Prickly pear (Opuntia ficus-indica
(L.) Mill.) is the most cultivated plant species in the Cactaceae family due to edible fruit production [1
]. It is a bushy-shaped, xerophytic, and crassulacean acid metabolism (CAM) plant originating from dry areas of Mexico [2
]. The annual global production of prickly pear is approximately 500,000 tons, and Italy supplies 12% of the total market, preceded by Mexico and followed by Israel [3
]. According to the Food and Agriculture Organization (FAO), in the last few years, prickly pear cultivation has gained a considerable amount of interest in relation to coping with food security issues in mostly dry and semi-dry regions, such as in South America, Africa, and the Mediterranean basin, thanks to its high resistance to drought and the important nutritional compounds present in the fruits [2
]. Despite numerous species of the Opuntia
genus being mainly cultivated to produce fresh fruit, this cultivation can play a key role in other contexts, such as environmental defense, forage and bioenergy production, the medicine and cosmetics sectors, and human health [1
]. For instance, in some tropical agroforestry systems, Opuntia elatior
(Mill.) and O. ficus-indica
are cultivated in association with other crops [9
], as a productive living fence guarding against desertification [11
]. In Africa and South America, the association of Opuntia robusta
(J.C. Wendl) and O. ficus-indica
provide both living fences and livestock fodder [7
]. Species such as Opuntia maxima
(Mill.), Opuntia heliabranoava
(Scheinvar), and O. ficus-indica
are currently studied for biogas and fertilizer production, especially when associated with domestic plants in rural areas that are situated off of the energy grid [8
]. O. ficus-indica
has also been recently studied for medical and nutritional purposes since its juice has been found to show nutraceutical activities [17
] and beneficial properties against specific types of cancer cells (bladder, ovarian, and leukemia) [18
The Opuntia ficus-indica
fruit is a false berry with an average weight of approximately 100–120 g, of which 2–10% is seeds and 60–70% is pulp [21
]. Opuntia ficus-indica
fruits present polyembrionatic seeds and 40–45% of them are aborted [22
], while the remaining 60–55% are viable hard-coated seeds. This represents one of the main production challenges, which can influence the market price, because fruits with few or abortive seeds are more appreciated by consumers [24
]. Abundant hard-coated seeds also complicate industrial processes, negatively impacting the transformation of fruit into such products as juice, nectars, jam, and food coloring [25
], and potentially affecting consumer health by causing constipation [28
]. However, whilst in other species such as citrus, the pulp development is not strictly linked with seed presence, in Opuntia ficus-indica
, fruit pulp development depends on the funiculus of the seeds [31
]. The funiculi are needed to produce a commercially acceptable pulp volume; however, a higher number of abortive seeds, characterized by their smaller size, would be more acceptable to consumers. The creation of hybrid types characterized by a good balance between these two latter aspects (i.e., high volume pulp—less number of seeds) would be feasible primarily through genetic breeding programs and agronomic techniques. The application of the first approach [31
] is not economically viable and has resulted in poor fruit performance. By contrast, agronomic techniques, such as spring flushing or growth regulator treatments to inhibit seed growth, especially with auxin and/or gibberellins, may easily and more quickly provide well-formed and seedless fruits [2
]. In the last decades, a few studies investigated the use of a phytoregulator on prickly pear [35
]. For instance, Gil et al. [36
] showed that the treatment of emasculated floral buds using gibberellic acid (GA3
) at 200 ppm increased both the development of ovular tissue and the funiculus, but also the hard-coated abortive seeds. Barbera et al. [35
] indicated that at least 200 ppm of GA3
injected into Opuntia
’s stem (cladode) underneath the fruit was able to decrease the percentage of regular seeds. Mejía et al. [38
], comparing the use of GA3
by injection and spray application at different maturation stages, indicated the best performances using a 100 ppm GA3
injection in pre- and postblooming. Kaaniche-Elloumi [23
] also reported that the number and timing of GA3
applications can affect fruit and seed development.
In this study, we tested the application of two methods (injection and spraying) of gibberellic acid (GA3) on cactus prickly pear both at pre- and postblooming in order to obtain well-formed seedless fruits in emasculated flowers. Increased GA3 concentrations and floral-bud emasculation techniques were also applied to evaluate fruit weight, length, and diameter; and seed weight, the total number of seeds, and the number of hard-coated viable seeds per fruit.
3.1. Fruits Characterization
In general, the lowest average values for diameter, length, and weight were those of EM plants (Table 1
). Considering the combined effect of only INJ/SPY and EM/IN (not GA3
level) on fruit diameter, the highest mean value was found in the IN+SPY group. No significant difference in diameter was found between IN+SPY and IN+INJ combined treatment (−3%), while lower diameters were detected using the combined treatments EM+SPY (−16%) and EM+INJ (−18.5%). The GA3
levels somewhat affected the results. Higher diameters were found in IN+SPY under all GA3
levels and the IN+INJ control, while the lower values were found in the EM group control using both methods (i.e., INJ and SPY). However, the highest statistical significance was observed between control levels of emasculated fruits (INJ: 3.35 ± 0.33 cm, SPY: 3.29 ± 0.33 cm) and control levels of intact fruits (INJ: 4.95 ± 0.24 cm, SPY: 4.74 ± 0.32 cm).
Considering the combined effect of only INJ/SPY and EM/IN (without GA3 levels) on fruit length, the highest mean was found within the IN+SPY combined treatment, while the lowest was observed using EM+INJ. The application of GA3 resulted in statistically significant differences in fruit lengths between intact and emasculated fruits. Specifically, greater fruit lengths were found in the intact fruits under all GA3 levels and application methods. In contrast, low statistical significance was observed in emasculated fruits among all GA3 treatments and methods, and the only exception was for the highest level of the SPY method in line with the results of intact fruits.
Considering the combined effect of INJ/SPY and EM/IN (without GA3 level) on fruit weight, the highest mean was found using the IN+SPY combined treatment, while the lowest was observed using EM+INJ. No significant difference in weight was found between EM+INJ and IN+INJ combined treatment (−6%), while with respect to the remaining combinations (EM+SPY and EM+INJ), considerable weight differences were observed (−38% and −42%, respectively). The application of GA3 resulted in statistical significance between intact and emasculated fruits. The higher values were found using IN+SPY at all GA3 levels, with similar weights for all treatments, and in the IN+INJ control group. In contrast, the lowest values were found specifically for the control of the emasculated fruits using both INJ and SPY. The highest statistical significance was observed, indeed, between the control group of the IN+INJ and control group of EM+INJ combination treatment.
3.2. Fruit Defects
At harvest time, 39% of total fruit displayed defects. These defects were defined as (Table 2
, Figure 1
) (a) lignification on pulp tissue; (b) lignification of ovular tissue; (c) recalcitrant fruits (i.e., fruits that have not reached maturity).
Lignification on pulp tissue was only found in EM+INJ and IN+INJ combined treatments, while under EM+SPY and IN+SPY, this defect was not observed. Specifically, this defect was observed when GA3 treatments were applied. In particular, the highest and lowest defect percentages were found in the intact fruits using the injection method for low and the high GA3 levels, respectively.
Lignification of ovular tissue was observed in three of the four combined treatments, specifically EM+INJ, EM+SPY (10.10%), and IN+INJ. Only within the IN+SPY group was this defect not found. This defect was observed to a greater extent in the EM fruits, with the highest percentage observed within the control group of both the INJ (20.00%) and SPY methods. In contrast, in intact fruit, lignification of ovular tissue was observed only for the INJ method for the low GA3 level (6.70%).
Finally, recalcitrant fruits were found under EM+INJ, EM+SPY, and IN+INJ, while no defect was found using IN+SPY. Fruit recalcitrance was observed in the EM group, with the highest percentage observed in the control groups of both the injected and sprayed methods. The lignification of ovular tissue in IN fruits was observed only in the low GA3 group using the INJ method (13.30%).
The only group free of defects was IN+SPY under all GA3 levels. In contrast, the highest presence of defects was observed using the EM+INJ combined treatment, where only 15.70% of harvested fruits were healthy.
3.3. Seed Characterization
In all groups, the EM flowers showed the lowest average values for all seed variables: the number of seeds was 83.83 ± 8, the number of hard seeds was 12.17 ± 2, and the weight of seeds was 0.16 ± 0.02 g (Table 3
Without considering the GA3 levels, the combination treatment IN+INJ showed the highest average number of seeds. Only a slight difference was found between IN+INJ and the IN+SPY combined treatment (−1.3%), while, on average, a considerably lower number of seeds was found with EM+SPY (−52%) and EM+INJ (−57.5%). The GA3 levels resulted in statistical significance between intact and emasculated fruits. Specifically, more seeds were found in the IN fruits under most GA3 levels and application methods, with the only exception being the low GA3 level using the INJ method. In contrast, low values were observed using EM fruits under all GA3 treatments and methods, particularly in the control groups.
Regardless of GA3 levels, the highest number of hard seeds per fruit was in the IN+SPY combined treatment. Few differences were observed between IN+SPY and IN+INJ (−13.3%), while large differences were found under EM+SPY (−94.1%) and EM+INJ (−92%) combined treatments. The GA3 levels resulted in statistical significance between intact and emasculated fruits. In particular, the highest level of significance was found in the control groups of both injection and sprayed methods.
Finally, the average highest seed weight per fruit, without considering GA3 levels, was within IN+SPY. The lower average weight of seeds per fruit was found using IN+INJ (−32%) with respect to IN+SPY, while large differences in seed weight were observed using EM+SPY (−93.5%) and EM+INJ (−91.7%). Similarly, the GA3 levels for the seed weight per fruit resulted in statistical significance between intact and emasculated fruits. In particular, the highest level of significance was found under the control of both the INJ and SPY methods.
3.4. ANOVA Results
ANOVA assumptions showed that only the seed variables needed to be transformed. Afterwards, the statistical analysis showed the significant effects of fixed factors and their interactions on both fruit and seed variables (Table 4
). Emasculation treatment significantly influenced each variable for both fruits and seeds. The combined treatment SPY/INJ influenced fruit size, seed weight, and viability, but not the number of seeds per fruit. GA3
levels strongly influenced all variables and only moderately impacted the seed number per fruit. Interaction between all factors (EM/IN × SPY/INJ × GA) revealed that there was a strong effect among factors for all the variables.
The use of different application methods and GA3 concentrations in prickly pears to obtain well-formed and seedless fruits in O. ficus-indica (L.) Mill. “Gialla” provided several curious results.
The objective of obtaining well-formed fruits with few seeds was only partially achieved in this study. More well-formed fruits were obtained from the IN rather than the EM treatment, but the IN treatment produced a higher seed content. This was also observed in the aforementioned study by Mejía and Cantwell [38
], which found the emasculated fruits were generally smaller and had lower numbers of hard seeds (viable seeds) than the intact ones. This difference could be explained by the lack of stamens (emasculation), which contributed to the lack of or low development of the flower tissues. The external tissue of the anthers is the main gibberellin biosynthesis site, and thus the main regulating factor for the development of the remaining floral parts. This was deduced from Inglese et al. [37
], who observed that Opuntia ficus-indica
flowers with removed anthers show lower levels of endogenous gibberellin than pollinated ones. This behavior has been observed in other crops such as rice [43
] and Arabidopsis
The emasculated fruits showed a general decrease in all the analyzed variables (i.e., diameter, length, and weight) compared to the intact ones, regardless of the GA3
treatment applied. In the prickly pear, pulp development originates from the funiculus, which connects the seed to the ovular tissue, and thus seeds are needed for fruit development [32
]. On this basis, if the development of the ovular tissue and funiculus was inhibited, the Opuntia
’s fruit may have difficulty in developing properly [23
]. Despite the plausibility of the hypothesis, the gibberellin transport mechanism in the developing organs of the flower—from the male part (stamen) to the female part (ovary tissue and funiculus)—is not currently fully understood [46
Generally, it has been observed that treatment with GA3
may improve pulp development and reduce seed numbers in emasculated prickly pear fruits as a result of the replacement of endogenous gibberellins. This was also suggested by the fact that in Opuntia ficus-indica
, the highest levels of GA3
in the flowers were found during blooming [37
], and these, in turn, are responsible for the development of fruits and natural pollination [42
]. This has also been observed in other plant species, i.e., the Citrus
], where although gibberellic acid is not the only factor emulating the effects of natural pollination, the contribution of both pollination and exogenous GA3
application can improve fruit development.
However, the response of fruits and seeds to growth regulator treatments in this study depended on the specific application method. More specifically, while the spraying of GA3
(both low and high levels) generally enhanced the performance of all the analyzed variables of the treated fruits, the injection method showed the opposite pattern, especially for the intact fruits. One of the effects caused by gibberellic acid is control over the elongation of cellular tissues in plants [49
]. Generally, the exogenous sprayed GA3
is able to spread through the elongation of cells in plant tissues, thus facilitating the absorption and avoiding the direct negative effect of gibberellic acid within the parenchyma tissue [42
Whilst several studies have demonstrated that GA3
application can increase the presence of defects in Arabidopsis
], Coriadrum sativum
], Oryza sativa
], Zea mays
], and Daucus carota
], to our knowledge, only a few studies have investigated the effects of GA3
]. In this study, the main defects were lignification of the ovular and pulp tissue, and the presence of recalcitrant fruits. Lignification on pulp tissue was observed in both EM and IN fruits, particularly in the INJ groups. Specifically, most pulp tissue lignification was found corresponding to the needle entry hole for the GA3
injection. This condition may have been caused by two different factors. First, the needle was not able to reach the ovary, thus spreading the GA3
solution into the pulp. It is also possible that the injected compounds returned to the entry hole. This was partially deduced by Nobel et al. [56
], who observed that in Opuntia ficus-indica
tissues, injected gibberellic acid likely came into contact with expanding parenchyma tissue, thus leading to an excessive accumulation of dry matter in the tissue around the needle entry hole. This has been confirmed in several studies [42
], which proposed that the gibberellic acid in prickly pear fruits can cause a sink effect promoting dry matter accumulation in parenchyma tissue. This pattern was also indirectly supported by Jedidi Neji et al. [57
], in which injection of gibberellic acid into the ovary of the Opuntia
flower through the stigma and not the pulp did not result in lignification effects on the pulp tissue in fruits.
The lignification on ovular tissue, mostly recorded in EM fruits, was highest when the GA3
was sprayed rather than injected. Ortiz Hernandez et al. [58
] reported a similar behavior in an O. amyclaea
study: when the flowers were emasculated and treated with different growth regulators, the fruits showed ovarian tissue lignification. This result was likely driven by the flower emasculation rather than the method of GA3
application since the absence of stamens can cause a lack or little development of the remaining flower tissues. Gupta et al. [47
] suggested that, generally, even a short-distance movement of GA3
from the stamen to the other floral organs and the pedicel may be sufficient for flower development.
Recalcitrant fruits were found mostly in EM fruits. The highest incidence was observed within the control groups for EM+INJ (80%) and EM+SPY (53%). This result was likely because emasculation does not allow for the development of full fruit maturity, thus creating smaller fruits. These findings are consistent with a similar study carried out by Kaaniche-Elloumi et al. [23
], in which prickly pear emasculated fruit reached maturity after GA3
treatment. Besides, when comparing the IN control group fruits with the different GA3
treatments, an overall decrease in the number of seeds, the number of hard-coated seeds, and the weight of seeds was observed. This may confirm that exogenous GA3
can reduce the number of hard-coated seeds [35
]. Furthermore, seed abortion could be related to the effect of GA3
on chromosomal DNA, which may lead to the incomplete development of the endosperm [57
Prickly pear cultivation is important in several dry and semi-dry areas of the world owing to its diverse uses (e.g., as fresh fruit, in bioenergy, cosmetics, and medicine production, and as forage). The results of GA3 application on fruits indicated that 500 ppm of GA3 sprayed on emasculated floral buds was the most effective technique for reducing the number of seeds within prickly pear fruits. The spraying of the GA3 (both low and high levels) enhanced the growth performance of all the analyzed variables of the treated fruits, while the injection method, though capable of reducing the number of seeds, can increase the presence of defects, making the fruit unmarketable. The results suggested the need to further investigate the impact of higher GA3 concentrations on fruit production, and particularly, GA3 application methods, especially regarding industrial production. The GA3 spraying method would indeed be easier to apply in large-scale production than the injection method, whilst manual emasculation may be better replaced by chemical emasculation, which provides similar results. Given the scarcity of studies on prickly pear cultivation and the repercussions of its industrial processes, future studies should focus on these aspects by conducting experiments that directly address the application of these treatments in industrial-scale processes. Moreover, further studies should focus on the maximum thresholds of GA3 applicability and the physiological effects of the gibberellic acid pathway through productive tissue, thus elucidating the economic viability of this cultivation technique and the changes in fruit quality and organoleptic properties.