Introduction

Mandevilla Lindl. (Apocynaceae, Apocynoideae) includes approximately 150 species in the Neotropical region, and new species are continually being described. Approximately 70 species of Mandevilla are found mainly in the Amazon and southeastern regions of Brazil (Rapini et al. 2010). This genus has several ethnobotanical citations related to uses in traditional folk medicine (Schultes 1979), but its pharmacological potential originates from the presence of an underground system consisting of a xylopodium with tuberous roots. This xylopodium has an extremely stiff consistency and the ability to produce buds, which have medicinal properties (Metcalfe and Chalke 1950; Appezzato-da-Glória and Estelita 2000; Sales et al. 2006).

Interest in Mandevilla was spurred by pharmacological studies reporting the use of infusions or alcoholic extracts of xylopodium, either from Mandevilla velutina (Mart. ex Stadelm.) Woodson or Mandevilla illustris (Vell.) Woodson, as a snake antivenom. However, unsustainable harvesting practices have led to reduced natural populations of these species. Therefore, strategies for in vitro conservation have been applied in order to maintain the genetic diversity of these populations and allow research on their pharmacological properties, including the production of secondary metabolites with pharmacological activities (Calixto et al. 1985; Handro et al. 1988; Maraschin et al. 2000, 2002; Biondo et al. 2004, 2007; Bertoni et al. 2010). Mandevilla has endemic species in impacted areas by predatory extraction and irregular occupation in southeastern Brazil (Wiersema and León 1999; Cordeiro et al. 2012). Although these species are promising from both pharmacological and ornamental perspectives, the genus is poorly studied; hence, little is known about the best conditions for seed storage and preservation of collections. Mandevilla moricandiana (A.DC.) Woodson has been reported in the restinga and rocky grassland areas of some northeastern Brazilian states, as well as in the southeastern region. It is a woody vine with twining and latescent branches. The ornamental potential originates from its inflorescence, which has very showy flowers with a pink and white funnel-shaped corolla and a yellow coralline tube (Woodson 1933; Cordeiro et al. 2012), while the pharmacological potential originates from its very well-developed xylopodium.

Germplasm conservation of native species with high ornamental or pharmacological potential has been used to preserve their genetic variability and allow the study of their properties in a controlled environment and is supported by in vitro culture techniques (Ford-Lloyd and Jackson 1991; Vasil 1991; Villalobos et al. 1991; Bertoni et al. 2010). The establishment of an in vitro protocol for the conservation of a wild species allows for the creation of reference databases for future studies of other species (Cordeiro et al. 2012).

Basically, in vitro plant conservation involves changing the culture environment in order to slow or suppress the growth of cells, tissues, and organs. This practice, in turn, maximizes the interval between subcultures, thus reducing manpower, storage space, expense, and possible contamination. The conservation method should allow immediate access to the germplasm of interest and maintain genetic diversity without compromising genetic stability (Engelmann 2004; Shibli et al. 2006; Rai et al. 2009). In vitro conservation methods depend upon the type of culture employed and the laboratory conditions available. Accordingly, several alternative protocols have been developed to adapt to the species conserved, type of disposable explant, and storage time in order to maintain the recovery capacity of the culture (Engelmann 2004; Shibli et al. 2006).

The aim of this study was to establish and compare strategies for in vitro conservation of M. moricandiana to maintain cultivars and germplasm for ornamental and pharmacological uses. To accomplish this, the ideal culture medium for slow-growth germplasm storage was determined, and, as an alternative to cryopreservation, encapsulation–dehydration techniques were tested to establish encapsulation, preculture, and dehydration conditions for storing nodal segments of M. moricandiana at low temperatures. Both methods were used in an attempt to expand the options for storage of M. moricandiana, since they permitted the full recovery of explant viability.

Materials and Methods

Plant material.

In vitro-grown plantlets of M. moricandiana were used as sources of nodal segments in all experiments. The in vitro cultures were obtained from seeds collected in the Restinga de Jurubatiba National Park (Rio de Janeiro, Brazil). The disinfestation of seeds and establishment of in vitro cultures, as well as the collection authorization and species identification, were performed as in Cordeiro et al. (2012).

Culture media and conditions.

Slow-growth storage. The semisolid basal media consisted of Murashige and Skoog (MS) (Murashige and Skoog 1962) salts at full or half strength (MS and ½MS, respectively), supplemented with MS vitamins and 3% sucrose (w/v), and Woody Plant Medium (Lloyd and McCown 1980) salts at full or half strength (WPM and ½WPM, respectively), supplemented with WPM vitamins and 2% sucrose (w/v). All culture media were prepared from stock solutions of macro- and micronutrients according to Chawla (2002) and were solidified with 0.75% (w/v) agar. For slow-growth storage, the osmotic agents glucose, mannitol, and sorbitol were added individually or in pairwise combinations at a concentration of 2% (w/v) each. The salts, vitamins, sucrose, osmotic agents, and agar used in culture media composition were from Sigma-Aldrich (Saint Louis, MO). The pH of each medium was adjusted to 5.8, and the molten media were dispensed into glass bottles (7.5 × 13.5 cm) and autoclaved for 15 min at 121°C. The medium for growth recovery consisted of semisolid MS basal medium. All cultures were maintained in a growth room at 25 ± 1°C, under a 16/8 h light/dark photoperiod, with irradiance of 23 μmol m−2 s−1 provided by daylight fluorescent lamps.

Encapsulation and preculture. The gelling medium was prepared with liquid MS basal medium without calcium and supplemented with 3% (w/v) sodium alginate (Sigma-Aldrich A-2033, medium viscosity). The polymerizing medium was prepared with liquid MS basal medium supplemented with 0.1 M calcium chloride. The preculture of capsules was performed in liquid MS basal medium supplemented with 0.40 or 0.75 M sucrose. The pH of each medium was adjusted to 5.8. All media were autoclaved for 15 min at 121°C. The medium for growth recovery consisted of semisolid MS basal medium. All cultures were maintained in a growth room under the same conditions as described above.

Rooting. The rooting medium consisted of MS salts, supplemented with MS vitamins, 3% sucrose (w/v), and 2.0 mg L−1 indoleacetic acid (IAA) (Cordeiro et al. 2012). The medium was solidified with 0.75% agar (w/v). The pH of the medium was adjusted to 5.8, and the molten medium was dispensed into glass bottles (7.5 × 13.5 cm) and autoclaved for 15 min at 121°C. All cultures for rooting were maintained in a growth room under the same conditions as described above.

Effect of basal medium and carbon source on slow-growth storage and growth recovery.

Nodal segments (1.0 cm) from in vitro cultures of M. moricandiana were excised and placed in glass flasks containing 100 mL of semisolid MS, ½MS, WPM, or ½WPM medium, with or without glucose, mannitol, or sorbitol. The experimental design was fully randomized in a 4 × 7 factorial arrangement, consisting of 4 culture media (MS, ½MS, WPM, or ½WPM) with 7 different osmotic treatments: no osmotic agent, one osmotic agent [2% (w/v) glucose, mannitol, or sorbitol], or a combination of two osmotic agents [sorbitol + mannitol, sorbitol + glucose, or mannitol + glucose] at 2% (w/v) each. Each treatment consisted of five replicates, where each replicate was a flask with six nodal segments (n = 30). After 6 mo in the growth room, plant development was evaluated by measuring survival, plant height, and nodal segments per explant (multiplication rate). At the end of the 6-mo period, plants from the media that allowed >50% survival were selected for the recovery phase. Nodal segments were excised from these plants and placed in semisolid MS basal medium. After 3 mo in the growth room, recovery capacity was evaluated by measuring survival and multiplication rate. The requirement that plant survival under slow-growth conditions be greater than or equal to 50% to pass to the recovery phase was chosen assuming that plants grown under conditions giving ≥50% survival would provide better results in the recovery phase. Low survival in the slow-growth phase might indicate some problem in explant maintenance and, consequently, more difficulty in explant recovery.

Effect of alginate matrix, storage temperature, and storage time on plantlet conversion.

Approximately 420 nodal segments (0.5 cm) from in vitro cultures of M. moricandiana were excised and divided into two groups: nonencapsulated and encapsulated. In the nonencapsulated group, one lot consisting of 30 nodal segments was immediately placed in semisolid MS basal medium after excision. In the encapsulated group, nodal segments were placed in a glass flask with 50 mL MS medium without calcium and supplemented with 3% sodium alginate. Aliquots of the alginate solution, each of which contained one nodal segment, were taken up with a micropipette, dropped into 100 mL MS medium supplemented with 0.1 M calcium chloride, and maintained under agitation for 30 min to polymerize the capsules. After hardening, the alginate capsules were washed in distilled water and dried on filter paper. Thirty capsules were placed in semisolid MS basal medium. The remaining nonencapsulated nodal segments and capsules were placed into sterile cryogenic tubes and stored in darkness for 30 or 60 d at 4, 15, or 25°C. After storage, the nonencapsulated nodal segments and capsules were placed in semisolid MS basal medium and evaluated to determine the percentage of plant conversion after 1 mo in the growth room. The experimental design was fully randomized, consisting of both encapsulated and nonencapsulated nodal segments stored at three different temperatures (4, 15, and 25°C) and then sampled after 30 and 60 d, with three replicates per treatment. Each replicate was a cryogenic tube with 10 nodal segments (n = 30).

Effect of different moisture contents on plantlet conversion from nonprecultured capsules.

The determination of moisture content (MC%) of capsules was made as described by Subaih et al. (2007) and Melo et al. (2011), with modifications. One hundred capsules, prepared as described above, were divided into lots of 10 capsules each, placed in paper muffin cups with a blue silica gel indicator, and allowed to gradually dehydrate for 8 h at 25°C. After each 1-h period, the lots were weighed and returned to the paper muffin cup. After 8 h, all lots were dried in an oven at 70°C and then reweighed. The MC% obtained after each hour of dehydration was calculated using the following formula:

$$ MC\%=\left[\frac{\left( fresh\kern0.5em weight- dry\kern0.5em weight\right)}{ fresh\kern0.5em weight}\right]\times 100. $$

To check the effect of moisture content on plantlet conversion, 30 capsules with nodal segments were prepared and placed in semisolid MS basal medium; these were identified as nonprecultured capsules with 100% MC. The remaining capsules were maintained for 1, 4, or 7 h in paper muffin cups with a blue silica gel indicator and allowed to gradually dehydrate, respectively reaching 80, 40, and 20% MC, as established above. After each period, capsules in lots of 30 each were placed in semisolid MS basal medium. After 1 mo in the growth room, the capsules were evaluated to determine the percentage of plant conversion under each condition.

Effect of sucrose concentration during preculture and moisture content on plantlet conversion from capsules.

Capsules were placed in liquid MS basal medium with 0.40 or 0.75 M sucrose and then incubated for 2 d on a rotary shaker (100 rpm) in the growth room. After incubation, the capsules were washed in distilled water, and 30 capsules were placed in semisolid MS medium; these were identified as precultured capsules with 100% MC. The remaining capsules were subjected to dehydration, reaching 80, 40, and 20% MC as described in the previous section, and for each MC%, one lot consisting of 30 capsules was placed in semisolid MS basal medium. The experimental design was fully randomized, consisting of two different sucrose concentrations of liquid MS basal medium at four different MC%, with three replicates per treatment. Each replicate consisted of 10 nodal segments (n = 30) in glass flasks. All capsules placed in MS medium were evaluated after 1 mo to determine the percentage of plant conversion under each condition.

Effect of preculture time, storage temperature, and storage time on plantlet conversion from capsules.

This experiment was carried out under the conditions that produced the highest percentage of plantlet conversion in the previous experiment using both 2 and 7 d of preculture. After preculture in liquid MS with 0.40 M sucrose and dehydration until reaching 40% MC, capsules in lots of 30 each were stored in sterile cryogenic tubes and subjected to different storage conditions: −80°C for 24 h; 0°C for 30, 60, and 90 d; and 4°C for 30, 60, and 90 d. The experimental design was fully randomized, with three replicates per treatment, where each replicate was a cryogenic tube with 10 capsules (n = 30). At the end of each storage time, the cryogenic tubes were soaked in warm water for 90 s, and the capsules were rehydrated with 1 mL of liquid MS basal medium. The rehydrated capsules were placed in semisolid MS basal medium and maintained in the growth room for 1 mo. At the end of this time, the percentage of plant conversion was evaluated.

Rooting and acclimatization.

After 6 mo, all plantlets (3–5 cm) with well-developed shoots that had regenerated from encapsulated, stored nodal segments were transferred to glass flasks containing 50 mL semisolid MS medium supplemented with 2 mg L−1 IAA for rooting and maintained for 3 mo in a growth room under the same conditions described above. After rooting, the plants were washed in tap water to remove excess medium and carefully transferred to plastic tubes (3 × 10 cm) containing autoclaved vermiculite. The tubes were placed in a plastic box covered with plastic film to maintain high humidity. The box was maintained in a greenhouse at 28 ± 2°C for acclimatization. Over 1-mo time, the plastic film cover was removed gradually. After 3–4 mo, plants were transferred to garden soil under field conditions.

Statistical analysis.

Slow-growth storage and recovery data were subjected to two-way analysis of variance (ANOVA; P < 0.05). Carbon source data were subjected to one-way analysis of variance (ANOVA), and comparisons of means were carried out with the Tukey–Kramer test (P < 0.05). The significance of differences between means of percentage of plant conversion from alginate capsules was evaluated by the Student’s t test or Duncan’s multiple range test (P < 0.05). All statistical analysis was performed using the SigmaPlotTM software for Windows, version 11.0.

Results and Discussion

Conservation under slow-growth conditions.

Under slow-growth culture conditions, plants developed within 6 mo. Recovery of nodal segments from these plants was made in semisolid MS basal medium within 3 mo. Control plants developed in semisolid MS basal medium displayed a height of 5.23 cm and a multiplication rate of 6; however, it was considered desirable that plants developed under slow-growth conditions should display height and multiplication rates lower than those of the control (Table 1).

Table 1. Survival, height, and multiplication rate of M. moricandiana after 6 mo of culture on slow-growth storage medium, and survival and multiplication rate after 3 mo of recovery in MS medium
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Two-way ANOVA (Table 2) showed that basal medium and carbon source did not interact to improve any of the three evaluated parameters (height and multiplication rate in slow-growth and multiplication rate in recovery). The basal medium (MS, ½MS, WPM, and ½WPM) did not affect either the slow-growth storage or the recovery of M. moricandiana under the tested conditions. The utilization of media with salt concentration reduced to half the usual level has been recommended for the conservation of germplasm as a means of decelerating plant growth (Collin and Edward 1998). However, in a unique study reporting on Mandevilla under slow-growth maintained on media with reduced salt concentration and supplemented with osmotic agents (Biondo et al. 2007), these conditions contributed to accelerated plantlet growth.

Table 2. Two-way ANOVAs for effects of slow-growth storage medium on growth characteristics of M. moricandiana after 6 mo of slow-growth storage and after 3 mo of recovery in MS medium
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According to Bekheet (2011), the addition of osmotic agents to in vitro culture was an efficient method of growth inhibition, causing reduction of the plant’s metabolic activities without affecting viability and also increasing the storage life of many in vitro-grown tissues of different plant species. In the present study, carbon source had a significant effect on slow-growth storage and recovery. Sucrose, the original carbon source of all tested media, promoted the highest values of height and multiplication rate under slow-growth storage, so it was considered unsuitable for this purpose. Sorbitol and glucose, separately or combined, promoted development of plants with low multiplication rate during the slow-growth period and with high multiplication rate during recovery (Fig. 1), demonstrating their suitability for use in culture medium for slow-growth storage of M. moricandiana.

Figure 1.
figure 1

Average values of height (A) and multiplication rate (B) of M. moricandiana after 6 mo of culture in slow-growth storage and multiplication rate (C) after 3 mo of recovery in MS medium, according the carbon source used in the slow-growth storage medium. In each graph, different letters indicate statistical differences by the Tukey–Kramer test (P < 0.05). GLU glucose, MAN mannitol, SUC sucrose, SOR sorbitol.

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All media that gave survival rates lower than 50% had mannitol as a supplement. The other media supplemented with mannitol gave more than 50% survival under slow-growth conditions, but the plants were short and had low multiplication rates. These plants were tested in the recovery phase because of the possibility that they would display high multiplication rates, but this did not occur (Table 1). Mannitol as a carbon source promoted high mortality in slow-growth storage and low height and multiplication rates in the recovery phase, demonstrating its ineffectiveness for use in conservation medium (Fig. 1). Although high mannitol concentration may be harmful and cause plant death (Silva and Scherwinski-Pereira 2011), this carbon source can enhance survival of plant germplasm conserved in vitro (Sarkar and Naik 1998). Other studies with Apocynaceae have also utilized mannitol. The deleterious effects of mannitol in slow-growth storage were shown in Hancornia speciosa (Sá et al. 2011) and Macrosyphonia velame (Martins et al. 2011). The use of mannitol resulted in low multiplication rates in M. velutina, even though the plants had not senesced (Biondo et al. 2007).

In vitro conservation of M. moricandiana under slow-growth permits storage of at least 6 mo and rapid recovery of cultures without developmental difficulties. Unexpectedly, high survival rates of the explants during the slow-growth phase did not ensure their development during the recovery phase. The present results indicate that in addition to high survival rates, the explants require a minimum multiplication rate for recovery; indicating that time in slow-growth did not affect their regenerative capacity (Fig. 2).

Figure 2.
figure 2

M. moricandiana after 3 mo of recovery in semisolid MS basal medium, following 6 mo in slow-growth storage on ½WPM supplemented with 2% sorbitol and 2% glucose.

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Conservation by encapsulation.

After nodal segment excision and before storage, the nonencapsulated nodal segments showed 100% plantlet conversion after 2 wk of culture, while the encapsulated nodal segments showed only 83.3% plantlet conversion after 1 mo of culture. The encapsulated nodal segments showed conversion into plantlets after storage for 30 d at 15°C, reaching 33.3% survival, but did not show conversion at either 4 or 25°C. After 60 d, no encapsulated nodal segments at any storage temperature showed conversion. On the other hand, storage at 4 and 15°C was lethal to the nonencapsulated nodal segments of M. moricandiana, which showed high resistance and gave 66.6% conversion into plantlets after 30 d of storage at 25°C, decreasing to 16.6% after 60 d of storage at the same temperature (Table 3).

Table 3. Percentage of plantlet conversion from encapsulated and nonencapsulated nodal segments of M. moricandiana before storage and after storage at 4, 15, and 25°C for 30 and 60 d
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Dehydration of encapsulated nodal segments from 100 to 20% MC without preculture decreased the plant conversion percentage in semisolid MS basal medium from 86.6 to 56.6% after 30 d (Table 4). For precultured encapsulated segments, all MC levels and both sucrose concentrations in the preculture medium gave >40% conversion. Plantlet conversion rates above 80% were obtained from nonprecultured encapsulated nodal segments at 100% MC and from encapsulated nodal segments precultured in liquid MS medium with 0.40 M sucrose and at 40–100% MC. Encapsulated nodal segments precultured in medium containing 0.40 M sucrose and dried to 40% MC gave the best conversion rate (93.3%), so these conditions were chosen for a test of different storage times and temperatures. Although the nodal segments precultured in liquid MS medium with 0.40 M sucrose and at 100% MC had an 86.6% conversion rate, low moisture content is recommended for storage at low temperatures (Engelmann 2004; Melo et al. 2011) so this condition was not tested further.

Table 4. Plantlet conversion after 30 d from encapsulated nodal segments of M. moricandiana without preculture and after 2 d of preculture in MS with different sucrose concentrations and moisture content (MC) levels
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When encapsulated nodal segments were precultured for 2 or 7 d in liquid MS medium with 0.40 M sucrose (Table 5), encapsulated nodal segments before storage showed ca. 90% plantlet conversion, with no significant difference between the two preculture periods. After 30 or 60 d of storage, only the encapsulated nodal segments stored at 4°C showed plantlet conversion. The encapsulated nodal segments precultured for 2 d resulted in 26.6% conversion after storage for 30 d, decreasing to 3.33% after 60 d, while for encapsulated nodal segments precultured for 7 d, the survival rate after 30 d was 33.3%, decreasing to 6.66% after 60 d. The conversion rates for each storage time showed no significant difference between the two preculture periods. After 90 d, no encapsulated nodal segments survived. Regardless of preculture time, none of the encapsulated nodal segments stored for any length of time at −80 or 0°C were recovered, indicating that preculture did not enable encapsulated nodal segments to convert into plants after storage at temperatures lower than 4°C.

Table 5. Percentage of plantlet conversion from encapsulated nodal segments of M. moricandiana precultured in MS medium with 0.40 M sucrose, dehydrated to 40% moisture content, and stored under various conditions
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Nodal segments encapsulated in sodium alginate matrix are shown in Fig. 3a . After emergence of the shoot from the capsule, plantlet conversion and development was easy and rapid (Fig. 3b ). The sequence of development of plantlets converted from encapsulated nodal segments 4 wk after placement in semisolid MS basal medium is illustrated in Fig. 3c .

Figure 3.
figure 3

Plantlet regeneration from encapsulated nodal segments of M. moricandiana. a, Nodal segments encapsulated in sodium alginate capsules. b, Shoot emergence in semisolid MS basal medium. c, Sequence development of plantlets converted from encapsulated nodal segments during a period of 4 wk. d, Well-hardened plants of M. moricandiana in pots after 1 yr of acclimatization. Bar = 5.0 cm.

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Before storage, the recovery rate of nonencapsulated nodal segments was higher than that of encapsulated nodal segments. On the other hand, reports in literature have indicated that the sodium alginate matrix offers protection against dehydration and supplies nutrients to explants, resulting in a conversion rate of encapsulated nodal segments equal to or greater than that of nonencapsulated nodal segments (Singh et al. 2009, 2010). The before-storage results show that the sodium alginate matrix consistency may have impeded shoot emergence of M. moricandiana. Difficulties and delay in shoot or root emergence were reported for alginate beads with sodium alginate concentrations higher than 4–5% (Singh et al. 2010). Sodium alginate concentrations of 2.5–4% and 0.1 M calcium chloride, as used in this study, have been considered most suitable for the formation of beads by allowing optimally sized, uniformly spherical, firm capsules (Kumar et al. 2010; Sundararaj et al. 2010; Sharma and Shahzad 2012), thus reducing the effects of salt stress resulting from exposure of the nodal segments to the sodium alginate solution (Faisal et al. 2006; Singh et al. 2009; West and Preece 2009). In the absence of preculture, the sodium alginate matrix only protected nodal segments under storage at 15°C, showing negative effects at 4 and 25°C. In a study with Rauvolfia tetraphylla, encapsulated nodal segments stored at 4°C showed conversion rates close to 90% after 1 mo and close 30% after 2 mo (Faisal et al. 2006).

The encapsulation–dehydration method used in the present study is based on the method of producing synthetic seeds reported by Engelmann (2004). In this protocol, explants are encapsulated in a sodium alginate matrix, precultured on medium supplemented with sucrose from 1 to 7 d, dehydrated under airflow or by exposure to silica gel to reduce the moisture content, and quickly frozen. According to Shibli et al. (2006), this procedure has been successfully used for conservation of large structures, such as apical and lateral buds, and for a variety of species in which other methods have been ineffective. The methodology used for M. moricandiana was the same as suggested by Engelmann (2004), except that the quick-freezing step was omitted. Storage at −80, 0, and 4°C was tested to enable alternative conservation methods, because cryopreservation requires more sophisticated conditions than those available in a typical laboratory.

The test of encapsulated nodal segments precultured in liquid MS medium with two different sucrose concentrations was aimed at selecting the most suitable concentration to ensure survival under subsequent storage at different temperatures. Since the accumulation of sugar by cells increases the stability of the membrane when exposed to dehydration, the preculture step was intended to induce resistance to dehydration and freezing (Sakai 2000).

Encapsulated nodal segments precultured for 2 d in liquid MS medium with 0.40 M sucrose reached the highest conversion rates after gradual drying to 40% MC on silica gel, but the conversion rate of the nonprecultured encapsulated nodal segments decreased in proportion to the decrease in MC%. Similar results were presented by Melo et al. (2011) in studies with cane sugar. These authors concluded that high concentrations of sucrose may result in osmotic shock, which would explain the decrease in conversion rates of the encapsulated nodal segments precultured in liquid MS medium with 0.75 M sucrose. According to Wang et al. (2002), progressive increase in sucrose concentrations can reduce the sensitivity of explants to high concentrations of sugar, but that procedure was not tested here.

After establishing the optimal concentration of sucrose in the preculture medium and MC% of the encapsulated nodal segments, the preculture time was extended in an attempt to increase the resistance of explants to dehydration and freezing. Nonstored encapsulated nodal segments reached the highest conversion rate when precultured for 7 d; however, increasing the preculture time did not significantly affect the conversion rates of encapsulated nodal segments. Irrespective of preculture time, only encapsulated nodal segments stored at 4°C survived, with ca. 30% survival after 30 d and ca. 5% after 60 d. According to Melo et al. (2011), the optimal time of preculture seems to be closely related to the concentration of sucrose used and the species studied.

All plantlets regenerated from encapsulated nodal segments stored for different periods at different temperatures had well-developed shoots and were placed in a rooting medium. After rooting, plants were successfully acclimatized in plastic pots and transplanted into field conditions, with 100% survival (Fig. 3d ).

Although encapsulation–dehydration techniques are commonly applied to cryopreservation, the results reported here show the possibility of storing M. moricandiana encapsulated nodal segments at alternative temperatures for 1 mo. Adjustments in sucrose concentration, duration of preculture, and MC% can provide higher conversion rates and increased storage time. This is the first report of using encapsulation–dehydration techniques for Mandevilla.