Plant Regeneration Protocol for Recalcitrant Passionﬂower ( Passiﬂora quadrangularis L.)

: This research was designed to provide the ﬁrst protocol to establish an efﬁcient solution for direct organogenesis regeneration in Passiﬂora quadrangularis using nodal explants from young shoots. Passiﬂoraceae tissue culture has been associated with problems such as recalcitrance, sensitivity to ethylene accumulation and browning of explants due to the presence of phenols in the tissues. Due to the high rate of endogenous contamination of the explants, a preliminary experiment was performed. The best results of surface sterilization were obtained using the pretreatment with 70% EtOH, 1 min and 50% NaOCl, 10 min along with the treatment of Rifampicin 15 µ g/mL and Benomyl 2 g/L. The effects of plant growth regulators on the induction of direct organogenesis, multiplication of shoots in subcultures and in vitro rooting were evaluated. Additional compounds such as AgNO 3 and Pluronic F-68 were added to the culture media in order to reduce the effects of phenols and the sudden browning of the explants. Shoot proliferation increased to the sixth subculture after which it decreased. A maximum of 7.17 shoots were obtained from one shoot on Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine and 1 mg/L thidiazuron. Supplementation of 1 / 2 MS medium with 1 mg/L 1-naphthaleneacetic acid was conducing to root formation in 61.11% of shoots. After acclimatization, the plants showed vigorous growth, green leaves, and well-developed roots. Although this species has previously shown difﬁculty in in vitro propagation, this protocol established based on the results proved to be efﬁcient and reproducible.


Introduction
The genus Passiflora is the largest of the Passifloraceae family [1], consisting of 576 species [2], of which only 50 are edible [3]. Passiflora quadrangularis (giant granadilla) native to the Andean Region, South America [4] is a vine that produces big fruits [5]. In recent years, growers have paid attention to the species P. quadrangularis due to its ornamental value, fragrant flowers [6], special fruit aroma, phytocompounds present in various organs of the plant [7][8][9] and phytotherapeutic properties [10], being used in traditional medicine [11,12]. Passiflora is an allogamous plant with self-incompatible flowers for most species belonging to the genus [13].
The production of planting material of P. quadrangularis by conventional methods has proven to be problematic due to the low germination rate of the seeds. At the same time, the presence of disease-causing microorganisms significantly reduces the efficiency of propagation by cuttings [14,15]. Passiflora spp. secretes a sweet sap that is a nutrient medium for many microorganisms [14]. Passionflower is often infected with Passion fruit Woodiness Virus [16][17][18] and Xanthomonas axonopodis pv. passiflorae [19,20], two pathogens that decrease their exploitation duration and decorative utility. Regarding the possibilities discs and axillary buds, and the present research was preceded by a preliminary study in which four culture media and two types of explants (nodal segments and leaf fragments) were tested [52].
The present research aims to establish an efficient solution for regeneration by direct organogenesis, using nodal explants from young shoots and the characterization of morphogenic responses, results confirmed by anatomical analysis.
The objectives of this study were to establish the optimal disinfection treatment of explants from nodal segments in P. quadrangularis, to inspect the influence of cytokinins and, as the case, auxins, at each stage of micropropagation. The study also analyzed whether the supplementation of culture media with AgNO 3 or PF-68 reduces recalcitrance, manifested by in vitro tissue browning in P. quadrangularis.

Source of Plant Materials and Explants
The biological material came from one-year-old P. quadrangularis plants, which were in the vegetative growth stage. The experimental plants were grown in the Didactic and Experimental Greenhouse of the discipline of Ornamental Plants, at the University of Agricultural Sciences and Veterinary Medicine (UASVM) Cluj-Napoca, Romania. These plants were obtained from cuttings from Naples Botanical Garden, USA.
The biological material used to initiate the in vitro culture were nodal segments taken from nodes 3-8 in the apical part of the young shoots.

Culture Media and Growth Conditions
In the case of each experiment, the explants were cultured in MS medium including vitamins, 30 g/L sucrose and 2.5 g/L Gelrite. Depending on the purpose, plant growth regulators (PGRs) ( Table 1) or phenolic compound inhibitors have been added. Thus, AgNO 3 [0, 1 or 2 mg/L)] was added to the culture medium from the stock solution before autoclaving and PF-68 ((0, 0.2 and 0.4% (w/v)) by filter sterilization using Millipore filters (0.22 µm), described below for each step.  The pH was adjusted with 1N NaOH or 1N HCl to 5.8. Distribution of the solution for culture initiation was performed in test tubes (10 mL/tube) and for subculturing and rooting in culture flasks (30-50 mL). The sterilization was performed by autoclaving at 121 • C, under a pressure of 101,325-121,590 Pa, for 20 min.
All cultures were maintained in the growth chamber at a photoperiod of 16 h light/8 h dark and a temperature of 25 ± 2 • C. Gro-Lux F36W/GRO fluorescent tubes (Sylvania, Germany) with an irradiance capacity of 100-112 µmol/m 2 /s were used.

Explant Asepsis
For asepsis of explants from nodal segments, 15 treatments were tested (Table 2). A total of 450 explants were initiated in order to identify the optimal disinfection treatment. The stems were shaped to prepare the nodal explants and washed in a continuous stream of water for 20 min. Then, in the case of T1-T9 treatments (Table 2), they were kept in a 70% EtOH solution with 2-3 drops of Tween 20 for 1 min and rinsed with sterile distilled water three times. This was followed by treatment with NaClO solution in different concentrations (15%, 25%, 50%) at three exposure times (10, 15 and 20 min) then three rinses with sterile distilled water were performed. In the case of T10-T15 treatments, surface disinfection of explants was preceded by treatment with the antibiotic Rifampicin 15 µg/mL, or with the fungicide Benomyl 2 g/L or a combination of Rifampicin + Benomyl at two exposure times, 15 and 30 min. The aseptic treatment consisted of maintaining the explants in 70% EtOH for 1 min, then in 50% NaClO solution, 10 min applying the protocol described above. In the preparation of pretreatments, Rifampicin was first solubilized using dimethylsulfoxide, followed by dilution in sterile distilled water. Benomyl fungicide was solubilized directly in sterile distilled water. After rinsing with autoclaved water, the stem fragments were shaped into 1 cm nodal segments with latent axillary buds. The explants were inoculated on MS-0 medium (without phytohormones). All operations were performed in aseptic conditions, in the laminar flow hood. Contamination data were registered 2-14 days after inoculation of the explants.

Initiation and Stabilization of In Vitro Plantlets
The experimental model of the initiation phase aims to determine the influence of cytokinins, ethylene inhibitors and the position of the explant on the regeneration axillary shoots. A total of 1020 nodal explants were initiated in order to stabilize the in vitro culture of P. quadrangularis in the form of three bifactorial experiments. All these explants were surface sterilized using the T14 treatment. The first study was on the influence of cytokinins on axillary shooting, then a study on the influence of the explant position and the culture medium, and the third experiment was on the influence of culture medium and additional compounds on axillary shooting. From an experimental point of view, the initiation phase was divided into two stages. In the preliminary initiation step (identified as the first experiment), the MS culture medium was supplemented with 6-benzylaminopurine (BAP) only Because in this first stage explants were regenerated in very low percentages, the experiment was continued with a secondary initiation stage, using the cytokinin concentrations that obtained the highest regeneration rate. Thus, MS + 2 mg/L BAP became MS-1, and MS + 2 mg/L BAP + 1 mg/L KIN became MS-2. Additional compounds such as AgNO 3 (0, 1 or 2 mg/L) and PF-68 (0, 0.2 and 0.4% (w/v)) were added to MS-1 and MS-2, in order to reduce the effects of leached phenols and sudden browning of explants. The explants from the nodal segments, 10 mm in thickness, were inoculated on the medium in a vertical and horizontal position. After 6 weeks, the following parameters were determined: regeneration frequency (%), callus incidence (%), browning (%), phenolic secretion (%), number of shoots per explant and shoot length (cm).

Shoot Subculturing
In vitro regenerated shoots were multiplied by repeated subcultures on fresh culture medium every 4 weeks. In the multiplication stage, three types of culture medium indicated in the literature were tested [53] for good results in the multiplication of passionflowers. Multiplication was continued on the MS-2 (MS + 2 mg/L BAP + 1 mg/L KIN) medium, on MS-3 represented by MS medium supplemented with 2 mg/L BAP and 1 mg/L TDZ and MS-4 being the MS medium with 2 mg/L BAP and 0.5 mg/L 1-naphthaleneacetic acid (NAA).
All three culture media were supplemented with PF-68 0.2% (w/v) in order to reduce the effects of leached phenols. The antibiotic Kanamycin was added to the culture medium at concentrations of 50-150 mg/mL to control endogenous infections. Callusing frequency and biometric parameters were determined for each subculture, statistical analysis was performed for subcultures 2 and 6.

In Vitro Rooting of Regenerants
The microshoots obtained after the sixth subculture represented the biological material used for the study of the in vitro rooting capacity. To induce rooting, the culture medium MS was reduced to half concentration, supplemented with auxins: 1 mg/L NAA and 1 mg/L indole-3-butyric acid (IBA). Each medium was supplemented with PF-68 0.2% (w/v). After 4 weeks, the regenerative response was determined (expressed as the number of days from inoculation to the appearance of the first root), the rate of rhizogenesis (expressed as a percentage) and the number of roots (calculated average value/plant).

Acclimatization of Regenerants
After in vitro rooting, the seedlings were removed from the culture flasks and washed with distilled water to remove residual culture media from the roots. These were transferred into pots containing a mixture of peat and perlite 1:1 or peat and vermiculite 1:1. Initially, the plants were covered with polyethylene bags to maintain 100% relative humidity. Then, for 2 weeks at 25 ± 2 • C, 70% humidity and a photoperiod of 16 h light/8 h darkness were maintained in climate-controlled storage rooms for 2 weeks. Further, the plants were transferred to greenhouse conditions and watered every 2 days. After a period of 4 months of acclimatization, the plants' survival rate was determined.

Experimental Design and Data Analysis
To determine the importance of disinfection treatments, growth phytoregulators and additional compounds (AgNO 3 and PF-68), a completely randomized experimental design was chosen. Each treatment was applied in 3 repetitions and each repetition consisted of 10 samples/replicate. To test the degree of endogenous infection, simple tests (smears) were performed. A sample from an infected plantlet was fixed on a microscope slide in the presence of a coloring agent (basic fuchsin). Specimens of bacteria and fungi from infected plants were then viewed under a binocular microscope (Motic B1-252 Binocular Microscope, with 100X objective lenses).
Histological studies were performed to determine the regenerative cell layers. For this purpose, the nodal regenerative tissues were fixed in Carnoy no. 2 solution [54] (6:3:1 EtOH:chloroform:acetic acid) for 3 h. The fixed material in Carnoy was washed and dehydrated in 90% EtOH, then in absolute EtOH twice. Clarification was performed in a mixture of absolute EtOH + chloroform (3:1; 1:1 and 1:3) than in pure chloroform followed by inclusion in paraffin. The vegetal parts were left in the above mixtures and pure chloroform until they became submerged [55]. Sections of 7-8 µm thickness were cut with a rotative microtome (Sakura Accu-Cut SRM 200) and fixed on glass slides then observed under an Olympus CKX41SF microscope with a 40X objective. The observations regarding the formation of the shoots were made with a binocular stereo magnifier Motic DM 143.
Data were tested using the analysis of variance procedure in the POLIFACT (UASVM Cluj-Napoca, Romania) statistical software, and the Duncan's Multiple Range Test (Duncan's MRT, p < 0.05) was used as a post hoc test for comparison among treatment means.

Explant Asepsis
Following the application of nodal explant asepsis treatments to P. quadrangularis (Table 3), the most effective formula proved to be T14: the pretreatment with Rifampicin 15 µg/mL in combination with Benomyl 2 g/L, followed by the treatment with EtOH 70%, 1 min and NaClO 50%, 10 min.
Through the T14 method of disinfection, 61.67% of the explants remained uncontaminated and viable for regeneration. Treatments based on 15% and 25% NaClO for 10-20 min (T1-T6) were shown to be ineffective (Figure 1a-c). A longer exposure time (20 min) caused phytotoxicity ( Figure 1f). The incubation period of the pathogens represents the time when contamination was first observed and it varies on average from 2 to 4 days in the case of T1-T4, T7, and T8 treatments and 5 to 7 days in the case of T12 and T13 treatments. This shows the influence of fungicide treatment on the number of days until the first symptoms of contamination. The pathogens have been shown to be both fungi and bacteria (Figure 1d,e). Through the T14 method of disinfection, 61.67% of the explants remaine taminated and viable for regeneration. Treatments based on 15% and 25% NaCl 20 min (T1-T6) were shown to be ineffective (Figure 1a-c). A longer exposure min) caused phytotoxicity (Figure 1f). The incubation period of the pathogens r the time when contamination was first observed and it varies on average from 2 in the case of T1-T4, T7, and T8 treatments and 5 to 7 days in the case of T12 treatments. This shows the influence of fungicide treatment on the number of d the first symptoms of contamination. The pathogens have been shown to be b and bacteria (Figure 1d,e).

Initiation and Stabilization of In Vitro Plantlets
The development of in vitro axillary shooting began with swelling of the nodal explant. The organogenic structure proliferated after about 3 weeks (Figure 2a,b). Table 4 summarizes the data on the in vitro regeneration of shoots on nodal explants of P. quadrangularis under conditions where different concentrations of cytokinins were applied. The best results regarding the regeneration rate and the number of shoots per explant were registered on the culture medium MS supplemented with 2 mg/L BAP (MS-I. 4) and, respectively, the one with 2 mg/L BAP + 1 mg/L KIN (MS-I.8) (Figure 3a). Thus, on the MS-I.4 medium, the regeneration frequency was 33.33% of the explants, with an average number of 2.52 shoots per explant. On the MS-I.8 medium, the regeneration frequency was 24.44%, which is lower than that on MS-I.9 (25.55%), but the regenerants formed an average of 3.19 shoots per explant. The longest shoots, with an average of 2.54 cm and a higher absolute value of 3.8 cm, were obtained on the MS-I.4 medium. The callus incidence was over 50% in the case of MS-I.4 and MS-I.5 treatments, with the callus obtained being friable and green.
summarizes the data on the in vitro regeneration of shoots on nodal explants of P. quad rangularis under conditions where different concentrations of cytokinins were applied The best results regarding the regeneration rate and the number of shoots per explan were registered on the culture medium MS supplemented with 2 mg/L BAP (MS-I.4) and respectively, the one with 2 mg/L BAP + 1 mg/L KIN (MS-I.8) (Figure 3a). Thus, on th MS-I.4 medium, the regeneration frequency was 33.33% of the explants, with an averag number of 2.52 shoots per explant. On the MS-I.8 medium, the regeneration frequency was 24.44%, which is lower than that on MS-I.9 (25.55%), but the regenerants formed an average of 3.19 shoots per explant. The longest shoots, with an average of 2.54 cm and higher absolute value of 3.8 cm, were obtained on the MS-I.4 medium. The callus inci dence was over 50% in the case of MS-I.4 and MS-I.5 treatments, with the callus obtained being friable and green.    To improve the regeneration frequency (%), the influence of the explant position was tested. Nodal segments were inoculated in either a vertical or horizontal position on the culture medium. Horizontally initiated explants obtained statistically higher regeneration rates compared to vertically initiated explants on each culture medium analyzed (Table 5). Additionally, biometric parameters registered higher average values in the case of explants inoculated in a vertical position, but without statistically significant differences. The callus incidence was higher in the case of explants inoculated in a horizontal position, of 82.22%, 71.11% and 70%, the differences being statistically significant.   To reduce the effects of leached phenols and sudden browning of explants, the media that obtained the best regeneration rates were supplemented with AgNO 3 and PF-68. The results of the present research demonstrated the essential role of AgNO 3 and PF-68 surfactant in the regeneration by direct organogenesis of nodal explants of P. quadrangularis, with specific cytokinin ratios (Table 6). The additional compounds act synergistically and beneficially in order to induce organogenesis, with the highest regeneration rate of 84.44% for the treatment with PF-68 0.2% on MS medium supplemented with 2 mg/L BAP (Figure 3d), which is statistically superior to the other experimental treatments. The growth-stimulating mechanism of PF-68 is concentration-dependent, and the incorporation of PF-68 at 0.2% or 0.4% supports plant growth. Thus, the treatment with PF-68 0.4% registered a regeneration rate of 76.67% and the one with PF-68% 0.2% of 73.33% on the MS-2 culture medium supplemented with BAP and KIN, the differences being statistically significant. On this culture medium, too, the treatment with 2 mg/L AgNO 3 registered the best shooting rate of 71.11%.
The regenerative response occurs on average after 3 days, but in some explants, it can be seen after as many as 5 days. AgNO 3 treatments demonstrated higher values than the control treatment, with the differences being statistically significant (Figure 3b,c). Regarding the browning of the explants, the treatment with PF-68 0.4% controlled this phenomenon best, leading to an average browning rate of only 8.52%, a statistically significant value. This treatment showed statistically significant positive differences in the number of shoots per explant (4.71 on MS + BAP + KIN and 4.36 on MS + BAP (Figure 3e)) and in the length of the main shoot (3.06 cm on MS + BAP). Analyzing the unilateral influence of the treatments, it appears that the anionic surfactant PF-68 led to the optimization of the in vitro culture initiation, with an efficiency of over 50% (Figure 4). DS 5% = 15. 80-18.91 No. of shoots/explant MS-0 (Ct) 0.00 g 0.00 g 0.00 g 0.00 g 1.

Shoot Subculturing
For mass propagation, the shoots were propagated by subculturing clusters of proliferated shoots. The shooting rate increased until the sixth subculture, after which it decreased. Table 7 summarizes the data on the subcultivation of shoots in the second and sixth subcultures under the influence of PGRs. In the first subculture, the shoots showed insignificant proliferations (Figure 3f). The shoots subculturing leads to obtaining a green, friable callus. The incidence of callus is much reduced in the sixth subculture compared to the second subculture. In the second subculture, there was an average callus incidence of over 69.99%, while in the sixth subculture only 22.22%. In the second subculture, on the MS-2 medium (Figure 3g), an average of 4.45 shoots were obtained in a cluster, compared to the MS-3 medium (Figure 3h), where the clusters were on average composed of 5.01 shoots. A maximum of 7.17 shoots could be obtained from one shoot on MS medium supplemented with 2 mg/L BAP and 1 mg/L TDZ (MS-3) in the sixth subculture (Figure 3k). Here, too, on the MS-2 medium, an average of 5.83 shoots were obtained (Figure 3j), the differences between the treatments being statistically significant. On the MS-3 culture medium, in the analyzed subcultures, average shoot lengths of 3.76 cm and 4.48 cm, respectively, were registered, which is statistically superior to the other experimental treatments. Even after the third subculture, the presence of endogenous bacterial infections continued to occur, affecting up to 30% of the explants. This infection is manifested in the form of haloes dispersed in the culture medium. The addition of Kanamycin to the culture medium at concentrations of 50, 100, and 150 mg/mL, respectively, did not affect the viability of the micro-shoots. Rhizogenesis began after the sixth subculture on the MS culture medium supplemented with 0.5 mg/L NAA (MS-4) ( Figure 5), when 48.89% of the shoots developed roots, a statistically superior value to the other treatments. Because root vigor was low, the in vitro rooting induction step prior to the acclimatization step was required.

In Vitro Rooting of Regenerants
Supplementation of ½ MS medium with 1 mg/L NAA (MS-5) proved to be prolific for stimulating root formation in 61.11% of shoots (Table 8). The root quadrangularis shoots on the ½ MS culture medium in the absence of auxins led percentage of only 11.11%, and lengths of only 2-3 mm. Supplementing the ½ dium with 1 mg/L IBA (MS-6) led to an average of 9.61 roots per seedling (Figu with the differences being significantly positive, but not statistically assured. In the number of days until the onset of rhizogenesis, the two auxins proved to ha the same degree of effectiveness. The roots obtained at this stage were of low vig large number were broken when the plants were washed or planted in the acc tion substrate. Additionally, the regenerated plants showed reduced vigor.

In Vitro Rooting of Regenerants
Supplementation of 1 /2 MS medium with 1 mg/L NAA (MS-5) proved to be the most prolific for stimulating root formation in 61.11% of shoots (Table 8). The rooting of P. quadrangularis shoots on the 1 /2 MS culture medium in the absence of auxins led to a low percentage of only 11.11%, and lengths of only 2-3 mm. Supplementing the 1 /2 MS medium with 1 mg/L IBA (MS-6) led to an average of 9.61 roots per seedling (Figure 3m,n), with the differences being significantly positive, but not statistically assured. In terms of the number of days until the onset of rhizogenesis, the two auxins proved to have about the same degree of effectiveness. The roots obtained at this stage were of low vigor and a large number were broken when the plants were washed or planted in the acclimatization substrate. Additionally, the regenerated plants showed reduced vigor.

Acclimatization of Regenerants
Well-developed and well-rooted plants survived in the acclimatization phase. For this stage, the substrate has an important role in the survival rate of regenerated plants.
On the peat + vermiculite (1:1) mix substrate (Figure 3o), the percentage of seedlings that survived was 73.33%, while it was 66.67% on the peat + perlite (1:1) substrate. Regarding plant growth, the substrate did not show any influence. The acclimatized plants showed vigorous growth, green leaves, and well-developed roots (Figure 3p).

Discussion
The main objective of this study was to establish a complete in vitro direct organogenesis regeneration protocol in P. quadrangularis using nodal explants. Among the previous investigations of other authors, we did not find any reports containing results regarding a complete in vitro regeneration protocol in P. quadrangularis from nodal explants.
To date, the micropropagation of Passiflora species has been a field of research of interest, with a significant number of studies being conducted. The first study was performed in 1966 on P. caerulea, using nodal explants as the initiation material [56]. Since then, studies showing various micropropagation techniques in Passiflora have been on the rise [22,29,39,42,[57][58][59][60][61]. All these studies show that organogenesis remains the main regeneration path for this species [53].
Several studies have reported difficulties in the decontamination of explants [61,62], which is the most important step in the micropropagation protocol. Generally, there are four possible sources of contaminants: internal or external contamination of the parent plant, insufficiently sterilized nutrient media, laboratory air, and inaccuracies on the part of the researcher [63]. Contamination with bacteria, fungi, yeasts, or viruses has been recognized as the most important cause of in vitro culture failure [64,65]. One of the most widely used disinfectants is NaClO [66][67][68][69], applied as a disinfection treatment in research on P. caerulea [70], P. edulis [40] and P. suberosa [31]. The treatment with NaClO solution preceded by EtOH [71] has also been used in the case of the P. miniata species [59]. To eliminate the possibility of contamination of explants, several experiments have been performed to evaluate the efficacy of various antibiotics and fungicides [72,73] during the sterilization stage of explants. In the present study, in the case of P. quadrangularis, since the sterilizing agents did not succeed in achieving total decontamination, the presence of endogenous saprophytic microorganisms could be involved. These pathogens, which have been shown to be both fungi and bacteria, could be responsible for the prolonged contamination of P. quadrangularis explants in the current experiments. Thus, after the subsequent subcultures, the appearance of endogenous infections persisted, with the rate of aseptic explants decreasing by up to 30% after the third subculture. Kanamycin has a general spectrum of action, so an accurate identification of microorganism species would allow the use of an antibiotic with a specific range of action and, consequently, with improved control of endogenous infections. The control of endogenous fungal or bacterial infections has been a problem in many plant micropropagation studies.
In the preliminary study conducted by Boboc et al. [52], the initiation was performed on four culture medium treatments: MS + 0.5 mg/L indole-3-acetic acid (IAA) + 1 mg/L BAP, 1/2 MS without PGRs, Anderson Rhododendron Medium without PGRs, and McCown Woody Plant without PGRs. Starting from nodal segments, a regeneration rate of 23.3% on the McCown Woody Plant culture medium was obtained. The leaf fragments did not obtain any regenerant. In their study, Otahola and Diaz [25] showed that a dose of 2 mg/L BAP led to the highest survival rate of the explants, 76.33% in P. quadrangularis and 80% in P. edulis f flavicarpa. At a concentration of 2 mg/L BAP, P. quadrangularis resulted in an average of 7.37 shoots per explant, compared to P. edulis f. flavicarpa, which resulted in the formation of 1.83 shoots per explant. Initiation of tissue cultures in Passiflora has been shown to be effective on media supplemented with BA in various concentrations, alone or in combination with other PGRs [30]. Nodal explants or leaf fragments are most often used as initiation explants. The initiation of other types of explants in a horizontal position has been recommended in P. tenuifila and P. setacea [58], and P. edulis [42].
Inorganic substances such as AgNO 3 and PF-68 have been used to improve the regenerative capacity of explants. The way in which AgNO 3 acts on the in vitro culture of plants is still ambiguous, but it has been shown that this compound can antagonize the action of ethylene by reducing the potential of the receptor to bind the gas molecule to ethylene [46]. Currently, scientific research indicates that AgNO 3 has beneficial effects on the regeneration and clonal propagation of several economically important plants [46,56]. The properties of AgNO 3 , such as water solubility, easy availability, specificity, and stability, have been found to be effective for in vitro regeneration. Silver ions in the form of nitrate play an important role in germination, rhizogenesis and somatic embryogenesis, being able to block the action of ethylene which causes abscission, senescence, and growth retardation [40]. The results obtained by Pinto et al. [47] demonstrated that AgNO 3 treatment is essential for the induction of adventitious buds in P. alata. The culture medium supplemented with cytokinins and AgNO 3 at a photoperiod of 16 h favored direct organogenesis in this species.
To minimize the negative effect of phenols on the regeneration of P. edulis f. flavicarpa explants and to induce robust shoot growth, Huh et al. [40] tested several antioxidant compounds. AgNO 3 was shown to be effective in reducing browning and sudden death of explants. Also in this species, Trevisan et al. [39] reported that induction of axillary shoots was improved with AgNO 3 on MS culture media supplemented with TDZ. Several researchers have reported the effect of AgNO 3 and PF-68 on the regeneration of recalcitrant species. The incorporation of AgNO 3 into the initiating medium led to improvements in the regeneration of the species Tagetes erecta [74] and Prosopis cineraria [75].
In the research conducted by Prammanee et al. [76], in the repeated subcultures of P. edulis, the highest number of shoots was obtained on the culture media supplemented with 1 mg/L BA and 1.5 mg/L BA. For P. tenuifila and P. setacea species, the subculture medium contained 2.5 mg/L IBA and 2 g/L Phytagel. Subcultures were performed at 8 weeks, for 24 months without reducing the proliferation of shoots [58]. In the subculture of P. edulis shoots, changing the position of the explants in the subculture on fresh medium led to a decrease in the browning rate due to polyphenols [77].
Numerous studies have shown that supplementation of culture media with auxins plays an important role in rhizogenesis. Moreover, the efficiency of different auxins differs from species to species. Effective rooting on 1 /2 MS medium with 1 mg/L IBA was registered for P. caerulea [70,71], Spemacoce hispida [78] and Lupinus albus [79]. Supplementation with NAA has been shown to be effective in rooting P. caerulea [23,26] and P. edulis f. flavicarpa [80] species. To reduce costs and minimize time to acclimatization, numerous studies have developed the possibility of ex vitro rooting, while acclimatizing using in vitro microshoots. In the case of P. setacea, shoots at least 30 mm long were removed and then immersed in a 9.94 mg/L IBA solution for 10 s to stimulate rooting. The shoots were then transferred to plastic cups containing a substrate for acclimatization [30]. This technique has also been applied to Pseudostellaria heterophylla [81] and Lawsonia inermis [82].
The results obtained in the acclimatization stage are consistent with the results of other studies performed on P. caerulea [26,70] and P. edulis [38]. Additionally, upon the acclimatization of P. foetida plants, a survival rate of 85% was registered on 1:1 vermiculite substrate and sterilized soil [83]. This protocol can be used for the regeneration of shoots, elongation, rooting and acclimatization of other species of interest in the genus Passiflora.

Conclusions
The present study led to the first establishment of a complete regeneration protocol by direct organogenesis in P. quadrangularis. Starting from nodal segments, the protocol proved to be efficient and reproducible. For the asepsis of nodal explants the most effective formula proved to be the pretreatment with Rifampicin 15 µg/mL in combination with Benomyl 2 g/L, followed by the treatment with EtOH 70%, 1 min and NaClO 50%, 10 min. Regarding the initiation and stabilization of the culture, the best morphogenic responses were obtained on the MS culture medium supplemented with 2 mg/L BAP. Supplementation of the culture medium with PF-68 0.2% (w/v) proved to have a multiple shooting capacity of 84.44%, and PF-68 0.4% (w/v) led to the maximization of biometric parameters like shoot length and the number of shoots per explant. Supplementing the basal medium with AgNO 3 and PF-68 decreased phenolic secretion, and improved plantlet quality and survival rate. Biometric parameters registered higher average values in the case of explants inoculated in a horizontal position on the analyzed culture media. The addition of phytoregulators BAP and TDZ in subcultures induced the maximum multiplication of shoots. To stimulate rhizogenesis, supplementation of 1 /2 MS medium with 1 mg/L NAA was shown to be the most prolific. For acclimatization, the substrate proved to be important, thus, peat + vermiculite 1:1 mixing substrate registered the highest plant survival rate. Although this species has been shown to be recalcitrant, in vitro propagation is recommended as a method of propagation on an industrial scale, especially in areas where this plant is not found in the natural area, as in Romania. On the basis of this study, it is recommended to apply the protocol both for the large-scale production of genetically uniform plant material, due to the ensured genetic stability, and also for the germplasm collections of P. quadrangularis species.