Parthenocarpic Cactus Pears (Opuntia spp.) with Edible Sweet Peel and Long Shelf Life

Abstract

The fruits of the nopal (Opuntia spp.), cactus pears, are considered functional foods due to their content of nutritive and bioactive substances. Its pulp is generated by numerous seeds that limit their consumption due to their size and hardness and detract from their quality. Other undesirable fruit characteristics are its inedible peel and its short shelf life. In the case of the cactus pear cactus (Opuntia spp.), no cultivar has been reported that produces quality parthenocarpic fruits, nor have they been obtained by artificially inducing parthenocarpy. The objectives of this research were to evaluate the response of 11 genotypes to the induction of parthenocarpy, to characterize the fruits of the genotypes with the best response, and to determine their postharvest life. To induce parthenocarpy, floral buds were used in the pre-anthesis stage, from which the style-stigma, stamens, and tepals were removed, generating a cavity in which a solution of growth regulators was applied (250 mg L⁻¹ AG₃ + 75 mg L ⁻¹ BA + 15 mg L⁻¹ of AIB). A similar number of buds was used as a control, without treatment, and in free pollination. Only two genotypes, MX CP-30 Red and MX CP-40 Yellow, produced parthenocarpic fruits since their empty integuments produced pulp, remaining small, empty, and soft. Their peel was sweet (10–14 °Brix) and edible, and they had a longer shelf life than the corresponding fruits with seeds.

1. Introduction

The Opuntia genus is the most diverse of the Cactaceae family, with 181 species [1], of which 78 are native to Mexico [2]. In Mexico, the nopal (Opuntia spp.) has historical, social, cultural, medicinal, ecological, and economic importance and is part of its patriotic symbols [3], and the country is the world’s leading producer of tunas (cactus pears) and vegetable cactus (locally known as “nopalitos”). In 2022, 69,605 ha of nopal were cultivated, of which 45,033 ha produced 444,080 tons of tuna, and 24,571 ha were used to produce nopalitos and forage [4]. The second country in the area cultivated with cactus pear is Italy, with 8300 ha producing 87,000 t [5].

Tuna is considered a functional food due to its nutritional properties and content of bioactive substances that help maintain and promote health [6,7]. The pulp contains vitamins (C, E, A, B1, and B2), minerals (calcium, potassium, magnesium, iron, and phosphorus), carotenoids, polyphenolic compounds, betalains, flavonoids (quercetin, isorhamnetin, kaempferol, and indicaxanthin), and amino acids (asparagine, alanine, and arginine) [8]. Some of these bioactive substances are also found in the peel, such as the phenolic compound isorhamentin-O-(deoxyhexosyl-hexoside) and betacyanins [9]. However, the peel is not consumed and, depending on the Opuntia species, represents 33 to 63% of the fruit weight [10,11]. In addition to the ample documented evidence of the health benefits of cactus pear, there is also evidence of the plant’s usefulness for medicinal uses, such as a colorant for food, juices, liquors, confectionery, and animal nutrition, as well as in biofuel production and soil phytoremediation [8,12,13].

The cactus pear is a unilocular, polyspermous, and fleshy berry that originates from a flower with an ovary with ovules of parietal placentation [3,14], whose locule is surrounded by the receptacle [15], and whose tissues are anatomically similar to the cladodes, so it is considered a modified stem [14,16]. The seed comes from the fertilized ovule, and its hard covering corresponds to the integuments covering the ovule that lignify during its development [17]. The pulp of the cactus pear originates from the dorsal epidermal cells of the funicular sheath covering the ovule [14], which requires the stimulation of ovule fertilization [18]. Per capita consumption of tunas in Mexico has not grown significantly in recent years [19], and one of the causes is the refusal of consumers to swallow the seeds, which are between 200 and 400 per fruit [10,11], with a hardened cover, popularly called bones [14]. To break this cover requires pressures of 0.2 to 4.6 kN [20,21] so chewing them would put the teeth at risk, and to avoid this, the consumer chooses to swallow them with the pulp. This causes some people to avoid the consumption of cactus pears and consider them inappropriate for small children [10,19,22]. Tuna seeds are of two types, normal and abortive; normal seeds are 4.5 mm long, 3.5 mm wide, and 1.6 mm thick, on average, while abortive seeds are smaller [21]. The number of seeds per fruit has been reduced during the domestication process of Opuntia, since wild species have more seeds than domesticated ones [23], although Chapman et al. [24] have pointed out that the higher the number of seeds, the larger the fruit tends to be.

To prevent seeds from limiting the consumption of fresh tunas, it is desirable that there be cultivars with few small seeds or that could produce parthenocarpic tunas without seeds [25]. Natural parthenocarpic seedless fruits have been observed in cucumber, eggplant, watermelon, and tomato [26]. In citrus, four parthenocarpy types have been described: obligatory parthenocarpy when varieties always produce seedless fruit; facultative parthenocarpy if seedless fruit is produced when cross-pollination with compatible sources of pollen is prevented; vegetative parthenocarpy when seedless fruit develops without requiring any external stimulus; and stimulative parthenocarpy that requires the pollination stimulus for the seedless fruit set [27]. Parthenocarpy, whether natural or artificially induced to produce seedless fruits, is a complex trait governed by multiple genes and hormone-signaling pathways that regulate fruit development [28]. Although parthenocarpic cultivars of some species are commercially cultivated (cucumber, watermelon, eggplant, tomato, orange, and mandarin, among others), parthenocarpy can result in small, low-quality seedless fruits [29]. To the best knowledge of the authors of this work, there are no parthenocarpic cultivars in commercial use in Opuntia. In Israel, a genotype of O. ficus-indica with abnormal, lignified, and semi-lignified seeds due to vegetative parthenocarpy was identified [30], whose fruits were of poor quality for the market [24].

High-quality seedless fruits produced with natural or artificial parthenocarpy are preferred by consumers and by the agroindustry because they are easier to eat and process [31]. As regards the induction of parthenocarpy with the application of growth regulators, it is a common agricultural practice for producing seedless fruits and reducing the cost of manual pollination in cucurbit crops [28], citrus [27], oil palm [32], and other crops. In this regard, Pimienta [10] pointed out that it would not be possible to obtain seedless Opuntia fruits as in other fruit trees such as apples, pears, or peaches, because in these the edible part derives from the floral parts surrounding the ovules (e.g., ovary and floral tube) and not from fertilized ovules as it occurs in tunas. The first studies on hormone induction were those of Trippi and del Frari [33], who tested different doses of naphthaleneacetic acid applied to open flowers of O. ficus-indica but did not prevent seed formation. Subsequently, gibberellic acid (GA₃) was applied to flowers at anthesis [34] or to emasculated flower buds at pre-anthesis [19,35,36], and even combining AG₃ with other growth regulators [25], but without achieving the expected success because the fruits presented important defects such as reduced size and weight, little pulp, and insipid, relatively large, and hard seed residues. In some cases, repeated applications of growth regulators were evaluated to increase fruit size without achieving seedless-quality fruit. Conversely, it is a common agricultural practice to induce parthenocarpy in other crops such as tomato [37], apple [38], cucurbits [28], and interspecific hybrids of oil palm with the exogenous application of naphthalene acetic acid (NAA). Romero et al. in cucumber [32], using cytokinin, auxin, and GA4 + 7 [29], and other crops. Current evidence supports the major role of three fertilization-induced hormones, auxin, gibberellin, and cytokinin, in the regulation of fruit development. Individually, any of these hormones can stimulate parthenocarpic growth to some extent when applied ectopically; combined, they can induce normal fruit growth even in the absence of fertilization [38].

The development of good-quality parthenocarpic tunas would be an approach to overcoming fruit refusal due to seeds. Under the hypothesis that the response to hormonal induction of parthenocarpy in cactus pear (Opuntia spp.) depends on genotype, this research was carried out to: (1) evaluate the response to parthenocarpy induction of 11 cactus pear genotypes; (2) characterize parthenocarpic fruits for their quality; and (3) postharvest life.

2. Materials and Methods

2.1. Study Area

This research was carried out in a private orchard located in Otumba, State of Mexico, Mexico, located at 98°45′22.97″ W and 19°41′59.46″ N, an altitude of 2350 masl. The climate is semi-dry temperate [BS1K + C(N)], with summer rainfall, precipitation, and a mean annual temperature of 623 mm and 14.9 °C, respectively. During the years in which most of this research was conducted (2019, 2021, and 2022), mean annual rainfall and temperatures were 583, 630, and 599 mm and 15.4, 16.6, and 17.1 °C, respectively. In the orchard, the five year old nopal plants are planted at a distance of 2.5 m between plants and 4 m between rows. Pests, diseases, and weeds were controlled; pruning was performed for formation, health, and fruiting, and organic fertilization with bovine manure was applied to the plants as recommended [39].

The study was conducted in three phases: (1) evaluation of the genotypic response to hormonal induction of parthenocarpy; (2) characterization and comparison of parthenocarpic (PF) vs. non-parthenocarpic (control) fruits (CF); and (3) measurement of postharvest life. The specifications are detailed below.

2.2. Evaluation of the Response to Parthenocarpy Induction

It was performed in 2019 with 11 genotypes from the Opuntia germplasm collection of the Genetics Program of the Colegio de Postgraduados, Campus Montecillo, as described in

Table 1. Opuntia genotypes were evaluated for their response to parthenocarpy induction.

Genotype	Pulp Color	Species
1. CP-CC	White	O. albicarpa
2. CP-RA	White	O. albicarpa
3. CP-RL	Red	O. ficus-indica
4. CP-CV	Red	O. ficus-indica
5. CP-30 Red	Red	O. ficus-indica
6. CP-ORTPM	Red	O. robusta
7. CP-ORTP	Red	O. robusta
8. CP-Amaroax10	Yellow	O. ficus-indica
9. CP-ORSELR	Red	O. robusta
10. CP-40 Yellow	Yellow	O. ficus-indica
11. CP-ORM-VL *	Pulp free	O. robusta

* Genotype 11 is a male clone (does not form female sex organs).

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Three treatments were applied to flower buds in pre-anthesis to the 11 genotypes, according to the following procedures: (1) total emasculation of 30 flower buds for each genotype by manual removal of the style and its stigma, together with the tepals and stamens, which generated a cavity in which a solution containing gibberellic acid (GA₃), benzyl adenine (BA), and indole butyric acid (IBA). The following mixture of growth regulators was applied only once: 250 mg L⁻¹ GA₃ + 75 mg L⁻¹ BA + 15 mg L⁻¹ IB acid, avoiding runoff; (2) natural pollinated control; 30 control buds were neither emasculated nor had the solution applied to them (they were left to flower and pollinate freely); and (3) emasculated control without application of regulators; another 30 buds were emasculated, but the regulator mixture was not applied.

All flower buds used were duly marked on different plants per treatment. The fruits were harvested when they presented their characteristic color of maturity in all the peels. The response to parthenocarpy induction was evaluated visually and with photographs, classifying genotypes into two classes: (1) with a positive response (PR) to those genotypes whose treated fruits formed pulp, and (2) with a negative response (NR) to those genotypes whose fruits could not form pulp. The integuments, i.e., the seed coatings without embryo or endosperm, were classified as hard or soft to the touch.

2.3. Comparison of Parthenocarpic Fruits (PF) vs. Non-Parthenocarpic Controls (CF)

In the years 2020 and 2021, the quantitative assessment of PF and CF (from untreated and open-pollinated flower buds) of the genotypes CP-30 Red and CP-40 Yellow was carried out because they were the ones that gave the best positive response to parthenocarpy induction. The eight treatments resulting from a complete factorial arrangement with three factors (year, genotypes, and parthenocarpy-inducing treatment) and two levels for each factor were distributed in a completely randomized experimental design. The experimental unit consisted of one flower bud or fruit, with 15 fruits as replicates. All buds, or flower buds, were marked on different plants per treatment.

The fruits were harvested when they presented the characteristic maturity pigmentation of the genotype in the whole peel, cutting them with a knife. The gourds, or glochids, were removed with a self-made de-spinning machine.

The fresh weight of the whole fruit (FW) and of the pulp (PuW) was determined in each fruit with a Scout^® Pro digital scale with an accuracy of 0.01 g; the weight of the peel (PW) was obtained from the difference FW-PuW when fresh. With these data, PuW/FW and PW/FW ratios were calculated. With a digital vernier, the equatorial (ED) and polar diameters (PD) of each fruit were measured, and the ED/PD ratio was calculated. The volumes of fruit (FV) and pulp (PuV) were measured by the displacement of water in a graduated cylinder of 1000 mL. The volume of the peel (PV) was calculated by the difference FV-PuV. Peel thickness (PT) was measured with a digital vernier in the middle part of the fruit after a longitudinal cut and separation of the pulp. Total soluble solids (TSS) in degrees Brix (°Bx) were determined in three drops of juice obtained directly from the pulp(TSSPu) and peel(TSSP) with an ATAGO^® PAL-1 (ATAGO Co., Ltd., Tokyo, Japan) digital refractometer.

From each normal fruit (CF), the normal (NS) and abortive (AS) seeds were weighed and counted, and in the PF, the empty integuments without embryos were counted, called seminal residues (SR). For this purpose, the pulp of the fruit was placed in a plastic strainer and washed with pressurized water until all the pulp was eliminated; the strainer with seeds was placed on a paper napkin for 30 s, and then the fresh weight (FWS = fresh weight of seeds or seminal residues) and the fresh volume (FVS, fresh volume of seeds or seminal residues) were measured. Weights were recorded with a Sartorius H51 analytical balance, and volume was measured by water displacement using a graduated cylinder. The seeds, or SR, were placed on paper, dried at room temperature, and then counted for each fruit measured. In CF, the pulp weight ratio was calculated by dividing PuW by the total number of normal and abortive seeds (PuW/TS), and in PF, by the total seminal residues (PuW/TS).

2.4. Shelf Life (SL)

In the summer of 2020, the postharvest life of normal and parthenocarpic fruits of the varieties CP-30 Red and CP-40 Yellow was evaluated in an environment (E1) with an air temperature of 18 ± 2 °C and a relative humidity of 50 ± 5%. The experimental unit was one fruit, with 15 fruits as replicates. In 2021, the same evaluation was made with an air temperature of 25 ± 2 °C and 60 ± 5% relative humidity (E2) with the same experimental unit and 28 fruits as replicates. The eight treatments resulting from a complete factorial arrangement with three factors (environment, genotypes, and parthenocarpy-inducing treatment) and two levels for each factor were distributed in a completely randomized experimental design. The useful postharvest life was determined as the number of days from fruit harvest until the presence of spots on the peel caused by pathogens or the appearance of wrinkles due to turgor loss.

The data obtained were subjected to analysis of variance for each variable with the SAS (Statistical Analysis System) package, version 9.3. In cases where the F test detected significant differences between treatments, the Tukey’s multiple means test was applied with α = 0.05.

3. Results

3.1. Parthenocarpy

Of the eleven genotypes treated to induce parthenocarpy, nine did not produce pulp (Figure 1); only two genotypes, CP-30 Red and CP-40 Yellow, showed a positive response because their empty seminal residues (SR) showed a capacity to produce pulp (Figure 2), remaining small, non-lignified, and soft to taste and touch (Figure 3). In the 11 genotypes, the fruits that were emasculated, but without the application of regulators, stopped growing and detached from the cladode after approximately 15 days. In the CPGen-VL genotype, its flower buds had no ovules, so both its fruits treated and not treated with regulators did not form pulp (Figure 1). In contrast, the other eight untreated genotypes (controls) developed their fruits and were harvested at the consumption maturity stage, when their peels acquired their characteristic color.

3.2. Comparison of Parthenocarpic (PF) vs. Non-Parthenocarpic (CF) Fruits

In the years 2020 and 2021, the PF of genotypes CP-30 Red and CP-40 Yellow had similar characteristics to those of 2019; their empty, small, and soft seminal residues (Figure 1, Figure 2 and Figure 3) were able to produce pulp by the induction treatment. The PFs of these two genotypes produced smaller fruits than CF in equatorial (ED) and polar diameters (PD), fruit weight, volume, and pulp production. The PFs were more elongated in shape (their ED/PD ratio was lower than that of the CF) and had a thicker peel. The contents of TSS solutes in peel and pulp were lower in PF (

Table 2. Effects of years 2020 and 2021, genotype, and the presence or absence of seeds in cactus pears. Comparison of means.

			VARIABLES RESPONSE X
Year	Genotype	Seed	ED (mm) Y	PD (mm)	ED/PD (mm)	TSSP (°B)	TSSPu (°B)
Main effects
			Pr > F: < 0.0001	Pr > F: 0.0086	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001
2020			52.5 ± 3.8 A	78.4 ± 6.1 A	0.67 ± 0.06 A	11.3 ± 1.2 B	12.8 ± 1.1 B
2021			46.3 ± 6.1 B	75.7 ± 7.5 B	0.61 ± 0.08 B	13.2 ± 1.5 A	15.0 ± 0.9 A
			Pr > F: 0.0016	Pr > F: 0.0388	Pr > F: 0.8387	Pr > F: 0.8889	Pr > F: 0.6548
	Yellow		48.5 ± 5.7 B	75.9 ± 6.9 B	0.64 ± 0.09 A	12.7 ± 1.7 A	14.3 ± 1.4 A
	Red		50.4 ± 6.1 A	78.2 ± 6.8 A	0.64 ± 0.06 A	12.5 ± 1.6 A	14.2 ± 1.4 A
			Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: 0.1133	Pr > F: 0.0126
		With	53.1 ± 4.8 A	79.5 ± 7.7 A	0.67 ± 0.08 A	12.9 ± 1.8 A	14.6 ± 1.5 A
		Without	45.9 ± 4.7 B	74.7 ± 5.2 B	0.62 ± 0.07 B	12.2 ± 1.4 B	13.9 ± 1.3 B
Interactions
Year x genotype			Pr > F: 0.5587	Pr > F: 0.1787	Pr > F: 0.4778	Pr > F: 0.3503	Pr > F: 0.6021
2020	Yellow		51.7 ± 3.8 a	78.0 ± 7.0 ab	0.67 ± 0.07 a	11.1 ± 1.0 b	12.8 ± 1.0 b
2020	Red		53.2 ± 3.7 a	78.8 ± 5.1 a	0.68 ± 0.05 a	11.4 ± 1.4 b	12.8 ± 1.2 b
2021	Yellow		45.3 ± 5.5 b	73.8 ± 6.3 b	0.62 ± 0.10 b	13.4 ± 1.5 a	15.1 ± 0.9 a
2021	Red		47.4 ± 6.7 b	77.6 ± 8.2 ab	0.61 ± 0.05 b	13.0 ± 1.5 a	14.8 ± 1.0 a
Year x seed			Pr > F: 0.0026	Pr > F: 0.5900	Pr > F: 0.0072	Pr > F: 0.2218	Pr > F: 0.9109
2020		With	55.6 ± 2.8 a	81.5 ± 6.4 a	0.69 ± 0.07 a	11.4 ± 1.0 bc	13.2 ± 1.0 b
2020		Without	49.8 ± 2.2 b	75.8 ± 4.4 bc	0.66 ± 0.05 a	11.2 ± 1.5 c	12.5 ± 1.1 b
2021		With	50.9 ± 5.1 b	77.8 ± 8.4 ab	0.66 ± 0.08 a	13.7 ± 1.6 a	15.3 ± 1.1 a
2021		Without	41.6 ± 2.2 c	73.4 ± 5.7 c	0.57 ± 0.05 b	12.7 ± 1.1 ab	14.6 ± 0.6 a
Genotype x seed			Pr > F: 0.7520	Pr > F: 0.0217	Pr > F: 0.0568	Pr > F: 0.5026	Pr > F: 0.6282
	Yellow	With	52.1 ± 4.7 a	77.0 ± 8.2 b	0.68 ± 0.09 a	13.1 ± 1.8 a	14.6 ± 1.5 a
	Yellow	Without	45.0 ± 4.3 b	74.8 ± 5.4 b	0.60 ± 0.07 c	12.2 ± 1.6 a	14.0 ± 1.3 a
	Red	With	54.3 ± 4.7 a	82.1 ± 6.2 a	0.66 ± 0.06 ab	12.8 ± 1.9 a	14.6 ± 1.4 a
	Red	Without	46.8 ± 5.0 b	74.6 ± 5.1 b	0.63 ± 0.06 bc	12.1 ± 1.2 a	13.8 ± 1.3 a
Year x genotype x seed			Pr > F: 0.0751	Pr > F: 0.0061	Pr > F: 0.3156	Pr > F: 0.1104	Pr > F: 0.8521
2020	Yellow	With	55.3 ± 1.9 a	81.3 ± 8.0 ab	0.69 ± 0.08 a	11.6 ± 0.7 abc	13.1 ± 0.9 cd
2020	Yellow	Without	48.6 ± 1.8 c	75.1 ± 4.6 bc	0.65 ± 0.05 ab	10.6 ± 1.2 c	12.6 ± 1.2 d
2020	Red	With	55.9 ± 3.6 a	81.6 ± 4.7 ab	0.69 ± 0.05 a	11.1 ± 1.2 bc	13.3 ± 1.2 bcd
2020	Red	Without	51.0 ± 1.8 bc	76.4 ± 4.3 abc	0.67 ± 0.05 a	11.8 ± 1.7 abc	12.4 ± 1.2 d
2021	Yellow	With	49.2 ± 4.6 bc	73.3 ± 6.5 c	0.68 ± 0.10 a	13.8 ± 1.8 a	15.4 ± 1.1 a
2021	Yellow	Without	41.1 ± 2.1 d	74.4 ± 6.2 c	0.55 ± 0.05 c	13.1 ± 1.1 ab	14.8 ± 0.4 ab
2021	Red	With	52.7 ± 5.1 ab	82.6 ± 7.5 a	0.64 ± 0.05 ab	13.7 ± 1.6 a	15.2 ± 1.1 a
2021	Red	Without	42.1 ± 2.3 d	72.5 ± 5.2 c	0.58 ± 0.04 bc	12.3 ± 1.0 abc	14.4 ± 0.7 abc

^X Yellow = CP 40 Yellow, Red = CP 30 Red, ED = equatorial diameter, PD = polar diameter, ED/PD = ratio equatorial diameter/polar diameter, TSSP = total soluble solids in the peel, TSSPu = total soluble solids in flesh. ^Y Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05). Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for means of the main effect.

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Table 3. Effects of the years 2020 and 2021, genotype, and the presence or absence of seeds in cactus pears. Comparison of means.

			VARIABLES RESPONSE X
Year	Genotype	Seed	FW (g) Y	PW (g)	PuW (g)	PuW/FW (g)	PW/FW (g)
Main effects
			Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: 0.0868	Pr > F: 0.0006	Pr > F: 0.0006
2020			114.5 ± 20.9 A	57.9 ± 10.7 A	57.4 ± 23.4 A	0.48 ± 0.14 B	0.52 ± 0.14 A
2021			98.3 ± 26.6 B	44.3 ± 10.0 B	54.6 ± 27.5 A	0.52 ± 0.14 A	0.48 ± 0.14 B
			Pr > F: 0.0027	Pr > F: 0.6669	Pr > F: 0.1141	Pr > F: 0.4843	Pr > F: 0.4843
	Yellow		102.1 ± 23.1 B	49.7 ± 12.4 A	53.5 ± 24.6 B	0.50 ± 0.14 A	0.50 ± 0.14 A
	Red		110.8 ± 26.5 A	50.6 ± 12.2 A	57.9 ± 26.8 A	0.51 ± 0.15 A	0.49 ± 0.15 A
			Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001
		With	124.3 ± 21.8 A	44.6 ± 10.7 B	80.7 ± 13.7 A	0.64 ± 0.07 A	0.35 ± 0.07 B
		Without	90.1 ± 14.7 B	55.1 ± 11.5 A	33.7 ± 5.7 B	0.38 ± 0.05 B	0.62 ± 0.05 A
Interactions
Year x genotype			Pr > F: 0.5000	Pr > F: 0.4481	Pr > F: 0.1824	Pr > F: 0.7671	Pr > F: 0.7671
2020	Yellow		111.1 ± 20.8 a	57.9 ± 9.5 a	57.5 ± 25.2 a	0.48 ± 0.14 a	0.52 ± 0.14 a
2020	Red		117.7 ± 20.8 a	57.9 ± 12.0 a	57.3 ± 22.1 a	0.48 ± 0.14 a	0.51 ± 0.14 a
2021	Yellow		93.1 ± 22.1 b	43.4 ± 10.7 b	50.3 ± 24.1 a	0.51 ± 0.14 a	0.48 ± 0.14 a
2021	Red		103.6 ± 30.0 ab	45.2 ± 9.4 b	58.4 ± 30.1 a	0.53 ± 0.15 a	0.47 ± 0.15 a
Year x seed			Pr > F: 0.1234	Pr > F: 0.0065	Pr > F: 0.3881	Pr > F: 0.7497	Pr > F: 0.7497
2020		With	131.0 ± 18.2 a	49.3 ± 6.9 b	81.3 ± 6.9 a	0.62 ± 0.03 a	0.38 ± 0.03 c
2020		Without	100.5 ± 10.0 c	65.3 ± 7.4 a	36.6 ± 5.6 b	0.36 ± 0.04 c	0.64 ± 0.04 a
2021		With	118.0 ± 23.4 b	41.1 ± 11.8 c	80.3 ± 17.3 a	0.66 ± 0.08 a	0.34 ± 0.08 c
2021		Without	78.6 ± 9.8 d	47.2 ± 7.0 bc	31.4 ± 4.9 b	0.40 ± 0.04 b	0.60 ± 0.04 b
Genotype x seed			Pr > F: 0.3897	Pr > F: 0.1283	Pr > F: 0.2719	Pr > F: 0.0973	Pr > F: 0.0973
	Yellow	With	118.6 ± 20.1 a	45.6 ± 12.3 bc	77.3 ± 14.9 a	0.63 ± 0.08 a	0.37 ± 0.08 b
	Yellow	Without	86.7 ± 12.8 b	53.1 ± 11.7 ab	33.3 ± 5.3 b	0.39 ± 0.06 b	0.61 ± 0.06 a
	Red	With	129.9 ± 22.3 a	43.7 ± 9.3 c	83.7 ± 12.1 a	0.66 ± 0.04 a	0.34 ± 0.04 b
	Red	Without	93.4 ± 15.9 b	57.0 ± 11.2 a	34.0 ± 6.2 b	0.37 ± 0.03 b	0.62 ± 0.03 a
Year x genotype x seed			Pr > F: 0.0227	Pr > F: 0.4219	Pr > F: 0.0075	Pr > F: 0.1418	Pr > F: 0.1418
2020	Yellow	With	129.6 ± 13.7 a	51.6 ± 7.2 b	82.6 ± 7.2 ab	0.62 ± 0.04 a	0.38 ± 0.04 b
2020	Yellow	Without	94.9 ± 8.6 bc	63.5 ± 7.8 a	34.7 ± 4.2 c	0.35 ± 0.05 b	0.64 ± 0.05 a
2020	Red	With	132.3 ± 22.2 a	47.0 ± 6.1 bc	79.9 ± 6.6 ab	0.63 ± 0.02 a	0.37 ± 0.02 b
2020	Red	Without	105.7 ± 8.5 b	67.0 ± 6.8 a	38.4 ± 6.2 c	0.36 ± 0.04 b	0.64 ± 0.04 a
2021	Yellow	With	108.3 ± 20.0 b	40.6 ± 13.6 c	72.9 ± 18.3 b	0.64 ± 0.11 a	0.36 ± 0.11 b
2021	Yellow	Without	77.9 ± 10.5 c	45.6 ± 7.4 bc	32.3 ± 5.9 c	0.42 ± 0.05 b	0.58 ± 0.05 a
2021	Red	With	127.7 ± 23.0 a	41.5 ± 10.6 bc	86.3 ± 14.4 a	0.68 ± 0.04 a	0.32 ± 0.04 b
2021	Red	Without	79.4 ± 9.3 c	48.9 ± 6.4 bc	30.5 ± 3.6 c	0.39 ± 0.02 b	0.61 ± 0.02 a

^X FW = fruit weight, PW = peel weight, PuW = flesh weight, PuW/FW = flesh weight/fruit weight ratio, and PW/FW = peel weight/fruit weight ratio. ^Y Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05). Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for means of the main effect.

,

Table 4. Effects of years 2020 and 2021, genotype, and presence or absence of seeds in cactus pears. Comparison of means.

			VARIABLES RESPONSE X
Year	Genotype	Seed	PT (mm)	NS (number)	AS (number)	SR (number)	ST (number)
Main effects
			Pr > F: < 0.0001	Pr > F: 0.5075	Pr > F: 0.9928	Pr > F: 0.5696	Pr > F: 0.8864
2020			6.2 ± 1.8 A	95.6 ± 101.4 B	30.2 ± 35.4 A	142.4 ± 155.7 A	274.9 ± 45.3 A
2021			4.1 ± 1.4 B	122.4 ± 98.9 A	40.9 ± 44.1 A	106.6 ± 147.1 B	269.9 ± 65.3 A
			Pr > F: 0.0648	Pr > F: 0.3400	Pr > F: 0.0456	Pr > F: 0.5288	Pr > F: 0.3612
	Yellow		4.9 ± 1.9 B	122.7 ± 94.0 A	50.2 ± 46.5 A	103.2 ± 151.4 B	276.1 ± 64.8 A
	Red		5.4 ± 1.9 A	102.9 ± 106.5 B	22.7 ± 29.0 B	135.8 ± 148.1 A	266.1 ± 52.5 A
			Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: 0.0360
		With	3.8 ± 1.1 B	194.5 ± 32.0 A	64.0 ± 34.9 A	0.0 ± 0 B	258.5 ± 52.5 B
		Without	6.4 ± 1.7 A	0.1 ± 0.4 B	0.0 ± 0 B	290.4 ± 64.4 A	290.5 ± 64.3 A
Interactions
Year x genotype			Pr > F: 0.9124	Pr > F: 0.1862	Pr > F: 0.6989	Pr > F: 0.9186	Pr > F: 0.7754
2020	Yellow		5.9 ± 1.9 a	96.9 ± 107.2 a	36.1 ± 42.1 a	153.2 ± 162.0 a	286.2 ± 39.1 a
2020	Red		6.4 ± 1.8 a	94.4 ± 101.1 a	24.4 ± 28.1 a	130.4 ± 157.1 a	262.4 ± 50.6 a
2021	Yellow		3.9 ± 1.3 b	134.4 ± 87.5 a	56.6 ± 47.9 a	80.4 ± 144.3 a	271.5 ± 74.0 a
2021	Red		4.3 ± 1.6 b	107.7 ± 112.0 a	21.7 ± 30.3 a	138.5 ± 148.0 a	268.0 ± 54.7 a
Year x seed			Pr > F: 0.0029	Pr > F: 0.5182	Pr > F: 0.9928	Pr > F: 0.5696	Pr > F: 0.5104
2020		With	4.5 ± 0.6 b	191.3 ± 37.1 a	60.5 ± 24.6 a	0.0 ± 0.0 b	251.8 ± 44.1 a
2020		Without	7.6 ± 1.1 a	0.0 ± 0.0 b	0.0 ± 0.0 b	300.7 ± 31.8 a	300.7 ± 31.2 a
2021		With	3.2 ± 1.2 c	195.8 ± 30.4 a	65.4 ± 38.6 a	0.0 ± 0.0 b	261.2 ± 56.1 a
2021		Without	5.0 ± 1.1 b	0.1 ± 0.5 b	0.0 ± 0.0 b	284.3 ± 78.3 a	284.4 ± 78.2 a
Genotype x seed			Pr > F: 0.2213	Pr > F: 0.3484	Pr > F: 0.0456	Pr > F: 0.5288	Pr > F: 0.9785
	Yellow	With	3.8 ± 1.1 b	187.0 ± 32.8 a	76.5 ± 35.3 a	0.0 ± 0.0 b	263.5 ± 55.1 a
	Yellow	Without	6.0 ± 1.9 a	0.0 ± 0.0 b	0.0 ± 0.0 c	300.2 ± 77.3 a	300.2 ± 77.3 a
	Red	With	3.9 ± 1.2 b	205.8 ± 28.2 a	45.3 ± 25.3 b	0.0 ± 0.0 b	251.1 ± 49.3 a
	Red	Without	6.8 ± 1.5 a	0.1 ± 0.5 b	0.0 ± 0.0 c	282.1 ± 53.0 a	282.3 ± 52.8 a
Year x genotype x seed			Pr > F: 0.2165	Pr > F: 0.1917	Pr > F: 0.6989	Pr > F: 0.9186	Pr > F: 0.6717
2020	Yellow	With	4.3 ± 0.7 c	193.8 ± 48.7 a	72.2 ± 27.0 a	0.0 ± 0 b	266.0 ± 44.9 a
2020	Yellow	Without	7.4 ± 1.2 a	0.0 ± 0.0 b	0.0 ± 0.0 b	306.4 ± 19.9 a	306.4 ± 19.9 a
2020	Red	With	4.7 ± 0.5 b	188.8 ± 26.7 a	48.8 ± 17.1 a	0.0 ± 0 b	237.6 ± 43.1 a
2020	Red	Without	7.8 ± 1.0 a	0.0 ± 0.0 b	0.0 ± 0.0 b	293.5 ± 45.2 a	293.5 ± 45.2 a
2021	Yellow	With	3.3 ± 1.2 c	184.9 ± 27.9 a	77.8 ± 38.2 a	0.0 ± 0 b	262.7 ± 59.3 a
2021	Yellow	Without	4.5 ± 1.0 bc	0.0 ± 0.0 b	0.0 ± 0.0 b	295.0 ± 107.6 a	295.0 ± 107.6 a
2021	Red	With	3.2 ± 1.2 c	215.2 ± 25.5 a	43.4 ± 29.8 ab	0.0 ± 0 b	258.7 ± 53.3 a
2021	Red	Without	5.5 ± 0.9 b	0.2 ± 0.7 b	0.0 ± 0.0 b	277.1 ± 57.9 a	277.3 ± 57.7 a

^X PT = peel thickness, NS = number of normal seeds, AS = number of abortive seeds, SR = number of seminal residues, and ST = total number of seeds and/or seminal residues. Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for means of the main effect.

and

Table 5. Effects of years 2020 and 2021, genotype, and the presence or absence of seeds in cactus pears. Comparison of means.

			VARIABLES RESPONSE X
Year	Genotype	Seed	PuV (ml)	PV (ml)	FV (ml)	FWS (g)	FVS (ml)	GRPu/ST (g)
Main effects
			Pr > F: 0.0265	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: 0.0030	Pr > F: < 0.0001	Pr > F: 0.2319
2020			58.9 ± 26.9 A	56.3 ± 10.5 A	110.9 ± 16.9 A	5.09 ± 2.7 A	4.97 ± 2.5 A	0.24 ± 0.12 A
2021			54.2 ± 26.8 B	43.1 ± 9.5 B	97.3 ± 28.4 B	4.62 ± 2.7 A	3.98 ± 2.4 B	0.22 ± 0.11 A
			Pr > F: 0.0809	Pr > F: 0.0668	Pr > F: 0.0015	Pr > F: 0.0336	Pr > F: 0.0071	Pr > F: 0.2942
	Yellow		54.8 ± 24.1 A	47.0 ± 14.3 B	104.4 ± 21.7 B	4.94 ± 2.1 A	4.21 ± 2.1 B	0.22 ± 0.10 A
	Red		58.3 ± 29.6 A	52.2 ± 8.4 A	110.3 ± 20.5 A	4.60 ± 3.1 A	4.73 ± 2.8 A	0.23 ± 0.13 A
			Pr > F: < 0.0001	Pr > F: 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001	Pr > F: < 0.0001
		With	80.5 ± 12.5 A	44.2 ± 10.3 B	122.4 ± 18.6 A	6.78 ± 1.5 A	6.68 ± 1.2 A	0.31 ± 0.08 A
		Without	31.2 ± 4.7 B	55.1 ± 11.1 A	94.6 ± 13.8 B	1.98 ± 0.7 B	2.14 ± 0.6 B	0.12 ± 0.03 B
Interactions
Year x genotype			Pr > F: 0.0265	Pr > F: 0.1180	Pr > F: 0.0170	Pr > F: 0.1660	Pr > F: 0.2454	Pr > F: 0.8896
2020	Yellow		59.5 ± 27.2 a	56.0 ± 12.6 a	110.0 ± 19.3 a	4.5 ± 2.3 a	4.5 ± 2.2 a	0.22 ± 0.11 a
2020	Red		58.3 ± 28.1 a	56.7 ± 8.6 a	111.8 ± 14.1 a	5.6 ± 3.0 a	5.4 ± 2.8 a	0.25 ± 0.13 a
2021	Yellow		50.4 ± 21.3 a	38.8 ± 10.5 b	89.3 ± 21.6 b	5.1 ± 2.1 a	3.9 ± 2.1 a	0.22 ± 0.10 a
2021	Red		58.3 ± 32.6 a	47.8 ± 5.5 ab	106.1 ± 33.3 ab	4.2 ± 3.1 a	4.1 ± 2.9 a	0.22 ± 0.13 a
Year x seed			Pr > F: 0.5367	Pr > F: 0.0687	Pr > F: 0.0045	Pr > F: 0.7826	Pr > F: 0.6157	Pr > F: 0.2557
2020		With	84.6 ± 6.7 a	48.9 ± 6.4 b	124.8 ± 14.3 a	7.6 ± 1.2 a	7.3 ± 0.9 a	0.34 ± 0.05 a
2020		Without	33.2 ± 4.3 b	63.8 ± 8.2 a	100.3 ± 9.3 b	2.6 ± 0.2 b	2.6 ± 0.2 c	0.12 ± 0.02 b
2021		With	76.8 ± 15.5 a	40.0 ± 11.6 b	116.8 ± 25.9 a	6.5 ± 1.6 a	6.1 ± 1.1 b	0.30 ± 0.09 a
2021		Without	29.3 ± 4.5 b	46.5 ± 5.1 b	75.8 ± 8.1 c	1.7 ± 1.7 b	1.6 ± 0.3 d	0.12 ± 0.03 b
Genotype x seed			Pr > F: 0.0054	Pr > F: 0.1595	Pr > F: 0.0449	Pr > F: 0.0634	Pr > F: 0.0052	Pr > F: 0.2076
	Yellow	With	74.9 ± 14.2 b	40.1 ± 12.4 b	118.5 ± 21.1 a	6.3 ± 0.9 a	6.1 ± 1.0 b	0.29 ± 0.07 a
	Yellow	Without	32.6 ± 4.9 c	54.6 ± 12.6 a	91.0 ± 11.7 b	2.1 ± 0.3 b	2.1 ± 0.4 c	0.12 ± 0.03 b
	Red	With	86.7 ± 6.5 a	48.8 ± 4.6 ab	127.2 ± 14.1 a	7.3 ± 1.9 a	7.3 ± 1.1 a	0.34 ± 0.08 a
	Red	Without	29.9 ± 4.4 c	55.7 ± 10.0 a	98.0 ± 14.9 b	1.9 ± 0.8 b	2.1 ± 0.7 c	0.12 ± 0.02 b
Year x genotype x seed			Pr > F: 0.0129	Pr > F: 0.2590	Pr > F: 0.0001	Pr > F: 0.7652	Pr > F: 0.5990	Pr > F: 0.6465
2020	Yellow	With	84.8 ± 7.1 a	48.2 ± 8.7 bc	126.4 ± 14.5 a	6.68 ± 0.8 ab	6.65 ± 0.7 b	0.33 ± 0.04 a
2020	Yellow	Without	34.2 ± 4.1 c	63.8 ± 11.2 a	95.6 ± 8.1 cd	2.39 ± 0.1 c	2.46 ± 0.2 c	0.12 ± 0.02 b
2020	Red	With	84.4 ± 7.2 a	49.6 ± 3.6 ab	122.7 ± 14.5 ab	8.48 ± 0.6 a	8.00 ± 0.7 a	0.36 ± 0.05 a
2020	Red	Without	32.2 ± 4.8 c	63.8 ± 5.1 a	104.7 ± 8.2 bc	2.82 ± 0.1 c	2.79 ± 0.1 c	0.12 ± 0.03 b
2021	Yellow	With	66.7 ± 13.7 b	33.3 ± 11.2 c	100.0 ± 23.7 c	6.17 ± 1.0 b	5.60 ± 1.0 b	0.27 ± 0.08 a
2021	Yellow	Without	31.0 ± 5.5 c	45.4 ± 4.6 bc	76.4 ± 9.3 de	1.86 ± 0.3 c	1.84 ± 0.3 c	0.12 ± 0.04 b
2021	Red	With	89.0 ± 5.5 a	48.0 ± 5.7 bc	137.0 ± 6.7 a	6.90 ± 2.0 ab	6.68 ± 1.1 ab	0.33 ± 0.10 a
2021	Red	Without	27.6 ± 2.9 c	47.6 ± 5.9 bc	75.2 ± 7.8 e	1.60 ± 0.7 c	1.46 ± 0.3 c	0.11 ± 0.02 b

^X PuV = pulp volume, PV = peel volume, FV = fruit volume, FWS = fresh weight of seed and/or seminal residues, FVS = fresh volume of seed and/or seminal residues, GRPu/ST = ratio of pulp grams per seed and/or seminal residues. Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for means of the main effect.

). The number of seeds in CF was similar to the number of seminal residues (SR) in PF, and these were of lower weight and volume. SR did not lignify and produced less pulp than the average of normal and abortive seeds from normal fruit, with no effect of year or genotype on this variable (Table 5). The main effects and interactions were as follows.

3.2.1. Main Effects

The year factor is statistically significant for most of the variables under study. The year 2020 had a significant main effect on most of the variables related to larger fruits. The reason could be the greater precipitation recorded that year, which in turn caused the dilution of the TSS in peel and pulp. In 2021, the fruits had a content of TSS, both in peel and pulp, significantly higher than in 2020. There were no significant differences in the comparison of means for PuW, AS, TS, FWS, and GRPu/TS between both years. As regards the genotype factor, CP-30 Red has larger and heavier fruits and produces more pulp than CP-40 Yellow, with significant differences in ED, PD, FW, AS, FV, FWS, and FVS, while the CP-40 Yellow genotype is only higher in the variables NS and AS. In the variables PW, PuW/FW, PW/FW, TS, PuV, FWS, and GRPu/TS, there is no significant difference between the means of both genotypes. Considering the seed factor, it is statistically significant as a main effect in all variables, except TSS. In the comparison of means, parthenocarpic fruits have higher values in PW, PW/FW, PT, SR, TS, and PV. In the rest of the variables, the control fruits presented higher values.

3.2.2. Interactions

Regarding the year × genotype interaction, the ANOVA shows that the two genotypes tend to behave in the same way in both years. There was significance just for PuV and FV, but comparing the means of the four year genotype combinations for PuV, there is no significant difference between them. In 2021, the combination 2021-CP 30 Red shows a value statistically equal to the values of the combinations with 2020 for FV. For year × seed, the effect of the presence of seeds is observed in both years; fruits with seeds were heavier. In 2020 and 2021, they weighed 131 g and 118 g, respectively, while the seedless fruits weighed 100.5 g and 78.6 g. The ANOVA shows significance for ED, ED/PD, PW, and PT. In the main effects, it is detected that it is the year 2020 and CF where the highest values occur. However, for the ED variable, the 2020-PF combination shows a statistically lower value than the 2020-CF combination and is equal to the 2021-CF combination. For ED/PD, the combination 2021-CF appears statistically equal to 2020-CF. For the variables PW and PT, the combination 2020-CF appears statistically lower than 2020-PF and equal to 2021-PF. Regarding the genotype x seed interaction according to the ANOVA, in each genotype, the fruits with seeds had a higher fruit weight, a lower peel weight, and a higher pulp weight. There was also significance for the variables PD, AS, PuV, FV, and FVS. In the main effects, it is shown that the PD, FV, and FVS of CP-30 Red and CF have the highest values in these variables. Considering FV, the CF are larger than the respective seedless ones, and the red fruits are larger than the respective yellow ones; however, the value of the combination CF-RED is statistically equal to CF-YELLOW, and the combination PF-RED is statistically equal to PF-YELLOW. Regarding PuV, the values of the combinations with seeds are greater than the corresponding ones without seeds; however, the combination CF-RED is greater and statistically different from the combination PF-YELLOW. As for the year × genotype x seed interaction, there is significance for the variables PD, FW, PuW, PuV, and FV (

Table 6. Effects of years 2020 and 2021, genotype, and the presence or absence of seeds in cactus pears. Significative year x genotype x seed interaction. Comparison of means.

			VARIABLES RESPONSE X
Year	Genotype	Seed	PD (mm) Y	FW (g)	PuW (g)	PuV (ml)	FV (ml)
year x genotype x seed interaction			Pr > F: 0.0061	Pr > F: 0.0227	Pr > F: 0.0075	Pr > F: 0.0129	Pr > F: 0.0001
2020	Yellow	With	81.3 ± 8.0 ab	129.6 ± 13.7 a	82.6 ± 7.2 ab	84.8 ± 7.1 a	126.4 ± 14.5 a
2020	Yellow	Without	75.1 ± 4.6 bc	94.9 ± 8.6 bc	34.7 ± 4.2 c	34.2 ± 4.1 c	95.6 ± 8.1 cd
2020	Red	With	81.6 ± 4.7 ab	132.3 ± 22.2 a	79.9 ± 6.6 ab	84.4 ± 7.2 a	122.7 ± 14.5 ab
2020	Red	Without	76.4 ± 4.3 abc	105.7 ± 8.5 b	38.4 ± 6.2 c	32.2 ± 4.8 c	104.7 ± 8.2 bc
2021	Yellow	With	73.3 ± 6.5 c	108.3 ± 20.0 b	72.9 ± 18.3 b	66.7 ± 13.7 b	100.0 ± 23.7 c
2021	Yellow	Without	74.4 ± 6.2 c	77.9 ± 10.5 c	32.3 ± 5.9 c	31.0 ± 5.5 c	76.4 ± 9.3 de
2021	Red	With	82.6 ± 7.5 a	127.7 ± 23.0 a	86.3 ± 14.4 a	89.0 ± 5.5 a	137.0 ± 6.7 a
2021	Red	Without	72.5 ± 5.2 c	79.4 ± 9.3 c	30.5 ± 3.6 c	27.6 ± 2.9 c	75.2 ± 7.8 e

^X Yellow = CP 40 Yellow, Red= CP 30 Red, PD = polar diameter, FW = fruit weight, PuW = flesh weight, PuV = pulp volume, FV = fruit volume. ^Y Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05).

). The different combinations between the levels of each factor allow us to see that the best results in obtaining parthenocarpic fruits of the CP 30 Red and CP 40 Yellow genotypes occurred in 2020. The mean values of the variables PD, FW, FV, PuW, and PuV were higher in 2020, but for PD, the difference was not significant, as was the case for PuW. PuV is not statistically different from those obtained in 2021. Apparently, under conditions of water limitation as observed in 2021, the pulp production was not significantly affected by the two genotypes.

3.3. Shelf or Postharvest Life

In both test environments, the PF reached a longer shelf life (SL) than the CF (

Table 7. Shelf life (days) ⁺ of normal fruit (CF) and parthenocarpic fruit (PF) of two genotypes stored at 18 ± 2 °C and 55 ± 5% relative humidity (E1).

Type of Fruit	Genotype
	CP-40 Yellow	CP-30 Red	Mean
CF	22.7 ± 12.2 b	34.5 ± 17.4 ab	29.3 ± 16.0 B
PF	49.2 ±15.0 a	44.3 ± 15.8 a	46.7 ± 15.2 A

⁺ Shelf life: days from harvest to the appearance of wrinkles or spots on the fruit. Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05). Lowercase letters (a, b) are for comparing the means of individual treatments, and uppercase letters (A, B) are for the means of the main effect.

and

Table 8. Shelf life ^X of normal fruit (CF) and parthenocarpic fruit (PF) of two Opuntia genotypes stored at 26 ± 2 °C air temperature and 50 ± 5% relative humidity (E2).

Type of Fruit	Genotype
	CP-40 Yellow	CP-30 Red	Mean
CF	7.5 ± 0.8 c	7.6 ± 1.0 c	7.6 ± 0.9 B
PF	13.1 ± 5.3 b	16.8 ± 1.5 a	14.9 ± 4.3 A

^X Shelf life: time from harvest to the appearance of wrinkles or stains in the indicated environment. Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05). Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for comparing means of the main effect.

). In the environment with an air temperature of 18 ± 2 °C and 55 ± 5% relative humidity (RH) (E1), the postharvest life of the PF on average of the two genotypes increased by 60% in relation to the CF (Table 7), but the PF of CP-40 Yellow doubled its SL in comparison with the CF. As regards the interactions (

Table 9. Effects of storage environment (E), genotype, and the presence or absence of seeds in prickly pear fruit shelf life or postharvest life. Comparison of means.

Environment X	Genotype	Seed	Postharvest Life Y
Main effects			Pr > F: 0.0001
E1			38.5 ± 17.4 a
E2			12.2 ± 5.0 b
			Pr > F: 0.0832
	Yellow		18.4 ± 15.7 a
	Red		19.1 ± 14.7 a
			Pr > F: < 0.0001
		With	14.1 ± 33.2 b
		Without	22.0 ± 26.9 a
E x genotype interaction			Pr > F: 0.6297
E1	Yellow		37.4 ± 19.1 a
E1	Red		39.4 ± 16.9 a
E2	Yellow		11.5 ± 5.2 b
E2	Red		12.7 ± 4.8 b
E x seed interaction			Pr > F: 0.0006
E1		With	29.3 ± 16.0 b
E1		Without	46.7 ± 15.2 a
E2		With	7.6 ± 0.9 d
E2		Without	14.9 ± 4.3 c
Genotype x seed interaction			Pr > F: 0.0365
	Yellow	With	13.0 ± 10.3 b
	Yellow	Without	21.1 ± 17.3 ab
	Red	With	14.7 ± 14.8 ab
	Red	Without	22.9 ± 13.6 a
E x genotype x seed interaction			Pr > F: 0.0012
E1	Yellow	With	22.7 ± 12.3 c
E1	Yellow	Without	49.2 ± 15.0 a
E1	Red	With	34.5 ± 17.4 b
E1	Red	Without	44.3 ± 15.8 a
E2	Yellow	With	7.5 ± 0.7 d
E2	Yellow	Without	13.0 ± 5.3 d
E2	Red	With	7.6 ± 1.0 d
E2	Red	Without	16.8 ±1.5 cd

^X Environment: E1: 18 ± 2 °C and 55 ± 5% relative humidity; E2: 26 ± 2 °C air temperature and 50 ± 5% relative humidity). ^Y Different letters indicate significant differences between means, using Tukey’s test (p ≤ 0.05). Lowercase letters (a–c) are for comparing means of individual treatments, and uppercase letters (A, B) are for means of the main effect.

), in the second environment, 26 ± 2 °C and 50% RH ± 5% (E2), the SL of the PFs of the two genotypes was twice that of the CFs (Table 8). The genotype CP-30 Red had a 1 day longer SL than CP-40 Yellow, on average, of the treatments with growth regulators.

Interactions

For environment x genotype interaction, the ANOVA does not present significance; year x seed was statistically significant, and PF has the longer SL. For genotype x seed interaction, the ANOVA presents significance. The PF of CP 30 Red has a longer SL. In the environment x genotype x seed interaction, the different combinations between the levels of three factors allow us to see that the longer SL was in the lower temperature environment (E2), that PF had longer SL, and that CP-30 Red had the longest (Table 9).

4. Discussion

4.1. Effect of Growth Regulators on Opuntia Fruit Development

Our results confirm that the response to hormonal induction of parthenocarpy in Opuntia depends on the genotype, as pointed out by Varela et al. [19] and Montalt et al. [27]. The characteristics of parthenocarpic tunas produced by CP-30 Red and CP-40 Yellow are better than those reported to date by different researchers. [19,25,36], such as small size, low weight, low pulp production, low solids content [35], or large and hard SR [25,40] that detract from their attractiveness for human consumption. The parthenocarpic fruits of the CP-30 Red and CP-40 Yellow genotypes obtained over three years show that the present results obtained with this method of parthenocarpy induction were effective in producing pulp without troublesome SR. Elimination of competing tissues vs. the ovary for photosynthates, application of the growth regulators over a larger absorbing surface area (Figure 2), and protection from the sun and wind the regulators applied may help to explain the superiority of this technique compared to emasculation methods previously used by various researchers that only removed the stigma or stamens and because the regulators were only sprayed or injected into the ovary [31]. With our method, it is not necessary to repeat the application more than once, as several researchers have undertaken [25,34,35,36]. Another key factor for the success recorded here in CP-30 Red and CP-40 Yellow may be the mix of regulators used, which differs from those evaluated by other researchers using just AG₃ [35,41]. Gil and Espinoza [40] mixed AG₃ with auxin 2,4,5-T or Fenoprop. These authors reported that it was possible to produce parthenocarpic fruits of prickly pear by spraying 100 or 500 ppm AG3 or 50 to 500 ppm Fenoprop 15 days before anthesis to immature emasculated flowers, plus two sprays of AG₃ after anthesis; but in those fruits, the severe problem of having hardened and troublesome abortive seeds persisted. In O. amyclaea, Aguilar [34] applied AG₃ (20 to 150 ppm) periodically from anthesis to 90 days later and thus achieved an increase in abortive and hardened seeds, but the fruits were small and had a low total soluble solids content. Ortiz et al. [25] studied the effect of AG₃ at doses of 300, 400, and 500 ppm and combined it with the auxins naphthalene acetic acid (NAA) and indole butyric acid (IBA), both at doses of 10, 20, and 50 ppm, sprayed on emasculated and intact flowers of O. amyclaea cv. Copena-5 on a single application date or several successive dates (15 days before anthesis, at anthesis, and 15, 30, and 45 days after anthesis); these authors were able to harvest PF in the treatment with AG₃ applied to emasculated flowers 15 days before anthesis, but with the serious drawback that the SR was rigid (lignified) and generated discomfort when consuming the fruit. Other researchers also used AG₃ (from 50 to 500 ppm) applied in emasculated flowers of Opuntia [42,43,44] or with an injection of AG₃ into the ovary without emasculation [45]. None of them managed to obtain PF of good size and quality for the market.

In their work, Varela et al. [19] emasculated flower buds in preantesis of four cultivars belonging to three species: Amarilla Montesa (O. megacantha Salm-Dick), Burrona and Cristalina (both O. albicarpa Sheinvar), and Rojo Liso (O. ficus-indica L.), to which they applied AG₃ at doses of 0, 50, 100, and 200 ppm; they obtained parthenocarpic fruits with the three gibberellin doses, but with very little weight and very little pulp. Marini et al. [36] applied five concentrations of AG₃ (0, 100, 200, 250, and 500 ppm) to flower buds of O. ficus-indica cv. Gialla by injection or spray to emasculated or intact flower buds; they found that the 500 ppm dose of AG₃ sprayed to emasculated flower buds was the most effective method to reduce to 46% the number of seeds in the fruits; injection also reduced the number of seeds (−50.7%), but no seedless fruit was useful for the market due to low pulp and having other undesirable characteristics.

It is evident that the most widely used hormone in recent research with Opuntia has been AG₃ [19,36], but it has not led to success. In the present study, a mixture of three regulators—a gibberellin, an auxin, and a cytokinin—was used. In apple (Malus domestica Borkh), the application of gibberellin before flowering allowed the formation of parthenocarpic fruit, but subsequent fruit growth also required cytokinin and auxin [46]. According to Pandolfini [47], seed and fruit development are intimately synchronized and controlled by plant hormones. Similarly, Lu et al. [48] found that in pre-anthesis emasculated grapevine flowers, the fruit set is induced by auxins and cytokinins and subsequently requires gibberellins for fruit growth. To use the growth regulator mix 250 mg L⁻¹ AG₃ + 75 mg L⁻¹ BA + 15 mg L⁻¹ IB, we consider that developing seeds are a source of phytohormones and stimulate the fruit growth and development [49]; so, this function must be replaced in either natural or artificial parthenocarpy as pointed out by various researchers [28,29,38] who also emphasize that current evidence supports the major role of three fertilization-induced hormones, auxin, giberellin (GA), and cytokinin in the regulation of fruit development and that, individually, any of these hormones can stimulate parthenocarpic growth to some extent when applied ectopically and when combined, they can induce normal fruit growth even in the absence of fertilization [38].

The GEN-OR-VLm male genotype of O. robusta did not form ovules in either untreated or treated buds because it lacks female function [50] due to a two-step process: first, the development of the ovarian wall and placenta is arrested, and egg initiation is suppressed, resulting in an empty locule; and in the second phase, a programmed cell death (PCD) occurs in the ovarian wall, placenta, and in the few complete and underdeveloped eggs that have formed, rendering them unviable [50]. In the present study, it is worth noting that the application of the hormonal mixture 15 days before anthesis did not influence the expression of this characteristic, since the male flowers of O. robusta started as hermaphrodites but then lost their female function [50]; this loss had already occurred when in our research the sexual organs were removed to generate the cavity and apply the growth regulators. Another important aspect to highlight in our results is that they demonstrate that Pimienta [17] was right in pointing out that with the elimination of the seeds (as occurs naturally in the male genotype GEN-OR-VLm), it would be impossible to obtain parthenocarpic tunas because it lacks the funiculus and the seminal covers (integuments), which is where the pulp is produced. In other words, the key to producing parthenocarpic fruits in Opuntia is to conserve their ovules, avoid their fertilization, and ensure that their SR keeps the capacity to form pulp in response to the application of growth regulators, as happened with CP-30 Red and CP-40 Yellow.

4.2. Parthenocarpic vs. Normal Fruits (Controls) in Opuntia

The parthenocarpic fruits (PF) of the two genotypes that responded positively to the growth regulators were smaller in volume and weight than the intact control fruits (CF). This was because they produced less pulp as the SR lost some of their ability to produce pulp compared to the normal (fertilized) seeds. On average, of the two genotypes studied here, an SR is estimated to produce one-third of the pulp generated by a normal seed. However, there may be genotypes whose SR can produce more pulp in response to growth regulator treatments. The Opuntia cultivars with a higher degree of domestication were found to form larger fruits with fewer seeds than the small fruits of genotypes of wild or less domesticated species [23], which implies that the seed coatings of cultivated species produce more pulp. It follows then that the genus Opuntia possesses genetic diversity in the ability to produce pulp from the integuments of both normal seeds and SR, and the latter is essential for producing parthenocarpic prickly pears. The small, smooth, empty SR of CP-30 Red and CP-40 Yellow FPs are not bothersome to consumers, who often do not even notice them, so these would be desirable characteristics in cultivars with natural parthenocarpy, which to date no one has found or created. It can also be assumed that the ability of RSs to produce pulp is a complex genetic trait, whose degree of expression will depend on the genotype, the type and dose of the regulators used, and their method of application, among other factors. This complexity is inferred because numerous genes that regulate ovule development [51] have been identified, as well as other genes encoding transcription factors that direct ovule spacing, identity, and tegument formation. In addition, the genetic control of natural or induced parthenocarpy has been revealed to be complex because it involves numerous genes [31], and seeds are known to provide the hormones necessary to initiate and maintain ovary development to form fruit [31].

The parthenocarpic fruits obtained in this study compensate for their smaller size, weight, and quantity of pulp with their thicker, sweeter, and more edible peel. Various investigators [19,52,53,54] had already pointed out that it would be desirable for parthenocarpic tunas to have sweet and edible peel. In the peel of three Opuntia cultivars, Amaya-Cruz et al. [55] identified sixty-eight extractable polyphenols, fifteen hydrolyzable polyphenols, forty-one betalains, sixteen carotenoids, and five phytosterols, noting that such peels can be used as functional ingredients rich in bioactive compounds. This agrees with what is indicated later [56]. In this study, it was possible to combine thick, edible peel with sufficient pulp formation and prolonged shelf life in two genotypes. The prolonged shelf life of parthenocarpic cactus pears agrees with that found in a genotype of O. ficus indica [53] and in tomato [57]. Extending shelf life while maintaining the quality required by the consumer is the major challenge for plant breeders, and researchers are focused on the search for better-quality fruits, as these traits seem to have a negative genetic correlation in many crops [57].

5. Conclusions

The cactus pear genotypes studied here gave contrasting responses to the parthenocarpy-inducing procedure with the growth regulator mix 250 mg L⁻¹ AG₃ + 75 mg L⁻¹ BA + 15 mg L⁻¹ IB. Only two genotypes, CP-30 Red and CP-40 Yellow, responded positively to the exogenous application made before anthesis in the cavity of the floral buds generated by the extirpation of the style-stigma, stamens, and tepals. The parthenocarpy is associated with three important traits: pulp with small and soft seminal rudiments, sweet edible peel, and a longer shelf life that corresponds to seeded fruits. As a result, the edible part increased from 50 to 95%. This is the first report of parthenocarpic tunas (cactus pear) with these characteristics of tasting quality. The parthenocarpy issue in “nopal tunero” (cactus pear cactus) is wide open for research. Future research could focus on searching for and generating natural parthenocarpy, efficiently inducing parthenocarpy, and investigating the genetic and molecular mechanisms of parthenocarpy. An important step is the finding of genotypes with contrasting parthenocarpic responses.