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Many different species of Bromeliaceae are endangered and their conservation requires specific knowledge of their growth habits and propagation. In vitro culture of bromeliads is an important method for efficient clonal propagation and in vitro seed germination can be used to maintain genetic variability. The present work aims to evaluate the in vitro growth and nutrient concentration in leaves of the epiphyte bromeliads Vriesea friburguensis Mez, Vriesea hieroglyphica (Carrière) E. Morren, and Vriesea unilateralis Mez, which exhibit slow rates of growth in vivo and in vitro. Initially, we compared the endogenous mineral composition of bromeliad plantlets grown in half-strength Murashige and Skoog (MS) medium and the mineral composition considered adequate in the literature. This approach suggested that calcium (Ca) is a critical nutrient and this was considered for new media formulation. Three new culture media were defined in which the main changes to half-strength MS medium were an increase in Ca, magnesium, sulfur, copper, and chloride and a decrease in iron, maintaining the nitrate:ammonium rate at ≈2:1. The main difference among the three new media formulated was Ca concentration, which varied from 1.5 mm in half-strength MS to 3.0, 6.0, and 12 mm in M2, M3, and M4 media, respectively. Consistently, all three species exhibited significantly higher fresh and dry weight on M4, the newly defined medium with the highest level of Ca (12 mm). Leaf nitrogen, potassium, zinc, magnesium, and boron concentrations increased as Ca concentration in the medium increased from 1.5 to 12 mm.
Bromeliaceae are known for their diverse forms, which range from soil-rooted terrestrials to epiphytes, which rely on their “tanks” and/or absorptive trichomes for water and nutrient absorption (Crayn et al., 2004). Epiphytic bromeliads are commonly well adapted to arid environments not only as a result of morphological modifications, but also for the presence of the crassulacean acid metabolism (CAM), a photosynthesis adaptation common in many Bromeliaceae. Although CAM photosynthesis improves water use efficiency as a result of limited water vapor exchange with the atmosphere during the day (Crayn et al., 2004), the limitation of CO2 capture only during the nighttime results in slower plant growth rates (Cushman and Bohnert, 1999). This type of metabolism has important implications for the mineral nutrition of these species. In addition, nutrient absorption in “atmospheric” (e.g., Tillandsia spp.) or in “tank” bromeliads (e.g., Aechmea spp. and Vriesea spp.) frequently occurs in the organic form by absorptive leaf trichomes, making the process of mineral nutrition in bromeliads unique (Benzing, 1990). These specificities related to growth and nutrient absorption of bromeliads have raised interest in the use of specific culture conditions for bromeliad micropropagation (Endres and Mercier, 2001; Hosoki and Asahira, 1980; Nievola et al., 2001) and have been the subject of a growing number of studies on different ornamental bromeliad genera such as Guzmania (Lin and Yeh, 2008), Noeregelia (Carneiro et al., 1999), Dyckia (Pompelli and Guerra, 2005), Cryptanthus (Carneiro et al., 1998; Mathew and Rao, 1982), Tillandsia (Pickens et al., 2000, 2003, 2006), and Vriesea (Alves et al., 2006; Mercier and Kerbauy, 1992, Rech Filho et al., 2005).
In addition to factors that influence in vitro growth such as plant growth regulators, temperature, and light quality, mineral nutrition plays an important role (Morard and Henry, 1998; Niedz and Evens, 2007). For an efficient micropropagation process, it should be noted that the ideal mineral composition of the culture medium may vary considerably with each species or genotype (Gonçalves et al., 2005; Williams, 1991). Although there are many reports on medium optimization, growth regulator type and concentration are the most frequent objects of study. In contrast, little has been done in terms of in vitro mineral nutrition, especially for nonconventional species (Morard and Henry, 1998) such as bromeliads (Endres and Mercier, 2001; Nievola et al., 2001). Only recently, as mentioned by Niedz and Evens (2007), have a number of different approaches been used by researchers attempting to define the adequate balance of mineral nutrients with positive results. The availability of new media formulation for different species is important for a better response in vitro and has not yet been attempted for bromeliads.
Designing culture media according to the specific mineral composition of the plant tissues has been reported mainly with perennials such as olive (Cozza et al., 1997), passion fruit (Monteiro et al., 2000), eucalypt (Gribble et al., 2002), hazelnuts (Nas and Read, 2004), pear (Sotiropoulos et al., 2006), and apple (Sotiropoulos, 2007). However, the most common medium, used in 50% to 75% of the micropropagated plants, is still Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), which was formulated for tobacco callus cultures (Gribble et al., 2002). The interaction among mineral elements creates challenges for the definition of new culture media (Nas and Read, 2004, Niedz and Evens, 2007).
Slow growth of Vriesea spp. in vitro, when compared with other bromeliads, was readily detected in several experiments in our laboratory. Comparisons of liquid versus solid medium and the use of a temporary immersion system showed very limited increases in Vriesea growth in vitro compared with results observed for Aechmea species, both “tank” epiphyte bromeliads (Aranda-Peres, 2005). The fact that several Vriesea species, endemic to the Brazilian Atlantic Forest, have been listed as species under risk of extinction (Aranda-Peres and Rodriguez, 2006) led us to intensify our efforts to improve their micropropagation. A similar approach successfully used to define a micropropagation medium for passion fruit (Monteiro et al., 2000) was attempted. The approach was to determine the endogenous mineral nutrient composition of micropropagated plants grown in different culture media.
The aim of the present study was to improve the growth and mineral composition of three epiphyte bromeliad species, Vrisea hieroglyhica, V. unilateralis, and V. friburguensis (Fig. 1), by adjusting the mineral composition of MS medium according to in planta nutrient levels and nutritional recommendations for bromeliads.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.106
For a preliminary definition of culture media, Aechmea nudicaulis (L.) Griseb, a more abundant and easy-to-propagate bromeliad species, was used. Seeds of A. nudicaulis were germinated in vitro on MS medium (Murashige and Skoog, 1962) with the concentration of macronutrients reduced to one-fourth. When seedlings reached ≈1 cm in height, they were transferred into Magenta boxes containing MS medium with the concentration of macronutrients reduced to half-strength, full-strength MS micronutrients and vitamins, 30 g·L−1 sucrose, and solidified with phytagel (2 g·L−1). Every 3 weeks, plantlets were subcultured to fresh medium and after 60 d in culture, leaf mineral analysis was done according to Malavolta et al. (1992; see leaf mineral analysis subsequently). Nutrient levels were compared with those available in the literature for bromeliads (Mills and Jones, 1996; Table 1).
Based on these observations, and as a result of the importance of calcium (Ca) in so many mechanisms in plant growth and metabolism, the following media were defined varying the Ca concentration: 1.5 mm (half-strength MS), 3.0 mm (M2), 6.0 mm (M3), and 12.0 mm (M4), which corresponds to 1 ×, 2 ×, 4 ×, and 8 × the Ca concentration in half-strength MS medium (Tables 2 and 3). Additionally, magnesium (Mg) and copper (Cu) concentrations were doubled and iron (Fe) was reduced by half in the new media (Tables 2 and 3). As a consequence of the salts used to increase Mg and Cu concentrations, sulfur levels were also increased (1.69×). The reduction of the source of Fe (FeSO4.7H2O + Na2EDTA) caused a reduction in sodium concentration. Besides calcium chloride, we also used calcium nitrate, which is not normally present in MS medium, to increase the concentration of Ca. This also increased the level of chlorine in culture media M3 and M4 but maintained the nitrate:ammonium ratio only slightly different in M2 (2.4:1.0), M3 (2.2:1.0), and M4 (2.2:1.0) compared with half-strength MS (2.0:1.0).
Vriesea friburguensis Mez, V. hieroglyphica (Carrière) E. Morren, and V. unilateralis Mez seeds were germinated in vitro on MS medium with the concentration of macronutrients reduced to one-fourth. When plantlets were ≈1 cm in height, they were transferred to Magenta boxes containing MS medium with the concentration of macronutrients reduced to half-strength and MS micronutrients and vitamins kept at full strength, 30 g·L−1 sucrose, and solidified with phytagel (2 g·L−1). Cultures were subcultured every 3 weeks until the seedlings reached ≈3 cm in height with average fresh weight of 0.15 g (V. friburguensis and V. hieroglyphica) and 0.10 g (V. unilateralis). These plantlets were then selected for the experiment and transferred to the four previously defined culture media: half-strength MS, M2, M3, and M4 (Table 2) supplemented with MS vitamins, 30 g·L−1 sucrose, 0.5 mg·L−1 BAP, and solidified with phytagel (2 g·L−1).
A completely randomized 3 × 4 factorial design with three species and four culture media with six replications was used. Each replication was composed of one magenta box with six plantlets.
The pH of all media was adjusted to 5.8 before autoclaving using HCl and KOH solutions. All cultures were maintained in a growth room under a 16-h photoperiod, temperature of 27 ± 2 °C, and light intensity of 40 μmol·m−2·s−1. Cultures were subcultured every 3 weeks and the experiment was evaluated after 12 weeks through measurements of fresh and dry weight and leaf mineral analysis.
The plantlets were thoroughly washed in tap water followed by the separation of roots from aerial parts. Fresh weight of plantlets from each replication was taken by weighting individual plants and then the samples were dried for 72 h at 62 °C, when weight stabilized, for dry weight measurement of individual plantlets. For fresh and dry weight determination, an average of 25 plantlets was taken. Means were compared by Tukey test using the Assistat Package (www.assistat.com).
The total dried aerial parts of the plantlets were analyzed for the determination of leaf macro and micronutrients using the methodology for mineral analysis described by Malavolta et al. (1992). Briefly, for nitrogen (N) determination, samples were subjected to a sulfuric digestion and analyzed by the micro-Kjeldahl method (Keys, 1938) using a nitrogen distillator (Tecnal TE 036/1, Piracicaba, SP/Brazil). For potassium (K), Ca, Mg, sulfur (S), phosphorous (P), and the micronutrients Cu, manganese (Mn), Fe, and zinc (Zn), samples were subjected to nitric–perchloric digestion. For boron (B), samples were incinerated. After digestion, Ca, Mg, Cu, Mn, Fe, and Zn were determined by atomic emission spectrometry (Spectrometer Perkin Elmer 3110, Norwalk, CT). K was determined by flame photometry (Photometer Micronal B262, São Paulo, Brazil). A digital spectrophotometer (Micronal B 34211, São Paulo, Brazil) was used for determination of S by barium turbidimetry and P and B by colorimetry. Chloride (Cl) was determined by titration with AgNO3 (0.05N) using potassium chromate (5%) as an indicator.
Leaf mineral analysis of Aechmea nudicaulis plantlets cultured in half-strength MS medium (Table 1) suggested that the plantlets were deficient in Ca, Mg, and Cu and that the levels of N and Fe were above those recommended by Mills and Jones (1996) (Table 1). Comparisons of the leaf nutrient levels of Aechmea nudicaulis in half-strength MS medium with the recommendations for Aechmea fasciata (Mills and Jones, 1996) and adequations of the medium components resulted in three newly defined media (Tables 2 and 3). These new media were used for culturing the three Vriesea species, which were the object of this work. Levels of the macronutrients Ca, Mg, and S and micronutrients Fe, Cu, and Cl varied between the evaluated media (Table 3). Visual observations of Vriesea plantlets after 12 weeks of in vitro culture show that all three species (V. friburguensis, V. unilateralis, V. hieroglyphica) cultured on medium containing the highest level of Ca (M4) had enhanced plantlet growth compared with half-strength MS, M2, and M3 media (Fig. 2).
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.106
Fresh and dry weight of all three species showed similar results when grown on half-strength MS, M2, and M3 media (Fig. 3). However, plantlets of all three species grown in M4 medium exhibited significantly (P < 0.05) higher fresh and dry weight (Fig. 3). V. friburguensis showed the highest growth, especially in M4 medium. However, V. unilateralis showed the highest increase in dry weight when plantlets cultured in half-strength MS versus M4 were compared with increase in dry weights of 92%, 76%, and 52% observed for V. unilateralis, V. friburguensis, and V. hieroglyphica, respectively. This indicates that although V. unilateralis had lower growth rates, this species benefited the most from the nutrient composition changes from half-strength MS to M4.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.106
Macro- (Fig. 4) and micro- (Fig. 5) nutrient concentrations in leaves of plantlets growing on M4 medium were very similar for all three species and presented striking differences when compared with those on half-strength MS medium. Higher levels of nutrients were observed in plants grown on M4 (Figs. 4 and 5), including some nutrients supplied equally in both media such as N, K, Zn, Mn, and B (see Table 3). The only nutrient showing a discrepancy in concentration among the three species was P, which was supplied in the same amount in half-strength MS and M4 media yet had foliar concentrations higher on half-strength MS medium for V. friburguensis, higher on M4 medium for V. unilateralis, and to a lesser extent for V. hieroglyphica. For the macronutrients Ca, Mg, and S, and the micronutrient Cu, which were higher on plantlets cultivated on M4 medium than on half-strength MS (Table 3), the differences in concentration in leaf tissues in the three species (Figs. 4 and 5) tended to be proportional to the differences in the supplement between the two media. Exceptions are Cl and Fe. Although the supplement of Cl in M4 was 6.7 times greater than that in half-strength MS, the difference in tissue concentration did not exceed 2.5 times in any of the three species (Fig. 5). This is consistent with the negative electrochemical gradient of plant cells (Higinbotham et al., 1967), which usually hampers the absorption of anions such as Cl−. Fe concentration in tissues was also not proportional to the quantity added in the medium. In half-strength MS medium, Fe supply was twice as much as in M4 (Table 3); however, Fe concentration in tissues cultured on the two media tended to be similar for all three species (Fig. 5). Moreover, the concentration of both Fe and Cl in tissues grown in M4 and half-strength MS was apparently excessive, although no obvious symptoms of toxicity were observed in any of the three species studied (Fig. 2).
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.106
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.106
In the present work, a similar methodology to that used for passionfruit (Monteiro et al., 2000) was used to design a more adequate medium for the in vitro culture of bromeliads. This was based on the fact that bromeliads present considerable particularities in terms of mineral nutrition in nature (Benzing, 1973), which can affect their behavior in vitro.
The mineral analysis of Aechmea nudicaulis, cultivated on half-strength MS medium (Table 1), suggested the need for a finer adjustment of some of the minerals in the culture medium, especially Ca. Vriesea plantlets grown on M4 medium, had fresh and dry weights significantly superior to those in all media tested and only differed from M2 and M3 in the levels of Ca and Cl (see Table 3). Thus, we suggest that the effect of M4 on biomass accumulation (Fig. 3) can be attributed to these two nutrients. Considering that Cl is a micronutrient, and thus not expected to be needed in high levels, it is very reasonable to assume that the growth-promoting effect of M4 medium was the result of an extra supplement of the macronutrient Ca. In fact, this nutrient accumulated in all three Vriesea species in ranges close to those recommended for another tank bromeliad (Mills and Jones, 1996; see Table 1) only when cultured on medium M4 (12.5 to 14.0 g·kg−1; see Fig. 4) with accumulation in half-strength MS (1.9 to 2.5 g·kg−1) even lower than that found in naturally occurring conditions for other tank bromeliads (Benzing, 1973).
The higher levels of Ca in M4 not only allowed an enhanced growth in all three species of Vriesea (Fig. 2), but also indicate that Ca may have facilitated the absorption of other nutrients. This seems to be the case for N, K, Zn, Mn, and B in that the concentration of the nutrients was not increased in the M4 medium composition (Table 3) but was present in higher levels in plantlets grown on M4 versus half-strength MS medium (Figs. 4 and 5). A positive effect of Ca supplementation on N, Zn, Mn, and B accumulation but hardly any change in the concentrations of K was reported for tobacco (Lopez-Lefebre et al., 2001).
Calcium has an important role in cell signaling (Reddy, 2001) acting as a secondary messenger together with signal transduction proteins such as calmodulin and Ca2+-dependent protein kinases and also maintains the integrity of the plasmalemma. Ca2+ stabilizes cell membranes by connecting various proteins and lipids on membrane surfaces (Hirschi, 2004). This integrity of the plasmalemma leads to a greater turgor pressure (higher water content) and nutrient retention in cells (Fenn and Feagley, 1999). To protect the plasma membrane, Ca2+ must always be present in the external solution (apoplast), where it can regulate the selectivity of ion uptake. Thus, both effects of Ca in the protection of cell membranes (e.g., regulation of ion uptake and nutrient retention in cells) may account for the enhanced foliar concentration of different nutrients observed when plants are grown in media with higher Ca levels (Figs. 4 and 5). Furthermore, Ca can also directly influence the content of other nutrients acting as a secondary messenger. For instance, it is known that the opening of potassium channels in leaves, especially in guard cells, is affected by Ca2+ as a secondary messenger (Schroeder et al., 2001).
The enhanced growth of bromeliads in M4 medium can be the result of a better nutritional status related to the accumulation of various minerals favored by Ca as discussed previously. However, an additional cause may be the direct effect of Ca on cell and organ growth. Ca is involved in cell elongation and cell division, influences cellular pH, and also acts as a regulatory ion in the source-sink translocation of carbohydrates through its effects in cells and cell walls (Hirschi, 2004). Furthermore, plants need Ca2+ to strengthen cell walls and provide biotic and abiotic stress protection. This last effect of Ca may have had limited importance for the in vitro growth but may have favored plantlets propagated in M4 medium during acclimatization and further ex vitro growth.
When the recommended levels in the literature are considered (Mills and Jones, 1996), some elements such as Mg and Cu were still in low concentration, whereas an apparent excess of Fe and N was detected in the leaves of plants cultured on M4 medium. Although the results in terms of growth were considerably positive, fine adjustments to the medium can be further attempted to improve culture conditions. Studies on the interaction between mineral elements and growth regulators such as cytokinins should also be attempted, because it is known that this class of substances may act as a negative signal to some nutrient starvation in plants (Martin et al., 2000).
The present work has shown the importance of making adjustments to the mineral composition of the culture medium, which resulted in higher levels of mineral elements in plantlets and improved growth during the micropropagation process of three Vriesea species. Particular changes were related to the calcium levels in the culture medium; however, other elements that were not present in the recommended levels were also adjusted.
Contributor Notes
We thank Dr. Gustavo Martinelli from the Botanical Garden of Rio de Janeiro, Brazil, and Dr. Shoey Kanashiro from the Botanical Garden of São Paulo, Brazil, for providing plants and seeds for introduction in vitro and Dr. Hazel Y. Wetzstein, from the University of Georgia, for reviewing this manuscript. Thanks to João Geraldo Brancalion, CENA/USP, for graphics assistance; to FAPESP, Brazil, for doctoral scholarship to ANAP (2000/07987-5) and CNPq, Brazil, for research fellowship to APM (304659/2005-3).
In memoriam.
To whom reprint requests should be addressed; e-mail adriana@cena.usp.br
