Effect of sucrose concentrations on in vitro growth and subsequent acclimatization of the native bromeliad Vriesea inlata ( Wawra ) Wawra

Using sucrose is crucial for in vitro plant growth, due to decreased photosynthesis under this condition. However, ornamental epiphytic species, which have slow growth, have shown better development in nutrient media with low concentrations of this carbohydrate. Vriesea inlata (Wawra) Wawra is an epiphytic bromeliad native to the threatened Atlantic Rain Forest and it has a high ornamental value. This study aimed to evaluate the effects of various sucrose concentrations in the culture medium on in vitro growth and subsequent ex vitro acclimatization. Plants were cultured in vitro in basal media without carbohydrates; and in media containing 1.0; 1.5; 3.0; 4.5; and 6.0% (w/v) of sucrose for 60 days. After, plants were acclimatized for 90 days. Plants cultured in sucrose concentrations over 3.0% had the smallest development after in vitro culture and acclimatization.


Introduction
The bromeliad Vriesea inlata (Wawra) Wawra is native to the threatened Atlantic Rain Forest, and it has morphological traits of high ornamental value (PEDROSO et al., 2010) (Figures 1a;1b).This species and many other bromeliads are extracted from their original habitat to supply the ornamental trade, something which can pose a signiicant threat to the Bromeliaceae biodiversity (NUNES, 2002;NEGRELLE et al., 2012).Many in vitro culture protocols have been developed for bromeliad species to effectively propagate them, aiming both at conservation and commercialization (SILVA et al., 2012;SANTA-ROSA et al., 2013;KURITA et al., 2014).
The development of plants in vitro depends to a large extent on the supply of carbon sources in the culture media, since morphogenesis and growth are high energy requiring processes.However, the required carbohydrate concentration varies according to species, something which demands the deinition of speciic protocols (YASEEN et al., 2013).Sucrose is one of the more frequently used carbon sources on in vitro cultivation, where concentrations from 2 to 4% usually lead to optimal growth, as reported by Hazarika (2003).Accordingly, bromeliads are mostly cultured in vitro with 3% sucrose in the medium (DAL VESCO et al., 2011;CARVALHO et al., 2013;MARTINS et al., 2013).
Vriesea inlata has been propagated in vitro, as previously reported by our research group (PEDROSO et al., 2010).Studies report that the species have slow growth, even when cultured in vitro.Therefore, it is important to optimize the in vitro V. inlata growth in order to obtain vigorous plants that can acclimatize in a more effective way.It has been reported that lower concentrations of carbon sources in the culture medium can improve acclimatization (JO et al., 2009), which have been associated with higher photosynthetic ability (LANGFORD; WAINWRIGHT, 1987;HUYLENBROECK;DEBERGH, 1996).Lower sucrose concentrations in the nutrient medium were applied to other epiphytic bromeliads and high survival rates after acclimatization were obtained, as described for Vriesea gigantea Gaudich.(BENCKE; DROSTE, 2008) and Acanthostachys strobilacea (Schult.f.) Link, Klotzsch & Otto (SANTOS et al., 2010).
This study aimed to evaluate the effects of various sucrose concentrations on in vitro and subsequent ex vitro V. inlata growth, in order to verify if lower concentrations improve the species' acclimatization.

In vitro culture at different sucrose concentrations
One month after the seeds germinated (Figure 1c), 10 plants were transferred to each 250 mL lask containing 40 mL of basal media, without sucrose and supplemented with 1.0, 1.5, 3.0, 4.5 and 6.0% (w/v) sucrose (Synth ® ).Flasks were kept in growth room under the same conditions described above.After 60 days of culture, 10 plants from each treatment were evaluated regarding the biometric features of survival rate, number of leaves and roots, length of the longest leaf and root, fresh and dry shoot mass.

Ex vitro acclimatization
After 60 days of culture under different sucrose concentrations, 10 additional seedlings from each treatment were transferred from the lasks to trays containing ine sterilized commercial medium-sized Pinus bark (100%) (Figure 1d).Plants from each treatment were fertilized biweekly with 10 mL of a solution with 50% macronutrient salts as proposed by Murashige and Skoog (1962).Trays containing plants were kept in growth room under the same conditions cited above for 90 days, then plants were evaluated regarding the features described in the previous section.C. Freitas et al.

Statistical analysis
The experiments' design was completely randomized.For the in vitro culture and acclimatization experiment, the number of samples was 10 per treatment (n = 10).Shoot mass was determined in triplicates (n = 3).Data underwent analysis of variance, followed by regression analysis at p < 0.05 by using the software SISVAR.

Results and Discussion
Vriesea inlata plants cultured in vitro without sucrose had low survival rates (20%), perhaps because most plants could not photosynthesize in an effective way under this condition to generate energy for other metabolic processes (VON ARNOLD, 2008).However, varying sucrose concentration from 1.0 to 6.0% enabled total survival of V. inlata plants (100%).Those treatments did not signiicantly inluence the development of new leaves (Figure 2a).On the other hand, the number of roots, leaf and root length increased linearly with the reduction of sucrose in the medium, therefore, plants at 1.0% sucrose showed the highest mean values (Figure 2a; 2b).Similar responses were noticed for banana and apple plants, where both had higher development when cultured at lower sucrose concentrations (< 3.0%) (YASEEN et al., 2009;WAMAN et al., 2014).
Fresh and dry shoot mass showed a quadratic response to the varying concentrations, where plants kept at 1.0% sucrose also had the highest values (Figure 2c).The alteration in V. inlata biometric features must derive from the inluence sugars such as sucrose have on growth and development, because they have high nutritional value and control over many genes related to morphogenetic and metabolic processes (KOCH, 1996;SOTIROPOULOS et al., 2006).
All plants cultured in vitro at 1.0-6.0%sucrose after ex vitro acclimatization (100%).The varying sucrose concentrations had signiicant effects on all biometric characters of acclimatized plants, in which they showed a quadratic response (Figure 3).The maximum average of leaf and root growth were 1.6 and 1.5% sucrose, respectively, whereas plants from higher concentrations had smaller values (Figure 3a; 3b).The results are in accordance with those observed before acclimatization (Figure 2).According to Rolland et al. (2006), carbon sources are essential for the production of growth hormones (auxins), which induce root development.However, the carbohydrate concentration required to produce auxins depends on the species.In fact, high sugar concentrations can lead to reduced osmotic potential in the media, resulting in lower availability of water for the plants, which in turn hampers growth (CALVETE et al., 2002;JO et al., 2009).Sucrose concentration affects bromeliad's acclimatization Fresh and dry mass of acclimatized plants were maximum at 1.9 and 1.7% sucrose, respectively (Figure 3c), similar to leaf and root growth.In average, the maximum growth after acclimatization was observed in plants cultured in vitro at concentrations circa 1.7%, something which suggests that such sucrose levels generate in vitro plants more adapted to grow under ex vitro conditions for longer periods.These results might be due to the signiicant root development observed in plants cultured in vitro under 3.0% sucrose (Figure 2a; 2b).Likewise, Galdiano Junior et al. (2013) noticed that cultivating the orchid Cattleya loddigesii Lindley under 2.0% sucrose effectively induced in vitro root growth, which resulted in higher survival after acclimatization in comparison with plants obtained from 3.0-4.0%sucrose.Nievola and Mercier (1996) have shown that not only leaves but also roots of the bromeliad Vriesea fosteriana L.B. Sm., which can be found in epiphytic conditions, are important for nitrogen assimilation.Considering that V. inlata is also an epiphytic bromeliad, the best developed root system of plants obtained from < 3.0% sucrose concentrations may have led to a more effective nutrient assimilation, producing vigorous plants after acclimatization.
According to regression analysis, cultivating V. inlata plants in vitro at 1.7% sucrose for 60 days promotes signiicant root growth, which leads to more vigorous plants after 90 days of ex vitro acclimatization when compared to higher sucrose concentrations.

FIGURE 1 :
FIGURE 1: Vriesea inlata as seen in the natural environment, its ornamental use, and the stages of in vitro culture: a) adult plant located at the "Alto da Serra de Paranapiacaba" Biological Reserve; b) lowering plants in vases indicating ornamental value; c) 1-month-old in vitro seedlings (bar: 1 cm); d) general aspect of 4-month-old in vitro plants after acclimatization for 60 days (bar: 2 cm).

FIGURE 2 :
FIGURE 2: Regression analysis of biometric features of Vriesea inlata plants cultivated in vitro for 60 days under different sucrose concentrations in the nutrient media: a) number of leaves and roots; b) leaf and root length; c) fresh and dry shoot mass (*: 5% signiicant (p ≤ 0.05)).