 70%, leaf temperature at 24 °C, leaf-to-air vapor pressure deficit of 1.0 ± 0.1 kPa and light intensity at 400–600 μmol·m⁻²·s⁻¹ PAR.

2.10. Chlorophyll Traits

To estimate Chlorophyll a, b, and total Chlorophyll, three plants were randomly chosen from each treatment. Three fully expanded leaves were taken at 60 days post-transplanting. A fresh leaf sample weighing 5 milligrams was collected and put into tiny test tubes with 10 mL of acetone at a concentration of 100%. Samples were wrapped in aluminum foil and stored in a dark place for seven to ten days. The absorption was measured at wavelengths of 662 for Chlorophyll and 646 for Chlorophyll b using a spectrophotometer (UV-6705, Jenway Co., London, UK). Chlorophyll a, chlorophyll b, and total chlorophyll content were calculated as follows:

$$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} \mathrm{a}={\displaystyle \frac{\left[\right(11.75\times \mathrm{A} 662)-(2.35\times \mathrm{A} 645\left)\right]\times \mathrm{V}}{1000\times \mathrm{W}}}$$

$$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} \mathrm{b}={\displaystyle \frac{\left[\right(18.61\times \mathrm{A} 645)-(3.96\times \mathrm{A} 662\left)\right]\times \mathrm{V}}{100}}$$

$$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{C}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l}={\displaystyle \frac{\left[\right(20.2\times \mathrm{A} 645+(8.02\times \mathrm{A} 662)]\times \mathrm{V}}{1000\times \mathrm{W}}}$$

A: specific wavelength. V: extract’s volume (mL). W: fresh weight of leaves (g) [42].

2.11. Statistical Analysis

Plants were randomized in complete block design (RCBD) with three replications and two-factor experiment. Collected data were subjected to analysis of variance (ANOVA). Means were compared by Fisher’s least significance difference test (LSD) at p < 0.05 using SAS software package 9.2 for windows [43].

3. Results and Discussion

3.1. Vegetative Growth Traits

The results show that CO₂ at 600 ppm (enrichment) significantly improved the phenotyping growth traits of strawberry at 60 days post-transplanting (

Table 1. Effect of the two CO₂ concentrations, 400 ppm (ambient) and 600 ppm (enrichment), on vegetative growth traits of strawberry plants at 60 days post transplanting for the three F1 strawberry varieties, D: Delizzimo, E: Estavana and S: Soraya. Means, in the same column, marked by the same lower-case letter are not significantly different (p < 0.05).

CO2 (ppm)	Variety	Plant Height (cm)	No. of Crowns	Crown Diameter (mm)	Leaf Area (cm2)	No. of Leaves	Leaves FW (g)	Leaves DW (g)
400		30.6 ± 0.61 a	2.4 ± 0.21 b	19.7 ± 0.80 b	847 ± 89.19 b	31.6 ± 1.12 b	41.6 ± 3.27 b	8.8 ± 0.64 b
600		31.5 ± 0.78 a	3.3 ± 0.44 a	25.1 ± 1.25 a	1378 ± 199.13 a	53.6 ± 2.74 a	95.5 ± 9.92 a	13.3 ± 0.95 a
	D	29.7 ± 0.54 a	2.2 ± 0.18 c	19.8 ± 0.14 bc	816 ± 58.34 cd	29.2 ± 1.01 e	42.8 ± 1.80 d	9.1 ± 0.46 b
400	E	30.4 ± 1.24 a	2.9 ± 0.44 b	17.0 ± 0.00 c	1005 ± 261.29 c	34.7 ± 2.40 d	46.3 ± 9.82 d	9.3 ± 2.02 b
	S	31.6 ± 1.36 a	2.1 ± 0.29 c	22.3 ± 0.80 b	720 ± 53.61 d	30.6 ± 0.70 de	35.8 ± 0.57 d	8.1 ± 0.32 b
600	D	29.1 ± 0.52 a	2.9 ± 0.33 b	23.1 ± 0.60 b	2068 ± 144.53 a	61.2 ± 1.4742 a	90.2 ± 0.98 b	15.6 ± 1.28 a
	E	31.5 ± 1.03 a	3.5 ± 0.05 a	22.0 ± 0.80 a	745 ± 98.36 d	44.3 ± 2.3643 c	64.7 ± 2.02 c	10.0 ± 0.70 b
	S	33.9 0.56 a	3.2 ± 0.44 a	29.8 ± 0.54 a	1321 ± 70.70 b	55.7 ± 2.8591 b	131.9 ± 4.87 a	14.1 ± 0.95 a

). However, the three grown F1 varieties show slight significant differences. Under the two CO₂ concentrations, CO₂ at 600 ppm caused significant increases in terms of leaf area (153.4%), number of leaves (93.7%) and leaves DW per plant (71.4%) of variety D, while varieties E and S presented significant improvements regarding the number of crowns per plant (20.6%, 52.35) and crown diameter (29.4%, 33.65), respectively. In addition, FW leaves of the S variety recorded a superior value in comparison to other varieties grown under 600 ppm of CO₂. Similar results were obtained by [28], who indicated that CO₂ enrichment significantly promoted the leaf growth of strawberry plants. The authors of [44] found that the number of leaves, leaf area and dry matter content of watermelon plants were significantly increased at 800 ppm of CO₂. In bell paper, total biomass was increased by 35% when increasing the CO₂ concentration to 800 μmol·mol⁻¹ [24]. Likewise, plant height, stem diameter, number of leaves, root length, leaf width, and lettuce leaf area were positively affected by CO₂ enrichment, over a 30-day period, compared to ambient CO₂ [45]. Similarly, [12] mentioned that CO₂ enrichment at 700 ppm increased the leaf area of tomato plants by 42.4% compared to ambient CO₂ conditions. These significant responses in strawberry growth may be due to the stimulation of net photosynthesis on leaf area expansion under elevated CO₂ conditions [26,46]. This explanation is confirmed in

Table 2. Effect of the two CO₂ concentrations, 400 ppm (ambient) and 600 ppm (enrichment), on photosynthesis traits at 60 days post cultivation, for the three F1 strawberry varieties, D: Delizzimo, E: Estavana and S: Soraya. Pr: Photosynthetic rate, Gs: stomatal conductance, Ci: intercellular CO₂, Tr: Transpiration rate. Means, in the same column, marked by the same lower-case letter are not significantly different (p < 0.05).

CO2	Variety	Pr (μmol. m−2. S−1)	Gs (mmol H2O.m−1. s−1)	Ci (μmol CO2.mol−1)	Tr (mmol H2O.m−1. s−1)	Chlorophyll a	Chlorophyll b	Chlorophyll (a+b)	Chlorophyll (a/b)
(ppm)
400		7.45 ± 0.62 b	0.440 ± 0.03 a	343 ± 4.61 b	9.21 ± 0.31 a	3.01 ± 0.01 a	3.45 ± 0.16 a	6.46 ± 0.15 a	0.89 ± 0.05 a
600		17.11 ± 1.73 a	0.446 ± 0.02 a	493 ± 7.71 a	9.82 ± 0.32 a	2.96 ± 0.02 a	3.85 ± 0.22 a	6.81 ± 0.19 a	0.78 ± 0.05 a
400	D	9.74 ± 0.40 b	0.453 ± 0.09 a	329 ± 7.43 d	9.20 ± 0.93 b	3.07 ± 0.33 a	2.87 ± 0.04 b	5.94 ± 0.04 b	1.07 ± 0.02 a
	E	5.67 ± 0.16 c	0.443 ± 0.05 a	356 ± 2.16 c	9.82 ± 0.31 ab	2.99 ± 0.02 ab	3.59 ± a 0.21 b	6.59 ± 0.19 ab	0.84± 0.06 ab
	S	6.95 ± 0.36 c	0.426 ± 0.02 b	344 ± 2.29 cd	8.63 ± 0.20 b	2.97 ± 0.38 ab	3.89 ± 0.09 a	6.86 ± 0.8 a	0.76 ±0.20 b
600	D	17.74 ± 1.07 a	0.430 ± 0.37 b	475 ±5.74 b	9.83 ± 0.11 ab	2.95 ± 0.03 b	3.84 ± 0.35 ab	6.48 ± 0.29 ab	0.88 ± 0.08 ab
	E	17.21 ± 1.24 a	0.466 ± 0.01 a	482 ± 5.33 b	10.71 ± 0.33 a	2.91 ± 0.52 b	4.26 ± 0.42 a	7.17 ± 0.38 a	0.70 ± 0.09 b
	S	16.60 ± 0.90 a	0.443 ± 0.26 a	520 ± 7.81 a	8.92 ± 0.22 b	3.01 ± 0.04 ab	3.47 ± 0.33 ab	6.80 ± 0.32 ab	0.78 ± 0.07 b

, which shows the photosynthetic rate was raised by 129.7% with CO₂ at 600 ppm rather than 400 ppm. A similar conclusion was reported by [47], who explained that the positive effect of CO₂ enrichment is linked to increased photosynthesis in annual plants. However, many reports pointed out that CO₂ enrichment promotes the growth of C3 plants by raising photosynthesis [48,49]. Despite the obvious effect of the CO₂ enrichment (600 ppm) on the three grown strawberry varieties, the interaction shows a clear cultivar-dependent characteristic. Thus, understanding the response of each cultivar to CO₂ enrichment is important to support selecting suitable greenhouse strawberry varieties towards improving crop yield [50]. However, non-consistent patterns of the used three hybrids according to growth traits might be related to the time of sampling, 60 days, which is a powerful growth period that minimizes the difference among the cultivars. Also, under the constant growth conditions in the highly controlled greenhouse, the cultivars may react similarly to light, temperature and the nutrient status of soil, as well as available metabolites [51] and their allocation to the aboveground plant parts, which might restrict CO₂, as a single factor, to strongly affect the growth of the cultivars [52].

3.2. Photosynthesis and Chlorophyll Traits

Mostly, variations in photosynthetic induction are obvious between the crops, and they seem minor among cultivars of the same crop. Ref. [53] found that time until reaching 20–90% of full net photosynthesis rate induction varied by 40–60% through genotypes, while the time needed for photosynthetic induction is a limiting actor and may increase the yields of crops [54]. In addition, increasing the CO₂ level cannot always improve photosynthesis. Therefore, selecting the appropriate CO₂ supplement based on the photosynthesis characteristics is crucial [55]. To verify the physiological response of the three F1 hybrid strawberries to CO₂ enrichment under the greenhouse condition, leaf gas exchange measurements and chlorophyll content were detected (Table 2). The photosynthetic rate (Pr) and intercellular CO₂ (Ci) were significantly higher (129.7%, 43.7%), respectively, in leaves of strawberry exposed to 600 ppm than 400 ppm of CO₂. A comparable pattern was observed when plants were grown under both CO₂ concentrations for the three grown strawberry varieties. Particularly, 600 ppm of CO₂ increased the values of the photosynthetic rate (Pr) for varieties D, E and S, while the height values of stomatal conductance (Gs) and intercellular CO₂ concentration (Ci) in strawberry leaves were recorded with variety S at 600 ppm of CO₂. Ref. [56] showed that elevated CO₂ increased the net photosynthesis rate of Albion and San Andreas strawberry cultivars at 650 µmol mol⁻¹ of CO₂ with no increase at 900 µmol mol⁻¹. Moreover, there was no significant difference in chlorophyll components between the two CO₂ concentrations. Moreover, varieties S and E responded significantly to 400 and 600 ppm of CO₂, respectively, in terms of transpiration rate (Tr), chlorophyll b and chlorophyll a+b. Ref. [57] confirmed that CO₂ enrichment usually resulted in increased chlorophyll content. Regardless of the significant increase in (Pr) at 600 ppm for the three varieties, no clear pattern under both CO₂ concentrations was observed in terms of chlorophyll a and a/b. A similar result was reported by [50], who found a significant increase in photosynthetic rate and intercellular CO₂ concentration in leaves of watermelon, while transpiration rate, stomatal conductance and chlorophyll content did not differ at 800 ppm of CO₂ enrichment, if compared to the ambient CO₂. In tomato, net photosynthesis was increased significantly with increasing CO₂ concentration at all light intensities [58]. For cucumber, short-term CO₂ enrichment significantly increased the net photosynthetic rate, while long-term CO₂ enrichment led to photosynthetic acclimation and a decrease in stomatal conductance [59]. The net photosynthetic rate of young lettuce plants was much higher with 700 ppm of CO₂ enrichment than at the ambient concentration [60]. Likewise, with strawberry, photosynthesis under elevated CO₂ was decreased above 600 µmol.mol⁻¹ [61]. However, quite a few studies have revealed that elevated CO₂ highly stimulates the carboxylation rate in the Calvin cycle [62] and enhances leaf photosynthesis under greenhouse conditions [24], indicating that CO₂ enrichment affected the plant growth of strawberry. Ref. [63] pointed out that elevated CO₂ improves the efficiency of Rubisco CO₂ assimilation over the alternate RuBP oxygenation, which enhanced plant growth and yield.

3.3. Fruit Yield

With the increase in CO₂ concentration, the number of strawberry fruits per plant and total fruit yield resulted in a significant increase for plants treated with 600 ppm of CO₂ compared to ambient up to 27.5% and 42.2%, respectively, (

Table 3. Effect of the two CO₂ levels at 400 ppm (ambient) and 600 ppm (enrichment) on yield and its components for the three F1-strawberry cultivars, D: Delizzimo, E: Estavana and S: Soraya. Means, in the same column, marked by the same lower-case letter are not significantly different (p < 0.05).

CO2	Variety	Fruit Length (mm)	Fruit Diameter (mm)	Fruit FW (g)	No. Fruits (Plant)	Yield
(ppm)						(g/Plant)
400		37.1 ± 2.86 a	26.3 ± 1.79 a	19.2 ± 0.73 a	34.9 ± 2.15 b	678 ± 52.83 b
600		37.5 ± 2.51 a	26.9 ± 1.90 a	21.1 ± 1.53 a	44.5 ± 2.55 a	964 ± 114.34 a
400	D	41.1 ± 1.95 a	29.8 ± 0.71 ab	20.0 ± 0.68 bc	37.6 ± 0.88 b	750 ± 10.17 d
	E	26.3 ± 1.99 b	19.4 ± 0.98 c	16.8 ± 0.11 cd	28.0 ± 3.79 c	471 ± 5.25 f
	S	44.0 ± 0.60 a	30.1 ± 0.53 b	20.7 ± 1.23 b	39.3 ± 1.58 b	813 ±11.84 c
600	D	39.9 ± 1.91 a	29.3 ± 0.72 b	22.8 ± 0.67 b	46.8 ± 1.83 a	1067 ± 24.86 b
	E	29.4 ± 4.18 b	19.6 ± 0.83 c	15.2 ± 0.63 d	34.8 ± 2.09 b	528 ± 4.04 e
	S	43.3 ± 1.29 a	31.9 ± 0.74 a	25.0 ± 1.96 a	51.9 ± 0.79 a	1297 ± 27.13 a

). Clearly, the application of 600 ppm of CO₂ increased fruit number, length and diameter for D and S varieties. Furthermore, total yield production increased by 30% and 37.3% for the same varieties, respectively. In terms of variety E, 600 ppm of CO₂ did not significantly reflect the fruit size or diameter. Our results are in harmony with [26], who cited that the number of fruit, average fruit weight and total yield of strawberry were significantly higher by 20% and 31% with 800 μmol·mol⁻¹ of CO₂ in comparison to 400 μmol·mol⁻¹. In addition, [27] found that strawberry fruit yield and average fruit number were significantly increased by 28.3% and 35.7%, respectively, with CO₂ enrichment compared to control, while no significant difference in terms of average fruit weight was observed. The authors of [12] indicated that 700 ppm of CO₂ exhibited a significantly higher number of tomato fruit, fruit weight and better yield compared to plants grown under ambient CO₂ conditions. The authors of [64] proved that elevated CO₂ has a direct effect on mulberry growth, development, nutritional status and yield. Previous reports mentioned that elevated CO₂ increased tomato yield from 7% to 125% [65], strawberry total fruit yield from 9.9% to 33.4% [13], and 17.6–38.5% increase in single strawberry fruit weight [65], compared with ambient CO₂ conditions. These findings might be attributed to the significant increase in growth traits and photosynthetic apparatus as well as the high carbohydrate accumulation in fruit during the fruit development stage, which in turn resulted in maximum total yield [66]. In harmony, [28] reported that CO₂ enrichment results in an increase in vegetable productivity inside the greenhouse up to 32%. Obviously, variety E showed constant lower yield and a smaller number of fruits either under ambient or enrichment CO_2, indicating that the genotype of D and S hybrids is more superior once the agroclimatic conditions are equal. A related conclusion was reported by [67], who explained that the difference between cultivars depends upon the agroclimatic conditions, and the best cultivars can only be selected by evaluating for yield under different climatic conditions. In this respect, it could be concluded that the variations in yield might be attributed to the production potential of the experimented hybrids and the differences with respect to climatic requirements [68].

3.4. Fruit Quality

With respect to the effect of CO₂ enrichment, the present findings revealed no significant change in total acidity (TA) either between varieties or the CO₂ concentrations (Figure 3,

Table 4. Assessment of strawberry fruit quality, 90 days post cultivation, as affected by the three F1 strawberry varieties, D: Delizzimo, E: Estavana, S: Soraya, and the two CO₂ concentrations 400 ppm (ambient) and 600 ppm (enrichment). TSS: Total soluble solids. Means, in the same column, marked by the same lower-case letter are not significantly different (p < 0.05).

CO2 (ppm)	Variety	TSS (Brix %)	Vitamin C (mg/100 g FW Fruit)	Total Acidity (gm/100 mL Citric Acid)
400	D	9.533 ± 0.08 ab	66.67 ± 1.09 d	0.7067 ± 0.65 a
	E	8.733 ± 0.27 b	82.93 ± 2.53 c	0.71 ± 0.06 a
	S	10.2 ± 0.36 a	80.27 ± 1.18 c	0.6233 ± 0.09 a
600	D	10.167 ± 0.52 a	82.4 ± 1.28 c	0.6267 ± 0.03 a
	E	10.067 ± 0.13 a	102.8 ± 1.66 a	0.6333 ± 0.02 a
	S	10.3 ± 0.10 a	94 ± 1.38 b	0.6733 ± 0.02 a

). However, the results show that elevated CO₂ at 600 ppm promotes total soluble solids accumulation and vitamin C in fruits. This effect could be explained based on the fact that CO₂ enrichment improves the synthesis of glyceraldehyde 3-phosphat in leaves into glucose, fructose, and sucrose [69], which, in turn, increases carbohydrate content in plant organs [70]. In addition, the enhanced photosynthetic rate under CO₂ enrichment (Table 2) might be connected to the enhancements in sucrose content [71]. This explanation was investigated by [24], who found that doubling the CO₂ concentration from 400 to 800 μmol·mol⁻¹ significantly enhanced the content of sucrose, fructose, and soluble sugars in leaves of bell pepper. Related reports confirmed that CO₂ enrichment enhanced soluble sugars in radish, turnip and carrot [16] and increased total soluble sugar in strawberry by 20% in comparison to ambient conditions [72]. The authors of [65] stated that elevated CO₂ results in a 7–35% increase in major sugars combined with a 10.2% increase in vitamin C in strawberry fruit. In our study, the interaction between the two CO₂ concentrations and the three grown strawberry F1 varieties did not differ in terms of brix content at 600 ppm of CO₂. Our results show that vitamin C is improved for all varieties for plants treated at 600 ppm compared to 400 ppm of CO₂, while some reports indicate that CO₂ enrichment reduced vitamin C content in barely, tomato and potato, with a nonsignificant increase in tomato fruit acidity [73,74,75]. These results suggest that CO₂ enrichment could contribute to increasing crop nutritional value; otherwise, species do not have comparable responses to CO₂, enrichment, even though the yield increases [76].

3.5. Macronutrient Content

In terms of CO₂ enrichment’s influence on mineral content in plant tissue, 600 ppm of CO₂ enrichment significantly decreased nitrogen, phosphorus, potassium, and magnesium accumulation in the leaves of exposed plants in comparison to 400 ppm of CO₂, except for calcium (

Table 5. Nutrient content in strawberry leaves, 60 days post-cultivation, for the three F1-strawberry cultivars, D: Delizzimo, E: Estavana and S: Soraya, as affected by the two CO₂ concentrations 400 ppm (ambient) and 600 ppm (enrichment). Means, in the same column, marked by the same lower-case letter are not significantly different (p < 0.05).

CO2 (ppm)	Variety	N %	P %	K %	Ca %	Mg %
400		2.79 ± 0.03 a	0.98 ± 0.10 a	0.60 ± 0.02 a	1.94 ± 0.21 b	0.55 ± 0.03 a
600		2.37 ± 0.02 a	0.80 ± 0.06 a	0.47 ± 0.03 a	2.55 ± 0.23 a	0.42 ± 0.02 a
400	D	2.81 ± 0.05 a	0.92 ± 0.03 ab	0.54± 0.04 b	1.46 0.04 c	0.51 ± 0.02 b
	E	2.75 ± 0.01 a	1.03 ± 0.39 a	0.62 ± 0.02 a	2.36 ± 0.52 b	0.64 ± 0.04 a
	S	2.83 ± 0.04 a	1.01± 0.03 a	0.66 ± 0.04 a	2.00 ± 0.09 b	0.50 ± 0.04 b
600	D	2.39 ± 0.02 a	0.65 ± 0.03 c	0.39 ± 0.02 c	1.86 ± 0.34 bc	0.37 ± 0.04 c
	E	2.36 ± 0.10 b	0.86 ± 0.02 b	0.48 ± 0.06 ab	2.94 ± 0.03 a	0.48 ± 0.06 ab
	S	2.38 ± 0.85 b	0.91 ± 0.16 ab	0.54 ± 0.04 b	2.83 ± 0.07 ab	0.43 ± 0.03 b

). In respect to the effect of CO₂ enrichment on the three grown varieties, the determined nutrients revealed significant increases with varieties E and S, especially, while lower content was found in the leaves of variety D treated with 600 ppm CO₂ treatment. Thus, it could be indicated that CO₂ enrichment caused a reduction in the concentration of minerals in strawberry leaves, while the variation among varieties might be attributed to the genotype [77]. Several reports documented that elevated CO₂ is expected to reduce concentrations of nutrients because of various factors [78]. For instance, elevated CO₂ could reduce element concentrations, even if the nutrient uptake is enhanced, but dry matter accumulation outpaces uptake. Likewise, elevated CO₂ consistently lowers transpiration and increases water use efficiency and, therefore, may reduce the nutrient uptake for element transfer from soil to plant roots, which is dependent on mass flow [79,80]. Also, [81] showed that elevated CO₂ reduced nitrogen content due to nitrogen dilution from increased carbohydrate concentrations, lower nutrient and water uptake from the soil and a reduction in stomatal conductance [82]. Moreover, phosphorus accumulation showed several patterns of either decreased, increased or no-change status [83]. On the other hand, [84] found that potassium and magnesium significantly increased, though phosphorus decreased in asparagus grown under elevated CO₂ compared to ambient conditions. Previously, [85] found no effect of elevated CO₂ on potassium and magnesium content in tomatoes, despite a nitrogen decrease and calcium increase in rice, as well as potassium decrease in wheat [86]. Therefore, it could be proposed that, under CO₂ enrichment, plants might change their nutrient allocation patterns, and nutrient elements might behave differently depending on their chemical properties [87]. Thus, differential responses of N, P and K content because of elevated CO₂ might be observed [88]. In conclusion, the response of grown plants under elevated CO₂ on mineral uptake and utilization are contradictory and diverse among crop species and experimental capabilities [89].

4. Conclusions

The world of agriculture is witnessing the emergence of new techniques and innovations aimed at improving crop production efficiency and sustainability. One of these innovations is using F1 hybrid strawberry varieties as an alternative to traditional runner-propagated cultivation. This method offers several advantages, including a shorter production period and increased production. CO₂ enrichment is considered a sustainable tool to increase production in greenhouses. A significant improvement in the growth traits of the strawberry plants was found when plants were exposed to CO₂ enrichment at 600 ppm. The results show that CO₂ enrichment significantly enhances the yield and quality of strawberries, which has important relevance for greenhouses in arid areas and can provide feasible technical solutions for local agricultural production. The varieties’ morpho-physiological response to increased CO₂ levels demonstrated notable enhancements in photosynthetic rate and intercellular CO₂ concentrations in leaves. This, in turn, led to a substantial increase in the number of fruits per plant and total fruit yield. In addition, the elevated CO₂ levels promoted the accumulation of total soluble solids and vitamin C in the fruit, while the levels of nitrogen, phosphorus, potassium, and magnesium in the leaves of the plants exposed to higher CO₂ concentrations decreased. These findings emphasize the complex interplay between CO₂ levels and nutritional content in plants. However, it is crucial to tailor the CO₂ enrichment strategy based on the specific requirements of each strawberry variety to maximize the yield and quality. In conclusion, this investigation indicates new possibilities for enhancing strawberry cultivation practices in arid zones and highlights the potential of innovative techniques to revolutionize sustainable agricultural production.