Effects of different culture conditions ( photoautotrophic , photomixotrophic ) and the auxin indole-butyric acid on the in vitro acclimatization of papaya ( Carica papaya L . var . Red Maradol ) plants using zeolite as support

Effects of different culture conditions (photoautotrophic, photomixotrophic) and the auxin indole-butyric acid on the in vitro acclimatization of papaya (Carica papaya L. var. Red Maradol) plants using zeolite as support Laisyn Posada Pérez, Yenny Padrón Montesinos, Justo González Olmedo, Romelio Rodríguez Sánchez, Osvaldo Norman Montenegro, Raul Barbón Rodriguez, Ortelio Hurtado Ribalta Rene Carlos Rodriguez Escriba, Dion Daniels and Rafael Gómez-Kosky*


INTRODUCTION
In Cuba, the most commercially important variety is the 'Red Maradol' and crop production exceeded 1.7 million tons in 2013 (FAOSTAT, 2014).In addition, it is sown in other countries in the Caribbean and Central America.Regeneration of papaya plants via somatic embryogenesis has been successful; however, the somatic embryos in germination have problems with root development due to the presence of a basal callus, which prevents the formation of roots or its connection to the stem, besides the low percentage of acclimatization of rooted plants (Fitch and Manchardt, 1990;Dhekney et al., 2007;Sekeli et al., 2013).Another critical aspect has been the adaptation to environmental conditions because of the high relative humidity that this species need to achieve high survival rates (Chen et al., 1991).The biggest problem that exists globally in in vitro papaya culture is the ex vitro acclimatization of regenerated plants, where more than 70% of in vitro plants produced are lost before being planted in the field (Malabadi et al., 2011).A plant that originated in vitro, differs in many aspects from those formed in vivo (Pierik, 1990), since its environmental conditions, substrate, light, and nutrition, are very different.It is also important to note that the growth in vitro is heterotrophic, while the conditions in vivo are autotrophic.The in vitro atmosphere, with a high relative humidity, low or zero gas exchange, shortage of CO 2 during most of the period, ethylene production and low photosynthetic rate, induce changes in plants grown under these conditions.After transferring the plants to ex vitro environment, plants have to correct all of these abnormalities in order to acclimatize to the new environment, either in greenhouse or into the field (Kadleček et al., 2001).
Furthermore, the anatomy of the leaves is influenced by light and humidity, differing anatomically from those originated from in vivo conditions (Brainerd et al., 1981).Because of this, the acclimatization is an important factor in the subsequent survival rates of the plants, since it is a critical stage in the process, in which the larger loss occurs.In this stage, the relative humidity should begin to decrease gradually, to allow in addition to stomata closure, better cuticle formation and reduced water loss.Moreover, for best results in in vivo establishment, it is necessary to have root in vitro development (Pierik, 1990).Decreasing the relative humidity inside the culture vessel and thus the increased ventilation, appear to have a greater effect on the adaptation of grape plants (Vitis vinifera L.), enhancing the stomata function and thereby enable better control of water loss from the leaves (Gribaudo et al., 2001).
In vitro photoautotrophic can be induced excluding carbohydrates from the culture medium and increasing gas exchange in the culture vessel (Kozai, 2010;Xiao et al., 2011).Photoautotrophic micropropagation is defined as micropropagation without sucrose in the culture medium, where the accumulation of carbohydrates in in vitro tissues cultured and their subsequent growth is completely dependent on photosynthesis and inorganic nutrients (Zobayed et al., 2004;Kozai, 2010).Therefore this may also be called photosynthetic micropropagation in culture medium devoid of sugar (Xiao et al., 2011).In photoautotrophic micropropagation, acclimatization can also be completed in the culture vessel, which is called in vitro acclimatization (Kozai et al., 2005).
Although the growth and physiological changes in some plant species with photoautotrophic growth have been studied (Norikane et al., 2010;Badr et al., 2011;Shin et al., 2013), there are very few reports of studies on the in vitro propagation of papaya and none specifically on its most critical phase -rooting.For this reason, this study aims to evaluate the effects of different concentrations of sucrose and the auxin indole-butyric acid to achieve in vitro acclimatization in a growth chamber with sunlight, greater ventilation of the culture vessels and using zeolite as a support for increased survival rates ex vitro of papaya plants obtained by somatic embryogenesis.

Plant material and culture media
As plant material, in vitro shoots of papaya -variety Maradol Roja were used.These were regenerated from somatic embryos, originating from the fourth subculture in the elongation culture medium proposed by Posada-Perez et al. (2007).This culture medium contained Murashige and Skoog (MS) (1962) salt at 100% concentration supplemented with 1 mg L -1 of thiamine, 1.2 µM of 6benzyl aminopurine (BAP), 1.5 µM of naphthaleneacetic acid (NAA), 100 mg L -1 of myo-inositol, 30 g L -1 of sucrose, 1 µM of riboflavin and 5 g L -1 of Agargel (Sigma Co.) and adjusted to a pH of 5.8.Shoots with a size between 3.0 to 5.0 cm in length, of which only the last three leaves were left, were placed in culture vessels containing three concentrations of sucrose (0, 10 and 20 g L -1 ) combined with the presence or absence of the growth regulator, indole-butyric acid (IBA) at a concentration of 9.8 µM for in vitro rooting and acclimatization of shoots, using as support to the mineral zeolite (natural aluminum-silicate with excellent ionic exchange properties and a high absorption power) 1 to 3 mm granulation (Table 1).To each glass culture vessel, 97 g of zeolite *Corresponding author.E-mail: kosky@ibp.co.cu, kosky2015@gmail.com.Density of the solid phase (γ) 1.77 g cm -3
previously sterilized in an oven at 180°C for 2 h were added.Glass culture vessels with a total volume of 250 mL with 30 mL of liquid culture medium were used.They were covered with aluminum foil of 20 µm thickness.After three days of culture, the ventilation of the culture vessels was increased by opening holes on the aluminum foil covering the culture vessels in the different treatments.A second hole was made three days after the opening of the first (Figure 1A).As the control treatment, a modified version of the culture medium for rooting proposed by Posada-Perez et al. (2007) was used.This culture medium was composed of MS salts at 50% concentration, 9.8 µM of IBA, 0.4 mg L -1 of thiamine, 1.0 µM of riboflavin, 40 g L -1 of sucrose, 7.0 g L -1 of agar and pH of 5.8 prior to sterilization.The culture vessels and the volume of culture medium were the same as previously mentioned, but these were covered with plastic lid (polycarbonate).Forty five vessels were used with two shoots each per each variant of culture medium.Of each variant 15 culture vessels were randomly selected every 10 days, from 17 to 37 days of culture for evaluations of the morphological and physiological indicators of plants, including contamination.Survival (%) in ex vitro acclimatization conditions was done with plants after 17 days of in vitro culture conditions.This evaluation was done 20 days after being transplanted.

Culture conditions
The culture vessels with the shoots were placed in growth rooms at a temperature of 27 ± 2°C with sunlight and a photoperiod of 13 / 11 h, light / dark with a light intensity ranging between 48.0 and 62.5 µmol m -2 s -1 ; measured with a light meter EXTECH 401,025 (USA).The experiments were repeated twice.The relative humidity inside the culture vessel covered with aluminum foil and two holes was between 72 to 68% and in control with culture vessel lids with plastic was 90 to 85%.

Evaluation of morphological and physiological variables
After 17, 27 and 37 days of culture, in vitro shoots and plants were evaluated for the following morphological variables: length of the plant (cm), number of leaves, number of internodes, fresh mass of in vitro plant (gFM), leaf area (by the method proposed by Cardona et al. (2009) to estimate the leaf area of papaya plants), presence or absence of basal callus, number of roots, length of the roots (cm) and presence of roots (% ).For the evaluations of the physiological indicators of in vitro shoots and plants, the contents of the pigments chlorophyll a, b and carotenoids were determined at 17, 27 and 37 days of culture using the same plants which had been used to assess the morphological variables.To determine the net photosynthetic activity and total transpiration, in vitro plants at 17 days of culture were used.

Chlorophyll and carotenoid pigments measurement
At 27 days, chlorophyll a, chlorophyll b and total carotenoid pigments were determined in the leaves from in vitro plants using the Meyer-Berthenrath´s method, modified by Stirban (1985).The absorbance of the extracts was measured at 663, 645 and 472 nm by spectrophotometry (GENESYS 6; Thermo Electronic Corporation Visionlite Vision 2.1),

Photosynthetic activity, total transpiration and stomatal conductance
For these measurements, fully extended leaves of the same position (leaves 2 and 3) of in vitro plants at the end of the experiment, 4 to 5 h after the start of the photoperiod were used.All measurements were performed with three leaves from different plants.The maximum photosynthetic capacity (µmol CO2 m -2 s -1 ), the total transpiration (mmol H2O m -2 s -1 ) and stomatal conductance (mmol m -2 s -1 ) were measured with the equipment CIRAS-2 (Portable Photosynthesis System, UK), coupled to a universal bucket PLC6 2.5 cm 2 .The area of the tray was completely covered with the leaf (1.7 cm 2 ).The concentration of CO2, air temperature and relative humidity (80 to 90%) were environmental values taken into consideration.The light equipment, intensity was set at 900 µmol m -2 s -1 .Measurements were always done on all in vitro plants between 9:00 to 10:00 a.m.

Ex vitro acclimatization conditions
The environmental acclimatization conditions are characterized by averaged daytime temperature of 30 ± 2°C, 65 to 70% relative humidity and light intensity ranging between 224 and 457 µmol m -2 s -1 measured with a light meter EXTECH 401 025.Plastic trays with cover were used and 100% zeolite as substrate with 15 in vitro plants per treatment, which had been cultured for the first 17 days (Figure 1B, C).These were kept closed during the first first days after transplanting to ensure high relative humidity above 90% and thereafter it was slowly reduced by opening the lid.Irrigation was done manually with aspersion three times daily.The trays were placed in a culture house with an internal black Saran mesh cover with 70% shade.The survival rate (%) was determined by counting the plants that were alive at the time of evaluation after 15 days with respect to the total number of plants initially planted.

Statistical analysis
For the statistical analysis of the data, the package SPSS version 17.0 for Windows 2008 was used.For analysis of the normality of    the variables, Shapiro Wilk test was used.For the comparison between means, the nonparametric alternative analysis of variances of Kruskal Wallis test was applied.
For the comparison between pairs of groups, the nonparametric Mann Whitney test was used.A bi-factorial experimental design with four repetitions was performed, analyzing the data using a multi-factorial ANOVA.Differences between groups were detected by the test of least significant difference or LSD intervals.For the statistical analysis, the statistical program Stat-graphics Centurion 2007 version 15 software was used.

Effects of sucrose and auxin on rooting and in vitro acclimatization
Results on management leading to increased rooting and which favor photoautotrophic culture conditions are shown in Tables 2, 3 and 4, which reflect the response of the different treatments on morphological variables recorded with increased ventilation during 10, 20 and 30 days.From the earliest morphological evaluations, the positive effect of the combination that represented the treatment without sucrose and 9.8 µM IBA over other treatments was significant.For the three times that in vitro papaya plants were measured, the largest number of roots and increased root length were obtained with the treatment with 10 g L -1 of sucrose and 9.8 µM of IBA, at 17 and 27 days of culture.It is also noteworthy that in the evaluation at 27 days of culture, high values in fresh mass were achieved in the in vitro plants grown in zeolite without sucrose and 9.8 µM IBA, although there were no significant differences observed in the treatments without sucrose and without IBA; with 10 g L -1 sucrose and without AIB and the agar control.This was due to, in the case of agar control, the presence of big basal callus (Figure 1D).The callus did not form or their presence was minimal in the other treatments evaluated (photoautotrophic and photomixotrophic conditions) using zeolite as a support and with increased ventilation in culture vessels regardless of the presence or absence of auxin in the in vitro culture medium.In this regard Kozai et al. (2005) reported that the species Calla Lily (Zantedeschia elliottian L.) in photoautotrophic culture conditions prevented the formation of basal callus of in vitro shoots, which is the cause of the poor rooting and limited uptake of water and nutrients by the plants.Zhang et al. (2009) in the Chinese medicinal species (Momordica grosvenori Swingle) indicated that no callus was formed in those plants grown in culture medium without sucrose, free of growth regulators and photoautotrophic culture conditions.With respect to the leaf area at 27 and 37 days of culture (Figure 1E) in the treatment without sucrose (photoautotrophic culture conditions) and with the presence of IBA, the in vitro plants showed higher values with significant differences with the other treatments (Tables 3  and 4).
Teixeira de Silva (2014) shows that photoautotrophic culture of in vitro plants was possible in papaya in two varieties (Rainbow and Sunrise Solo) using plants from in vitro germinated seed and transferred to culture vessels Vitron® type and using as support rock wool.To the culture vessels, constant CO 2 at a concentration of 3,000 ppm was added.In the photoautotrophic conditions evaluated, plants of both varieties had a higher number of leaves and number of roots with respect to the photoheterotrophic and photomixotrophic treatments.Afreen- Zobayed et al. (2000) report that the photoautotrophic culture of sweet potato (Ipomoea batata L. (Lam)) significantly stimulated the growth of the leaves (leaf area) using vermiculite as substrate with respect to the control in agar.Also, Iarema et al. (2012) noted that the photoautotrophic conditions developed for the micropropagation of the Brazilian ginseg (Pfaffia glometata (Spreng.)Pedersen) appears to increase the leaf area of in vitro plants using culture medium solidified with agar.In Limonium spp.plants, Lian et al. (2002) report that growth in photoautotrophic conditions, the growth of the surface area of the leaf and the number had a superior effect.In Doritaenopsis orchid under photoautotrophic culture conditions and with increased CO 2 in the culture vessel, also achieved the best results with respect to heterotrophic culture conditions for the variables leaf area and length (Shin et al., 2013).This was also observed in the present study on papaya.However, photoautotrophic culture conditions are not also suitable for the growth of some in vitro plants cultured as in the case of coconut (Cocos nucifera L.), where without the presence of sucrose in the culture medium, plants had a smaller leaf area with respect to those grown with sucrose (Fuentes et al., 2005).
In the treatment without sucrose and with IBA after 17 days of culture, 50% of the papaya plants rooted, and the evaluation done at 37 days it reached 80.0%.Also, the treatment with 10 g L -1 of sucrose and IBA, similar results were obtained, but with lower values in all the evaluations done (Table 2).The rooting percentage previously expressed, reiterates the value of this auxin as an inducer of the process with enhanced effects through the lack of or low levels of sucrose in the culture medium.In addition, the results of this study suggest that continuous exposure to the auxin (IBA) improves the frequency of root formation, which is observed in the results obtained at 17, 27 and 37 days of culture (Tables 2, 3, 4 and Figure 1F).Plants grown without sucrose and with IBA also obtained the highest values in the variables height and fresh weight at 37 days of culture (Table 4).This behaviour is good evidence of the effectiveness of the culture conditions and zeolite as a substrate, ventilation of the culture vessels, IBA as a rooting inducer, and the absence of sucrose probably favored the source-demand effect so that photoautotroph would enhance the rooting stimulus of auxin.Several authors note the superior effect that IBA has with respect to other auxins for rooting of in vitro shoots in papaya (Yu et al., 2000;Teixeira da Silva et al., 2007;Kumar et al., 2012;Nzilani et al., 2013;Sekeli et al., 2013).These last authors reported that the best results in the rooting of in vitro shoots of transgenic papaya, Eksotika variety were achieved when first placing the shoots in culture medium with 9.8 µM IBA and in darkness for 4 days and thereafter were subcultured on MS culture medium at 50% of salt concentration, 10 µM of riboflavin and vermiculite as support.
According to George (2008), IBA easily becomes IAA causing a slow release of IAA and thus providing a continuous supply of the most common active auxin in concentrations, which may be more suitable for rooting.Calamar and de Klerk (2002) report the interaction between sucrose and auxin in the rooting of apple shoots.Increasing the sucrose concentration shifted the dose-response curve of auxin.The auxin concentration at the maximum response obtained, indicates that the cells require more auxin to give the response and / or that less free auxin reaches the target cells.There are reasons to believe that sucrose enhances the sensitivity to auxin.The results obtained in this study are contrary to reported by these authors.For sucrose concentrations studied (0, 10 and 20 g L -1 ) without auxin rooting percentages of papaya shoots were very low or zero.However in the presence of auxin (IBA) root formation was achieved, but the percentage of rooting were decreasing in the middle that sucrose concentration was increased.It seems that for these photoautotrophic culture conditions attached to the zeolite as porous support had a positive effect on the rooting of shoots than in mixotrophic culture condition with 10 and 20 g L -1 of sucrose.About it, Malamy and Ryan (2001) reported that when Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed.Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation.They isolated a mutant, lateral root initiation 1 (lin1) that overcomes the repressive conditions.This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios.The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues.Fujiwara and Kozai (1995) report that the use of porous support material improves the environment in the root zone and thereby increase rooting.A high porosity of the culture medium increases the concentration of oxygen around the rooting system, enhancing root development and improving water and nutrient absorption by the in vitro plants.In addition, the extensive root system produced in vitro, appears to contribute to the higher percentage of plant survival during acclimatization to greenhouse conditions and in the field.The results described in this study may be related to the characteristics conferred by the zeolite.According to Flores et al. (2007), zeolite drastically reduces the leaching of potassium cations (K +) and ammonium (NH 4 +), also it facilitates solubilization of phosphate by the available phosphorus to plants and therefore stimulates radical development.Zeolite is a crystalline hydrated aluminum silicate with three-dimensional structures, characterized by the ability to hold and release water and exchange ions without modifying their atomic structure, exchange cations such as Ca 2 +, Mg 2 +, K+ and NH 4 +; and various phosphate compounds, ammonium and organic matter components.It has a rigid three-dimensional structure formed by a network of interconnected tunnels creating a large surface area for the cation exchange and moisture adsorption.Similar results in terms of correlation between improved root system, improved growth and high survival rate were obtained in other plant species such as acacia (Acacia mangium) (Ermayanti et al., 1999); coffee (C.arabusta) (Nguyen et al., 1999); sweet potatoes (I.batata L. Lam.) (Afreen- Zobayed et al., 1999); Eucalyptus sp.(Zobayed et al., 2001); four Australian papaya varieties (Kaity et al., 2009) and in the variety Eksotika (Sekeli et al., 2013); Orchid (Doritaenopsis sp.) (Shin et al., 2013) using different types of porous materials (vermiculite, perlite and mixtures of both).This response was also reached in this work in papaya plants using the porous zeolite mineral as substrate.The presence of contaminants (bacteria and fungi) in all the treatments was quantified (Table 5).It is noteworthy that despite the increased exchange through the lid of the culture vessel, the visual presence of contamination in the treatments without sucrose was 0% for the total time of the experiment of 37 days.The treatments with sucrose showed no contamination during the assessments done at 17 and 27 days, but at 37 days, which shows the level of air cleanliness in the culture chamber and that the conditions and management developed can be used for further scaling.

Physiological parameters: Chlorophyll a, b and carotenoids
In terms of the quality of in vitro plants, the effects of the factors involved in the management of the contents of the active pigments in the photosynthetic process are analyzed.No significant differences were noticed for the variables chlorophyll a, chlorophyll b and carotenoids for the factors auxin and sucrose for in vitro shoots of papaya at 17 days of culture between the different treatments and the control (Table 6); significant differences were however observed in the assessments at 27 and 37 days as shown in Figures 2 and 3.At 27 days of culture there was a significant interaction between auxin and sucrose factors influencing the variable chlorophyll a.In Figure 2A as seen, when there was no sucrose in the culture medium without auxin, the plant produces significantly more chlorophyll a.Also, at 27 days there was a significant interaction between auxin and sucrose factors influencing the response of the variable chlorophyll b.As shown in Figure 2B when no sucrose was added to the culture medium, the plant produces significantly more chlorophyll b when there was no auxin than when the medium was supplemented with it.In the presence of sucrose, production levels of chlorophyll a and b decreased independently of the presence of auxin.With increasing levels of sucrose in the culture medium, production levels of chlorophyll a and b remain low regardless of the presence or absence of auxin.The response is quite similar for both molecules in this species.There was no interaction between the factors sucrose and auxin, only that sucrose was significant, influencing the response of the variable carotenoids content.When no sucrose was added to the culture medium, the plant produces significantly more carotenoids.As long as there is sucrose in the culture medium, this production was reduced significantly, although between the concentrations between 10 and 20 g L -1 of sucrose this decrease is not significant (Figure 2C).At 37 days of culture, there was a significant interaction between the factors sucrose and auxin influencing the response of the variable chlorophyll a (Figure 3A).When there is no sucrose in the culture medium, the plant produces significantly more chlorophyll a than when there is no auxin.In the presence of sucrose, the production levels of chlorophyll a decreased regardless of the presence of auxin, but this decrease is significant in the case that there is no auxin for the greater concentration for sucrose, without differences in the case of 10 g L -1 . Sucrose alone had a significant influence on the content of chlorophyll b (Figure 3B).When there was no sucrose added to the culture medium, the plants produced significantly more chlorophyll b.As long as there is sucrose in the culture medium, this production decreased, without being significant between the concentrations of 10 and 20 g L -1 sucrose.Sucrose alone had a significant influence on chlorophyll b at 37 days of culture.It is observed that when there is no sucrose in the culture medium, the plant produces significantly more chlorophyll b.Sucrose also had a significant effect influencing the variable carotenoids.When there was no sucrose in the culture medium, the plant produces significantly more carotenoids (Figure 3C).As long as there is the presence of sucrose in the culture medium, the production of these chlorophyll pigments decreased significantly, even though between the two concentrations of sucrose this decrease is not significant.

Physiological parameters: Photosynthesis and transpiration
Best photosynthetic values were achieved when the in vitro shoots were grown in culture medium with auxin and different concentrations of sucrose, even though they were also high in the treatment without the presence of IBA and without sucrose at 17 days of culture.In photomixotrophic culture conditions, transpiration levels were low with respect to the heterotrophic and photoautotrophic conditions.This is due to the presence of sucrose in the culture medium which the plant used as an energy source and it was not required for an increase in the photosynthetic activity and thus the opening and closing of the stomata, which made the transpiration levels so low.Nevertheless, the lowest levels of transpiration was obtained in plants grown in 20 g L -1 sucrose since the osmotic potential of the culture medium was higher, therefore for in vitro plants it is more difficult to take up water and hence transpiration rate was lower.For photoautotrophic conditions in the absence of the auxin, the plants did not have any roots at 17 days of culture, which resulted in a high rate of photosynthesis, but also high transpiration and having no roots to take up water for photosynthesis they had to have a greater stomata activity for the intake of CO 2 , causing a greater transpiration (Table 7).
Plants grown in photomixotrophic conditions and without auxin, presented the lowest values of photosynthesis.In this regard, Rolland et al. (2002), Amiard et al. (2005); Jo et al. (2009) refer to plants that were grown in culture medium with sucrose, exhibited reduced photosynthesis, probably due to the presence of a sufficient energy source (sugar) and other metabolic activities.Franck et al. (2006) reported that sucrose plays a central role in the mechanism mediating control of the regulation by decreasing photosynthesis.The low rate of substrate regeneration for the carboxylation of ribulose bisphosphate (RuBP) due to the accumulation of soluble sugars in the leaves is the possible result in the inhibition of photosynthesis (Azcon-Bieto, 1983).
However, results obtained by these authors indicate that a greater amount of starch granules found in the chloroplasts of leaves of plants grown in the greenhouse probably were part of the storage product.On the contrary, in in vitro seedlings they did not show any starch granules, probably because the rate of photosynthesis is low or exogenous sucrose caused a negative feedback on the enzyme level of the plastid for starch biosynthesis (Krapp and Stitt, 1994).However, plants grown in photomixotrophic conditions and auxin, had high photosynthetic rate equal to those grown in photoautotrophic conditions, this might be because these plants began to develop their rooting system, which offset the loss of water for photosynthesis, making efficient use of water (Table 7).
Photosynthesis in plants grown on agar (heterotrophic control) was very low compared with their high transpiration rate, a reason that adds to the justification for the zero survival assessed at 17 days after planting in the acclimatization phase.This demonstrates the low ability to control water loss of these plants in heterotrophic culture conditions.The levels of photosynthetic pigments also corresponded with this result given their involvement in the photosynthetic process.When sucrose was zero, the contents of carotenoids and chlorophylls were high and also appear to achieve good performances of the collecting antennas and the light producing complexes which integrate the photosystems involved and which constitute the pigments analyzed and other components.Although, the content of chlorophyll is not a direct indicator related to the photosynthetic capacity (Fujiwara et al., 1992); this is a good indicator of the state of the photosynthetic apparatus (Seon et al., 2000).This happened when the in vitro papaya plants were evaluated at 17 days of culture in the different treatments with and without photoautotrophic culture conditions where there were no significant differences observed among them; however, when determining the photosynthetic rate, there were significant differences between the different treatments as shown in Table 7.In this regard, Iarema et al. (2012) obtained the same response on analyzing the content of chlorophyll pigments and carotenoids of in vitro plants of Brazilian ginseg [P.glometata (Spreng.)Pedersen] cultured in the absence of sucrose and in the culture vessel with greatest level of exchange or ventilation and hence had an increase in the photosynthetic activity.
However, other authors reported the increase of photosynthetic pigments and increased photosynthetic activity in in vitro shoot cultured of Limonium spp.(Lian et al., 2002) and in Dendrobium candidum Wall.ex Lind (Xiao et al., 2007).The results obtained in this study support those reported by Kozai and Kubota (2005) on the benefits of photoautotrophic micropropagation over conventional micropropagation.The benefits from a biological point of view include: (1) promoting growth and photosynthesis; (2) high rates of survival and a smooth transition to environmental conditions ex vitro; (3) elimination of morphological and physiological disorders; (4) no callus formation at the base of the explant and (5) less plant lost due to contamination by microorganisms.

Ex vitro acclimatization
The treatment without sucrose and 9.8 µM IBA reached the highest percentage of survival which are suitable for rooting percentage that had in vitro papaya plants at 17 days of culture (Table 2 and Figure 1F).The treatments with the presence of auxin had the highest percentages of rooting, which corresponded to those of the greatest survival.The problem of very low survival is confirmed if appropriate management strategies are not performed that guarantee better quality of in vitro plants, with emphasis on their rooting pattern, and treatments without IBA at 17 days of culture (Table 2).Afreen- Zobayed et al. (2000) report that, in sweet potato 90% achieved survival of plants cultured in vitro in photoautotrophic conditions Pérez et al. 2633 compared to 73% of those grown on agar.Kozai et al. (2005) reported in the species Calla Lily (Zantedeschia elliottian L.), 95% survival (photoautotrophic conditions) at 12 days after transplanting to acclimatization phase relative to 60% of plants grown in heterotrophic conditions.Also, in the species China fir (Cunninghamia lanceolata (Lambert) Hooker) only 16% survival in in vitro plants cultured in heterotrophic conditions was obtained and 95% in photoautotrophic.However, Jo et al. (2009) report that the best results in ex vitro acclimatization was reached for Alocasia amazonica plants cultured with 3.0% sucrose and not those that were cultured in autotrophic conditions.

Conclusion
The management of papaya plants var.Red Maradol obtained through somatic embryogenesis during the transition in vitro-ex vitro integrated by using zeolite as a support, the combination of zero or low levels of sucrose, increased ventilation and use of auxin IBA (9.8 µM) as an inducer of rooting, improve the quality of the plants and thus their survival.
Abbreviations: IBA, Indole-butyric acid; BAP, 6-benzyl aminopurine; NAA, naphthaleneacetic acid.Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Figure 1 .
Figure 1.Rooting and in vitro acclimatization of papaya (Carica papaya L. var.Red Maradol) shoots obtained by somatic embryogenesis under different culture conditions.(A) Culture vessel with increased ventilation.(B-C) Plastic trays (polycarbonate) with zeolite as support used for ex vitro acclimatization plants after 17 days of in vitro culture.(D) Aspects of in vitro plants at the end of the experiment (37 days) in photoautotrophic culture conditions.(E) In vitro papaya plants with the formation of basal callus cultured in culture medium with agar and sucrose.(F) Stimulus of rhizogenesis in the presence or absence of auxin and sucrose at 37 days of culture.

Figure 1 .
Figure 1.Rooting and in vitro acclimatization of papaya (Carica papaya L. var.Red Maradol) shoots obtained by somatic embryogenesis under different culture conditions.(A) Culture vessel with increased ventilation.(B-C) Plastic trays (polycarbonate) with zeolite as support used for ex vitro acclimatization plants after 17 days of in vitro culture.(D) Aspects of in vitro plants at the end of the experiment (37 days) in photoautotrophic culture conditions.(E) In vitro papaya plants with the formation of basal callus cultured in culture medium with agar and sucrose.(F) Stimulus of rhizogenesis in the presence or absence of auxin and sucrose at 37 days of culture.

Figure 2 .
Figure 2. Effect of sucrose and IBA on the concentration of chlorophyll a, b, and total carotenoids content in the leave of in vitro papaya (Carica papaya L. var.Red Maradol) plants growing in culture vessel with increased ventilation and zeolite as support at 27 days de culture (A) Chlorophyll a (B) Chlorophyll b and (C) Carotenoids content.Reference: (0) without IBA and (1) 9.8 µM of IBA.Statistical difference between means according to the Fisher LSD test at p ≤ 0.05.

Figure 3 .
Figure 3.Effect of sucrose and IBA on the content of chlorophyll a, b, and total carotenoids content in the leave of in vitro papaya (Carica papaya L. var.Red Maradol) plants growing in culture vessel with increased ventilation and zeolite as support at 37 days of culture (A) chlorophyll a (B) chlorophyll b and (C) carotenoids content.Reference: (0) without IBA and (1) 9.8 µM of IBA.Statistical difference between means according to the Fisher LSD test at p ≤ 0.05.

Table 2 .
Effects of the interaction sucrose and IBA on growth and rooting of in vitro papaya (Carica papaya L. var.Red Maradol) plants growing in culture vessel with increased ventilation and zeolite as a support at 17 days of culture.

Table 3 .
Effects of the interaction sucrose and IBA on growth and rooting of in vitro papaya (Carica papaya L. var.Red Maradol) plants growing in culture vessel with increased ventilation and zeolite as a support at 27 days of culture.

Table 4 .
Effects of the interaction sucrose and IBA on growth and rooting of in vitro papaya (Carica papaya L. var.Red Maradol) plants growing in culture vessel with increased ventilation and zeolite as a support at 37 days of culture.

Table 5 .
Contamination in the culture vessel with increased ventilation at different days of culture during rooting and in vitro acclimatization of papaya plants.

Table 6 .
Effects of the interaction sucrose and IBA on the concentration of chlorophyll a and b and total carotenoids content in the leaves of in vitro papaya (Carica papaya L. var.Red Maradol) plants in culture vessels with increased ventilation and zeolite as a support at 17 days of culture.
a Different letters within a column indicate significant differences at p ≤ 0.05 by Tukey test (n=15).Values ± SD.

Table 7 .
Effects of the interaction sucrose AIB on the photosynthetic activity (mol CO2 m -2 s -1 ) and transpiration (mmol H2O m -2 s -1 ) in in vitro papaya plants (Carica papaya L. var.Red Maradol) cultured in culture vessels with increased ventilation and zeolite as a support at 17 days of culture.