Introduction

The ability of plants to produce new adventitious organs throughout their life cycle is the basis for the application of cell and tissue culture methods. The main plant regeneration methods rely on the de novo organogenesis and somatic embryogenesis pathways (Xu and Huang 2014; Rocha et al. 2016).

De novo organogenesis is a morphogenetic route based on the regeneration pathway in which shoots or roots are formed, in a direct or indirect way. The success in this regeneration pathway depends upon the type of explant and the growth regulators used, which are considered the main controllers of in vitro morphogenesis (Moura et al. 2001; Nick and Optamy 2014; Vieira et al. 2014; Rocha et al. 2016). Studies involving genetics, biochemistry, morphology, and cytology were important in revealing three distinct morphological patterns: first, competence is acquired and the cells are able to be induced towards a specific pathway. Then determination takes place, whereby compromised cells and tissues are induced by exogenous growth regulators and become permanently determined towards a particular pathway, for example the development of an organ (Duclercq et al. 2011). The morphological differentiation occurs last, and it is characterized by the independence of growth regulators (Duclercq et al. 2011).

The regeneration system in passion fruit is based mainly on de novo organogenesis (Zerbini et al. 2008; Silva et al. 2011; Otoni et al. 2013; Rocha et al. 2016; Rosa et al. 2016), though some recent studies have shown regeneration occurring via somatic embryogenesis (Silva et al. 2009, 2015; Paim Pinto et al. 2010; Rosa et al. 2015; Ferreira et al. 2015). Many studies have described regeneration via organogenesis in some Passiflora species using different types of explants such as node, internode, leaf segments (Garcia et al. 2011; Pacheco et al. 2012; Anand et al. 2012; Shekhawat et al. 2015), root segments (Silva et al. 2011; Vieira et al. 2014), hypocotyl segments (Vieira et al. 2014; Rocha et al. 2016), and zygotic embryos, as observed in P. foetida (Rosa et al. 2015).

Passiflora cristalina Vanderplank and Zappi (2011) is a recently described plant species first identified in Novo Mundo, MT, Brazil. Despite the little knowledge about its biology, this species has been outstanding due to its ornamental and commercial potential. It has bright-red flowers and variegated fruit with a mild sweet taste (Vanderplank and Zappi 2011). To the present date, research involving in vitro culture has not been conducted on the species yet. Thus, the present study aimed to establish an effective regeneration system of P. cristalina via de novo organogenesis from different types of non-meristematic explants and to characterize the development of morphogenetic responses obtained from each explant source using anatomical analysis. We believe that the results here presented will be useful in providing a reproducible and reliable micropropagation protocol to support conservation strategies for this wild Amazonian Passiflora species.

Materials and Methods

Plant materials and culture conditions

Mature seeds of P. cristalina were collected from wild populations located in Alta Floresta (S 09° 99′ 67.7″ w 56 12′33.6″)—MT, Brazil. The tegument of the seeds was removed with the aid of a mini vise (Reis et al. 2007) in order to facilitate the germination of the zygotic embryos. The surface sterilization of seeds was accomplished by immersion in 70% ethanol (v/v) for 3 min, followed by 25 min of immersion in a solution of commercial sodium hypochlorite 2.5% (v/v) added with two drops of Tween-20 dispersant 0.1% (v/v) per 100 mL of solution. The seeds were then subjected to four consecutive rinses in autoclaved distilled water. Following the sterilization, seeds were inoculated in 250 mL flasks containing half-strength MS medium (Murashige and Skoog 1962) and kept under in vitro cultivation for 30–40 d.

Leaf fragments (average 1 cm2) and hypocotyl and root segments (average 1 cm) were obtained from the seedlings germinated in vitro and used as explants. The cotyledons of zygotic embryos and endosperms were also used as explants (seed-derived explants). To facilitate their isolation, mature seeds were disinfected as previously described and kept overnight in sterile distilled water. All explants were cultivated in medium containing MS basal salts, MS vitamins, 100 mg L−1 myo-inositol, 3.0% sucrose (w/v), and 0.85% agar (w/v) (Acumedia®, MI). The medium was supplemented with Benzyladenine (BA), Thidiazuron (TDZ), and Kinetin (KIN) at the concentrations of 0.5, 1.0, 1.5, 2.0 mg L−1, respectively. A control treatment received no addition of growth regulators.

All the cultures were kept in a growth room under irradiance of 36 μmol m−2 s−1 provided by fluorescent lamps at a temperature of 26 ± 2 °C. Experiments were repeated at least once, and observations were recorded after 30 d.

Shoot elongation and rooting were achieved after up to 60 days in culture on MS medium without plant growth regulators in this period. All elongated and rooted shoots were transferred to greenhouse for acclimatization.

Microscopy

P. cristalina explants subjected to organogenesis induction conditions for 15 and 30 d were collected and fixed in Karnovsky solution (1965) [glutaraldehyde (2.5% w/v) and paraformaldehyde (4% w/v) in monobasic potassium phosphate buffer (pH 7.2) plus 5 mM calcium chloride]. After fixation, the samples were dehydrated through a graded ethanol series and embedded in acrylic resin (HistoResin, Leica®, Wehrheim, Germany). Transverse and longitudinal sections, 5-μm thick, were obtained using a motorized rotary microtome (Leica®, RM 2155, Nussoloch, Germany) and stained with toluidine blue (O’Brien et al. 1964) for anatomical characterization. The images were captured under a light microscope (Olympus AX70TRF, Olympus Optical, Tokyo, Japan) equipped with a digital camera (Diagnostic Instruments Inc., Spot 3.2.0 Insight Color, Sterling Heights, MI).

Statistical analysis

A completely randomized experimental design was adopted, with three replicates (three dishes) and ten explants per dish. Explants were cultivated in 13 different treatments corresponding to the growth regulators BA, TDZ, and KIN in four different concentrations each, plus a control treatment without growth regulators. The evaluated traits were number of shoots and callus production. The data were subjected to the Scott-Knott test with a significance level of 5% (p ≤ 0.05), using Sisvar software (Ferreira 2011).

Results

Shoot organogenesis from leaf explants

For leaf explants, morphogenetic responses were only observed in BA- or TDZ-supplemented treatments. In the presence of KIN or in the treatment without growth regulators (control), no shoots or calli were formed (Table 1). After 30 days of culture, BA induced the highest number of shoots both through direct organogenesis from leaf explants and indirectly through callus formation (Table 1). The highest rate of induced shoots of (368.33 average number) was observed in treatments supplemented with 2.0 mg L−1 BA, significantly different from the other treatments. Treatments supplemented with TDZ also induced formation of adventitious shoots, with the highest frequency (mean 77.3) observed in the treatment with 2.0 mg L−1 TDZ. However, this rate was approximately four times less than the treatment with 2.0 mg L−1 BA (Table 1). The frequency of callus formation in leaf explants did not show a significant difference among treatments supplemented with BA or TDZ (Table 2).

Table 1 Average number of shoots produced from P. cristalina explants after 30 d of in vitro culture with different growth regulators
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Table 2 Average number of callus produced from P. cristalina explants after 30 d of in vitro culture with different growth regulators
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The first morphogenetic responses observed in BA-supplemented treatments were visible after 15 d in the induction medium, when organogenic structures were observed at the explant edges (Fig. 1a). These structures proliferated throughout the periphery of the explant and then differentiated into adventitious shoots (Fig. 1b). The development of adventitious shoots from leaf explants was asynchronous, even in shoots originating from the same explant (Fig. 1b). After 30 days of culture, shoots with fully expanded leaves were observed in all BA-supplemented treatments.

Figure 1.
figure 1

De novo shoot organogenesis of Passiflora cristalina after 30 d cultivation. a–c Leaf explants; a Early development of shoots (arrow) at the border of leaf explant in 0.5 mg L−1 de KIN. b Asynchronous development (arrow) of shoots cultivated in 1.5 mg L−1 BA. c Shoots with leaf expansion after 30 d of cultivation in 1.5 mg L−1 BA. d–f Hypocotyls explants cultivated in 0.5 mg L−1 TDZ; d–e Organogenic structures distributed in the hypocotyl explant at 15 (arrow) and 30 d (respectively) of in vitro culture. f Differentiation of multiple shoots using 0.5 mg L−1 TDZ. g–i Root explants; g Development of shoots primordia throughout the root explant (arrow) cultivated in 0.5 mg L−1. h Development of shoots directly from the explant tissue and formation of potentially organogenic callus and shoot (arrows) of roots cultivated with 1.0 mg L−1 KIN. i Shoots with expanded leaves produced directly from the explant tissue, cultivated with 2.0 mg L−1 KIN. j–l Cotyledons of zygotic embryo explants cultivated 1.0 and 0.5 mg L−1 BA (k–l). j Development of organogenic structures at the border of the cotyledon (arrow) that will give rise to shoots. k Shoots at an early developmental stages. l Intense proliferation of shoots in the cotyledons of zygotic embryo. m–o Endospermic explants. m Early organogenic structures formed in the presence of 2.0 mg L−1 TDZ. n Organogenic structures in differentiation in the presence of 2.0 mg L−1 TDZ. o Shoots with expanded leaves cultivated in 2.0 mg L−1 BA. p Elongating regenerated plantlets. q Rooted seedling. r Acclimatized regenerated plants. Bars (a b–d) 70 μm, c 90 μm; e–f–l–n 60 μm, g–n 50 μm, h–j–m 80 μm, i 100 μm, k 110 μm, o 120 μm, p 10 mm, q 5 mm, r 15 mm.

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Shoot organogenesis from hypocotyl explants

Hypocotyl explants showed the greatest organogenesis response among the explants assessed. Morphogenetic responses were observed in all treatments, even in the absence of plant growth regulators (PGRs) (Table 1). The highest number of adventitious shoots formed (men = 744) was also found in BA-supplemented treatments. However, in hypocotyl explants, lower concentrations of BA (0.5, 1.0, and 1.5 mg L−1) induced significantly higher numbers of shoots among BA treatments (mean 651, 744, and 556, respectively) (Table 1). Growth media supplemented with KIN and lacking PGRs showed the lowest numbers of adventitious shoots (Table 1). As for callus formation, a low frequency was observed in the hypocotyl segments, without statistical differences between the treatments (Table 2).

Organogenic structures appeared as early as 7 d of in vitro culture in hypocotyl explants (Fig. 1d). Structures displayed a smooth surface and developed initially from the explant cut regions. Throughout the morphogenetic process, structures along the entire explant surface differentiated into multiple shoots (Fig. 1e). After 30 days of culture, it was possible to observe the growth and development of adventitious shoots, as well as leaf expansion (Fig. 1f).

Shoot organogenesis from root explants

For root explants, organogenic calli and adventitious buds were obtained only in KIN supplemented media (Tables 1 and 2). However, they have showed the lowest frequency of morphogenetic responses (calli or adventitious shoots) among the P. cristalina explants (Tables 1 and 2). At 6 days of in vitro culture, in all treatments with presence of KIN, the development of organogenic structures (Fig. 1g) like buds and calli was observed, in regions distant from the explant cut surface (Fig. 1h). Subsequently, they differentiated into adventitious shoots (Fig. 1i).

Shoot organogenesis from seed-derived explants

Adventitious shoots were observed only in the BA- and TDZ-supplemented treatments when cotyledons of zygotic embryos and endosperm were used as explants (Table 1). There were no significant differences between treatments using PGRs, except for cotyledons inoculated in medium supplemented with 1.0 mg L−1 TDZ, which produced the lowest number of shoots (71.33), and endosperms cultivated with 0.5 mg L−1 BA, which did not induce de novo shoot organogenesis (Table 1).

Organogenic calli formation was also observed in cotyledons of zygotic embryos and endosperms. For both explants, organogenic calli were observed in all treatments with BA and TDZ supplementation, except in the treatment with 0.5 mg L−1 BA for zygotic embryo explants (Table 2). In the endosperm explants, organogenic calli were also obtained in the medium supplemented with 1.0 mg L−1 KIN, however, in a low number (0.33) (Table 2).

For both cotyledon and endosperm explants, the formation of greenish-white organogenic structures was observed after 10 days of in vitro culture (Fig. 1j, m). Subsequently, these structures differentiated into adventitious shoots (Fig. 1k, n). After 30 days of culture, a large number of shoots (Fig. 1l, o) were observed (as large as 205.33), revealing the remarkable organogenic potential of these explant sources.

Shoot elongation and rooting were achieved after up to 60 d in culture on MS media (Fig. 1p). After this period, all elongated and rooted shoots (Fig. 1q) were transferred to greenhouse condition for acclimatization period. All acclimatized plants resume juvenile development (Fig. 1r).

Anatomical characterization

The anatomical analyses revealed that the morphogenetic responses observed in leaf, hypocotyl, cotyledon, and endosperm explants began after intense proliferation of cells from superficial layers of the explants (Fig. 2a, o). Conversely, organogenic structures obtained from root explants seem to develop from cell divisions of inner layers of the organ (Fig. 2i).

Figure 2.
figure 2

Histological analysis during the induction of the organogenesis in Passiflora cristalina using different types of tissue after 30 d cultivation. a–c Leaf explants, d–f hypocotyl explants, g–i root explants, j–l cotyledon explants of zygotic embryo, m–o endosperm explants. a Cellular divisions periclinal and anticlinal on fundamental parenchyma and meristemoids formation. b Meristemoids formation and leaf primordium (arrow). c Shoots with development of leaf and procambial region (asterisk) and apical dome (arrow). d–e Cell divisions in the epidermis and parenchyma at the base of the hypocotyl forming organogenic structures (arrow). f Organogenic structures with the initial development of shoots (arrow). g Endogenous origin of the meristemoid in root explants (arrow). h–i Shoots (arrows) arose from the proliferation of procambial cells (asterisk) observe the vascular connection with the explant of origin (arrow). j Primordium development of multiple shoots (arrow). k Development of the primordium shoots. l The leaf primordium (arrow) with trace of vascular system (asterisk) in shoots. m–n Small meristemoids distributed throughout the endosperm (asterisks). n Organogenic structures (arrows). Bars a 35 μm, b 60 μm, c–h–i–k–m–o 80 μm, d–g 110 μm, e 70 μm, f 100 μm, j–l 90 μm, n 50 μm.

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For leaf explants, regeneration occurred mainly at the border of the explants. At the beginning of the induction process, cell divisions were observed in layers of the parenchyma adjacent to the adaxial epidermis. In certain regions, the intense cell proliferation in the subepidermal layers of the explant originated cell clusters with cytological characteristics of meristemoids (Fig. 2a). Meristemoid cells are small, isodiametric, with a dense cytoplasm, and evident nucleus and nucleoli. Subsequently, these structures differentiated into adventitious shoots (Fig. 2b). Fully regenerated shoots were then observed after 25–30 days of culture (Figs. 2c).

In hypocotyl-derived explants, the morphogenetic process began also after cell divisions from the subepidermal parenchyma (Fig. 2d, f). The presence of meristemoids could be observed in this region in the first week of in vitro culture (Fig. 2d). In later developmental stages, these meristemoid regions remained under constant cell division (Fig. 2f, g), subsequently differentiating into multiple shoots.

In the root segments, the formation of adventitious shoots also occurred from meristemoids at the edge of the explants.

However, these structures arose from the proliferation of procambial cells. The continuous development of meristemoids into shoots culminated in the rupture of the outermost layers of the explants (Fig. 2h). Fully formed shoots were also observed after 30 days of culture, and they were connected to the vascular system of the explant, confirming the organogenic regeneration pattern (Fig. 2i).

In cotyledon of zygotic embryos and endosperm explants, the development of meristemoids in the peripheral layers of the explant was even more evident (Fig. 2j, m). In these explants, the meristemoids constituted small meristematic cell protuberances along the entire surface of the explants (Fig. 2m, n). These protuberances indicated the formation site of each adventitious shoot, produced from the continuous development of these structures (Fig. 2k, l, o).

The seedlings of the shoot explants converted (Fig. 1p) have produced intense proliferation of root (Fig. 1q) and after transferred to the greenhouse acclimatization successfully as observed in Fig. 1r.

Discussion

The induction of de novo shoot organogenesis from different explant sources was successfully established for P. cristalina. The regeneration ability in plants has been widely explored in modern agriculture (Sussex 2008), and tissue culture has revealed the pluri and totipotentiality of plant cells for the establishment of more responsive in vitro propagation and regeneration systems (Xu and Huang 2014).

De novo organogenesis is the predominant regeneration method for obtaining in vitro plants in the genus Passiflora (Otoni et al. 2013). In general, cytokinin supplementation in the growth medium is fundamental for shoot induction and development in passion fruit plants (Silva et al. 2011; Vieira et al. 2014; Rosa et al. 2016). The presence of cytokinin in the induction medium was essential for shoot regeneration in leaf segments of P. cristalina. The in vitro organogenesis studies in P. setacea described the essential role of BA in shoot formation from leaf explants (Vieira et al. 2014).

Likewise, TDZ has also been used for the regeneration of several Passiflora species (Trevisan and Mendes 2005; Pinto et al. 2010; Vieira et al. 2014), although for most of them, it has promoted the development of less shoots than BA (Pinto et al. 2010; Vieira et al. 2014) and confirmed here. On the other hand, when KIN was used in our study, no morphogenetic responses occurred and the P. cristalina leaf explant got senesced.

Shoot formation in the leaf segments occurred indirectly, and after 30 days of in vitro culture, they were already elongated and easily individualized. Leaf segments are usually employed for the regeneration of several passion fruit species, such as P. suberosa (Garcia et al. 2011), P. foetida (Komathi et al. 2011), P. cincinnata (Lombardi et al. 2007), P. alata (Pinto et al. 2010), and P. setacea (Vieira et al. 2014), to mention a few.

The organogenic potential of hypocotyl segments was observed even in the growth regulator-free medium; however, the BA and TDZ cytokinins were the most effective for these explants. A high number of shoots were produced at their lower concentrations, 1.0 mg L−1 BA and 1.0 mg L−1 TDZ yielded an average of 745 and 180 shoots, respectively. Contrarily, when using 0.5 mg L−1 KIN, a reduced number of shoots were produced (average 36.0). In general, this intense proliferation and production of shoots in hypocotyl segments and in the other types of explants used in the present study are not common for Passiflora species. Thus, P. cristalina revealed a very high organogenic potential, with superior production of adventitious buds when compared to other Passiflora species, e.g., P. cincinnata and P. edulis (Silva et al. 2011), P. setacea (Vieira et al. 2014), P. alata (Pinto et al. 2010), and P. suberosa (Pinto et al. 2010). These results suggest that P. cristalina could be a potential Passiflora model species for studies involving micropropagation, genetic transformation, and factors related to induction of the morphogenetic pathways.

In P. cristalina root explants, de novo organogenesis only occurred in the presence of KIN. The largest number of directly formed shoots was obtained at 1.0 mg L−1 KIN (mean 25.3). Interesting, the shoot regeneration from P. setacea root explants was obtained only in the presence of BA and TDZ (Vieira et al. 2014). The in vitro regeneration of Passiflora spp. is apparently dependent on the type of explant and PGR (Silva et al. 2011; Vieira et al. 2014; Rocha et al. 2015). In P. cristalina root explants, a potentially organogenic cell aggregate was formed consisting of large and elongated cells with prominent nuclei, from where the adventitious shoots have arisen, posteriorly. According to Rocha et al. (2012), root-derived shoots are formed from the differentiation of pericycle cells, which determine the formation of initial regions, the meristemoids, followed by the development of shoots. Meristemoid formation was also reported in direct and indirect regeneration in roots of P. cincinnata (Lombardi et al. 2007; Silva et al. 2011) and P. edulis (Silva et al. 2011; Rocha et al. 2012, 2016).

The P. cristalina endosperm revealed a great organogenic potential when cultivated in BA- and TDZ-supplemented medium showing the development of multiple shoots. Endosperm tissues are not commonly used as an explant source for the regeneration of passion fruit plants, but they may be considered as an alternative, with the possibility of obtaining triploid plants, since their cells have three chromosome sets as a result of double fertilization. Chaturvedi et al. (2003) reported that obtaining plants from endosperm culture is the basis of a modern approach for direct crop breeding, facilitating the regeneration of triploid plants that present problems with in vivo growth.

Overall, the de novo shoot organogenesis system for P. cristalina was successfully established from different explant sources, constituting the first report for this endemic passion fruit species. We believe that these data will be useful in future research for rapid P. cristalina micropropagation and conservation, and they might also facilitate the development of biotechnological tools to study such wild Amazonian species.