Adventitious shoot organogenesis from leaf explants of Portulaca pilosa L.

This study established, for the first time, shoot proliferation and plant regeneration protocols via shoot organogenesis from leaf explants of a medical and ornamental plant, Portulaca pilosa L. The optimal proliferation of axillary shoots was 6.2-fold within 30 days on Murashige and Skoog (MS) medium supplemented with 3.0 µM 6-benzyladenine (BA). Shoots could be induced directly from leaf explants, forming an average of 3.8 adventitious shoots per explant, on optimal MS medium supplemented with 1.0 µM thidiazuron (TDZ) and 0.1 µM α-naphthaleneacetic acid (NAA). A higher concentration of TDZ (3.0 µM), alone or in combination with 0.1 µM NAA, induced somatic embryo-like shoot buds and then developed into real shoots. Rooting was easier since roots were induced on all rooting media within one month. Half-strength MS medium free of plant growth regulators was best for rooting. Rooted plantlets were transferred to a sand: perlite (1:1, v/v) substrate, resulting in highest survival (90%). Plantlets showed more robust growth, however, on substrates of yellow mud: perlite (1:1, v/v) or peat soil: vermiculite: perlite (1:1:1, v/v).

Effects of plant growth regulators on axillary shoot proliferation. Axillary shoots were cut into single shoots (about 2 cm long) or multiple shoots were cut into smaller clumps and inoculated onto MS medium supplemented with different PGRs and concentrations. PGR-free MS served as the control (Table 1). Each treatment contained six jars with five shoots per jar. After culture for 30 days, the axillary shoot proliferation coefficient was calculated as: number of axillary shoots after proliferation/number of axillary shoots before proliferation.

Effect of plant growth regulators on leaf-induced adventitious shoots and somatic embryo-like
shoots. In vitro leaves (1.0 cm long) were used as explants that were inoculated onto MS medium supplemented with different PGRs and their combinations, with PGR-free MS medium serving as the control (Table 2). Each treatment contained six jars with five leaf explants per jar. After culture for 30 days, the number of adventitious shoots were induced were assessed.
Effect of auxins on root formation. Shoots 3-4 cm tall cut from the base were inoculated onto MS medium supplemented with different concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA), with auxin-free MS medium serving as the control (Table 3). Each treatment had 10 jars and three shoots were inoculated in each jar. After 30 days of culture, rooting percentage, number of roots and root length were assessed. Acclimation and transplantation. Jars with rooted plantlets were transferred to natural light for acclimatization for 7 days. Plantlets were then carefully removed from jars, and agar was rinsed off with tap water. Plantlets were transferred to several mixed substrates (Table 4). All the vermiculite and perlite substrates were bought from Guangzhou Shunxin Company, China. Every treatment has 30 plantlets and each plantlet was transplanted into a separate black plastic bag (12 cm high; 10 cm in diameter), and irrigated with tap water every morning. After transplantation for 30 days, survival percentage was assessed.
Data and statistical analysis. The experimental data were analyzed by SPSS17.0 software. Following mean separation by ANOVA, Duncan's multiple range test was used to assess significant differences (P < 0.05) between treatments. Experiments were repeated three times with 30 samples per treatment.

Results
Effect of plant growth regulators on the proliferation of axillary shoots. On PGR-free medium, a mean of one shoot could proliferate into 4.7 axillary shoots within 30 days. These usually developed roots, and no callus was visible (Fig. 1a). On medium supplemented with 1-5 µM kinetin (KIN), one shoot proliferated 5.1-5.3-fold into axillary shoots (Table 1), forming roots within 30 days, and no callus was visible (Fig. 1b). On medium supplemented with 1-3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), almost of all the single shoots did not develop new axillary shoots, and yellow compact callus was induced at the base of some shoots (Fig. 1c). On medium supplemented with 1-5 µM 6-benzyladenine (BA), one shoot proliferated 5.6-6.2-fold into multiple shoots within 30 days, but could not develop roots. Friable callus was induced at the base of multiple shoots (Fig. 1d).

Effect of plant growth regulators on the induction of adventitious shoots from leaf explants.
On PGR-free medium, some adventitious roots were induced at the leaf cut surface within 15 days (Fig. 2a), but even after culture for 30 days, no adventitious shoots were visible. On medium supplemented with 1.0-3.0 µM BA, callus and some adventitious shoot buds was induced from the callus surface, even from uncut surfaces,   (Fig. 2b). After culture for a total of 30-45 days, some adventitious shoots were induced on the callus surface. As the culture period was prolonged, more adventitious shoots became visible. A low concentration (1.0 µM) of BA induced more adventitious shoots (2.5/leaf explant) than at a high concentration (3.0 µM) (1.9/ leaf explant). No roots were visible. When culture was prolonged to 60 days, more adventitious shoots became visible (Fig. 2d). When BA (1.0 µM) and NAA (0.1 µM) were combined in medium, more adventitious shoots were greater (3.7/leaf explant) than when BA alone was used in the medium (2.5/leaf explant). When BA (3.0 µM) and NAA (0.1 µM) were combined in medium, more adventitious shoots were induced (2.4) than when BA was used singly (1.9/leaf explant) ( Table 2). On medium supplemented with 1.0 µM TDZ, some callus was induced from the leaf surface. After culture for 30 days, some adventitious shoots (3.5/leaf explant) were also induced on the surface (Fig. 2c). When TDZ (1.0 µM) and NAA (0.1 µM) were combined in the media, more adventitious shoots were induced (3.8/leaf explant) than when TDZ alone was used in the medium (3.5/leaf explant) ( Table 2). A similar outcome was observed with 3.0 µM TDZ, but only 3.1 somatic embryo-like shoot buds formed per leaf explant (Fig. 2e,f) within 30-40 days. In both cases, no roots were visible during shoot morphogenesis (Table 2). When TDZ (3.0 µM) and NAA (0.1 µM) were combined in the media, more somatic embryo-like shoot buds were induced (3.4 /leaf explant) ( Table 2). With the culture time prolonging, some more shoots occurred on the callus surface, no somatic embryo structure was found in the subsequent culture period. On medium supplemented with 1.0-3.0 µM 2,4-D, some yellow compact callus was induced within 7 days. After culture for a total of 30 days, callus turned black and tended to become necrotic within 45 days, and no adventitious shoots or roots were induced.
Effect of plant growth regulators on rooting. All shoots developed roots on all rooting media (Table 3).
On medium supplemented with NAA, the induced roots broke off more easily because they were thinner. On PGR-free and IBA-supplemented media, all roots were normal. Among all media, PGR-free medium induced the thickest and strongest roots showing best quality (Fig. 3a,b).  (Fig. 3c). Average plant height in the former substrate was 2.63 cm (Table 4). In the yellow mud: pearl rock (1:1) substrate, lowest survival (63%) was observed, but with taller plants on average (5.57 cm), with shoots occasionally developing branches.

Discussion
There are only a few reports on the tissue culture of members of the genus Portulaca. In P. oleracea, callus induction, direct shoot regeneration from nodes shoot tips and petioles was reported 16 . MS medium containing 10 µM IBA in combination with 5 or 10 µM BA could induce callus from leaf explants, shoots regenerated directly from shoot tips or petiole explants only in the presence of 10 µM IBA, while 8.88 µM BA was optimal for shoot regeneration from nodal segments 16 . An efficient and reliable in vitro regeneration and flower production protocol was developed for P. oleracea, and medium supplemented with KIN (2.3 µM) and GA 3 (0.58 µM) induced flowering in vitro 17 . Direct differentiation of somatic embryos from leaf explants of P. oleracea was observed on MS medium supplemented with 6.6 µM BA in the dark, but somatic embryos were then transferred to PGR-free MS medium under a 16-h photoperiod 18 .
In P. grandiflora, direct organogenesis was observed, with the induction of callus from petals requiring a combination of four PGRs (2 µM 2,4-D, 2 µM NAA, 2 µM KIN and 2 µM BA) and the presence of 10-100 µM gallic acid, a flowering inhibitor 19 . In P. grandiflora, the culture of stem sections on MS medium supplemented with 3.8 µM 2,4-D induced callus that could be continuously subcultured while numerous shoots plus leaves were induced on MS medium supplemented with 10% cocanut milk and 4.6 µM KIN 20 . From seedlings of two violet flowering, inbred lines of P. grandiflora, hypocotyls were isolated and then halved, and cell cultures derived from basipetal sections were more effective than acropetal sections in terms of betacyanin production 21 . Tyrosine hydroxylase was isolated from callus that had been induced from red and white lines of P. grandiflora on MS medium with 3.8 µM 2,4-D and 0.46 µM KIN and subcultured every 14 days by transferring to 40 ml of fresh liquid medium 22 . Nodal segments formed 56.55 shoots/explant (98% induction) in MS medium with 17.6 µM BA 23 . Cell suspension cultures of P. grandiflora in MS medium with 6.6 µM BA and 0.38 µM 2,4-D were used for the biotransformation of L-tyrosine into L-DOPA, an anti-Parkinson's drug, while callus cultures were an excellent source of tyrosinase 24 . Thus, in P. grandiflora, only shoot organogenesis was reported from hypocotyls, but no shoots or somatic embryos were induced from leaf explants.
The genus Portulaca is a potentially suitable model plant to carry out studies on drought and/or salinity 25 . Information on relevant mechanisms of tolerance to salt and water stress can be achieved by correlating the activation of specific defense pathways with relative stress resistance. Portulaca species may also serve as new www.nature.com/scientificreports www.nature.com/scientificreports/ economically important crops for culture in saline soils and arid environments, via sustainable agriculture, as medicinal plants, highly nutritious vegetable crops and ornamentals 26 .
No report on the tissue culture of P. pilosa exists. In this study, we established an axillary shoot proliferation system for P. pilosa that could be proliferated in PGR-free MS medium or in MS medium supplemented with moderate concentrations of KIN (1-5 µM). On these media, shoots not only proliferated but roots also formed naturally, i.e., in the absence of auxin (Table 1, Fig. 1a,b). In other media supplemented with BA, axillary shoots could be proliferated. However, some callus was induced at the shoot base (Fig. 1c). TDZ induced fewer shoots and cause leaf hyperhydricity (Fig. 1e). This fortified the existence of TDZ-induced abnormalities in tissue culture 27 . Our results show that new shoots could not develop in the presence of 2,4-D.
When testing the induction of morphogenesis from leaf explants, only MS media supplemented with cytokinins (1.0-3.0 µM BAP and TDZ) or combined with 0.1 µM NAA could all induce adventitious shoots or somatic embryo-like shoot buds (Fig. 2b-e). However, PGR-free medium or 1.0-3.0 µM 2,4-D could not induce adventitious shoot or somatic embryo. Therefore, only cytokinins could induce adventitious shoots and somatic embryo-like shoot buds from leaf explants in P. pilosa. We should emphasize that culture period has a distinct effect on adventitious shoot formation. When leaf explants were cultured on MS media supplemented with cytokinins (BA or TDZ) for 30 days, only several adventitious shoots were induced (Table 2, Fig. 2c). However, as the culture period was prolonged to 60 days, more adventitious shoots were induced (Fig. 2d). This indicates that culture period influences the quantitative outcome of shoot organogenesis 28 .
In a rare and endangered species, Primulina tabacum Hance, 5.0 µM BA induced shoots while 5.0 µM TDZ induced somatic embryos, and both somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of the two cytokinins supplemented in the culture medium 29 . A low concentration of TDZ www.nature.com/scientificreports www.nature.com/scientificreports/ (2.5 µM) induced shoots while a high concentration of TDZ (5-10 µM) induced somatic embryos in Saintpaulia ionantha Wendl 30 . In another rare and endangered species, Metabriggsia ovalifolia W. T. Wang, BA and TDZ at 5-10 μM could induce both shoots and somatic embryos, a higher concentration of TDZ (25 μM) induced only somatic embryos (39.8/explant) while BA (25 μM) induced both adventitious shoots (23.6/explant) and somatic embryos (9.7/explant) 31 . All these studies indicate that TDZ is able to alter the morphogenetic pathway from shoot organogenesis to somatic embryogenesis through a simple change in TDZ concentration 30,32 . However, among the PGRs, only TDZ induced somatic embryo-like shoot buds in P. pilosa. With the prolonging culture time, we could not observe the development of somatic embryos.
Our test showed that P. pilosa rooted easily, even in PGR-free MS medium and in MS medium supplemented with KIN (1.0-5.0 μM), which served for shoot proliferation. In rooting trials, all media could induce roots with 30 days. Taking into account the speed of root induction rooting quality, best rooting medium was PGR-free MS medium. Unlike our 100% rooting success, a maximum of 95% rooting on half-strength MS medium supplemented with 0.75 mg/l NAA was observed for P. grandiflora 23 . In P. oleracea, both IBA and NAA at 2.5 µM in MS medium was better than a higher concentration (5.0 µM) for root regeneration from shoots but with equimolar amounts of these auxins, IBA was more effective than NAA, although acclimatization or transplantation were not assessed 16 .
In the experiment related to plantlet acclimatization, P. pilosa plantlets showed a high survival rate that exceeded 90% after they were transplanted to any of four substrates: vermiculite: sand (1:1), peat soil: pearl rock (1:1), sand: pearl rock (1:1), peat soil: sand: pearl rock (3:2:1) (Fig. 3c). In contrast, survival rate was low (63%) in yellow mud: sand, but plants grew well and branched more, which also occurred in sand: pearl rocks (1:1). Taking into account the growth rate and branching of plants, the best substrate was peat soil: pearl rock (1:1). Rooted P. grandiflora plantlets were transferred to pots filled with a mixture of sterile soil, sand and vermiculite (1:1:1), and after hardening, plants were transferred to the field, showing 100% survival 23 . In P. oleracea, acclimatization or transplantation of in vitro-derived plantlets were not tested 16 . However, in a separate study, P. oleracea plantlets were transferred to plastic cups containing an autoclaved mixture of sand, soil and vermiculate (1:2:1), covered with transparent plastic cups to provide high relative humidity, then gradually successfully established under natural conditions, with a survival rate of 100% 18 .

Conclusion
Shoot proliferation and plant regeneration protocols via shoot organogenesis from leaf explants of Portulaca pilosa L. for the first time. The optimal proliferation of axillary shoots was 6.2-fold within 30 days on MS medium supplemented with 3.0 µM BA. Adventitious shoots could be induced directly from leaf explants, forming an average of 3.8 adventitious shoots per explant, on optimal MS medium supplemented with 1.0 µM TDZ and 0.1 µM NAA. A higher concentration of TDZ (3.0 µM), alone or in combination with 0.1 µM NAA, induced somatic embryo-like shoot buds and they developed adventitious shoots.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information Files.