Conservation of seeds of cactaceae species endemic to the caatinga biome: Pilosocereus pachycladus and Tacinga inamoena

ABSTRACT Ecosystems with dry soils are particularly sensitive to climate changes and anthropogenic actions, which represents a threat to the survival of many cactus species. Ex situ seed conservation strategies should be adopted to support in situ conservation. This study evaluated the effect of conservation conditions on germination and vigor of seeds of Pilosocereus pachycladus subsp. pernambucoensis and Tacinga inamoena. The seeds were packaged in paper bags and glass containers and stored under controlled (8 ± 1 °C; 56 ± 2% relative humidity) and non-controlled (24 ± 2 °C; 75 ± 5% relative humidity) environmental conditions for 0 (control), 12, 16, and 20 months. The experiment was conducted in a completely randomized design, in a 2 × 2 × 4 factorial arrangement (conservation environment × packaging × storage period) for each species. The variables evaluated were: water content (%), germination (%), germination speed index (GSI), and mean germination time (MGT). The physiological quality of seeds of both species were preserved when seeds were maintained under controlled environment, regardless of the packaging. However, when stored under non-controlled environment, the packaging in paper bags was more efficient for the conservation of seeds of P. pachycladus subsp. pernambucoensis, and the glass container was more efficient for T. inamoena. This information may be needed for ex situ conservation of these species and to support the recovery of degraded areas susceptible to desertification in the Caatinga biome.


INTRODUCTION
There are 484 cactus species (Cactaceae) distributed throughout the Brazilian territory, including Pilosocereus pachycladus subsp.pernambucoensis (Ritter) Zappi and Tacinga inamoena (K.Schum.)N.P.Taylor & Stuppy, which are two species endemic to dry tropical forests of the Caatinga biome (ZAPPI; TAYLOR, 2020).P. pachycladus subsp.pernambucoensis is an arboreal columnar cactus that can reach up to 6.0 m height, and T. inamoena is a subshrub cactus that presents plain cladodes and red-orangish flowers (BATISTA et al., 2018).Both species are found over almost the whole Semiarid region of Brazil and have a high importance for the sustainability and conservation of the Caatinga biodiversity, as their flowers and fruits serve as food for the local fauna (NASCIMENTO et al., 2015;SOUSA NETO;GOMES;QUIRINO, 2021;PAIXÃO;GOMES;VENTICINQUE, 2021).
Although Cactaceae species are an important component of the Caatinga flora (BARBOSA et al., 2020a), populations of many species have been subjected to predatory exploitation and degradation of their habitats (MEIADO; ALMEIDA, 2022), making Cactaceae one of the ten most threatened botanical families in the Brazilian flora (GOMES et al., 2020).Natural Cactaceae populations are subjected to strong anthropogenic pressures, which considerably increases risks of extinction (OLIVEIRA et al., 2020).Natural disturbances by herbivory or anthropogenic causes, environmental conditions, and availability of resources affect the populational structure and distribution of P. pachycladus subsp.pernambucoensis and T. inamoena (BARBOSA et al., 2020b;OLIVEIRA et al., 2020).Despite the current conservation status of these two species are "little worrisome", recent studies indicate a significant decrease in the distribution of these and other cactus species in their original sites of occurrence, mainly for the future scenario of climate changes (CARVALHO et al., 2021;CAVALCANTE;SAMPAIO, 2022).
Considering that Cactaceae species present long reproduction cycles, lasting several years to start their reproduction phenological phase, seed storage is an alternative for the maintenance of seed stocks for these species (SILVA; AMARIZ; KIILL, 2018).Understanding long-term effects of storage on seed germination is important to assess the dynamics of the soil seed bank regarding its preservation capacity.However, information about the family Cactaceae is scarce, although essential for conservation programs (GURVICH et al., 2021;ROJAS-ARÉCHIGA;GARCÍA-MORALES, 2022).
Researches correlating packaging and conservation environment for seeds of native Cactaceae species are incipient in Brazil, and some studies on the species Pilosocereus gounellei (Xiquexique gounellei) (ABUD et al., 2012) and Cereus jamacaru (ABUD et al., 2016;TARGINO et al., 2021) are highlighted for presenting important contributions about conservation methods.According to these studies, the seed viability was maintained when the seeds were packaged in paper, plastic, and glass containers and stored under low-temperature environments.However, under natural environment, the kraft paper bag provided the ideal conditions for conservation of seed viability for these species.
The storage of seeds in adequate temperature and relative air humidity conditions favors the maintenance of their physiological potential, decreasing the respiratory process and, thus, decreasing the seed deterioration process (CARVALHO; NAKAGAWA, 2000;MARCOS-FILHO, 2015).This deterioration also depends on the packaging used for seed storage, which are classified as permeable (porous), semipermeable (semiporous), and impermeable (hermetic), facilitating or inhibiting gas and water vapor exchanges between the seeds and the storage environment (SILVA; FERRAZ, 2015).
The increasing threats to the family Cactaceae show the need to seek safe alternatives for long-term preservation, which can be even simple ways that are within the reach of anyone who wants to preserve these seeds at home.In addition, despite some studies evaluated Cactaceae seeds in other areas of the Caatinga biome, it is important to investigate these seeds in the municipalities of Bananeiras and São João do Cariri, state of Paraíba, Brazil, mainly focused on their physiological potential, and whether they can maintain their viability and vigor over time.
The objective of this study was to evaluate the effect of conservation conditions on germination and vigor of seeds of P. pachycladus subsp.pernambucoensis and T. inamoena.

MATERIAL AND METHODS
The experiment was conducted at the Laboratory of Seed Technology of the Center for Human, Social, and Agricultural Sciences of the Federal University of Paraíba, Campus III, Bananeiras, Paraíba (PB), Brazil.The seeds of P. pachycladus subsp.pernambucoensis and T. inamoena used were from mature fruits collected from adult individuals from two populations of natural occurrence in the municipalities of Bananeiras and São João do Cariri, PB, Brazil, respectively (Figure 1).The predominant vegetation in these municipalities is formed by Deciduous and Semideciduous Forests typical of the Agreste, which is a spatial region between the Sertão (a semiarid region with predominance of caatinga vegetation) and the Zona da Mata region (consisted of Atlantic Forest).
The pulp was removed from the fruits through maceration in a fine-mesh metal sieve, with subsequent washing in running water.The seeds were then homogenized and shade dried (24 ± 2 °C) for four days.The seeds were then packaged in two package types (paper bags and glass containers), except those of the control group, and stored under controlled environment (refrigerator 8 ± 1 °C; 56 ± 2% relative humidity) and non-controlled environment (laboratory 24 ± 2 °C; 75 ± 5% relative humidity) for 0, 12, 16, and 20 months.Glass containers were filled with cotton up to the top to avoid empty spaces inside them and sealed with a rubber cover and adhesive tape.
A completely randomized statistical design was used, in a 2 × 2 × 4 factorial arrangement (conservation environment × packaging × storage period), resulting in 16 treatments for each species.
The seeds (four replications of 50 seeds) were germinated in paper substrate (between blotting papers) previously sterilized and moistened with distilled water at the volume (mL) of 2.5-fold the paper dry weight inside transparent plastic boxes with lids (11 × 11 × 3.5 cm).The seeds were maintained in a germinator under constant temperature (25 ± 1 °C) and light, based on preliminary tests.
The number of germinated seeds in each experimental test was counted daily for 21 days for P. pachycladus subsp.pernambucoensis, and for 30 days for T. inamoena; the root protrusion was the criterion adopted to consider the seeds as germinated.Water content (%), germination (%), and germination speed index (GSI) were evaluated according to equation proposed by Maguire (1962); and mean germination time (MGT) was calculated using the formula proposed by Labouriau (1983).The seed water content was determined by The statistical analyses were carried out using the softwares ESTAT/Jaboticabal ® and Microcal Origin ® 6.0.The data of normality and homogeneity of variances were assessed using the Shapiro-Wilk and Bartlett tests, respectively.The data were subjected to analysis of variance by the F test and the means were then compared by the Tukey's test (p≤0.05).First, the fit of the data to polynomial and linear equations was carried out, but the data was fitted according to the methodology used by Targino et al. ( 2021) due to the occurrence of negative estimates for some characteristics.

RESULTS AND DISCUSSION
The results obtained for the variables as a function of the two species studied and seed storage conditions are presented in Table 1.According to the analysis of variance, the effect of the interaction between the factors and the isolated factors were significant for germination (%) and MGT for seeds of P. pachycladus subsp.pernambucoensis (Table 1).However, only the effects of the interaction conservation conditions × storage period and the isolated effects of the factors conditions and storage period were significant for GSI.Regarding the species Tacinga inamoena, the effects of all factors, isolated and their interactions, were significant for MGT and GSI, except for the factor packaging.The effects of conservation conditions × storage period, and packaging × storage period, and factor conservation conditions were significant for germination (Table 1).placing them in an oven at 105 ± 3 °C for 24 hours, using two replications of approximately 100 seeds.The evaluations were carried out following the criteria established by the Rules for Seed Analysis (BRASIL, 2009).

Seed water content
The water contents before the experimental tests (period zero) were 12.79% and 11.48% for seeds of P. pachycladus subsp.pernambucoensis and T. inamoena, respectively.The water contents, ranging from 8.6% to 15.9% for P. pachycladus subsp.pernambucoensis seeds and from 7.4% to 12.4% for T. inamoena seeds over the storage period, depending on the packaging type and storage environment (Table 2). 1 The water content in seeds of P. pachycladus subsp.pernambucoensis was higher in the glass containers, ranging from 14% to 16% in the two storage environments (Table 2).The recommended seed water content for impermeable packages is lower than or equal to 10%.According to Marcos-Filho (2015), the seed moisture and the environment air temperature and relative humidity are factors that significantly affect the deterioration process over the storage period.The water content found in the present work were higher than that recommended for the storage period, which probably triggered changes in moisture over the storage period and accelerated the respiratory activity of seeds.

Conservation of seeds of Pilosocereus pachycladus subsp. pernambucoensis
The germination of seeds of P. pachycladus subsp.pernambucoensis at the beginning of the storage period under controlled environment was high (95%) (Figure 2A).However, it decreased over the storage period, reaching 80% for those seeds packaged in glass containers and 88% for those packaged in paper bags.This decrease in germination potential of seeds packaged in glass containers is probably connected to the initial seed water content (12.7%), which increased the seed metabolic activity over the storage period, as explained by Silva and Ferraz (2015).The data of seeds packaged in paper bags fitted to a quadratic equation and those of seeds packaged in glass containers fitted to a decreasing linear equation.The decrease in germination percentage of seeds stored under non-controlled environment (Figure 2B) was even more pronounced, mainly for those packaged in glass containers, whose germination percentage was zero for all tested periods, except for the control.The germination percentage of seeds packaged in paper bags and stored under non-controlled environmental conditions decreased 50%, compared to that obtained before storage (control), within only 11 months (Figure 2B).Germination speed index (GSI) of seeds stored under controlled environment presented small decreases for both packaging; the data fitted to the linear model (Figure 2C).GSI of seeds before storage was 11.08; after 20 months of storage, it was 7.14 and 7.34 for seeds packaged in paper bags and glass containers, respectively.Considering the equations obtained by the logistic-1 model, the storing of P. pachycladus subsp.pernambucoensis seeds packaged in glass containers and paper bags for 1.4 and 8.4 months resulted in a decrease of 50% in GSI (Figure 2D).According to Marcos-Filho (2015), a low germination speed is one of the indicators of low physiological potential, and one of the most frequent symptoms of the deterioration process.
The seeds with higher germination and GSI were those stored under controlled environment, regardless of the packaging and storage periods (Figures 2A and 2C).This response can be due to a decrease in seed metabolism and to degradation of reserve compounds in seeds under low temperatures (SANTOS; HASSEMER; MEIADO, 2018).
Regarding the mean germination time (MGT), the data of seeds stored under controlled environment fitted to a decreasing linear equation and the data of seeds stored under non-controlled environment fitted to the logistic-1 model, for both packaging.Seeds stored under controlled environment (Figure 2E) needed approximately 4 to 6 days to germinate, depending on the packaging type and storage period.The MGT data were significant only for seeds packaged in glass containers (Figure 2E).MGT of seeds packaged in glass containers and paper bags and stored under non-controlled environment increased during the storage period (Figure 2F) from four days (control) to 11 and 13 days after 12 and 16 months, respectively.
The paper bags were more efficient than the glass containers for storing seeds under non-controlled environment (Figures 2B,2D and 2F).This result reinforces the results found by Abud et al. (2016) andTargino et al. (2021), who evaluated the storing of seeds of Cereus jamacaru and reported that the germination and vigor of seeds stored under non-controlled environment decreased over the storage period, mainly for those packaged in glass containers.
The viability of seeds of Cactaceae species varies significantly, as the seeds of some species can remain viable for one or two years under non-controlled environment, whereas others remain viable for almost a decade (TRUJILLO et al., 2014;GURVICH et al., 2021).Seeds of Cephalocereus polylophus germinated above 90% under laboratory conditions, even two years after they were packaged in paper bags and stored under room conditions (ORTIZ-MARTÍNEZ et al., 2021).Other studies showed increases in germination of seeds of Ferocactus peninsulae after 10 months of storage under room temperature (20 ± 2 °C), with viability maintained for 48 months (ROJAS-ARÉCHIGA; GARCÍA-MORALES, 2022).According to these studies, the germination speed also increased over the storage time, denoting that the seed physiological dormancy was overcome over the post-maturation period, i.e., the germination can increase over the storage time under adequate conditions, maintaining the seed viability for long periods, depending on the cactus species.
Other studies on Cactaceae species reported decreases in seed viability over the storage period when the seeds were stored under non-controlled environment, regardless of the packaging used (ABUD et al., 2012;SANTOS;HASSEMER;MEIADO, 2018;GURVICH et al., 2021).Seeds of Pilosocereus and Cereus species lose their viability and vigor when stored under room temperature (25-27 °C) for 13 months, but when stored in low temperature environments (≤ 8 °C) they can have germination similar to that of freshlycollected seeds (SANTOS; HASSEMER; MEIADO, 2018).Similarly, Silva, Amariz, and Kiill (2018) found that the storage of seeds of Melocactus bahiensis and Harrisia adscendens in cold chamber (10 ± 2 °C) maintained their physiological quality for 7 years, despite the low germination percentage, which did not exceed 35% for any of the species or treatments evaluated.These findings indicate that ex situ conservation under low temperatures is the most efficient form to maintain the viability of seeds of most cactus species, mainly endangered species.

Tacinga inamoena seed conservation
The germination of T. inamoena seeds before storage was 14% (Figure 3A); however, this percentage varied from 14% to 28% under controlled conditions over the storage period, depending on the packaging type and storage period.Under non-controlled conditions, the germination after 12 months was 2% and 6% for seeds packaged in paper bags and glass containers, respectively, but after 16 and 20 months, only the seeds packaged in glass containers germinated, reaching 6% and 9%, respectively (Figure 3B).
The germination percentage of seeds packaged in paper bags fitted to quadratic (controlled environment) (Figure 3A) and logistic (non-controlled environment) (Figure 3B) equations.The germination data of seeds packaged in glass containers in the two conservation conditions were not significant (Figures 3A and 3B).According to the logistic equation, the xc value that resulted in 50% of the maximum value of the characteristic (G%) for seeds packaged in paper bags under non-controlled environment was approximately 10 months (Figure 3B), which is equivalent to 7% germination.Seeds stored under controlled environment expressed higher germination percentage than those under non-controlled environment; however, this percentage did not exceed 30% in any of the treatments evaluated.Meiado (2012) investigated the formation of cactus seed banks in areas with caatinga vegetation and found low germination for the species T. inamoena subsp.inamoena and T. palmadora in the first evaluation (≤ 10%), but a significant increase in germinability after 12 and 24 months (periods when the seeds remained buried in the soil), which did not exceed 50% for any of the species or treatments evaluated.Nascimento et al. (2015) found low germination (≤ 32%) for T. inamoena seeds from freshly-collected fruits and after passing through the digestive tract of Chelonoidis carbonaria; they reported that the low germinability of this species may be connected to the production of seeds without embryo or to presence of some type of dormancy, common characteristics in the subfamily Opuntioideae.

Table 2 .
Water contents (%) in seeds of Pilosocereus pachycladus subsp.pernambucoensis and Tacinga inamoena, packaged in two packaging types and stored in two conservation conditions for twenty months.