Determination of Total Carotenoids and β-Carotene in Germplasm of Pumpkin Caboclo (Cucurbita maxima)

Abstract

The genus Cucurbita has been mentioned as a good source of carotenoids, including β-carotene, which is a precursor of vitamin A. However, the variability observed for the genus denotes the individual need to invest in the species. An experiment was carried out with the aim of evaluating the content of total carotenoids and β-carotene in C. maxima germplasm. Treatments consisted of 23 accessions and two commercial cultivars of C. maxima. The content of total carotenoids, β-carotene, and the pulp color of the accessions were evaluated. Clustering was performed, and the components of genotypic variance, mean heritability, and accuracy were calculated. The grouping led to the formation of four groups where the total carotenoid content influenced the formation the most. There was no difference in color parameters. High variability was observed for total carotenoids in the studied genotypes (22.28 µg/g to 49.58 µg/g) and β-carotene (7.81 µg/g to 13.75 µg/g). Accessions CMAX-09, CMAX-10, CMAX-12, CMAX-13, and CMAX-14 showed the highest levels of total carotenoids and β-carotene.

1. Introduction

Germplasm analysis is a fundamental technique for identifying plants with specific nutritional characteristics for biofortification [1]. Biofortification is the process of nutritionally enriching foods by increasing the essential nutrients in their composition. This technique is vital to combat malnutrition and nutritional deficiencies, especially in developing countries [2]. Germplasm evaluation identifies specific nutritional characteristics, such as the content of vitamins, minerals, and proteins in seeds or the edible parts of plants. Based on this information, it is possible to select individual plants to cross with other plants and thus obtain new varieties with higher levels of nutrients [3].

Considering the characteristics of the nutritional importance of the genus Cucurbita, its levels of carotenoids have been studied, with an emphasis on β-carotene, which is a precursor of vitamin A [4], and this characteristic has been explored for the development of biofortified cultivars.

However, scientific literature describes wide variability for these species, including their morphological, agronomic, and nutritional characteristics [5,6]. Thus, it is denoted that the results obtained for a genotype cannot be extrapolated to others.

A study evaluated the diversity of 55 pumpkin accessions from different geographic regions and 3 commercial cultivars. They sought to evaluate the composition of carotenoids and identify parents with a high content of vitamin A precursors [7]. The authors identified wide genetic diversity among accessions and found three with high vitamin A content, including β-carotene, suggesting the potential for using these accessions in biofortification programs. In another study, the viability of indirect selection for determining the total carotenoids in 51 accessions of squash was verified, confirming the possibility of the indirect selection of accessions with higher levels of carotenoids and indicating an accession with a higher content of carotenoids [8].

Characterizing the nutritional constituents in the germplasm of cultures of interest in genetic biofortification is extremely important. Particularly, for crops such as pumpkins, which have documented germplasm rich in genetic variability [5,6], measuring nutritional characteristics may provide valuable information for breeders who work with the crop.

Among the pumpkin species is Cucurbita maxima Duchesne, which is a cucurbit that is native to the Americas and known worldwide for its nutritional value. It is characterized by being an allogamous species with an annual cycle and low growth. Its fruits have varied shapes, colors, and sizes, and it is considered one of the most diverse species of the Cucurbita genus [9].

For the species C. maxima, variability has been described regarding its morphological and nutritional characteristics with differences in nutritional characteristics present between genotypes and parts of the same genotype [10].

Given the above, the objective of this study was to evaluate the content of total carotenoids and β-carotene in accessions of C. maxima that were preserved in a cold chamber.

2. Materials and Methods

2.1. Location

The experiment occurred at the Rafael Fernandes Experimental Farm which belongs to the Federal Rural University of the Semi-Arid–UFERSA in Mossoró-RN and has an average temperature of 27.2 °C and an annual rainfall of 766 mm [11]. The soil in the experimental area is characterized as a Typical Dystrophic Red Argisol [12]. The average meteorological data comprising the period the experiments were carried out are presented in Figure 1.

2.2. Design and Treatments

The design used in this study was complete randomized blocks (DBC) with three replications and five plants per replication. The experiment consisted of 23 accessions, a commercial open-pollinated, and a hybrid cultivar, totaling 25 treatments. The accessions belonged to the Germplasm Collection of the Universidade Federal Rural do Semi-Árido (UFERSA), the Department of Agronomic and Forestry Sciences (DCAF), and the commercial cultivars acquired in the local market of Mossoró-RN (

Table 1. Origin and location of Cucurbita maxima germplasm evaluated for total carotenoid and β-carotene contents.

Treatments	Code a	Origin b	Location	Collection Date
T1	CMAX 01	Santa Rosa II-Apodi	5°39′40.4″ S 37°47′55.3″ W	12 August 2010
T2	CMAX 02	Sítio Totoro de Baixo-Currais Novos	6°13′30.4″ S 36°28′16.5″ W	15 March 2011
T3	CMAX 03	Sítio Boqueirão-Touros	5°11′43.5″ S 35°27′49.4″ W	20 December 2010
T4	CMAX 04	Loteamento Punaú-Rio do fogo	5°16′18.0″ S 35°23′00.3″ W	21 December 2010
T5	CMAX 05	Origin unknown	-	-
T6	CMAX 06	Origin unknown	-	-
T7	CMAX 07	Sítio Carpina (Apodi)	5°39′40.4″ S 37°47′55.3″ W	29 November 2013
T8	CMAX 08	Baraúna	5°04′41.6″ S 37°37′03.6″ W	18 August 2010
T9	CMAX 09	Caiçara do Rio do Vento	5° 45.435″ S 36° 00.094″ W	15 May 2014
T10	CMAX 10	Sítio Boqueirão (Vila Meire)-Touros	5°11′43.5″ S 35°27′49.4″ W	20 December 2010
T11	CMAX 11	Sítio Bico torto-Apodi	5°39′40.4″ S 37°47′55.3″ W	8 December 2010
T12	CMAX 12	Cobal-Mossoró	5°11′21.1″ S 37°20′15.5″ W	-
T13	CMAX 13	Sítio Bico torto-Apodi	5°39′40.4″ S 37°47′55.3″ W	12 August 2010
T14	CMAX 14	Vazia dos Bois-Currais Novos	6°13′30.4″ S 36°28′16.5″ W	15 March 2011
T15	CMAX 15	Loteamento Punaú-Rio do fogo	5°16′18.0″ S 35°23′00.3″ W	21 December 2010
T16	CMAX 16	Povoado Cruz-Currais Novos	6°13′30.4″ S 36°28′16.5″ W	28 October 2010
T17	CMAX 17	Santa Rosa II-Apodi	5°39′40.4″ S 37°47′55.3″ W	4 June 2010
T18	CMAX 18	Assentamento Sítios dos gois-Apodi	5°39′40.4″ S 37°47′55.3″ W	19 November 2010
T19	CMAX 19	Almino Afonso	6°08′49.5″ S 37°45′49.9″ W	-
T20	CMAX 20	Loteamento Punaú-Rio do fogo	5°16′18.0″ S 35°23′00.3″ W	21 December 2010
T21	CMAX 21	Sítio Boqueirão-Touros	5°11′43.5″ S 35°27′49.4″ W	20 December 2010
T22	CMAX 22	Origin unknown	-	-
T23	CMAX 23	Origin unknown	-	-
T24	CV. Comercial Moranga Coroa c		Comercio local de Mossoró (RN)	May 2019
T25	Cv. Hibrida Tetsukabuto c		Comercio local de Mossoró (RN)	May 2019

^a Access code in the Cucurbitaceae Germplasm Collection of UFERSA/CCA/DCAF. ^b Place of origin of access. ^c Commercial cultivars obtained from local businesses in Mossoró (RN).

).

2.3. Area Preparation and Conduction

The preparation of the area consisted of plowing and harrowing. The soil samples were collected at a depth of 0–20 cm, and the results of the chemical analysis were: pH in water = 6.90; P = 14.1 mg/dm³; K⁺ = 39.1 mg/dm³; Na⁺ = 7.3 mg/dm³; Ca²⁺ = 1.10 mg/dm³; Mg²⁺ = 0.40 cmolc dm⁻³; Al³⁺ 0,0; (H + Al) = 0.17 cmolc dm⁻³; SB = 1.63 cmolc dm⁻³; t = 1.63 cmolc dm⁻³; CTC = 1.80%; V = 91%; m = 0; and PS = 2%.

Considering the levels of nutrients in the soil and the needs of the crop, fertilization with nitrogen (N), phosphorus (P), and potassium (K) was carried out following the recommendation of the fertilizer manual of the Agronomic Institute of Pernambuco for the pumpkin culture [13]. It was applied in the following foundation: 30 kg ha⁻¹ of N, 60 kg ha⁻¹ of P₂O₅, and 30 kg ha⁻¹ of K₂O. Topdressing fertilization was 90 kg ha⁻¹ of N and 30 kg ha⁻¹ of K₂O twenty days after planting.

Direct sowing was performed using three seeds per hole with a spacing of 0.90 m × 2.00 m between rows. Thinning was performed, leaving only one plant per hole. The irrigation system used was a micro-sprinkler with a flow (volume of irrigation) rate of 1.58 m³/h [14]. The cultural treatments were carried out according to the needs of the culture.

2.4. Fruit Physicochemical Characteristics

When ripening, the fruits were taken to the DCAF/DCV/UFERSA post-harvest laboratory to evaluate the color, total carotenoids, and β-carotene content, and one fruit per plant was evaluated. The pulp colorimetry analyses were performed using a Konica Minolta Color Reader CR-400 tristimulus manual colorimeter with the following parameters: L* = luminosity; a* = the contribution from red, and b* = the contribution from yellow. Reading was performed at three different points of the pulp, and the average of the points was obtained. From the values of a* and b*, the parameters of chromaticity (C*) and hue (h°) were calculated based on the equations described by Itle and Kabelka (2009) [15].

2.5. Assessment of Total Carotenoids

2.5.1. Reagents

The experiment used acetone and hexane from Vetec Química Fina (Rio de Janeiro, RJ, Brazil) and methanol purchased from Merck KGaA (Darmstadt, Hesse, Germany).

2.5.2. Obtaining the Extract

The fruits were cut in half to obtain the extract, and cylindrical samples were taken from both ends with a coconut punch. Subsequently, the peels were discarded, and the samples were crushed in a food processor. One gram of processed pulp was weighed and added to a falcon tube with a capacity of 50 mL and then wrapped with aluminum foil to avoid contact with light. After this process, a 20 mL mix of methanol:acetone:hexane (1:1:2) was added. The samples were taken to an ultrasonic washer for 20 min and then centrifuged for 5 min, which formed two layers, including a denser one containing the plant material and a less dense one containing the carotenoid extract of the pipette.

The rest of the plant material (the denser layer) was macerated with another 10 mL of hexane and taken to the centrifuge again, and the process of forming the two layers was repeated, removing the less dense one. During the two extractions, maximum carotenoid extraction was obtained. The pulp resulting from the extraction was whitish. It resulted in samples with a final volume of 10 mL. The remainder of the hexane was evaporated during the extraction process.

2.5.3. Determination of Total Carotenoids

After obtaining the extract, a 3 mL aliquot was removed for reading in the UV/VIS spectrophotometer at a wavelength of 450 nm, according to the methodology described by Can-Cauicha et al. (2019) [16].

2.6. Evaluation of β-Carotene

2.6.1. Reagents and Standard Preparation

The experiment was conducted using liquid chromatography analytical grade reagents. The β-carotene standard was obtained from Sigma-Aldrich Brazil Ltd. (Cotia, SP, Brazil) with a purity greater than 93%, while the acetone and hexane were obtained from Vetec Química Fina (Rio de Janeiro, RJ, Brazil), and the acetonitrile was purchased from Merck KGaA (Darmstadt, Hesse, Germany).

A stock solution of 1000 mg/L of β-carotene was prepared in a binary solution of hexane and acetone (1:1, v/v). The standard stock solution was further diluted with hexane to obtain a working solution containing the standard at a concentration of 100 mg/L for method development and validation. The solutions were stored in an amber bottle at −18 °C.

2.6.2. Obtaining the β-Carotene Extract

From the extract obtained for the total carotenoids in the subitem above (Section 2.5.2), a 5 mL aliquot of the extract was concentrated in a rotary evaporator. After this process, 2 mL of acetonitrile was added to re-suspend the extract, which was then placed in an amber bottle to be quantified later.

2.6.3. Quantification of β-Carotene

β-carotene quantification was performed using the ultra-high performance liquid chromatography (UHPLC) system from Shimadzu Corporation, Kyoto, Japan. The UHPLC is equipped with a Restek column (Pinnacle DB AQ C18, size 50 × 2.1 mm, with 1.9 μm particles), two LC pumps-30AD, a DGU degasser-20A5R, an auto-sampler Sil-30AC, a CTO-30AC column furnace, a Shimadzu DAD (nexera model X2 SPD-M30A), and a CBM-20A controller.

The optimized chromatographic conditions to obtain the best resolution during the analysis were a binary mobile phase with mobile phase A (methanol) and mobile phase B (acetonitrile) in a 1:9 ratio with a flow rate of 0.15 mL·min⁻¹; additionally, the column oven temperature was adjusted to 35 °C, and the maximum wavelength was 450 nm.

2.6.4. Method Validation

The validation of the method was carried out with an evaluation of various performance parameters, such as selectivity, linearity, and the limit of detection, quantification, precision, and accuracy, to assess the reliability of the results provided by the method [17].

2.6.5. Selectivity, Linearity, Sensitivity, and Repeatability

The identification of β-carotene in the pumpkin samples was made by comparing the retention time with the standard and investigating the three-dimensional scans of the iodine arrays (ranging from 250 to 500 nm) for the maximum wavelength. Linearity was evaluated using eight calibration levels with concentrations ranging from 0.5 to 100 mg/L of β-carotene which were prepared by successive dilutions of the working solution.

The limit of detection (LD) and the limit of quantification (LQ) were calculated based on the parameters of the calibration curve, and the LD and LQ were, respectively, 3 and 10 times the value of the ratio of the standard deviation of the linear coefficient of the regression with the angular coefficient of the analytical curve. The study method’s repeatability was demonstrated by the relative standard deviation (RSD) which was calculated for six consecutive measurements at three concentration levels (1, 50, and 100 mg/L) of solutions containing the β-carotene standard.

2.6.6. Recovery

Method recovery studies were evaluated at three fortification levels, including 1, 10, and 100 µg/g of β-carotene. In 50 mL Falcon tubes, 1 g of pumpkin macerated with 1 mL of incorporation solution was added. These samples were left to rest in the dark for complete evaporation of the solvent, and the unfortified samples were subjected to the same conditions as the controls.

After extraction, the spiked and unfortified sample solutions were compared to assess recovery. Three replications were performed for each treatment.

2.7. Statistical Analyses

Statistical analysis was performed using SELEGEN-REML/BLUP software [18]. Model 2 was used, which corresponds to y = Xr + Zg + Wp + e, where y is the data vector, r is the vector of the repetition effects (assumed to be fixed) added to the general mean, g is the vector of the genotypic effects (assumed to be random), and p is the vector of plot effects. Is is the vector of the errors or residuals (random). The capital letters represent the incidence matrices for the above effects.

The variance components include Vg: the genotypic variance; Ve: the residual variance; h2mg: the genotype mean heritability; Acclon: the accuracy of the genotype selection; CVgi%: the coefficient of genotypic variation; CVe%: the coefficient of residual variation; and CVr = CVg/CVe = the coefficient of relative variation. The mean is the overall mean of the experiment.

The UPGMA (Unweighted Pair Group Method With Arithmetic Mean) [19] was used as a grouping method. The criterion of Mojena (1977) [20] was used for the cut-off point. We use Singh’s (1981) [21] criteria to estimate the relative contribution of color traits, the total carotenoids, and β-carotene in terms of divergence. The analyses were processed by the GENES program [22].

3. Results and Discussion

Suitable identification of the β-carotene present in the analyzed solutions was verified through the chromatographic conditions used in the elution of the samples. The β-carotene peak showed a purity index with values greater than 0.95. The separation factor between the peak of β-carotene and the interferents was more significant than 1.5 (Figure 2).

Linearity was evaluated for the concentrations of 0.5, 1, 2, 5, 10, 20, 50, and 100 mg/L, and linear regression was obtained by plotting the area of the β-carotene peak as a function of the concentration of each standard. The detector response at 450 nm was linear throughout the calibration interval. Good linearity was achieved by the high correlation coefficient value (R² = 0.9998).

The consecutive injection of six samples for concentrations 1, 10, and 100 mg/L revealed good accuracy since the values found for the relative standard deviation (RSD) ranged from 1.05 to 2.64 (

Table 2. Estimates of the variance components for the quantitative characteristics of pulp, lightness (L*), saturation (C*), hue (h°), total carotenoids (CAT), and β-carotene (BC) in 23 accessions and 2 cultivars of C. maxima. Mossoró-RN, 2020.

			Deviance
Effect	Lp*	Cp*	h°p	CAT	BC
Full	510.37	620.99	581.91	905.93	714.12
Access	510.4	620.99	581.92	912.99	714.87
LRT	0.03 ns	0 ns	0.01 ns	7.06 **	0.75 ns
Vg	0.123	0.08	0.04	75.57	7.75
Ve	8.331	22.01	18.69	245.72	76.51
h2m	0.09	0.04	0.03	0.82	0.55
Ac	0.3	0.19	0.18	0.9	0.74
CVg(%)	0.43	0.87	0.29	22.99	25.59
CVe(%)	2.37	7.72	2.75	18.96	39.95
CVr	0.18	0.11	0.1	1.21	0.64
Mean	81.23	33.04	71.15	37.82	10.88

LTR: the likelihood ratio test; Vg: the genotypic variance; Ve: the residual variance; h2mg (%): the mean genotype heritability, assuming complete survival; Ac: the accuracy of the genotype selection, assuming complete survival; CVe%: the coefficient of residual variation; CVr: the relative coefficient of variation; mean: the overall mean of the experiment; **: significant at the 1% probability level; and ns: not significant.

). The method effectively extracted the β-carotene as the β-carotene recoveries at the three concentration levels produced results with average percentages of 96.49, 99.78, and 100.70%.

When evaluating the estimates of the variance components for the traits of flesh color (L*, C*, and H*), total carotenoids (CT), and β-carotene (BC), the accession effects were significant at the 1% level for the total carotenoids (TC) content according to the chi-square test (Table 2).

Characteristics related to flesh color (Lp* brightness, Cp* saturation or chromaticity, hue or hue angle h°p) and β-carotene (BC) showed no difference according to the chi-square test. The genotypic variation (Vg) for total carotenoids variables was 75.57, and the average heritability (h2m) was 0.82. According to Resende’s classification (1995), this value is considered high because it is above 0.5; thus, it infers that it is possible to succeed in selecting new generations with good gains. Heritability is an essential parameter as it helps with decision-making in breeding programs for different cultures [23,24]. The average heritability for CT was also considered good as it was above 0.5 (Table 2).

The accuracy value (Ac) for the CT character was 0.9. This value is considered to have very high accuracy according to the classification by Resende and Duarte (2007) [25] and indicated that the method used was adequate. Additionally, the accuracy value for BC presented a high accuracy as it was above 0.7. The CT variable had a relative variation coefficient of 1.21, reinforcing the possibility of successful selection (Table 2).

According to the means for the quantitative color traits (

Table 3. Means and standard deviations of color descriptors, total carotenoids, and β-carotene. Mossoró-RN, 2020.

	Average (Evaluated Characters)
Germoplasm a	Lp*	Cp*	h°p	CT	BC
CMAX-01	81.18 ± 2.63	33.03 ± 1.56	71.12 ± 2.07	40.57 ± 1.97	10.54 ± 1.33
CMAX-02	81.21 ± 2.12	33.05 ± 1.65	71.15 ± 2.03	39.92 ± 1.93	11.45 ± 1.31
CMAX-03	81.27 ± 2.55	33.02 ± 2.21	71.15 ± 2.75	32.03 ± 2.65	9.09 ± 1.92
CMAX-04	81.27 ± 2.54	33.02 ± 1.78	71.12 ± 2.61	41.27 ± 2.51	11.56 ± 1.22
CMAX-05	81.25 ± 2.03	33.04 ± 2.54	71.14 ± 2.19	40.14 ± 2.09	10.69 ± 1.94
CMAX-06	81.23 ± 2.23	33.03 ± 1.94	71.15 ± 2.10	37.70 ± 2.00	11.60 ± 1.24
CMAX-07	81.13 ± 2.58	33.02 ± 2.37	71.15 ± 2.23	31.73 ± 2.13	10.09 ± 1.36
CMAX-08	81.28 ± 2.12	33.07 ± 2.17	71.19 ± 2.55	31.81 ± 2.45	9.87 ± 1.26
CMAX-09	81.22 ± 2.21	33.02 ± 2.18	71.11 ± 1.95	47.81 ± 1.85	13.49 ± 2.01
CMAX-10	81.35 ± 2.27	33.01 ± 1.79	71.14 ± 2.52	38.13 ± 2.42	13.75 ± 1.47
CMAX-11	81.21 ± 2.91	33.09 ± 2.08	71.14 ± 2.67	43.04 ± 2.57	12.59 ± 1.57
CMAX-12	81.16 ± 2.67	32.99 ± 2.24	71.15 ± 2.26	45.76 ± 2.16	11.65 ± 1.79
CMAX-13	81.24 ± 2.25	33.02 ± 1.87	71.15 ± 2.25	46.52 ± 2.15	13.42 ± 1.71
CMAX-14	81.15 ± 2.45	33.08 ± 1.83	71.14 ± 2.26	49.58 ± 2.16	12.02 ± 2.17
CMAX-15	81.27 ± 2.69	33.03 ± 2.17	71.16 ± 1.92	38.36 ± 1.82	10.82 ± 1.48
CMAX-16	81.32 ± 2.29	33.01 ± 2.09	71.15 ± 2.51	33.91 ± 2.41	10.00 ± 1.20
CMAX-17	81.16 ± 2.11	33.10 ± 1.86	71.15 ± 2.74	35.63 ± 2.64	9.97 ± 1.70
CMAX-18	81.23 ± 2.17	33.04 ± 2.00	71.14 ± 2.79	31.80 ± 2.69	10.01 ± 2.06
CMAX-19	81.28 ± 2.60	33.03 ± 2.55	71.14 ± 2.06	44.82 ± 1.96	12.37 ± 1.75
CMAX-20	81.33 ± 2.18	33.04 ± 1.91	71.16 ± 2.38	36.57 ± 2.28	9.85 ± 1.94
CMAX-21	81.22 ± 2.38	33.05 ± 2.10	71.15 ± 2.53	36.46 ± 2.43	9.98 ± 1.42
CMAX-22	81.30 ± 2.63	32.94 ± 2.16	71.21 ± 2.55	22.28 ± 2.46	7.81 ± 1.61
CMAX-23	81.18 ± 2.33	33.08 ± 2.27	71.11 ± 1.97	34.29 ± 1.87	10.77 ± 2.17
M. Coroa b	81.01 ± 2.32	33.03 ± 1.90	71.18 ± 2.47	25.68 ± 2.37	8.10 ± 1.26
Tetsukabuto b	81.34 ± 2.34	33.03 ±.1.87	71.14 ± 2.78	39.54 ± 2.68	10.51 ± 2.11

^a Germplasm of C. maxima: CMAX = accessions preserved in the Cucurbitaceae Germplasm Collection at UFERSA; ^b M. Coroa and Tetsukabuto = commercial cultivars obtained from the local trade in Mossoró, RN. Pulp lightness (Lp*), pulp saturation (Cp*), pulp hue (h°p), total carotenoids in µg/g (CT), and β-carotene in µg/g (BC).

), it can be seen that there was not much variation between the studied accessions. For Lp*, the accessions ranged from 81.01° in the commercial cultivar moringa corona to 81.35° in the CMAX-10 accession. Regarding the chromaticity, the accessions ranged from 32.94 to 33.10° for accessions CMAX-22 and CMAX-17, respectively. When evaluating the h°, the accessions ranged from 71.11° for the CMAX-09 accession to 71.21° for the CMAX-22 accession (Table 3).

The contents related to the fruit pulp color, luminosity (Lp*), saturation (Cp*), and hue represent a space of coordinates, or the space (L*C*H*), where L is the luminosity and varies from black 0 to white 100. The chroma or saturation represents the concentration of the color element or dye; in this way, the purest colors have a high saturation, while neutral colors have a low saturation and are less noticeable in human vision. The hue angle (h°) can be considered a quantitative color attribute. Its colors can be defined considering the angles of 0° red, 90° yellow, 180° green, and 270° blue [9].

According to the color space (L*C*H*) adapted from Ferreira and Spricigo (2017) [9], it was noted that in general, the accessions presented means around L* = 81, C* = 33, and h° = 71 and tended to be a light orange color (Table 3).

The average content of total carotenoids varied between 22.28 µg/g for the CMAX-22 accession and 49.58 µg/g for the CMAX-14 accession. In contrast, when considering the variability within the accession, it was observed that CMAX-22 had a range of 6.48 to 23.05 and accession CMAX-14 had a range of 27.78 µg/g to 75.37 µg/g (Table 3). Considering that the germplasm evaluated in the present work is of an allogamous species, it is also essential to highlight the variability within the accessions since considering only the average values may mask or even discard superior genotypes. Taking into account the amplitude, it can be seen that accession CMAX-09 obtained the highest content of total carotenoids and reached 94.12 µg/g. However, the average value of this accession was below the value found for accession CMAX-1 (Table 3). Priori et al. (2012) [26] evaluated the total carotenoids in accessions of C. maxima from the active cucurbit germplasm bank of EMBRAPA temperate climate and found higher values that ranged from 22.64 µg/g to 221.29 µg/g of total carotenoids. However, as already reported, the carotenoid content in vegetables can be variable due to climatic factors, such as temperature, radiation, and rainfall, which may have been the main factor behind the difference in carotenoid content in the previously reported study [26].

Mean β-carotene values ranged from 7.81 µg/g to 13.75 µg/g for accessions CMAX-22 and CMAX-10. In contrast, when considering the within-access variation, CMAX-22 ranged from 0.83 µg/g to 8.0 µg/g of β-carotene, while CMAX-10 ranged from 4.90 µg/g to 48, 88 µg/g of β-carotene. Notably, the maximum value observed for the accession was almost three times the average observed for this accession (Table 3). Pevicharova and Velkov (2017) [27] evaluated the sensory, chemical, and morphological characterizations of C. maxima and C. moschata genotypes from different geographic origins and found values that ranged from 7.3 to 61.12 µg/g for β-carotene in accessions of C. maxima.

The accession with the highest average content of total carotenoids (CMAX-14 = 49.58 µg/g) was not the accession with the highest average content of β-carotene (CMAX-10 = 13.75 µg/g), which showed that the ratio of total carotenoids and β-carotene was not constant for all accessions (Table 3).

Observing the average for accession CMAX-10, it can be seen that 36% of the total amount of carotenoids was β-carotene. However, when considering the amplitude of the averages for this accession, it is possible to notice that the fruit that obtained a content of 65.71 µg/g of total carotenoids is the same one that obtained 48.88 µg/g of β-carotene; that is, 74.38% of the total amount of carotenoids is β-carotene.

Considering that β-carotene has greater pro-vitamin A activity and C. maxima may have other carotenoids in its composition, the results emphasize the importance of evaluating the germplasm one intends to use to improve its direct use. The 23 accessions studied had higher means of total carotenoids and β-carotene than the commercial and hybrid cultivars (Table 3), demonstrating their high potential for the use and improvement of the species. Accessions CMAX-14, CMAX-09, and CMAX-12 stood out as they had higher levels of total carotenoids, while accessions CMAX-09, CMAX-13, and CMAX-10 had higher levels of β-carotene.

The dendrogram of genetic diversity based on the fruit characteristics (Lp* pulp luminosity, Cp* pulp chromaticity, and hp* hue angle), total carotenoids, and β-carotene allowed the formation of four distinct groups (Figure 3).

According to an analysis by Sinhg (1981) [21], the total carotenoid content was the characteristic almost entirely responsible for forming groups under the conditions in which the experiment was developed as it was responsible for 99.99% of the contribution.

The highest number of accessions formed the first group: CMAX-01, CMAX-04, CMAX-02, CMAX-05, the hybrid cultivar (CV.H), CMAX-11, CMAX-06, CMAX-17, CMAX-20, CMAX-21, CMAX-15, CMAX-10, CMAX-03, CMAX-07, CMAX-08, CMAX-18, CMAX-16, and CMAX-23. This group was formed mostly by accessions with total carotenoid content that ranged from 31.73 µg/g for accession CMAX-07 to 41.27 µg/g for accession CMAX-04 (Table 3).

The shortest distance between accessions in this group was 0.08 units between accessions CMAX-08 and CMAX-18 with a carotenoid content of 31.81 and 31.80 µg/g, respectively, and the most distant accession was CMAX-23, which was 161.02 units away from the CMAX-01 accession (Table 3).

The following accessions formed the second group with the highest levels of total carotenoids: CMAX-09, CMAX-12, CMAX-13, CMAX-19, and CMAX-14 (Figure 3). Accession CMAX-14 had the highest average of total carotenoids with an average of 49.58 µg/g. The smallest distances for this group were found between accessions CMAX-13 and CMAX-19 (5.21 units). The most significant distances were between accessesions CMAX-09 and CMAX-14 (35.09 units). The CMAX-14 accession was the most divergent for this group because it had the highest TC content among the others (Table 3).

4. Conclusions

The accessions of C. maxima from the UFERSA cucurbit germplasm collection showed high variability regarding their total carotenoids. Accessions CMAX-13 and CMAX-10 showed the highest levels of total carotenoids and β-carotene. The study identified significant genetic diversity among accessions and found two with potential for use in biofortification programs.