Effect of Sweet Pepper ( Capsicum annuum L.) Seedling Age and Cultivation Method on Seedling Quality, Photosynthetic Parameters and Productivity

: The age of seedlings affects not only the quality of the seedlings but also the yield. The age of seedlings of sweet peppers grown in a greenhouse and their cultivation method has been little studied. Therefore, the aim of this research was to determine the effect of agrotechnological tools (direct seeding or transplanting) on sweet pepper ( Capsicum annuum L.) seedling quality and crop yield. The research was carried out in a greenhouse covered with double polymeric ﬁlm at the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry. Two factors were investigated: seedling establishment method (direct seeding and transplanting) and their different age (60, 50, and 40 days). The 60-day-old seedlings (both sown directly and grown by transplanting) were taller, and had more leaves, a larger leaf area, and a thicker stem, than the 50-and 40-day-old seedlings. The highest content of dry matter was found in the leaves of 60-day-old seedlings that were grown by transplanting. The chlorophyll index was also highest in the leaves of these seedlings. The highest photosynthetic parameters (photosynthetic rate, stomatal conductance, intercellular CO 2 , transpiration rate) were found in the leaves of 40-day-old seedlings (both sown directly and grown by transplanting). Higher yields were demonstrated in sweet peppers whose seedlings were planted in the greenhouse at the age of 60 days and which were sown directly in cups.


Introduction
The age of vegetable seedlings, which is one indicator of the quality of the seedlings, is an essential factor in obtaining the highest yield. The age of the seedlings affects plant growth and yield [1]. The duration of seedling growth determines the degree of plant development, mass accumulation, the number of high-quality seedlings, adaptation, and resistance to unfavorable conditions [2,3]. The optimum seedling age depends on the type of vegetable, environmental factors (temperature, moisture), the time and method of cultivation, etc.
Sweet peppers are one of the most vitamin-rich vegetables, and leaders in provitamin A (carotene) and vitamin C content [4,5]. They are also rich in vitamins P, B 1 , and B 2 , and contain a lot of potassium, sodium, iron, phosphorus, and magnesium salts [6]. In Lithuania, sweet peppers are primarily grown in soil in greenhouses covered with stabilized polyethene film. Most growers grow this plant for their own needs [7]. Sweet pepper seedlings have a relatively long growing time and, depending on the growing conditions, variety, and hybrid, grow in 40-60 days. A few studies have been conducted to determine peppers' optimal age and their effect on plant productivity when growing them in the field. According to the data of various investigations, different optimum ages of pepper seedlings are indicated. Weston stated [8] that sixty-day-old "Yolo Wonder L" pepper seedlings transplanted into the field had greater early yield than younger ones. Montano-Mata and Nunez [9] observed that the older sweet pepper seedlings produced the maximum possible yield. Ibrahim et al. [10] stated that sweet plants whose seedlings were planted at 8 to 10 weeks of age produced higher fresh fruit yields per hectare than those transplanted at 12 and 14 weeks. Saxena and Singh [11] indicated that 33-36-day-old seedlings were the best for pepper growth and yield in the Uttarakhand Dehradun region. Conflicting results about the age of pepper seedlings can vary depending on the pepper species, the environment, and growing conditions, and where the plants are grown [1].
The successful establishment of seedlings is an essential first step in crop production and determines the success of future harvests. There are different opinions about the cultivation of seedlings. Transplanting is very often used in vegetable cultivation. However, some species (for example, maize) can hardly tolerate transplanting, and root regeneration is slower after transplanting [12]. Seedlings established by transplanting are more uniform, can tolerate or escape early environmental/biological stresses, and can achieve maturity earlier than direct-seeded plants [13]. Lescovar and Cantliffe [14] indicated that directseeded plants maintained a more balanced distribution of root, stem, leaf, and dry matter than those grown by transplanting, but other studies showed significant improvement in artichoke roots and shoots and yield compared to direct-seeded plants [15]. Most researchers have investigated the influence of pepper seedlings' age on the seedlings' biometric parameters and yield. Not much data has been found on the effect of seedling age on fruit quality. Saxena and Singh [11] indicated that the age of pepper seedlings did not affect the total soluble solids in the fruits. The internal quality of the fruit varies according to the variety. Sweet peppers develop more slowly compared to other vegetable seedlings. It is essential to estimate not only the optimum age of the seedlings, but also the cultivation methods used to grow the seedlings to obtain an earlier harvest of sweet peppers. A large amount of research has been carried out to determine the age of different types of pepper seedlings grown in the field, but there are few data on the optimum age of sweet peppers grown in the greenhouse or on whether they should be transplanted or sown directly. Therefore, the objective of this work was to evaluate the influence of various seedling age and establishment methods on the quality of sweet pepper seedlings and their physiological processes, and to determine the influence of these agrotechnological tools on plant yield in greenhouses.

Growing Conditions
The investigations were carried out at the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry (55 • 60 N, 23 • 48 E), Babtai, Lithuania. Sweet peppers (Capsicum annuum L.) variety "Reda" (Lithuania) were grown in a greenhouse covered with a double polymer film in 2020-2021. Two factors were investigated: factor Aseedling cultivation method (a 0 -sown directly, a 1 -transplanting) and factor B-seedling age (b 0 -60 days, b 1 -50 days, b 2 -40 days). The sweet peppers were sown at the beginning of March. One part of the seeds was sown directly into pots 12 cm in diameter on peat substrate (N 100-120, P 2 O 5 30-80, K 2 O 120-200 mg L −1 ; microelements Fe, Mn, Cu, S, Mo, Zn; pH 5.5-6.5) (Profi 1, Durpeta, Lithuania), and the other part of the seeds was sown in rows in a box, and after 10 days from germination, these seedlings were transplanted into pots. The pots were arranged on racks. The nursery was heated. The plants were watered when necessary. During seedling cultivation, the day/night temperature was 20-25/16-18 • C, and the relative air humidity was 50-60%. The seedlings were transplanted in the greenhouse and cultivated in soil ( Figure 1A,B) at the end of May. The scheme for planting sweet pepper seedlings in a greenhouse was 70 × 40 cm (70 cm between rows, 40 cm between plants). Soil conditions of the experiment were as follows: clay loam, pH 6.3, EC-1.7 mS/cm, humus 2.2%, nitrogen-60 mg L −1 , phosphorus-25 mg L −1 , potassium-167 mg L −1 , calcium-147 mg L −1 , magnesium-29 mg L −1 . The soil had been fertilized before planting with "Nutricomplex" (NPK-14 11 25 + 2MgO) (Tradecorp, Belgium). The sweet peppers in the greenhouse were fertilized every three weeks with "Nutrifol "(brown NPK-14 9 25 and green NPK 8 11 35) (Yara Poland Sp. Zo. o.). The end of the sweet pepper experiment was at the beginning of October. During the plant's growing season, the day/night temperature was 19-26/15-19 • C, and the relative air humidity was 60-80%. The plot area was 4.8 m 2 . Three replications were conducted in a randomized block design ( Figure 2).  Figure 2).  The experimental scheme: A factor: a0-sown directly, a1-transplanting, B factor: seedling age-b0-60 days, b1-50 days, b2-40 days. 1st replication; 2nd replication; 3rd replication.

Biometric Measurements
At the end of the seedling growth, biometrical measurement was conducted. The seedling height and the leaf area of the seedlings were measured. The leaf area was measured using a "WinDias" leaf area meter (Delta-T Devices Ltd., Cambridge, UK). These measurements were performed in ten replicates (n = 10).   Figure 2).  The experimental scheme: A factor: a0-sown directly, a1-transplanting, B factor: seedling age-b0-60 days, b1-50 days, b2-40 days. 1st replication; 2nd replication; 3rd replication.

Biometric Measurements
At the end of the seedling growth, biometrical measurement was conducted. The seedling height and the leaf area of the seedlings were measured. The leaf area was measured using a "WinDias" leaf area meter (Delta-T Devices Ltd., Cambridge, UK). These measurements were performed in ten replicates (n = 10).

Biometric Measurements
At the end of the seedling growth, biometrical measurement was conducted. The seedling height and the leaf area of the seedlings were measured. The leaf area was measured using a "WinDias" leaf area meter (Delta-T Devices Ltd., Cambridge, UK). These measurements were performed in ten replicates (n = 10).

Determination of Dry Matter
The dry matter content in the leaves of the seedlings was determined. The leaves of the seedlings were dried in a drying oven (Venticell, MBT, 2 Czech Republic) at 105 • C for 24 h. The measurements were performed in four replicates (n = 4).

Nondestructive Measurements
Leaf chlorophyll (CHL) index was measured using the Dualex 4 Scientific ® (FORCE-A, Orsay, France) meter. These measurements were performed in ten replicates (n = 10) in each variant.

Yielding of Plants
The sweet pepper yield was recorded at every harvest. Sweet pepper fruits were harvested once a week. Next, they were separated into marketable and non-marketable (small, irregularly shaped, damaged by disease, etc.) fruits. The total yield was calculated by aggregating each harvest.

Determination of Organic Acids and Carotenes (α and β) by High-Performance Liquid Chromatography (HPLC)
Organic acids (malic, citric, and ascorbic) contents were determined using the HPLC method [17] on a Shimadzu 10A (Japan) system with a diode-array detector (DAD). The sample was prepared by grinding plant material and diluting it with H 2 O 1:10 (w:v). Extraction was performed in a heated water bath (50 • C) for 30 min. The extract was clarified by centrifugation at 10,000× g rpm for 15 min and filtered through a 0.22 µm PTFE syringe filter (VWR International, Radnor, PA, USA). Separation was performed on a Lichrosorb RP-184.6 × 250 mm, 5 µm column (Altech). Mobile phase-0.05 M sulfuric acid, flow rate 0.5 mL min −1 , injection volume −10 µL. The calibration method (R 2 < 0.99) was used for each organic acid quantification (mg g −1 in FW).
Carotenes were evaluated using the HPLC method with diode array detection (DAD) on a Shimadzu 10A (Shimadzu, Kyoto, Japan). About 0.5 g of fresh plant tissue was ground and diluted with 80% glacial acetone. The extraction was carried out for 24 h at +4 • C temperature. The centrifugate was extracted at 10,000× g rpm for 15 min. Then, the solution was filtered over a 0.22 µm PTPE syringe filter (VWR International, Radnor, PA, USA). The sample separation was performed on Chromegabond C30 3 µ 120 Å, 15 cm × 2.1 mm column (ES Industries, West Berlin, NJ, USA). A 10 µm sample was injected; the column oven temperature was set at +20 • C. The pigments were eluted for 30 min with gradient solvent systems A (Methanol:water, 1:4) and B (Ethyl acetate) at a flow rate of 0.2 mL min −1 . Initial conditions were 20% B for 2.5 min, followed by a linear gradient to 30% B at 5 min, holding 30% B for 5 min, then elevated until 80% B for 2.5 min, until 87% B for 7.5 min, and until 100% for 5 min, and again 20% B until the end of the run. The calibration method was used for carotene quantification (mg g −1 in FW).

Statistical Analysis
Microsoft Excel 2016 and Addinsoft XLSTAT 2022.1 XLSTAT statistical and data analysis (Long Island, NY, USA) were used for the data statistical analysis. The data are presented through three replicates (n = 3) linked to the sampling points. Two-way analyses of variance (ANOVA), followed by Tukey's significant difference test (p < 0.05) for multiple comparisons, were used to evaluate differences between means of measurement.
LSD-Fisher's protected least, where * p < 0.05 shows significant differences, and nsno significant differences between factors. Multivariate principal component analysis (PCA) was performed to determine the statistical relations between the cultivation method and seedling age in the experiment according to organic acids, carotenes, yield, photosynthesis systems' response, and biometrical parameters.

Results
The biometric parameters for sweet pepper seedlings are presented in Table 1. The 60-day-old seedlings (both sown directly and grown by transplanting) were 1.1-1.7 times higher and had more leaves, a larger leaf area, and a thicker stem than the 50-day and 40-day-old seedlings (Table 1). Directly sown pepper seedlings of various ages were slightly shorter (except for 40-day-old seedlings) than seedlings grown by transplanting, but their stem was thicker, they had more leaves, and the leaf area was more significant. The 40-dayold seedlings (both sown directly and grown by transplanting) were the shortest, and their leaf area was the smallest compared to the older seedlings. Sweet peppers whose seedlings were planted at the age of 60 days and were sown directly began to flower 9-6 days earlier than peppers whose seedlings were planted at 50 and 40 days ( Table 2). These plants also started fruiting 7-10 days earlier. Sweet peppers whose seedlings were planted at the age of 40 days started flowering and fruiting last. The method of growing seedlings did not affect the early flowering and fruiting of pepper plants planted at the age of 50 and 40 days. Both plants started flowering and fruiting at the same time. Sweet pepper seedlings sown directly accumulated 2.5-10.3% more dry matter in the leaves than transplanted seedlings ( Figure 3A). Both the 60-day-old seedlings sown directly and transplanted accumulated more dry matter in the leaves than in the leaves of 50-day-old and 40-day-old seedlings. The 40-day-old seedlings (both sown directly and grown by transplanting) have accumulated minor dry matter content in the leaves.
Sweet pepper seedlings sown directly accumulated 2.5-10.3% more dry matter in the leaves than transplanted seedlings ( Figure 3A). Both the 60-day-old seedlings sown directly and transplanted accumulated more dry matter in the leaves than in the leaves of 50-day-old and 40-day-old seedlings. The 40-day-old seedlings (both sown directly and grown by transplanting) have accumulated minor dry matter content in the leaves.
The age of the seedlings did not affect the chlorophyll index of their leaves, and the chlorophyll index of the leaves of seedlings of all ages was similar ( Figure 3B). The seedlings' cultivation method had a negligible effect on the chlorophyll index in seedling leaves. As the age of the seedlings increased, the chlorophyll index in the leaves of transplanted seedlings increased. The highest leaf chlorophyll index was found in the 60-dayold seedlings that were transplanted. The chlorophyll index of the leaves of these seedlings was 2.3% higher than that of the leaves of seedlings sown directly at the same age.  The age of the seedlings did not affect the chlorophyll index of their leaves, and the chlorophyll index of the leaves of seedlings of all ages was similar ( Figure 3B). The seedlings' cultivation method had a negligible effect on the chlorophyll index in seedling leaves. As the age of the seedlings increased, the chlorophyll index in the leaves of transplanted seedlings increased. The highest leaf chlorophyll index was found in the 60-day-old seedlings that were transplanted. The chlorophyll index of the leaves of these seedlings was 2.3% higher than that of the leaves of seedlings sown directly at the same age.
The above-ground and root fresh masses of direct-sown seedlings were higher (except for the above-ground mass of direct-sown seedlings at 60 days old) compared with the above-ground and root masses of the transplanted seedlings. The above-ground mass of the 60-day-old transplanted seedlings was 1.1 times higher than of the direct-sown seedlings at the same age. Fresh above-ground and root masses of 40-day-old seedlings were the lowest ( Figure 4A,B). Both the seedlings' age and the sweet pepper seedlings' cultivation method affected the plants' fresh mass. As the age of the seedlings increased, the above-ground and root masses were higher.
The highest photosynthetic rate, intercellular CO 2 , and transpiration rate were recorded in the leaves of 40-day-old seedlings (both sown directly and grown by transplanting) ( Table 3). The photosynthetic parameters of 60-and 50-day-old seedlings (both sown directly and grown by transplanting) were lower compared to those of 40-day-old seedlings.
Not only the seedlings' age, but also the cultivation method, influenced the photosynthetic parameters in the leaves of the seedlings. The photosynthetic rate was 4.1% higher, the intercellular CO 2 was 1.3% higher, and the transpiration rate was 1.2 times higher in the leaves of the 40-day-old directly sown seedlings than in the leaves of transplanted seedlings at the same age.
Agronomy 2023, 13, x FOR PEER REVIEW 7 of 13 The above-ground and root fresh masses of direct-sown seedlings were higher (except for the above-ground mass of direct-sown seedlings at 60 days old) compared with the above-ground and root masses of the transplanted seedlings. The above-ground mass of the 60-day-old transplanted seedlings was 1.1 times higher than of the direct-sown seedlings at the same age. Fresh above-ground and root masses of 40-day-old seedlings were the lowest ( Figure 4A,B). Both the seedlings' age and the sweet pepper seedlings' cultivation method affected the plants' fresh mass. As the age of the seedlings increased, the above-ground and root masses were higher. The highest photosynthetic rate, intercellular CO2, and transpiration rate were recorded in the leaves of 40-day-old seedlings (both sown directly and grown by transplanting) ( Table 3). The photosynthetic parameters of 60-and 50-day-old seedlings (both sown directly and grown by transplanting) were lower compared to those of 40-day-old seedlings. Not only the seedlings' age, but also the cultivation method, influenced the photosynthetic parameters in the leaves of the seedlings. The photosynthetic rate was 4.1% higher, the intercellular CO2 was 1.3% higher, and the transpiration rate was 1.2 times higher in the leaves of the 40-day-old directly sown seedlings than in the leaves of transplanted seedlings at the same age. The age of the seedlings had almost no effect on the biochemical characteristics of the fruit (Table 4). Sweet peppers planted at 40 days of age, sown directly and transplanted, had a slightly higher content of ascorbic and malic acids in fruits (Table 4). Sweet peppers whose seedlings (seedlings of all ages) were transplanted had 1.3-5.2 times more malic acid in the fruit than in the fruit of plants whose seedlings were sown directly.
The yield data for sweet peppers are presented in Figure 5. Both the seedlings' age and the cultivation method influenced the yield of sweet peppers. As the age of the seedlings increased, the total yield increased. The highest yields were obtained from plants whose seedlings were grown within 60 days (both sown directly and grown by transplanting). Sweet peppers whose seedlings were planted at 40 days of age (both sown directly and grown by transplanting) had the lowest yield. The total and the marketable yield of peppers whose seedlings were grown by direct sowing were 1.1-2.5 times higher than those grown by transplanting. The cultivation method did not significantly affect the yield of peppers whose seedlings were planted at 40 and 50 days of age. The method of seedling cultivation had the most significant influence on 60-day-old seedlings. The total yield of these seedlings, which were sown directly, was 14.6% higher, and the marketable yield was 26.5% higher compared to that of the same age seedlings grown by transplanting.  All values in the table are expressed as mean, standard error (x ± SD, n = 3). According to Tukey's significant difference test, means with different letters are significantly different at the p < 0.05 level. LSD-Fisher's protected least: * p < 0.05 shows significant differences, ns-no significant differences between factors.
The principal component analysis (PCA) shows that the cultivation method and the seedling's age have a differential impact ( Figure 6A). The PCA scatterplot divides the data into two groups, depending on the factor, according to the cultivation method and the age of the seedlings. Factor F1 is partitioned into two groups, according to seedling age: one group is 40-day-old seedlings, and the second group is 50-and 60-day-old seedlings. There are no significant differences between 50-and 60-day-old seedlings. Meanwhile, factor F2 divided data into groups depending on the cultivation method into transplanted and direct seedings. According to factor loadings ( Figure 6B), 40-day-old sweet pepper seedlings differ from 50-and 60-day-old seedlings in terms of most of the measured indicators: height, number of leaves, stem diameter, leaf area, dry and green mass, photosynthetic indicators, and ascorbic acid. Meanwhile, transplanted and directly seeded seedlings differed in carotene and citric acid content ( Figure 6B). and grown by transplanting) had the lowest yield. The total and the marketable yield of peppers whose seedlings were grown by direct sowing were 1.1-2.5 times higher than those grown by transplanting. The cultivation method did not significantly affect the yield of peppers whose seedlings were planted at 40 and 50 days of age. The method of seedling cultivation had the most significant influence on 60-day-old seedlings. The total yield of these seedlings, which were sown directly, was 14.6% higher, and the marketable yield was 26.5% higher compared to that of the same age seedlings grown by transplanting. Figure 5. The effect of transplant growth stage and cultivation method of sweet pepper seedlings on the total (A) and marketable (B) yield. According to Tukey's significant difference test, means with different letters are significantly different at the p < 0.05 level. Error bars show standard deviation. LSD -Fisher's protected least: * p < 0.05 shows significant differences, ns-no significant differences between factors.
The principal component analysis (PCA) shows that the cultivation method and the seedling's age have a differential impact ( Figure 6A). The PCA scatterplot divides the data into two groups, depending on the factor, according to the cultivation method and the age of the seedlings. Factor F1 is partitioned into two groups, according to seedling age: one group is 40-day-old seedlings, and the second group is 50-and 60-day-old seedlings. There are no significant differences between 50-and 60-day-old seedlings. Meanwhile, factor F2 divided data into groups depending on the cultivation method into transplanted and direct seedings. According to factor loadings ( Figure 6B), 40-day-old sweet pepper seedlings differ from 50-and 60-day-old seedlings in terms of most of the measured indicators: height, number of leaves, stem diameter, leaf area, dry and green mass, photosynthetic indicators, and ascorbic acid. Meanwhile, transplanted and directly seeded seedlings differed in carotene and citric acid content ( Figure 6B).

Discussion
Our research has shown that the age of sweet pepper seedlings and the cultivation method influenced the biometric characteristics of the seedlings (

Discussion
Our research has shown that the age of sweet pepper seedlings and the cultivation method influenced the biometric characteristics of the seedlings (Table 1). Other researchers have found a different effect of seedling age on biometric parameters. Some scientists have suggested that seedling age influenced the biometric characteristics of peppers [8,10,18,19], while other authors have reported that seedling age only affected the height of seedlings [20]. It was found that plant height, number of leaves, and leaf area increased with the age of pepper seedlings [9,10,18]. This is due to a better-developed root system and better absorption of moisture and nutrients from the soil by the plants. However, other researchers have found the opposite, i.e., younger seedlings had more leaves and a more extensive leaf area [21,22]. Our research data confirmed the conclusions of those authors who stated that the age of sweet pepper seedlings affected their height, the number of leaves, the leaf area, and the fresh mass of the plant. According to our results, the 60-day-old seedlings (both sown directly and grown by transplanting) were taller, had more leaves, and had a larger leaf area than the 50-day and 40-day-old seedlings ( Table 1).
According to our data, the age of sweet pepper seedlings and the cultivation method positively affected the dry matter content and the chlorophyll index in leaves ( Figure 3A,B). Sixty-day-old seedlings accumulated more dry matter in leaves than 40-and 50-day-old seedlings ( Figure 3A). Other researchers have also confirmed that the dry matter content in pepper leaves increased as the age of the pepper seedlings increased [18,23]. With increasing seedling age, the dry matter content in the seedlings leaves increased, and the chlorophyll index increased ( Figure 3B). Leskovar and Othman [15] determined the effect of the cultivation method of artichoke seedlings on the chlorophyll index in their leaves. Globe artichokes transplanted had a higher leaf chlorophyll index than the leaves of directly sown plants. Meanwhile, our data showed that the cultivation method did not affect the chlorophyll index in the leaves of sweet pepper seedlings.
One of the parameters of seedling quality is a well-developed root system. In our study, both the age of the seedlings and the seedlings' cultivation method influenced the fresh weight of the roots. Leskovar and Cantliffe [14] indicated that bell pepper plants sown directly had a more developed root system and more dry matter in their leaves, stems, and fruits. Naz et al. [19] stated that the age of chili seedlings did not affect root mass. In agreement with Leskovar and Cantliffee, in our research, sweet pepper seedlings sown directly had a better-developed root system, and their leaves accumulated more dry matter ( Figure 4).
A plant's physiological status and health are indicated by the photosynthetic rate, chlorophyll content, and fluorescence [24,25]. These parameters show how the experimental conditions affect the plant [26]. Our data showed that the age of sweet pepper seedlings influenced the photosynthetic parameters in their leaves (Table 3). No literature data were found on the effect of age and cultivation method on the photosynthetic parameters of sweet pepper seedlings. Leskovar and Othman [15] investigated the cultivation methods of artichoke seedlings and established that they had no significant effect on leaf gas exchange (photosynthesis, stomatal conductance, and transpiration). In contrast, the sweet pepper seedling cultivation method affected leaf photosynthesis, transpiration, and stomatal conductance. It could be argued that this depends on the type of vegetable.
Determining the optimum transplanting age is one of the most critical factors influencing marketable yield [27]. Researchers have found that the optimum age of pepper seedlings to obtain the highest yields varies. However, pepper yield is not only influenced by the age of the seedlings; plant yields are influenced by the cultivation area, plant distance, etc. [8,23,28]. Researchers have found that the optimum age for pepper seedlings grown outdoors varies. According to Shukla et al. [29], 33-36-day-old transplants were best regarding the growth and yield of the pepper California Wonder. Others have concluded that the optimum age of pepper seedlings grown in the field was between 44 and 55 days [23,30]. Some authors have reported that higher peppers yields per hectare were obtained when planting four-week-old seedlings [28,31]. Conflicting research results about the optimum age of seedlings may be due to different environmental and cultural conditions [22]. Results also depend on the type of pepper.
There is limited information on the effect of the seedling age on plant growth and yield for sweet peppers grown in greenhouses. Jang and others [32] investigated the effect of the seedlings' age on the growth and fruit yield of peppers grown in greenhouses at different periods. The results showed that 50-, 55-, and 60-day-old seedlings gave the highest yields. Vázquez-Casarrubias et al. [33] studied the effect of seedling's age (15,30, and 45 days) on the growth and yield of apaxtleco chili grown in a greenhouse. It was found that plants whose seedlings were planted at 45 days were the most productive. However, most authors argued that older pepper seedlings produce higher yields. Our results confirmed the findings of these researchers, who found that older seedlings provided higher yields. The highest yields were obtained from sweet pepper plants whose seedlings were grown for 60 days ( Figure 5). The cultivation method of the seedlings also affected the yield of sweet pepper plants. Several investigations have been conducted to test the seedling growing method's effect on crop performance. Globe artichoke plants grown by transplanting had higher yields than plants sown directly [15]. According to Bouz and Favaro [34], different cultivation technologies significantly affected the productive characteristics and early maturity of sweet corn. The establishment of sweet corn crops through transplantation resulted in lower yields. Gavric and Omerbegovic [12] found that higher yields were obtained when the plants were transplanted. The influence of the age of tomato seedlings and the cultivation method on their yield was studied. Research data showed that direct seeding did not affect tomato yield [35]. According to Leskovar and Cantliffe [14], the yield of bell peppers grown by transplanting was significantly higher and earlier than that of directly seeded plants. Contrary to Leskovar's data, our results showed that the yield of sweet peppers whose seedlings were grown by direct sowing was higher than those grown by transplanting ( Figure 5). These seedlings with higher above-ground and root masses were better developed and, therefore, gave the highest yields.
The biochemical composition of peppers changes during ripening. Citric and ascorbic acids are the predominant organic acids in pepper. In our study, neither the age of the seedlings nor the method of cultivation affected the organic acid content in the sweet pepper fruit (Table 4).

Conclusions
Sweet pepper seedlings' age and growing method influenced the biometric and physiological parameters of the sweet pepper seedlings, phenological stage, and marketable and total yield of the plants. The 60-day-old seedlings (both sown directly and grown by transplanting) were 1.3-1.7 times taller, had more leaves, and had 1.4-1.9 times larger leaf area than the 50-and 40-day-old seedlings. The chlorophyll index and dry matter content were also highest in these seedlings' leaves. However, the highest photosynthetic parameters were found in the leaves of 40-day-old seedlings (both sown directly and grown by transplanting). Sweet peppers whose seedlings were planted at the age of 60 days, and were sown directly, began to flower and started fruiting earlier than peppers whose seedlings were planted at 50 and 40 days. For a higher yield of sweet peppers, it is recommended to grow the seedlings by sowing the seed directly into cups and planting them in the greenhouse at 60 days old.