A reliable methodology for assessing the in vitro

A reliable methodology for assessing the in vitro photosynthetic competence of two Brazilian savanna species: Hyptis marrubioides and Hancornia speciosa Alan Carlos Costa, Márcio Rosa, Clarice Aparecida Megguer, Fabiano Guimarães Silva, Flávia Dionisio Pereira & Wagner Campos Otoni Plant Cell, Tissue and Organ Culture (PCTOC) Journal of Plant Biotechnology ISSN 0167-6857 Plant Cell Tiss Organ Cult DOI 10.1007/s11240-014-0455-y

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Author's personal copy Plant Cell Tiss Organ Cult DOI 10.1007/s11240-014-0455-y

ORIGINAL PAPER

A reliable methodology for assessing the in vitro photosynthetic competence of two Brazilian savanna species: Hyptis marrubioides and Hancornia speciosa Alan Carlos Costa • Ma´rcio Rosa • Clarice Aparecida Megguer Fabiano Guimara˜es Silva • Fla´via Dionisio Pereira • Wagner Campos Otoni

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Received: 3 November 2013 / Accepted: 20 February 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Evaluation of photosynthetic efficiency is critical for studies on plant responses to environmental conditions as well as for genotype selection; however, there is a lack of reliable and functional protocols for such assessments of plants cultured in vitro. In this study, we aimed to adapt the conventional methodology for measuring gas exchange of plants grown in vitro to analyze the effects of irradiance, flow rate, and air humidity on the photosynthetic rate in cultured plantlets of two ‘Cerrado’ species, namely Hyptis marrubioides and Hancornia speciosa plantlets. Chlorophyll (chl) a fluorescence and chloroplastidic pigment content were also assessed. The highest photosynthetic rates were observed at a photon flux density of 600 lmol m-2 s-1, with tube inlet airflow rates between 100 and 300 mL min-1 and 80 % relative humidity in the inlet air. The electron transport rate curve, by means of chl a fluorescence, was similar to the photosynthetic rate response curve obtained with the infrared gas analyzer. These results demonstrate that both H. marrubioides and H. speciosa seedlings grown in vitro have a functional photosynthetic apparatus and respond to variations in measurement conditions, exhibiting substantial rates of CO2 assimilation under saturating irradiance

A. C. Costa (&) M. Rosa C. A. Megguer F. G. Silva F. D. Pereira Instituto Federal de Educaçaõ, Cieˆncia e Tecnologia Goiano, Campus Rio Verde, Rodovia Sul Goiana, km 01, Zona Rural, Caixa Postal 66, Rio Verde, GO 75901-970, Brazil e-mail: [email protected] W. C. Otoni Departamento de Biologia Vegetal, Ed. CCBII, Av. P. H. Rolfs s/n, Universidade Federal de Viçosa, Campus Universita´rio, Viçosa, MG 36570-900, Brazil

conditions. The methodology proposed here can be adapted and applied to other species growing in vitro. Keywords Brazilian Cerrado species Chlorophyll fluorescence Chloroplastidic pigments Light curve Photosynthesis

Introduction An accurate assessment of the photosynthetic rate is critical for analyzing the impact of environmental conditions on a plant’s photosynthetic apparatus and productivity (MillanAlmaraz et al. 2009). In previous studies, the photosynthetic rate of in vitro cultured plantlets was assessed by measuring the O2 evolution rate with the Clark electrode (Chen 2006). Gas chromatography also enables the quantification of variations in the CO2 concentration under in vitro conditions and, consequently, the determination of photosynthetic rates (Xiao and Kozai 2006; Cha-um et al. 2011). The measure of CO2 assimilation rates using infrared gas analyzers (IRGAs) is the method most commonly used in studies of ex vivo plants, including agricultural crops (Matos et al. 2013) and forest species (Pires et al. 2013); the use of this method has also been reported in some in vitro studies (Iarema et al. 2012; Sae´z et al. 2013). However, there is a lack of functional and reliable protocols for measuring the photosynthetic rates of in vitro cultured plants using methods that do not alter the in vitro environment, for example by preventing dehydration and minimizing injuries to the seedlings. In vitro propagation systems are characterized by low transpiration and photosynthetic rates, restricted water and nutrient absorption, and high dark respiration rates, all of which reduce the

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Author's personal copy Plant Cell Tiss Organ Cult

explant growth rate (Kozai et al. 1997; Xiao et al. 2011). In addition, the high relative humidity (RH) inside the culture vessel reduces the deposition of epicuticular waxes, as well as the development of functional stomata, which may lead to losses during acclimatization (Chandra et al. 2010). Chlorophyll (chl) a fluorescence can be used to determine the photochemical efficiency and photo inhibition in plants (Krause and Weis 1991), as this parameter reflects photosynthetic performance and capacity (Snel and Van Kooten 1990; Yusuf et al. 2010). By measuring chl a fluorescence, one can determine the maximal quantum yield of photosystem II (PSII) (Fv/Fm), the effective quantum yield of PSII (DF/Fm0 ) (Genty et al. 1989), the relative electron transport rate (ETR), and the extinction coefficients for photochemical and non-photochemical quenching (Maxwell and Johnson 2000). Two additional factors that reflect plant photosynthetic efficiency, and consequently growth and adaptability to different environments, are the chlorophyll and carotenoid contents. Leaf chlorophyll content can be used to estimate the photosynthetic potential of plants due to its direct relationship to light absorption and energy transfer (Reˆgo and Possamai 2006). The accurate quantification of chloroplastidic pigments is relatively simple and, when assessed in combination with other physiological parameters such as chl a fluorescence and gas exchange, can broaden our understanding of the photosynthetic apparatus and its function in plants. Hyptis marrubioides Epling (Lamiaceae) is an essential oil-producing herbaceous species, commonly known as bush mint, with several medicinal properties. The essential oil from plants in the Hyptis genus is traditionally used as an anesthetic and as an antispasmodic and anti-inflammatory agent; additionally, it can induce abortion in high doses (Botrel et al. 2010). Hancornia speciosa Gomez (Apocynaceae) is a woody fruit tree species known as mangaba. H. speciosa is used for medicinal purposes and in the fabrication of candies, ice cream, jams and liquors (Cabral et al. 2013); this tree also has the potential for rubber production (Paula 1992), being a native species of importance to the development of an economy based on the use and conservation of natural resources. Both species are native to the Brazilian savanna, specifically to the biome known as the Cerrado. The establishment of tissue culture techniques for the in vitro multiplication of these species is critical for preserving specific genotypes and for largescale production. Such culture conditions can be established and optimized using tailored methods to evaluate the physiological and photosynthetic performance of plantlets under various in vitro conditions. As the measurement of gas exchange by conventional methods can significantly alter the environmental conditions of plants growing in vitro, this study aimed to adapt

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the existing gas exchange methodology to minimize these effects. Therefore, we evaluated the effects of irradiation, flow rate, and humidity on the photosynthetic rate of H. marrubioides and H. speciosa seedlings. Additionally, we measured chlorophyll fluorescence and chloroplastidic pigment content.

Materials and methods Plant material In vitro seedlings were grown from seeds. H. marrubioides seeds were obtained from the savanna area in the county of Lumina´rias, Minas Gerais state, Brazil, and H. speciosa seeds were obtained from the county of Montes Claros de Goia´s, Goia´s state, Brazil. The species were identified and voucher specimens were deposited at IF Goiano Herbarium—Rio Verde Campus (IFRV), Goia´s, Brazil, and registered under number IFRV71 for H. marrubioides and IFRV72 for H. speciosa. Seeds from both species were sterilized and inoculated into test tubes containing 20 mL of culture medium. After inoculation, the tubes were sealed with polypropylene caps. For H. marrubioides, seeds were cultured onto half-strength MS (Murashige and Skoog 1962) medium, supplemented with 30 g L-1 sucrose and 3.0 g L-1 agar (VetecÒ, Duque de Caxias, RJ, Brazil), whereas woody plant medium (Lloyd and McCown 1980), supplemented with 30 g L-1 sucrose and 3.5 g L-1 agar (DinaˆmicaÒ, Diadema, SP, Brazil) was used for H. speciosa seeds. No growth regulator was added for either species. The pH of the culture media was adjusted to 5.7 ± 0.3. The media were autoclaved at 121 °C under 1.5 atm. pressure for 20 min. In vitro cultivation was carried out according to the protocols established by Sales (2006) for H. Marrubioides, and by Cabral (2012) for H. speciosa. Inoculated vials were maintained under a 16 h photoperiod at 25 ± 3 °C, an RH of 50 ± 5 %, and photosynthetically active radiation (PAR) of 50 ± 5 lmol m-2 s-1. The radiation was supplied by OSRAMÒ (Osasco, SP, Brazil) 40 W daylight fluorescent tubes. Radiation was assessed using a PAR sensor (QSO-S model; Decagon Devices, Pullman, WA, USA). Hyptis marrubioides and H. speciosa seeds were maintained in the original medium for 30 and 60 days, respectively, until they had attained growth of approximately 3.0 cm. At this point, the first subculture was performed. The nodal segments were excised and placed in the same type of medium used for the initial culture, although the salt concentration was reduced by half for the H. speciosa seedlings. The explants were then

Author's personal copy Plant Cell Tiss Organ Cult Fig. 1 Layout of the leaf chamber adapted to measure gas exchange in in vitro conditions: test tube containing a seedling (A, F); EPS compartment internally lined with aluminum foil (B, H); polystyrene vat filled with water for thermal insulation (C, I); light source (D); reference air inlet (E); analysis air outlet (G)

incubated for 50 days. Sufficient H. marrubioides seedlings were obtained from the first subculture for subsequent experiments on photosynthetic efficiency. A second subculture, lasting more than 50 days, was required for H. speciosa before the photosynthesis assays could be performed. Description and adjustment of the gas exchange assessment device There is no commercially available device specifically designed for measuring the photosynthetic characteristics of in vitro cultured seedlings; however, with careful adjustments and adaptations, it was possible to measure these characteristics using available devices. In the present study an IRGA (model S151; Qubit Systems, ON, Canada) was used to analyze the seedlings. To measure gas exchange, we replaced the original IRGA leaf chamber with a test tube used to raise the seedlings (Fig. 1A, F). The air used for the measurements came from a CO2/N2 cylinder with a standard CO2 concentration of 390 lmol mol-1 and was directed toward an air pouch. The air pouch was pumped at a constant flow rate to the tube containing the seedling. The reference value was obtained by passing air through the empty chamber. The procedure was then repeated with the seedling in range of the chamber. The differences between the seedling-exposed air and the reference air were used to calculate the gas exchange. To optimize and standardize the incident light, the tube containing the plant material was placed in a circular enclosure of expanded polystyrene (EPS) internally lined with aluminum foil (Figs. 1B, H, 2D). The reference air entered through a flexible hose fitted

to the tube cap (Fig. 1E). The end of this hose was placed at the seedling’s base so that the air would flow evenly across the whole shoot, exiting through another hose fitted to the tube cap (Fig. 1G). The light source was a LCi Light System (ADC Bioscientific, Herts, England), which consists of a ventilated support containing a 20 W dichroic halogen lamp. To avoid overheating the test tube, a polystyrene vat (140 mm high, 125 mm wide, and 5 mm thick, with 1 mm walls) was filled with water and placed between the test tube and the light source (Fig. 1C, I). The water in the vat was changed every 2 h or when heated. The adjustments made to the technique for assessing in vitro gas exchange were based on the photosynthetic light response curves (A/I curve), the gas exchange response curves of the air passing through the test tube, and the RH conditions of the inlet air (reference air) in the tube. The measurements were performed at room temperature (25 ± 1 °C). Both the temperature and RH of the reference and analysis air were monitored using the S161 sensor (Qubit Systems). The airflow rate was adjusted with the G265 digital flow monitor (Qubit Systems). Photosynthetic light response curves (A/I curve) The light curve was obtained by measuring the potential net photosynthetic rate of cultured seedlings under decreasing photon flux densities (PFD) of 1,350, 1,200, 950, 450, 250, 110, 50 and 0 lmol m-2 s-1. The reference air RH was raised to 80 ± 5 %, while the airflow rate was adjusted to 300 mL min-1. The seedlings were kept in the dark for 8 h prior to the experiment and then exposed to

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Fig. 3 Layout of the reference air moistening system. The air collected from a cylinder containing a standard mixture of CO2/N2 is stored in a pouch (A) and subsequently pumped, in distilled water, (B) through hoses directly into a sealed test tube (C). The moistened air is propelled through a hose placed in the tube cap and directed into another air collection pouch (D)

Gas exchange response curves of the airflow through the test tube

Fig. 2 Adaptation of the gas exchange analyzer setup for in vitro conditions: air pouch (A); airflow pump (B); G265 airflow rate monitor (C); test tube containing a seedling in the EPS compartment lined with aluminum foil (D); temperature and RH S161 sensor (E); desiccant tube (F); IRGA S151 (G); light source (H); and Interface Lab Pro (I)

50 lmol m-2 s-1 radiation for 60 min before the measurements were initiated. For each point of the curve, the irradiance was calculated using the variation in the distance between the light source and the test tube and adjusted using the PAR sensor. The seedlings were exposed for 10 min to each level of irradiance. Following the period of exposure, the CO2 concentration and the temperature of the air exiting the tube were recorded and subsequently used to calculate the seedling’s photosynthetic rate. The net photosynthetic light response curves were obtained by adjusting the data to the non-rectangular hyperbole model (Prioul and Chartier 1977). The saturation irradiance was obtained from the PFD value in which A = 90 % of Amax. The experiment was conducted using a completely randomized design with eight replicates of H. marrubioides seedlings and ten of H. speciosa seedlings. Each replicate consisted of a test tube containing one seedling.

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We tested five airflow rates (50, 100, 200, 300 and 400 mL min-1) by manipulating the digital flow monitor, aiming to obtain an adequate band of electrical signal in the sensors while causing minimal changes to the in vitro environment. Considering the preliminary results of the aforementioned light curve, these measurements were conducted under an irradiance of 600 lmol m-2 s-1, using air from a cylinder with a 390 lmol mol-1 CO2 concentration and an RH adjusted to 80 ± 5 %. This experiment was conducted as a completely randomized design, with four replicates for each species. Each replicate consisted of a test tube containing one seedling. The data were submitted to an analysis of variance using SAEG 9.1 (UFV, Viçosa, Brazil). Regression models were adjusted to the means, when necessary. Adjustment of the relative humidity of the reference air We tested two RH values for the inlet air: 20 ± 5 and 80 ± 5 %. The air with 20 % RH was directly obtained from the air cylinder with a 390 lmol mol-1 CO2 concentration. The air with 80 % RH was obtained by bubbling air from the cylinder into a test tube containing 30 mL of distilled water that had previously been boiled and cooled to room temperature. The boiling was conducted to eliminate CO2 from the water and maintain a stable original CO2 concentration. The moistened air was stored in plastic bags for use in the experiment (Fig. 3).


The measurements were performed using an airflow rate of 300 mL min-1 and an irradiance of 600 lmol m-2 s-1, in agreement with the preliminary results of the light curve analysis. The experiment was conducted as a completely randomized design, with ten replicates for each species. Each replicate consisted of a test tube containing one seedling. The data were submitted to analysis of variance using SAEG 9.1 (UFV, Viçosa, Brazil), and the differences between means were compared by an F test. Chlorophyll a fluorescence analysis Measurements of chl a fluorescence were taken with a MINI-PAM modulated fluorometer (Walz, Effeltrich, Germany), using the leaves of the same seedlings used for measuring gas exchange. Some adjustments to the protocol used for ex vitro conditions were necessary to minimize additional stress resulting from the measurement procedures. Prior to analysis, the sealed test tubes containing the seedlings were kept for 30 min in the dark under external air with 60 ± 5 % RH and a temperature of 25 ± 1 °C to allow the complete oxidation of the leaves’ electron transport system components. Subsequently, the seedlings (still unexposed to PAR) were removed from the tubes and clamped to allow the insertion of the optical fiber cable of the fluorometer. The leaf area under the clamp was then subjected to a low-intensity pulse (0.03 lmol m-2 s-1) of modulated red light, followed by a 0.8 s pulse of saturating actinic light ([6,000 lmol m-2 s-1). This protocol allowed us to calculate the potential quantum yield of PS II, i.e., Fv/Fm. The same seedlings used in the previous procedure were held in a wet chamber for approximately 20 min under an irradiance of 50 ± 5 lmol m-2 s-1 for subsequent measurements concerning the fluorescence parameters of light. The wet chamber was composed of a polyethylene tray (20 cm wide, 30 cm long and 6 cm deep) covered with a layer of cotton and two layers of soaked GermitestÒ paper. The tray was sealed with a transparent glass plate to allow transmittance and to minimize seedling dehydration prior to taking the measurements. Photosynthetic performance with respect to PFD was determined by programming the fluorometer with increasing levels of light (0–1,500 lmol m-2 s-1) over 4 min, specifically, eight levels lasting 30 s each. A saturating pulse was applied at the end of each level to determine the fluorescence parameters. The effective quantum yield of PSII was calculated as DF/Fm0 , which was subsequently used to calculate and design the irradiance response curve of the relative ETR (maximal ETR).

The experiment was conducted as a completely randomized design, with ten replicates for each species. Each replicate consisted of a test tube containing one seedling. The data were subjected to analysis of variance using SAEG 9.1 (UFV, Viçosa, Brazil). Regression models were adjusted to the means, when necessary. Extraction and assessment of chloroplastidic pigment concentration Three leaf discs (5 mm in diameter) were removed from H. marrubioides and H. speciosa seedlings after 50 days of in vitro culture. To determine their chloroplast pigment concentrations, the discs were incubated in 5 mL of dimethyl sulfoxide saturated with CaCO3 in sealed glass flasks wrapped in aluminum foil at temperatures of 25 and 65 °C. At each temperature, absorbance readings were performed after 4, 8, 12, 24 and 48 h of incubation. Extract absorbance was determined using the Evolution 60S UV–VIS spectrophotometer (Thermo Fisher Scientific, WI, USA). Determination of carotenoids and chl a and b followed Welburn (1994). Chlorophyll degradation was also assessed by spectrophotometry, using the pheophytinization index (PI = A435/A415) described by Ronen and Galun (1984). The experiment was arranged in a completely randomized design, with nine replicates of the H. marrubioides seedlings and twelve replicates of the H. speciosa seedlings. Each replicate consisted of a test tube containing one seedling. The data were subjected to analysis of variance. When necessary, the means were compared by a Tukey’s test with 5 % probability, and the regression models were adjusted using the SAEG 9.1 software (UFV, Viçosa, Brazil).

Results and discussion Photosynthetic light response curves (A/I curve) The present study demonstrates that seedlings grown in vitro possess a relatively functional photosynthetic apparatus. An increased rate of photosynthesis was shown to accompany increases in PFD in both H. marrubioides and H. speciosa seedlings. The photosynthetic light response curves indicated that saturation of CO2 assimilation occurred in both species near an irradiance level of 600 lmol m-2 s-1 (Fig. 4). These results are in accord with the light saturation points (LSPs) recorded for other species cultured in vitro. For instance, De La Vinã et al. (1999) reported an LSP of 600 lmol m-2 s-1 in a study involving Persea americana Mill. seedlings cultured in vitro under growth conditions similar to those used in the

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at the LSP observed in H. speciosa seedlings grown in vitro are also similar to those found in plants of the same species that were cultured in ex vitro conditions. Vieira Neto (2010) determined that H. speciosa seedlings, cultured for 98 days under varying phosphate fertilization conditions, displayed photosynthetic rates (A) between 8.9 and 12.4 lmol m-2 s-1. Lobo et al. (2008) found similar values in adult H. speciosa individuals subjected to varying levels of irrigation. Under saturating irradiance, the photochemical process is not limited, and the photosynthetic rate begins to stabilize. Further increases in the PFD have no effect on the photosynthetic rate, and factors such as the rate of enzymatic activity (RuBisCo) and triose phosphate metabolism become limiting (Lambers et al. 2008). Although the LSP results from the light curve analysis performed in the present study illustrate the potential net photosynthetic rates of these plants over the short term, they do not necessarily indicate that these species should be grown under high PFD. Long-term exposure to high irradiation levels, such as those near the LSP of the cultured seedlings, may lead to photo inhibition (Ruban 2009). Conversely, the results obtained at the extreme points of the curve more clearly illustrate the plants’ photosynthetic performance and thus clarify the differences between treatments. The light curve analysis revealed positive photosynthetic rates, even under low irradiance (PFD = 50 lmol m-2 s-1). Fig. 4 Net CO2 assimilation rates (A) of H. marrubioides (a) (n = 8) and H. speciosa (b) (n = 10) seedlings cultured in vitro under increasing PFD

Gas exchange response curves with respect to the airflow rate through the test tube

present study. Similar results were also observed in Asparagus officinalis L. seedlings grown for 5 weeks in culture; the LSP of these seedlings occurred at 450 lmol m-2 s-1 (De Yue et al. 1992). The LSP can change, however, depending on the in vitro culture conditions. In fact, the LSP of Actinidia deliciosa A. Chev. seedlings, grown for 40 days in vitro under 150 lmol m-2 s-1 of light, stabilized at 300 lmol m-2 s-1 when the plants were grown either in the absence of or with low levels of sucrose (Arigita et al. 2002). Plantlets of Castanea sativa Mill. cultivated in vitro reached an LSP value of 164 lmol m-2 s-1 in a non-ventilated environment under 50 lmol m-2 s-1 of irradiance; however, in the same study, a greater LSP value (414 lmol m-2 s-1) was observed under forced ventilation and 150 lmol m-2 s-1 of irradiance (Saéz et al. 2013). The photosynthetic rate-saturating PFD values recorded for H. marrubioides and H. speciosa cultured in vitro are similar to those of certain woody and shrubby species of the Cerrado biome, which have exhibited LSPs of approximately 600 lmol m-2 s-1 when measured under natural conditions (Palhares et al. 2010). The photosynthetic rates

The photosynthetic performance of both H. marrubioides (Fig. 5a) and H. speciosa (Fig. 5b) seedlings was affected by the tube airflow rate, with the extreme values (50 and 400 mL min-1) resulting in the lowest mean values for photosynthetic rate; however, the pattern was not significant under any of the regression models tested. Reading variations occurred at flow rates less than 100 mL min-1, possibly due to a loss of the sensor’s electrical signal. Most of the previous studies that assessed photosynthetic rates in vitro did not specify the airflow rate used. In the few studies that did provide this information, the airflow rates were highly variable. Triques et al. (1997) used a flow rate of 140 mL min-1 when measuring the photosynthetic rate of detached Cocos nucifera L. seedling leaves that were partially removed from the tube. In a similar procedure, Fila et al. (2006) used a flow rate of 330 mL min-1 for measuring grapevine seedlings. Flow rates of 500 mL min-1 were used to measure photosynthesis in Pfaffia glomerata (Spreng.) Pedersen (Iarema et al. 2012). Although the manufacturer’s manual (Qubit Systems 2009) recommends using a flow rate of 500 mL min-1 to assess plant gas exchange in ex vivo conditions, the in vitro

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Author's personal copy Plant Cell Tiss Organ Cult Fig. 5 Net CO2 assimilation rates (A) and transpiration rates (E) of H. marrubioides (a, c) and H. speciosa (b, d) seedlings cultured in vitro under increasing airflow rates. The transpiration rate data can be explained by a linear regression model in both species (n = 4). Significance: ** p \ 0.01

measurement chamber did not achieve the necessary stability in preliminary tests performed using flow rates higher than 400 mL min-1. An increase in airflow rate resulted in a rise in transpiration rate (E) in both H. marrubioides (Fig. 5c) and H. speciosa (Fig. 5d). This relationship could be explained by a linear regression model. However, changes in the transpiration rate, observed with increasing airflow rates, do not reflect the physiological performance of seedlings cultured in vitro, despite the fact that they could be explained by statistically significant regression models. The increase in transpiration rate is most likely due to the water vapor drag promoted by high airflow rates. Flow rates between 100 and 300 mL min-1 are the most suitable for measuring the rate of photosynthesis, as they permit high mean photosynthetic rates without causing large disturbances in the air of the seedling growth tubes. Adjustment of reference air relative humidity conditions Higher photosynthetic rates were observed at a reference air RH of 80 ± 5 % for both H. marrubioides (Fig. 6a) and H. speciosa (Fig. 6b)seedlings, although the difference relative to the values observed at 20 % RH was only significant for H. speciosa (Fig. 6b). The increased photosynthetic rate observed at 80 % RH may be related to the fact that this RH level is similar to that of the air in which the seedlings had been cultured and would thus cause less disturbance and desiccation than would the air with 20 % RH. According to Kozai and Kubota (2005), the RH inside the in vitro culture flask is

usually high, ranging from 80 to 100 %. Although plants grown in vitro do not develop layers of epicuticular wax (Majada et al. 2001; Alvarez et al. 2012), it is likely that, under 20 % RH, the high vapor pressure deficit may inhibit the photosynthetic rate through stomatal closure, as observed in ex vivo conditions. Indeed, significant decreases in stomatal conductance, and therefore in the net photosynthetic rate, occur under high vapor pressure deficits (Shirke and Pathre 2004). In contrast to the photosynthetic rate, lower values of E were found at 80 % RH. Regardless of the species, E decreased intensely at 80 % RH relative to 20 % RH. E values higher than 13 mmol m-2 s-1 were found in H. speciosa (Fig. 6d) seedlings but remained at approximately 6.9 mmol m-2 s-1 in H. marrubioides (Fig. 6c). These rates are higher than those recorded in ex vitro experiments. The high transpiration rates in plants subjected to 20 % RH are more likely related to differences in the vapor pressure deficit in the growth environment at the time of measurement than to the loss of water through the leaves. This result is likely an overestimate because, according to Kozai and Kubota (2005), the transpiration rate is proportional to the vapor pressure deficit. This assertion is supported by a comparison of the H. marrubioides and H. speciosa plants. Together with the free-water drag through the airflow in the tube, the high RH of the plant growth tube contributed to the accumulation of water vapor in the connective hoses of the device’s sensors. This condition likely led to an overestimation of the transpiration rate. Most notably, this condition produced entirely contradictory and unstable values of stomatal conductance, precluding their interpretation. The

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Author's personal copy Plant Cell Tiss Organ Cult Fig. 6 Net CO2 assimilation rates (A) and transpiration rates (E) of H. marrubioides (a, c) and H. speciosa (b, d) seedlings cultured in vitro under increasing RH in the air used for analysis. Means followed by the same letter are not significantly different in the F test at a 5 % significance level. Data are means and standard deviation (n = 10)

Fig. 7 Relative ETR and effective quantum yield of PSII (DF/Fm’) of H. marrubioides (a, c) and H. speciosa (b, d) seedlings cultured in vitro under increasing PFD. The performance of both species is explained by the square root model (n = 10). Significance: **p \ 0.01

stomatal conductance data were therefore excluded from the present study. Fluorescence assessments The Fv/Fm ratio reflects photochemical energy dissipation and expresses the capture efficiency of excitation energy by the PSII open reaction centers (Krause and Weis 1991; Baker 2008). According to Bolha`r-Nordenkampf et al. (1989), when a plant is not stressed, the Fv/Fm ratio should

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range between 0.75 and 0.85. The Fv/Fm ratio (0.79) of H. marrubioides and H. speciosa seedlings recorded in the present study indicates that, despite the limiting environmental conditions of in vitro culture, the seedlings possess a photosynthetic apparatus that can effectively capture light energy and transfer it to the photosynthetic pathway. With regard to the fluorescence curves, both H. marrubioides (Fig. 7a) and H. speciosa (Fig. 7b) seedlings showed increasing ETR as PFD was increased, saturating at an irradiance of 600 lmol m-2 s-1. A square root

Author's personal copy Plant Cell Tiss Organ Cult Fig. 8 Effects of temperature (25 and 65 °C) on chl a, chl b, carotenoid and total chlorophyll levels of cultured H. marrubioides (a–d) (n = 60) and H. speciosa (e–h) (n = 45) seedlings. Data are means and standard deviation. Means followed by the same letter are not significantly different in the F test at a 5 % significance level

regression model explained this result. The ETR increases with the light intensity until the electron carriers are saturated (Mohamed et al. 1995). The ETR curves determined in the present study are in accordance with the photosynthetic rate curves established previously for each species, in which the saturation points were approximately 600 lmol m-2 s-1. In Tetrastigma hemsleyanum Diels et Gilg plants grown under varying irradiance conditions in a greenhouse, the ETR curve was correlated with the maximal photosynthetic rate, thereby yielding the same pattern as the CO2 assimilation curve (Dai et al. 2009). As expected, DF/Fm0 progressively decreased with the increase in PFD in both species. Beginning at an irradiance

level of 600 lmol m-2 s-1, there was rapid decrease in DF/Fm0 , which was more pronounced in the H. speciosa seedlings (Fig. 7d). In both species, the changes in DF/Fm0 with increasing PFD were explained by a square root regression model (Fig. 7c, d). The decrease in DF/Fm0 observed with increasing PFD, especially above the ETR saturation irradiance, is an indication that not all the electrons involved in photochemical processes are used for CO2 fixation. Under conditions where CO2 fixation is limited, the electron flux can be redirected to other biochemical pathways, including oxygen reduction and photorespiration (Bussotti et al. 2011).

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Author's personal copy Plant Cell Tiss Organ Cult Fig. 9 The chl b content of H. marrubioides (a) (n = 18) and H. speciosa (c) (n = 24) seedlings cultured in vitro and extracted after different incubation times. The PI of H. marrubioides (b) (n = 9) and H. speciosa (d) (n = 12) seedlings cultured in vitro and extracted at two different temperatures after different incubation times. Significance: **p \ 0.01

Chloroplastidic pigment extraction In H. marrubioides and H. speciosa the temperature was more crucial in extracting chloroplastidic pigments than the incubation time, except for the PI. In H. marrubioides, chl a (Fig. 8a) and carotenoid (Fig. 8c) extractions were more efficient at 25 °C than at 65 °C. According to Santos et al. (2008), plants grown in vitro do not require an extended incubation time or temperatures higher than room temperature for leaf sample extractions because of their thin leaf lamina and low cutin ization levels. The extraction process, therefore, proceeds efficiently without significant degradation of leaf pigments. For both H. marrubioides and H. speciosa, however, higher levels of chl b were obtained at 65 °C than at 25 °C (Fig. 8b, f, respectively). Increased exposure times also increased the amount of chl b extracted from H. marrubioides, including under temperatures of 25 °C (Fig. 9a). According to Koca et al. (2006), chl b is less sensitive to degradation than chl a. This difference is due to the electron attraction effect of the aldehyde group at C-3 of chl b (Von Elbe 2000; Streit et al. 2005). The chl b content, however, was only 9.5 % higher at 65 °C than at 25 °C. In H. speciosa, the chl a (Fig. 8e) and carotenoid (Fig. 8g) contents were not affected by temperature or incubation time; however, more chl b was extracted at 65 °C (Fig. 8f). The presence of latex in the leaves may explain the lower extraction efficiency of this pigment in samples incubated at 25 °C. Both extraction temperature and incubation time influenced the PI of this species. For H. marrubioides, despite

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the decrease in the PI that began after 8 h of incubation at 25 °C, the index values remained acceptable for up to 48 h of incubation (Fig. 9b). According to Ronen and Galun (1984), the shift in the PI to values well below 1.40 is indicative of chlorophyll degradation, specifically of chl a. The interaction between temperature and incubation time in H. speciosa led to greater reductions in the PI at incubation periods longer than 12 h at 65 °C (Fig. 9d). Incubation of samples at 65 °C for up to 12 h allowed a satisfactory level of chl b extraction without reducing the ability to extract other chloroplast pigments. The higher chloroplast pigment content found in H. speciosa explains the higher photosynthetic rate in this species relative to H. marrubioides, because these pigments are closely involved in the process of capturing light energy.

Conclusion The methodology used in the present study was able to adequately measure photosynthetic responses in H. marrubioides and H. speciosa seedlings cultured in vitro, indicating a relative functionality of the photosynthetic apparatus of these species under the conditions of this study, and exhibiting substantial rates of CO2 assimilation under high irradiance levels. The highest photosynthetic rates were observed at a PFD of 600 lmol m-2 s-1 with tube inlet airflow rates between 100 and 300 mL min-1, and under 80 % RH in the inlet air. In addition, the chl a fluorescence analysis confirmed the photosynthetic


capacity of the cultured seedlings, as the ETR curve was similar to the photosynthetic rate response curve obtained with the IRGA. Overall, the methodology proposed here can be adapted and applied to other species growing in vitro. Acknowledgments The authors would like to thank the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and the Instituto Federal Goiano—Rio Verde Campus, GO, for financial support. They are also grateful to the Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES) for the scholarship provided to the master’s student involved in the research and to Prof. Francisco de Almeida Lobo, Universidade Federal do Mato Grosso, Brazil, for valuable suggestions regarding the methods applied in the present study. Conflict of interest The authors declare that there are no conflict of interests.

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