Pre-Germination Treatments, Temperature, and Light Conditions Improved Seed Germination of Passiflora incarnata L.

: Perennial medicinal and aromatic plants (MAPs) may represent interesting, environmentally friendly crops for the Mediterranean environments. Among MAPs, Passiﬂora incarnata L. (maypop) represents a very promising crop for its wide adaptability to diverse climatic conditions, low input requirements, and high added-value due to its unique medicinal properties. The main problem in P. incarnata large-scale cultivation is the poor seed quality with erratic and low seed germination, due to its apparent pronounced seed dormancy. Therefore, the aim of this work was to investigate different chemical and physical treatments for overpassing seed dormancy and enhancing seed germination rates of P. incarnata . The effects of (i) different pre-germination treatments (pre-chilling, gibberellic acid—GA 3 , leaching, scariﬁcation, non-treated control), (ii) light or darkness exposure, and (iii) temperature conditions (25, 30, and 35 ◦ C constant and 20–30 ◦ C alternating temperatures) have been examined in seed germination percentage and mean germination time of three P. incarnata accessions (F2016, FF2016, and A2016) grown in ﬁeld conditions in Central Italy. Data showed that the pre-germination treatments generally stimulated faster germination compared to the control, with the best results obtained in the dark and with high temperatures. These ﬁndings are useful for the choice of the most suitable seed pre-germination treatment that can facilitate stable, high and agronomically acceptable germination rates in P. incarnata. (iv) scariﬁcation, and (v) non-treated control. For each of these treatments, different light (or darkness) and temperature conditions (25, 30, and 35 ◦ C constant temperatures and 20–30 ◦ C alternating temperatures) were also examined. For each P. incarnata accession, four replications of 50 seeds each for every pre-germination treatment and for the control, were used.


Introduction
Among conservation agriculture practices, the introduction of perennials in crop rotations has been proposed as a viable opportunity to improve the long-term sustainability and productivity of systems thanks to the reduction in tillage, the protection of the soil surface, and the decrease in erosion and runoff. As a consequence, a considerable improvement in soil organic matter and nutrient cycling, as well as the overall physical and biological health of the soil, can be achieved. In this context, perennial medicinal and aromatic plants (MAPs) may represent interesting environmentally friendly crops for Mediterranean countries. In recent years, the attraction of MAPs as worthy farm crops has grown due to the demand created by consumer interest for these plants for culinary, medicinal, and other anthropogenic applications. Among MAPs, Passiflora incarnata could represent an interesting crop for Mediterranean systems, due to its perennial cycle and its potential agronomic benefits. Passiflora is a genus belonging to Passifloraceae's family, consisting of more than 500 species, which mostly live in tropical and subtropical regions, except for P. incarnata, which is native to temperate North America (southeast of the USA) and it has been introduced into Australasia, Bermuda, Europe, and Hawaii [1]. P. incarnata (maypop) is mainly cultivated for its pharmaceutical and cosmetic properties. It was historically used as a sedative and anxiolytic plant and for the treatment of insomnia in North America; as an analgesic, anti-spasmodic, anti-asthmatic, wormicide, and sedative

Plant Materials
The experiments were carried out at the Seed Research and Testing Laboratory of the Department of Agriculture, Food, and Environment (DAFE) of the University of Pisa.
The seeds of three P. incarnata accessions, namely F2016, FF2016, and A2016, were kindly supplied by F.I.P.P.O. (Federazione Italiana Produttori Piante Officinali) and by Aboca s.r.l. company (Sansepolcro, Arezzo, Italy). Mature fruits were collected during 2016 from plants grown in an open field in Central Italy (Tuscan-Umbrian, Val Tiberina, Italy), under the same pedo-climatic conditions and with an organic management system. The planting had been carried out in 2014 by transplanting the seedlings on a clay loamy soil. The crop was carried out without irrigation since rainfall in the area was able to satisfy crop water requirements.
During the growing period, between the flowering and ripening stages, until fruit harvesting (from August to October 2016), rainfall and temperatures (maximum and minimum air temperature) were recorded using a weather station located nearby the cultivation area ( Figure 1).
Agriculture 2021, 11, x FOR PEER REVIEW of three P. incarnata accessions grown in 2016 in Central Italy with different treatm (pre-chilling, GA3, leaching, scarification, non-treated control), different light or dark exposure, and different temperature conditions (25,30, and 35 °C constant tempera and 20-30 °C alternating temperatures) have been examined.

Plant Materials
The experiments were carried out at the Seed Research and Testing Laboratory o Department of Agriculture, Food, and Environment (DAFE) of the University of Pis The seeds of three P. incarnata accessions, namely F2016, FF2016, and A2016, kindly supplied by F.I.P.P.O. (Federazione Italiana Produttori Piante Officinali) an Aboca s.r.l. company (Sansepolcro, Arezzo, Italy). Mature fruits were collected du 2016 from plants grown in an open field in Central Italy (Tuscan-Umbrian Val Tibe Italy), under the same pedo-climatic conditions and with an organic management sy The planting had been carried out in 2014 by transplanting the seedlings on a clay lo soil. The crop was carried out without irrigation since rainfall in the area was able to isfy crop water requirements.
During the growing period, between the flowering and ripening stages, until harvesting (from August to October 2016), rainfall and temperatures (maximum minimum air temperature) were recorded using a weather station located nearb cultivation area ( Figure 1).

Pre-Germination Treatments
Fruits were harvested and soaked in tap water for a couple of days until macer occurred. At the end of maceration process, the seeds were separated from the washed with tap water at room temperature, air dried, and cleaned with sieves and f of air ( Figure 2). Subsequently, seeds were stored in darkness at 4-5 °C and 60% rel humidity for 6 months.

Pre-Germination Treatments
Fruits were harvested and soaked in tap water for a couple of days until maceration occurred. At the end of maceration process, the seeds were separated from the pulp, washed with tap water at room temperature, air dried, and cleaned with sieves and flows of air ( Figure 2). Subsequently, seeds were stored in darkness at 4-5 • C and 60% relative humidity for 6 months.
Different pre-germination treatments in a completely random block design have been examined. In detail, the treatments were: (i) pre-chilling, (ii) Gibberellic acid (GA 3 ), (iii) leaching, (iv) scarification, and (v) non-treated control. For each of these treatments, different light (or darkness) and temperature conditions (25,30, and 35 • C constant temperatures and 20-30 • C alternating temperatures) were also examined. For each P. incarnata accession, four replications of 50 seeds each for every pre-germination treatment and for the control, were used. Different pre-germination treatments in a completely random block design have been examined. In detail, the treatments were: (i) pre-chilling, (ii) Gibberellic acid (GA3), (iii) leaching, (iv) scarification, and (v) non-treated control. For each of these treatments, different light (or darkness) and temperature conditions (25,30, and 35 °C constant temperatures and 20-30 °C alternating temperatures) were also examined. For each P. incarnata accession, four replications of 50 seeds each for every pre-germination treatment and for the control, were used.
The seeds were placed in 12 cm Petri dishes and incubated in climatic cabinets. Preliminary tetrazolium tests, according to the International Seed Testing Association (ISTA) [12], were conducted to estimate the seed viability of each accession.
For pre-chilling treatment, seeds in groups of 25 were placed in Petri dishes between two sheets of filter paper and moistened with 5 mL of tap water, then they were put in a refrigerator at 4-5 °C for 4 days. The hormone treatment was conducted using GA3 (200 ppm or 0.2 g/L) prepared starting from gibberellic acid, 90% gibberellin A3 basis (TLC) (Fluka Biochemika, Buchs, Switzerland). Seeds were placed between two sheets of filter paper, moistened with 5 mL GA3 solution. The third treatment consisted of seed leaching in tap water for 8 h, which were then put in Petri dishes between two sheets of filter paper moistened with only 3 mL of tap water, because they were already partially imbibed. Finally, mechanical scarification was carried out by rubbing seeds manually on sandpaper to damage the hard outer layers, after which they were put in Petri dishes between two sheets of filter paper moistened with 5 mL of tap water. The dishes were put in climatized cabinets (

Germination Test and Measurements
Prior to the germination test, thousand seed weight, for each accession, was assessed according to ISTA [12]. Germination was monitored every two or three days up to 30 days as a function of temperature. Germination ended with the appearance of cotyledons. Germinated seeds were counted, and germination counts were stopped when final germination percentages were reached.
Germination percentage (G %) and mean of germination time (MGT) were calculated according to following equations:  The seeds were placed in 12 cm Petri dishes and incubated in climatic cabinets. Preliminary tetrazolium tests, according to the International Seed Testing Association (ISTA) [12], were conducted to estimate the seed viability of each accession.
For pre-chilling treatment, seeds in groups of 25 were placed in Petri dishes between two sheets of filter paper and moistened with 5 mL of tap water, then they were put in a refrigerator at 4-5 • C for 4 days. The hormone treatment was conducted using GA 3 (200 ppm or 0.2 g/L) prepared starting from gibberellic acid, 90% gibberellin A3 basis (TLC) (Fluka Biochemika, Buchs, Switzerland). Seeds were placed between two sheets of filter paper, moistened with 5 mL GA 3 solution. The third treatment consisted of seed leaching in tap water for 8 h, which were then put in Petri dishes between two sheets of filter paper moistened with only 3 mL of tap water, because they were already partially imbibed. Finally, mechanical scarification was carried out by rubbing seeds manually on sandpaper to damage the hard outer layers, after which they were put in Petri dishes between two sheets of filter paper moistened with 5 mL of tap water. The dishes were put in climatized cabinets (

Germination Test and Measurements
Prior to the germination test, thousand seed weight, for each accession, was assessed according to ISTA [12]. Germination was monitored every two or three days up to 30 days as a function of temperature. Germination ended with the appearance of cotyledons. Germinated seeds were counted, and germination counts were stopped when final germination percentages were reached.
Germination percentage (G %) and mean of germination time (MGT) were calculated according to following equations: -G (%) = S NG /S NO × 100; where S NG is the number of germinated seeds and S NO is the number of experimental seeds with viability, respectively. -MGT = Σ (n × d)/N, where n = number of germinated seeds per day; d = number of days needed for germination, and N = total number of germinated seeds.

Statistical Analyses
Data of both seed germination and MGT tests were subjected to an analysis of variance (ANOVA) using the statistical software Costat Cohort V6.201 (CoHort Software, Monterey, CA, USA). For each accession, the effect of pre-treatments (A, i.e., prechilling, GA 3 , leaching, scarification, and control) and the temperature/light treatments (B, i.e., different constant and alternating temperatures and light/dark conditions) and their reciprocal interactions Agriculture 2021, 11, 937 5 of 10 (A × B) were analyzed by two-way ANOVA. Means were separated on the basis of a least significant difference (LSD) test only when the ANOVA F-test per treatment was significant at the 0.05 probability level [21]. For germination percentage, data arcsin transformation [(x + 0.5)/n] 1/2 was performed before variance analysis.

Results
The 1000 seeds weights (TSW), evaluated before starting the experiment, are reported in Table 1. TSW significantly varied depending on accession, with the highest value reached by F2016, followed by A2016 and, finally, FF2016. The main results of the two-way ANOVA, performed to assess the effects of pregermination treatments (A), temperature/light (T&L) exposure (B), and A × B interaction on germination percentage and MGT, are reported in Table 2. The results showed that germination percentage was significantly affected by pre-germination treatments and T&L exposure, as well as by their reciprocal interactions, in all P. incarnata accessions. On the other hand, pre-germination treatments did not show any significant effect on MGT values of any accession. T&L exposure and the interaction between the two variability factors, instead, played a key role in affecting mean germination time in all three accessions.  Tables 3-5, the differences in germination percentage, separately for each P. incarnata accession, are reported. Regarding the effect of temperature and light/dark (L/D) conditions, no germination was achieved for the control at 25 • C both in light and dark conditions, confirming that this temperature value represents, for maypop, the below threshold for germination. In F2016 accession (Table 3), the highest germination rate was achieved at 35 • C/D, while the lowest one was observed at 25 • C/L. Considering the effect of pre-germination treatments, significantly higher germination percentages were registered with prechilling, GA 3 , and leaching in comparison with the control. Scarification worsened germination rate, with values equal to control. Taking into account the effect of AxB interaction, interestingly, the best conditions were found at 35 • C/D in control and after prechilling, GA 3 , and leaching. It is important to note that, in all other conditions (25 • C/D; 30 • C/D&L; 35 • C/L), the pre-germination treatments significantly increased the germination rates in comparison with the control, except for scarification.   For FF2016 accession (Table 4), the highest germination was obtained under both 35 • C/D and 30 • C/D, while, as observed for F2016, the lowest germination occurred by adopting the 25 • C/L condition. Differently to what was observed for F2016 accession, all the pregermination treatments significantly enhanced the germination percentage in FF2016 seeds. Considering AxB interaction, a significant improvement of germination, going from the control to pre-chilling under alternating temperatures of 20/30 • C (both photoperiods), as well as under 35 • C/L, was observed (Table 4). Furthermore, the combination between leaching and 35 • C/D conditions provided the highest germination percentage, followed by the seeds subjected to GA 3 and scarification treatments under the same T&L conditions. For A2016 accession (Table 5), a similar trend as described for FF2016 was detected. In fact, under 35 • C/D, the best germination conditions occurred, followed by 30 • C both in light and dark. On the contrary, as observed for the other accessions, under 25 • C (light and dark), the worst germination values were recorded. Once again, considering the AxB interaction, the best conditions able to enhance germination percentage were due to the combination of 20/30 • C 16/8 h and prechilling, and the combination of 35 • C/D and leaching.
All these findings revealed that, among accessions, the untreated/control seeds of F2016 had the highest germination rate (germination percentage up to 90%) when exposed to 35 • C under dark conditions. This behavior confirmed that, for P. incarnata, the optimal germination can be achieved at 35 • C in the dark. In such conditions, the untreated seeds of FF2016 and A2016 achieved lower germination percentages (around to 60%) than F2016. The higher values observed for control seeds of F2016 were expected on the basis of 1000-seed weight (Table 1). In FF2016 and A2016 accessions, pre-germination treatments were absolutely necessary in order to improve the germination process.
MGT values showed that germination peaks usually occurred within two weeks. Beyond this time, seeds sporadically sprouted. As a general trend, the time required for germination ( Figure 3) decreased progressively from light to dark conditions, depending on pre-germination treatments, accession, and temperature. In F2016, this behavior was particularly evident under 35 • C, while no differences were observed between light and dark at 30 • C. Conversely, in FF2016 and A2016, a strong decrease in MGT was observed from light to dark, both at 30 • C and 35 • C. MGT lasted roughly ten days in light conditions (as mean value among accessions and pre-germination treatments) but fell to about one week in dark conditions. Finally, considering the average values over the three accessions, all the pre-germination treatments generally stimulated a faster germination compared to control. Among the tested treatments, scarification seemed to lead to a quick germination process, even if germination rate was not elevated.

Discussion
In the effort to improve and promote the cultivation of P. incarnata, the effects of temperature, dark/light conditions, and pre-germination treatments on the germination percentage and MGT of its seeds were investigated. The 1000 seeds weight (TSW) was also evaluated as it represents one of the most important components in determining seed quality. In fact, it is generally reported that seed germinability is positively related to seed mass. Larger seeds germinated to a higher percentage thanks to their greater reserves, which enable a greater tolerance to a range of hazards, including shade, drought, and physical damage [22][23][24].
Definitively, the obtained results confirmed that P. incarnata seeds are photoblastically negative and have pronounced heat requirements for germination. Optimal germination percentages, in fact, were achieved with 35 • C in darkness, for each accession. In such conditions, a significant and strong decline in MGT was also detected, confirming the tropical origins of this species [25]. On the contrary, very low values were observed at 25 • C, more pronounced under light conditions, for each pre-germination treatment and seed accession. Data showed a significant interaction between complete light/dark exposition and temperatures, underlining the fact that the light exposition has an inhibitory effect on the germination of P. incarnata seeds. Among pre-germination treatments, pre-chilling, GA 3 , and leaching appeared to be the most effective in enhancing normal seedling germination. Only for A2016, scarification gave similar results to pre-chilling, GA 3 , and leaching treatments. On the contrary, in the other two accessions, under scarification, the dead seeds percentage considerably increased, probably due to embryo damaging. A significant interaction between pre-chilling and temperature was observed with significantly higher germination values than control (+330%) at 20/30 • C (16/8 h).
Previous studies investigated the effect of different combinations of light and different temperature regimes with the aim to improve seed germination in P. incarnata. In this regard, Benvenuti et al. [18] tested combinations of white light (or darkness) and temperature (20,25,30,35, and 40 • C), or subjected P. incarnata seeds to different sequences of light treatments (succession of red and far-red light, with 5 min each one) after 12 h of dark incubation at 30 • C. These authors found that the germination threshold was surprisingly high, both in darkness and light conditions (25.4 • C and 23.9 • C, respectively), while no germination was observed at 20 • C. In addition, these authors observed that a suboptimal temperature (lower than 35 • C) and far/far-red light both produced extremely low levels of germination (around 5%). Zucarelli et al. [19] investigated the effect of alternate temperatures of 20-30 • C and 30-20 • C for periods of 16 and 8 h, simulating the photoperiod and highlighting that alternating temperatures of 30-20 • C promoted the highest germination rates. The results of these studies also demonstrated that high temperatures progressively decreased the time required for germination, independently from light conditions. Similarly, in our study, MGT decreased as result of the combination of high temperatures (35 • C) and dark conditions, regardless of the pre-germination treatment.
In the literature, several studies have underlined the presence of dormancy in Passiflora spp. [16,26] and, specifically in P. incarnata, a combination of physical and physiological dormancy has been detected [27]. In general, all seed pre-germination treatments led to enhanced water and oxygen exchange across seed coat layers. However, scarification did not have the best result, as was supposed to be the case with physical dormancy. Otherwise, results obtained with GA 3 and leaching seem to confirm a possible physiological dormancy in P. incarnata, as hormones and inhibitors were stimulated/removed, respectively.
Interestingly, our study, for the first time, pointed out that pre-chilling is an efficient treatment to improve germination in P. incarnata seeds. Pre-chilling enhanced germi-nation as well, probably because this pre-treatment stimulated a variation in abscisic acid/gibberellic acid rate (ABA/GA 3 ) and free gibberellins biosynthesis [28]. Macchia et al. [29] found that prechilling for 7-15 days in light or in darkness hardly affected percentage germination of Echinacea angustifolia seeds, but significantly increased the rate of germination. On the contrary, GA 3 treatment was not useful for this species.
The time conclusively required for seed germination decreased progressively with increasing temperatures, but only under dark conditions, while in complete light conditions, no variation was observed and MGT values remained almost constant with increasing temperatures. Optimal germination times were achieved at 35 • C in dark conditions. Similarly to germination percentages, even in MGT, alternating temperature (20/30 • C) did not improve germination energy, except when combined with pre-chilling treatment.

Conclusions
The use of high-quality seeds is an important key factor in modern agriculture to obtain a successful nursery and crop production, and particularly to enhance food security. In fact, for a rapid and uniform crop establishment, the selection of good quality seeds with improved vigor is of primary importance in order to enhance this critical and yield-defining stage. Currently, in Italy, there are difficulties in the large-scale cultivation of Passiflora incarnata, starting from the seed, due to dormancy. An enhancement of seed performance during germination and seedling emergence is required for an efficient and competitive nursery production. Little is known about the conditions under which the germination process takes place and the treatments necessary to remove dormancy, since no guidelines have been reported by the International Seed Testing Association (ISTA) for this species. Thus, this study identified the main and most significant pre-germination treatments and environmental parameters for improving the seed germination percentage and germination energy of P. incarnata under controlled conditions. This makes possible to achieve stable and agronomically satisfactory germination rates, thereby reducing seed propagation costs for this species.