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Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andreanum Lind.
Scientia Horticulturae 249 (2019) 185–191
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Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andreanum Lind.
T
Eduardo Martínez-Estradaa, Bartolo Islas-Lunab, José Antonio Pérez-Satoa, ⁎ Jericó Jabín Bello-Belloc, a
Colegio de Postgraduados-Campus Córdoba, Carretera Córdoba Veracruz, km 348, Amatlán de los Reyes, 94946, Veracruz, Mexico Universidad Veracruzana-Facultad de Ciencias Biológicas y Agropecuarias, 94945, Municipio Amatlán de los Reyes, Veracruz, Mexico c CONACYT-Colegio de Postgraduados-Campus Córdoba, Carretera federal Córdoba-Veracruz Km. 348, Amatlán de los Reyes, 94946 Veracruz, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Temporary immersion Chlorophyll Stomatal index Acclimatization Micropropagation
A lack of automation and the high production costs entailed by the use of labor and a gelling agent limit micropropagation on a commercial scale. The aim of this study was to evaluate the temporary immersion (TI) technique in in vitro multiplication and acclimatization of Anthurium andreanum cv. Rosa. Nodal segments from in vitro-derived adventitious shoots were cultivated in different culture systems: semisolid medium, liquid medium with partial immersion and TI using an Ebb-and-Flow bioreactor. The effect of the culture system, immersion frequency, and culture medium volume per explant on shoot multiplication of A. andreanum was evaluated after 45 days of culture. In addition, chlorophyll content, stomatal index and survival rate during acclimatization were evaluated in different culture systems. A completely randomized experimental design was used for all experiments, with three replicates. An analysis of variance (ANOVA) and Tukey’s range test (p ≤ 0.05) were performed. The results showed significant differences in the variables evaluated among the different culture systems. The highest shoot production was obtained in TI with 31.50 ± 0.50 shoots per explant, followed by the partial immersion system and culture in semisolid medium, with 7.25 ± 0.16 and 4.50 ± 0.18 shoots per explant, respectively. Immersion frequency and the amount of culture medium per explant did not show significant differences, which allows us to recommend the immersion frequency of every 12 h and the culture medium volume of 25 mL per explant. TI favored an increase in chlorophyll content, a low stomatal index and a high percentage of closed stomata, suggesting an increase in the functionality of the stomata and probably a higher photosynthetic rate. The survival rate during acclimatization increased when using temporary immersion systems (TIS). This study shows for the first time an efficient TIS for commercial micropropagation of A. andreanum that produces plants with a high survival rate during acclimatization.
1. Introduction Anthurium (Anthurium andreanum Lind.) is a tropical species of ornamental importance as a pot plant and cut flower due to its extended vase life (Gantait et al., 2012). Due to its high demand, the creation of new, healthy and uniform cultivars is important (Gantait and Sinniah, 2011). In this sense, conventional methods for propagating this species are not very efficient; its sexual propagation by seeds results in a heterogeneous progeny (Bejoy et al., 2008) and its asexual propagation takes years to develop clones of commercial standard and can transmit pests and diseases (Martin et al., 2003). Micropropagation is an attractive alternative for mass propagation of plants with high genetic and phytosanitary quality. However,
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conventional micropropagation in semisolid media involves high production costs caused mainly by labor, the use of gelling agents and the lack of automation (Etienne and Berthouly, 2002; Georgiev et al., 2014). Therefore, scaled-up and automated systems are desirable to minimize production costs, increase multiplication rates and reduce the amount of handling during in vitro propagation (Watt, 2012). Currently, new methodologies are being developed for plant micropropagation, among which the TIS stand out. TIS are semi-automated bioreactors designed for the mass propagation of tissues, embryos or organs exposed to liquid medium for a determined time and frequency. There are several TIS models, which have been described by various authors (Etienne and Berthouly, 2002; Georgiev et al., 2014; Gatti et al., 2017); however, in general, the success of micropropagation
Corresponding author. E-mail address: [email protected] (J.J. Bello-Bello).
https://doi.org/10.1016/j.scienta.2019.01.053 Received 14 July 2018; Received in revised form 25 January 2019; Accepted 28 January 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 249 (2019) 185–191
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agent.
in TIS is largely determined by the immersion frequency to which the explants are subjected and the volume of initial liquid medium in relation to the number of explants inoculated (Watt, 2012). TI has become a very useful technique to achieve semi-automation, lower costs and increased multiplication rates during in vitro culture of plants (Ramos-Castellá et al., 2014; Mosqueda et al., 2017; Vives et al., 2017), favoring, in addition, the acclimatization process (Aragón et al., 2014). Although in vitro regeneration of A. andreanum has been reported by different authors using different types of explants (Gantait et al., 2012; Cardoso and Habermann, 2014; Texeira da silva et al., 2015; MartínezEstrada et al., 2016), in this species, a micropropagation system that allows scaling at a commercial level using the principle of TI has not been reported. The objective of this study was to evaluate the use of a TIS to improve the multiplication and acclimatization of Anthurium andreanum.
2.3. Evaluation of culture medium volume per explant After evaluating the immersion frequency, three culture medium volumes per explant were evaluated (50, 37.5 and 25 mL) at the immersion frequency of 2 min every 12 h. In all treatments, three bioreactors with 500 mL of medium each were used, inoculating the corresponding number of plants. The liquid medium and culture conditions in all treatments were the same described above. 2.4. Chlorophyll content Chlorophyll content was determined according to the methodology proposed by Harborne (1973) with slight modifications. Leaf tissue (500 mg) was placed in a 20 mL amber bottle, 5 mL of 80% acetone were added and then the sample was incubated for 24 h at −4 °C. After incubation the sample was macerated and the extract was placed in a funnel with filter paper (Whatman, grade 4). The filtrate was adjusted to 12.5 mL with 80% acetone and its absorbance was measured in a spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA) at 645 and 663 nm. Chlorophyll content was calculated using the following formulas:
2. Materials and methods 2.1. Evaluation of the different culture systems Young brown leaves (5–10 days after unfolding) were collected from mature, healthy Rosa cultivar plants maintained under greenhouse conditions. The leaves were submerged in 70% (v/v) ethanol for 1 min and in 10% and 5% commercial bleach (4–6% sodium hypochlorite) for 20 min and 10 min, respectively, and then rinsed three times with sterile distilled water. Finally, lamina segments (1.5 cm2) were planted in petri dishes containing 15 mL of MS (Murashige and Skoog, 1962) medium supplemented with 30 g L−1 sucrose, 1 mg L−1 6benzylaminopurine (BAP, Sigma Chemical Company, MO, USA) and 0.5 mg L 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma Chemical Company, MO, USA). The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide, and 0.25% (w/v) Phytagel® (Sigma Chemical Company, MO, USA) was added as a gelling agent before being autoclaved at 120 °C and 117.7 kPa for 15 min. The cultures were incubated at 24 ± 2 °C in dark conditions for 45 days, before being transferred to 16 h light photoperiod using white-light lamps with 25 μmol m-2 s−1 photosynthetic photon flux density. After 45 days of culture, nodal segments from in vitro-derived adventitious shoots were cultured 45 days in different culture systems: semisolid medium, liquid medium with partial immersion (˜5 mm of the shoot base was submerged in liquid medium), and TI using a 1000 mL Ebb-and-Flow bioreactor system (Tisserat and Vandercook, 1985; Ducos et al., 2007). All culture systems used MS (Murashige and Skoog, 1962) medium supplemented with 2 mg L−1 6-benzylaminopurine (BAP, Sigma Chemical Company, MO, USA). The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide and 0.25% (w/v) Phytagel® (Sigma Chemical Company, MO, USA) was added as a gelling agent for semisolid medium before being autoclaved at 120 °C and 117.7 kPa for 15 min. The cultures were incubated at 24 ± 2 °C and a 16 h light photoperiod using white-light lamps with 25 μmol m-2 s−1 photosynthetic photon flux density. A total of 15 explants were used for semisolid medium and partial immersion, three explants per Magenta box (Sigma Chemical Company, MO, USA), with 25 and 15 mL of medium per vessel, respectively. For the TIS, 10 explants per bioreactor were used with an immersion of 2 min every 4 h. Three bioreactors were used, each containing 500 mL of medium (50 mL per explant).
Chlorophyll a (C ) = ([(12.7 * A _663 ) − (2.59 * A _645)](V ))/((1000 * P )) Chlorophyll b (C ) = ([(22.9 * A _645 ) − (4.70 * A _663)](V ))/((1000 * P ))
Total Chlorophyll (C ) = ([(8.20 * A _663 ) − (20.2 * A _645)](V )) /((1000 * W )) Where: A = Absorbance C = Concentration (mg g−1 FW) V = Graduated volume (mL−1) W = Sample weight (g) 1000= Conversion factor 2.5. Effect of different culture systems on stomatal index, stomatal aperture and acclimatization To calculate the stomatal index and percentage of closed stomata, the third leaf was taken, from the apex to the base of the stem, of ten different shoots of each system after 45 days of in vitro culture. A thin layer of nail varnish was applied uniformly on the abaxial surface. After 3 min, the dried varnish was gently peeled off and mounted on a microscope slide. The leaf replicas were examined in an optical microscope (Axio Lab.A1, ZEISS). The number of open and closed stomata and the number of epidermal cells per mm2 were counted from three random fields of a leaf at 10X and 40X. The stomatal index was calculated using the formula suggested by Wilkinson (1980): SI=(NS/(EC+NS))*100s Where: SI = Stomatal Index NS: number of stomata EC: number of epidermal cells To evaluate the effect of the culture system on the acclimatization of A. andreanum, thirty shoots were taken from the different culture systems. In TI the evaluation was carried out in the three frequencies with 25 mL of medium per explant. The shoots were planted ex vitro, without passing through the rooting stage, in a peat moss and volcanic rock (particle sizes 5 to 10 mm) (1:1) substrate in 72- cavity plastic trays. Plantlets were watered twice a day for the first 15 days of culture and once a day after that. A foliar fertilizer (Nitrofoska® foliar PS, COMPO, Zapopan, Mexico) was applied weekly. Plants were maintained in a greenhouse, where the temperature was maintained at 27 ± 3 °C with
2.2. Evaluation of the immersion frequency To determine the appropriate immersion frequency for in vitro multiplication of A. andreanum, three frequencies were evaluated: 2 min every 4, 8 and 12 h. In all assays, 10 explants with 50 mL of medium per explant per bioreactor and three bioreactors per frequency were used. The culture medium and conditions in all the treatments were the same as that described for the previous experiment, but without gelling 186
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Table 1 Effect of culture system, immersion frequency and culture medium volume per explant on shoot multiplication of A. andreanum cv. Rosa after 45 days of culture. Micropropagation system
No. shoots
Shoot length (cm) c
No. leaves per shoot
Fresh weight per shoot (g)
c
b
b
Dry weight per shoot (g)
Semisolid culture medium Partial immersion Temporary immersion
4.50 ± 0.18 7.25 ± 0.16b 31.50 ± 0.50a
1.15 ± 0.04 1.39 ± 0.04b 1.76 ± 0.05a
3.00 ± 0.26 2.37 ± 0.18b 4.25 ± 0.16a
0.048 ± 0.003 0.059 ± 0.005b 0.082 ± 0.003a
0.004 ± 0.0002b 0.005 ± 0.0006b 0.008 ± 0.0003a
Immersion frequencies in TIS Frequency every 4 h Frequency every 8 h Frequency every 12 h
31.50 ± 0.50a 31.62 ± 0.67a 33.12 ± 0.97a
1.76 ± 0.05a 1.49 ± 0.03a 1.78 ± 0.04a
4.25 ± 0.16a 4.37 ± 0.18a 4.25 ± 0.16a
0.082 ± 0.003a 0.085 ± 0.005a 0.086 ± 0.003a
0.008 ± 0.0003a 0.006 ± 0.0005a 0.008 ± 0.0003a
Culture medium volume per explant (mL) 25 32.84 ± 0.74a 37.5 32.45 ± 0.82a 50 33.12 ± 0.97a
1.82 ± 0.03a 1.71 ± 0.03a 1.78 ± 0.04a
4.37 ± 0.18a 4.37 ± 0.32a 4.25 ± 0.32a
0.074 ± 0.003a 0.077 ± 0.005a 0.086 ± 0.003a
0.007 ± 0.0004a 0.007 ± 0.0003a 0.008 ± 0.0003a
Means ± standard error within a column followed by the same letter are not significantly different according to Tukey’s range test at p ≤ 0.05.
a photosynthetic photon flux density of 300 μmol m-2 s−1.
results showed no significant differences among the evaluated variables (Table 1). Similarly, in the chlorophyll content, no significant differences were observed among the different immersion frequencies assessed (Fig. 2b).
2.6. Experimental design and data analysis A completely randomized experimental design was used for all experiments, which were replicated three times. In all in vitro treatments, the number and length of shoots, fresh weight, dry weight, number of leaves and chlorophyll contents were evaluated after 45 days of culture. Stomatal index, stomatal aperture and acclimatization were also evaluated in the different culture systems and immersion frequencies. Plant survival was evaluated after 30 days. An analysis of variance (ANOVA) and Tukey’s range test (p ≤ 0.05) were performed for all variables using SPSS statistical software (version 22 for Windows).
3.4. Stomatal index, stomatal aperture and acclimatization The stomatal index and percentage of closed stomata showed significant differences among the culture systems. The highest stomatal index was found in the semisolid and partial immersion systems with 7.43 ± 0.21 and 7.13 ± 0.32%, respectively. A lower stomatal index was found in TI, without presenting significant differences among the frequencies evaluated. Regarding the percentage of closed stomata, shoots cultured on liquid medium systems presented the highest percentages (between 23–29%). On the other hand, shoots cultured in semisolid medium had the lowest percentage of closed stomata, with 2% (Fig. 3). Regarding acclimatization, the plantlets obtained from the TIS showed survival percentages higher than 90%.No statistical differences were found for survival regarding frequency treatments (Fig. 4). The lowest survival percentages were found in the plantlets obtained in semisolid medium, with 80.4. Fig. 5 shows plantlets obtained in the TIS at 30 and 90 days of culture under greenhouse conditions.
3. Results 3.1. Evaluation of the different culture systems The results show significant differences among the three culture systems evaluated (Table 1). The highest number of shoots was obtained in the TI treatment with 31.50 ± 0.50 shoots per explant, followed by the partial immersion system and culture in semisolid medium, with 7.25 ± 0.16 and 4.50 ± 0.18 shoots per explant, respectively. Regarding shoot length, the shoots in TI obtained the longest length (1.76 ± 0.05 cm) in comparison with the explants in partial immersion and in semisolid medium (1.39 ± 0.4 and 1.15 ± 0.4 cm, respectively). For the variables fresh weight, dry weight and number of leaves, the shoots obtained in TI had the highest values compared to the semisolid medium and partial immersion (Fig. 1). As for chlorophyll content, the results showed significant differences among the different culture systems evaluated. The highest chlorophyll contents were found in the liquid medium culture systems, while the lowest chlorophyll values were obtained in the semisolid medium culture system (Fig. 2a).
4. Discussion 4.1. Evaluation of the different culture systems The evaluation of the different in vitro culture systems demonstrated the usefulness of TI to increase the quantity and quality of shoots produced with respect to the conventional micropropagation system in semisolid medium. The liquid culture media in partial and TI improved shoot production, which reduces the production costs of the culture medium and labor. According to Dewir et al. (2014), liquid culture systems offer many potential advantages over solid cultures, such as faster growth rates, rapid uptake of nutrients and growth regulators by tissues and dilution of exuded growth inhibitors i.e. phenolics released by explants. These advantages have allowed the use of liquid media to be an alternative for commercial-scale propagation, especially when using TIS. In this study, the Ebb-and-Flow bioreactor proved to be the most efficient system for the micropropagation of A. andreanum. In other reports for this species, Lee-Espinosa et al., 2003 obtained a maximum of 10 and 15 shoots per explant in the cvs Midori and Kalapana, at 75 and 90 d, respectively. Similarly, Gantait et al., 2008 reported for the cv CanCan a production of ten shoots per explant at 50 d of culture. On the other hand, Bejoy et al., 2008 reported in cv Agnihothri a maximum of 9.7 shoots by indirect organogenesis. Gantait et al., 2012 reported that in cv CanCan it is possible to regenerate 17.2 shoots per protocorm like bodies (PLBs). In another study, Gu et al.,
3.2. Evaluation of the immersion frequency Regarding the evaluation of immersion frequencies, no significant differences were found in the morphological variables evaluated (Table 1). Shoot production in the immersion frequencies every 4, 8 and 12 h were 31.50 ± 0.50, 31.62 ± 0.67 and 33.12 ± 0.97, respectively. Chlorophyll content also showed no significant differences (Fig. 2b). However, these results allow us to recommend the frequency of 2 min every 12 h for the micropropagation of A. andreanum due to the lower use of electrical energy. 3.3. Evaluation of culture medium volume per explant When evaluating the initial culture medium volume per explant, the 187
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Fig. 1. Effect of culture system and immersion frequency in TIS on in vitro multiplication of A. andreanum. a) semisolid medium, b) liquid medium in partial immersion, c) temporary immersion for 2 min every 4 h, d) temporary immersion for 2 min every 8 h, and e) temporary immersion for 2 min every 12 h. Bar = 1 cm.
Fig. 3. Effect of culture system and immersion frequency in TIS on stomatal index and percentage of closed stomata during the multiplication stage of A. andreanum cv. Rosa after 45 days of in vitro culture. Bars represent the mean ± standard error. Bars followed by a different letter denote significant statistical differences (Tukey, P ≤ 0.05). TI: Temporary immersion.
production with respect to the indirect organogenesis micropropagation system. Similar to the results obtained in this study, the use of TIS has recently been reported in Stevia rebaudiana (Ramírez-Mosqueda and Iglesias-Andreu, 2016; Vives et al., 2017), Quercus robur (Gatti et al., 2017), Gerbera jamesonii (Mosqueda et al., 2017) and Dianthus caryophyllus (Ahmadian et al., 2017). Roels et al., 2005 reported that in Musa AAB the use of a Temporary Immersion Bioreactor (TIB) increased the multiplication rate with respect to the semisolid medium. The same results were reported by Farahani and Majd (2012) and Vives et al. (2017) in Musa spp and Stevia rebaudiana, respectively. In ornamental plants, Hahn and Paek (2005) reported in Dendranthema grandiflorum that the use of TI resulted in greater fresh weight, dry weight, shoot length and leaf area compared to semisolid culture. In the same way, Daquinta et al. (2007) reported that the multiplication rate of Caladium × hortulanum was more than 12 times higher than under a semisolid culture. Similar results were reported by Ahmadian et al. (2017), who showed that TI
Fig. 2. Chlorophyll contents during the multiplication stage of A. andreanum cv. Rosa after 45 days of in vitro culture. Effect of a) culture system and b) volume of medium per explant. Bars represent the mean ± standard error. Bars followed by a different letter denote significant statistical differences (Tukey, P ≤ 0.05). IT: Temporary immersion.
2012 obtained 24.9 and 24.7 shoots through indirect organogenesis in cvs Alabama and Sierra, respectively. More recently, Bhattacharya et al., 2015 regenerated up to 25.6 shoots per explant in cv Tinora. To date, our micropropagation system in TIS, compared to the previously mentioned reports, suggests the possibility of doubling or tripling the production of shoots per explant compared to direct organogenesis systems from shoots in semi-solid medium and even improving 188
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4.2. Evaluation of the immersion frequency Immersion frequency is an important aspect when using TIS since it plays a vital role in nutrient intake, growth regulators and hyperhydricity control (Etienne and Berthouly, 2002; Watt, 2012). It has been shown that immersion frequency varies among species. Mordocco et al. (2009) reported a higher percentage of regeneration in explants of three cultivars of Saccharum spp. (cv: Q117, Q165 and Q205) when using an immersion frequency of 1 min every 24 h. Akdemir et al. (2014) decreased hyperhydration in Pistacia vera by prolonging the immersion frequencies, from 16 min every 8 h to 16 min every 24 h. This study is the first reported evaluation of different immersion frequencies to optimize the production of shoots in A. andreanum. Although no significant differences were found in the variables evaluated, our results allow us to recommend an immersion frequency of every 12 h due to the fact that less electrical energy is required per day for the operation of the compressor and solenoid valve that make up the immersion system. Similar to our results, Mosqueda et al. (2017) found no significant differences in the multiplication rate of Gerbera jamesonii among the immersion frequencies; however, they reported that the frequencies of 4 min every 8 and 12 h produced longer shoots with fewer hyperhydration symptoms compared to the frequency of 4 min every 6 h.
Fig. 4. Effect of culture system on survival percentage of A. andreanum cv. Rosa after 30 days of acclimatization under greenhouse conditions. Bars followed by a different letter denote significant statistical differences (Tukey, p ≤ 0.05).
produces more than 10 times the shoot production in Dianthus caryophyllus in comparison to semisolid culture. One of the main advantages of using TI is that it promotes ventilation within the culture vessel, allowing the removal of volatile compounds such as ethylene (Roels et al., 2006), recirculating the carbon dioxide necessary for photosynthesis (Aragón et al., 2014) and increasing stomatal functionality in the leaves compared to those obtained in semisolid environments (Afreen, 2008). These factors, together with the loss of apical dominance, could explain the increase in the multiplication rate of A. andreanum. In this sense, our results showed that total chlorophyll content is higher in culture systems in liquid medium than in semisolid medium. Similar results were reported by Arencibia et al. (2013) in the micropropagation of Rubus spp. Probably the increase in the content of chlorophylls in liquid medium is due to a greater availability of nutrients and other compounds, which can be taken practically throughout the explant through leaves and tissues while, in semisolid medium, the explants obtain the components of the culture medium only from the area in contact with the medium.
4.3. Evaluation of culture medium volume per explant The initial medium volume per explant should be a variable to optimize in TI. Although it is a factor that is usually omitted, several authors have shown its importance. Lorenzo et al. (1998) showed that 50 mL of medium per explant resulted in a higher multiplication rate than lower or higher amounts of medium (27.5 or 72.5 mL per explant) during the micropropagation of Saccharum spp. Ramos-Castellá et al. (2014) reported an increase in shoot production by optimizing the medium volume at 25 mL per explant in Vanilla planifolia. According to Etienne and Berthouly (2002), large volumes of medium per explant are usually inefficient because the in vitro cultures produce extracellular chemicals that stimulate shoot formation, which would be diluted when large volumes of medium are used. In addition to this, the initial medium volume determines the quantity of explants inside a bioreactor that, once developed, will compete for the space, light and nutrients of the culture medium. In this sense, the greater the number of explants per bioreactor, the greater the chances the explants could lack the optimal conditions for their development. The results of this study suggest that 25 mL of medium per explant is sufficient to maintain a high multiplication rate in A. andreanum.
Fig. 5. Acclimatized plantlets of A. andreanum cv. Rosa obtained from Ebb-And-Flow Bioreactor at a) 30 days and b) 90 days of ex vitro culture under greenhouse conditions. Bar = 10 cm. 189
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4.4. Stomatal index, stomatal aperture and acclimatization
References
According to Zobayed (2005), by applying ventilation, some anatomical and physiological changes that are common in conventional micropropagation can be prevented, including the production of malfunctioning stomata, which remain permanently widely open. Closed stomata and a low stomatal index generate a low transpiration rate, avoiding the loss of water and dehydration, one of the most frequent causes of plant death, during acclimatization (Aragón et al., 2014; Vieira et al., 2015). The results obtained in this study show that the TIS favors a low stomatal index and a high percentage of closed stomata compared to the system in semisolid medium, suggesting the functionality of the stomata, thereby promoting a lower transpiration rate and probably a higher photosynthetic rate. It also demonstrated a greater accumulation of photosynthetic pigments. The success of a micropropagation protocol not only depends on the number of shoots obtained by the explants but also depends on the morphological quality and vigor present in the plantlets produced (Martre et al., 2001). Therefore, acclimatization is an important stage that determines the success of micropropagation. Reports similar to this study have demonstrated that the TIS environment prepares the plantlets for the stress of acclimatization (Yang and Yeh, 2008; Aragón et al., 2010; Regueira et al., 2018). Escalona et al. (2003) reported that TI bioreactor-derived Ananas comosus plantlets showed remarkable nutrient uptake, indicating a higher photo-mixotrophic metabolism. Ahmadian et al. (2017) reported that TIS have a suitable effect on enhancing the elongation and root induction of Dianthus caryophyllus plantlets during acclimatization in comparison with plants from solid culture. Recently, Zhang et al. (2018) reported that Bletilla striata plants obtained using TIS showed uniform growth, enhanced leaf and stem development and substantially improved pseudobulbs. There are great differences in the physiology of the plants between the semisolid medium culture and TIS. The results show that among the factors that can contribute to improve the acclimatization of the plants obtained in TIS in comparison with those obtained in semisolid medium are a greater accumulation of photosynthetic compounds, a lower stomatal index and a higher percentage of closed stomata. In this regard, Yang and Yeh (2008) reported that, during ex vitro acclimatization, Calathea orbifolia plants produced in TIS had higher photosynthetic rates and subsequently a greater leaf area and fresh and dry weights compared to those obtained in semisolid medium. Likewise, Aragón et al. (2014) showed that TIS-derived plantlets exhibit better growth and stomatal function regulation and can therefore have better water loss control and even accumulate more starch in the leaves, which could be used during the first days of ex vitro acclimatization. Finally, the evaluation of the immersion frequencies and the medium volume per explant showed no significant differences in shoot production; however, they are important because they allow obtaining a high multiplication rate without decreasing quality, reducing production costs with respect to the traditional system in semisolid medium. Therefore, the use of TIS with an immersion frequency of 2 min every 12 h with an initial volume of 25 mL of culture medium per explant is recommended. In conclusion, it is herein reported for the first time that the TI technique is an alternative for the commercial micropropagation of A. andreanum. In addition to increasing the multiplication rate, these systems improve the quality of the shoots without a previous rooting stage and promote an increased survival rate during acclimatization.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andreanum Lind.
T
Eduardo Martínez-Estradaa, Bartolo Islas-Lunab, José Antonio Pérez-Satoa, ⁎ Jericó Jabín Bello-Belloc, a
Colegio de Postgraduados-Campus Córdoba, Carretera Córdoba Veracruz, km 348, Amatlán de los Reyes, 94946, Veracruz, Mexico Universidad Veracruzana-Facultad de Ciencias Biológicas y Agropecuarias, 94945, Municipio Amatlán de los Reyes, Veracruz, Mexico c CONACYT-Colegio de Postgraduados-Campus Córdoba, Carretera federal Córdoba-Veracruz Km. 348, Amatlán de los Reyes, 94946 Veracruz, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Temporary immersion Chlorophyll Stomatal index Acclimatization Micropropagation
A lack of automation and the high production costs entailed by the use of labor and a gelling agent limit micropropagation on a commercial scale. The aim of this study was to evaluate the temporary immersion (TI) technique in in vitro multiplication and acclimatization of Anthurium andreanum cv. Rosa. Nodal segments from in vitro-derived adventitious shoots were cultivated in different culture systems: semisolid medium, liquid medium with partial immersion and TI using an Ebb-and-Flow bioreactor. The effect of the culture system, immersion frequency, and culture medium volume per explant on shoot multiplication of A. andreanum was evaluated after 45 days of culture. In addition, chlorophyll content, stomatal index and survival rate during acclimatization were evaluated in different culture systems. A completely randomized experimental design was used for all experiments, with three replicates. An analysis of variance (ANOVA) and Tukey’s range test (p ≤ 0.05) were performed. The results showed significant differences in the variables evaluated among the different culture systems. The highest shoot production was obtained in TI with 31.50 ± 0.50 shoots per explant, followed by the partial immersion system and culture in semisolid medium, with 7.25 ± 0.16 and 4.50 ± 0.18 shoots per explant, respectively. Immersion frequency and the amount of culture medium per explant did not show significant differences, which allows us to recommend the immersion frequency of every 12 h and the culture medium volume of 25 mL per explant. TI favored an increase in chlorophyll content, a low stomatal index and a high percentage of closed stomata, suggesting an increase in the functionality of the stomata and probably a higher photosynthetic rate. The survival rate during acclimatization increased when using temporary immersion systems (TIS). This study shows for the first time an efficient TIS for commercial micropropagation of A. andreanum that produces plants with a high survival rate during acclimatization.
1. Introduction Anthurium (Anthurium andreanum Lind.) is a tropical species of ornamental importance as a pot plant and cut flower due to its extended vase life (Gantait et al., 2012). Due to its high demand, the creation of new, healthy and uniform cultivars is important (Gantait and Sinniah, 2011). In this sense, conventional methods for propagating this species are not very efficient; its sexual propagation by seeds results in a heterogeneous progeny (Bejoy et al., 2008) and its asexual propagation takes years to develop clones of commercial standard and can transmit pests and diseases (Martin et al., 2003). Micropropagation is an attractive alternative for mass propagation of plants with high genetic and phytosanitary quality. However,
⁎
conventional micropropagation in semisolid media involves high production costs caused mainly by labor, the use of gelling agents and the lack of automation (Etienne and Berthouly, 2002; Georgiev et al., 2014). Therefore, scaled-up and automated systems are desirable to minimize production costs, increase multiplication rates and reduce the amount of handling during in vitro propagation (Watt, 2012). Currently, new methodologies are being developed for plant micropropagation, among which the TIS stand out. TIS are semi-automated bioreactors designed for the mass propagation of tissues, embryos or organs exposed to liquid medium for a determined time and frequency. There are several TIS models, which have been described by various authors (Etienne and Berthouly, 2002; Georgiev et al., 2014; Gatti et al., 2017); however, in general, the success of micropropagation
Corresponding author. E-mail address: [email protected] (J.J. Bello-Bello).
https://doi.org/10.1016/j.scienta.2019.01.053 Received 14 July 2018; Received in revised form 25 January 2019; Accepted 28 January 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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agent.
in TIS is largely determined by the immersion frequency to which the explants are subjected and the volume of initial liquid medium in relation to the number of explants inoculated (Watt, 2012). TI has become a very useful technique to achieve semi-automation, lower costs and increased multiplication rates during in vitro culture of plants (Ramos-Castellá et al., 2014; Mosqueda et al., 2017; Vives et al., 2017), favoring, in addition, the acclimatization process (Aragón et al., 2014). Although in vitro regeneration of A. andreanum has been reported by different authors using different types of explants (Gantait et al., 2012; Cardoso and Habermann, 2014; Texeira da silva et al., 2015; MartínezEstrada et al., 2016), in this species, a micropropagation system that allows scaling at a commercial level using the principle of TI has not been reported. The objective of this study was to evaluate the use of a TIS to improve the multiplication and acclimatization of Anthurium andreanum.
2.3. Evaluation of culture medium volume per explant After evaluating the immersion frequency, three culture medium volumes per explant were evaluated (50, 37.5 and 25 mL) at the immersion frequency of 2 min every 12 h. In all treatments, three bioreactors with 500 mL of medium each were used, inoculating the corresponding number of plants. The liquid medium and culture conditions in all treatments were the same described above. 2.4. Chlorophyll content Chlorophyll content was determined according to the methodology proposed by Harborne (1973) with slight modifications. Leaf tissue (500 mg) was placed in a 20 mL amber bottle, 5 mL of 80% acetone were added and then the sample was incubated for 24 h at −4 °C. After incubation the sample was macerated and the extract was placed in a funnel with filter paper (Whatman, grade 4). The filtrate was adjusted to 12.5 mL with 80% acetone and its absorbance was measured in a spectrophotometer (Genesys 10S, Thermo Scientific, MA, USA) at 645 and 663 nm. Chlorophyll content was calculated using the following formulas:
2. Materials and methods 2.1. Evaluation of the different culture systems Young brown leaves (5–10 days after unfolding) were collected from mature, healthy Rosa cultivar plants maintained under greenhouse conditions. The leaves were submerged in 70% (v/v) ethanol for 1 min and in 10% and 5% commercial bleach (4–6% sodium hypochlorite) for 20 min and 10 min, respectively, and then rinsed three times with sterile distilled water. Finally, lamina segments (1.5 cm2) were planted in petri dishes containing 15 mL of MS (Murashige and Skoog, 1962) medium supplemented with 30 g L−1 sucrose, 1 mg L−1 6benzylaminopurine (BAP, Sigma Chemical Company, MO, USA) and 0.5 mg L 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma Chemical Company, MO, USA). The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide, and 0.25% (w/v) Phytagel® (Sigma Chemical Company, MO, USA) was added as a gelling agent before being autoclaved at 120 °C and 117.7 kPa for 15 min. The cultures were incubated at 24 ± 2 °C in dark conditions for 45 days, before being transferred to 16 h light photoperiod using white-light lamps with 25 μmol m-2 s−1 photosynthetic photon flux density. After 45 days of culture, nodal segments from in vitro-derived adventitious shoots were cultured 45 days in different culture systems: semisolid medium, liquid medium with partial immersion (˜5 mm of the shoot base was submerged in liquid medium), and TI using a 1000 mL Ebb-and-Flow bioreactor system (Tisserat and Vandercook, 1985; Ducos et al., 2007). All culture systems used MS (Murashige and Skoog, 1962) medium supplemented with 2 mg L−1 6-benzylaminopurine (BAP, Sigma Chemical Company, MO, USA). The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide and 0.25% (w/v) Phytagel® (Sigma Chemical Company, MO, USA) was added as a gelling agent for semisolid medium before being autoclaved at 120 °C and 117.7 kPa for 15 min. The cultures were incubated at 24 ± 2 °C and a 16 h light photoperiod using white-light lamps with 25 μmol m-2 s−1 photosynthetic photon flux density. A total of 15 explants were used for semisolid medium and partial immersion, three explants per Magenta box (Sigma Chemical Company, MO, USA), with 25 and 15 mL of medium per vessel, respectively. For the TIS, 10 explants per bioreactor were used with an immersion of 2 min every 4 h. Three bioreactors were used, each containing 500 mL of medium (50 mL per explant).
Chlorophyll a (C ) = ([(12.7 * A _663 ) − (2.59 * A _645)](V ))/((1000 * P )) Chlorophyll b (C ) = ([(22.9 * A _645 ) − (4.70 * A _663)](V ))/((1000 * P ))
Total Chlorophyll (C ) = ([(8.20 * A _663 ) − (20.2 * A _645)](V )) /((1000 * W )) Where: A = Absorbance C = Concentration (mg g−1 FW) V = Graduated volume (mL−1) W = Sample weight (g) 1000= Conversion factor 2.5. Effect of different culture systems on stomatal index, stomatal aperture and acclimatization To calculate the stomatal index and percentage of closed stomata, the third leaf was taken, from the apex to the base of the stem, of ten different shoots of each system after 45 days of in vitro culture. A thin layer of nail varnish was applied uniformly on the abaxial surface. After 3 min, the dried varnish was gently peeled off and mounted on a microscope slide. The leaf replicas were examined in an optical microscope (Axio Lab.A1, ZEISS). The number of open and closed stomata and the number of epidermal cells per mm2 were counted from three random fields of a leaf at 10X and 40X. The stomatal index was calculated using the formula suggested by Wilkinson (1980): SI=(NS/(EC+NS))*100s Where: SI = Stomatal Index NS: number of stomata EC: number of epidermal cells To evaluate the effect of the culture system on the acclimatization of A. andreanum, thirty shoots were taken from the different culture systems. In TI the evaluation was carried out in the three frequencies with 25 mL of medium per explant. The shoots were planted ex vitro, without passing through the rooting stage, in a peat moss and volcanic rock (particle sizes 5 to 10 mm) (1:1) substrate in 72- cavity plastic trays. Plantlets were watered twice a day for the first 15 days of culture and once a day after that. A foliar fertilizer (Nitrofoska® foliar PS, COMPO, Zapopan, Mexico) was applied weekly. Plants were maintained in a greenhouse, where the temperature was maintained at 27 ± 3 °C with
2.2. Evaluation of the immersion frequency To determine the appropriate immersion frequency for in vitro multiplication of A. andreanum, three frequencies were evaluated: 2 min every 4, 8 and 12 h. In all assays, 10 explants with 50 mL of medium per explant per bioreactor and three bioreactors per frequency were used. The culture medium and conditions in all the treatments were the same as that described for the previous experiment, but without gelling 186
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Table 1 Effect of culture system, immersion frequency and culture medium volume per explant on shoot multiplication of A. andreanum cv. Rosa after 45 days of culture. Micropropagation system
No. shoots
Shoot length (cm) c
No. leaves per shoot
Fresh weight per shoot (g)
c
b
b
Dry weight per shoot (g)
Semisolid culture medium Partial immersion Temporary immersion
4.50 ± 0.18 7.25 ± 0.16b 31.50 ± 0.50a
1.15 ± 0.04 1.39 ± 0.04b 1.76 ± 0.05a
3.00 ± 0.26 2.37 ± 0.18b 4.25 ± 0.16a
0.048 ± 0.003 0.059 ± 0.005b 0.082 ± 0.003a
0.004 ± 0.0002b 0.005 ± 0.0006b 0.008 ± 0.0003a
Immersion frequencies in TIS Frequency every 4 h Frequency every 8 h Frequency every 12 h
31.50 ± 0.50a 31.62 ± 0.67a 33.12 ± 0.97a
1.76 ± 0.05a 1.49 ± 0.03a 1.78 ± 0.04a
4.25 ± 0.16a 4.37 ± 0.18a 4.25 ± 0.16a
0.082 ± 0.003a 0.085 ± 0.005a 0.086 ± 0.003a
0.008 ± 0.0003a 0.006 ± 0.0005a 0.008 ± 0.0003a
Culture medium volume per explant (mL) 25 32.84 ± 0.74a 37.5 32.45 ± 0.82a 50 33.12 ± 0.97a
1.82 ± 0.03a 1.71 ± 0.03a 1.78 ± 0.04a
4.37 ± 0.18a 4.37 ± 0.32a 4.25 ± 0.32a
0.074 ± 0.003a 0.077 ± 0.005a 0.086 ± 0.003a
0.007 ± 0.0004a 0.007 ± 0.0003a 0.008 ± 0.0003a
Means ± standard error within a column followed by the same letter are not significantly different according to Tukey’s range test at p ≤ 0.05.
a photosynthetic photon flux density of 300 μmol m-2 s−1.
results showed no significant differences among the evaluated variables (Table 1). Similarly, in the chlorophyll content, no significant differences were observed among the different immersion frequencies assessed (Fig. 2b).
2.6. Experimental design and data analysis A completely randomized experimental design was used for all experiments, which were replicated three times. In all in vitro treatments, the number and length of shoots, fresh weight, dry weight, number of leaves and chlorophyll contents were evaluated after 45 days of culture. Stomatal index, stomatal aperture and acclimatization were also evaluated in the different culture systems and immersion frequencies. Plant survival was evaluated after 30 days. An analysis of variance (ANOVA) and Tukey’s range test (p ≤ 0.05) were performed for all variables using SPSS statistical software (version 22 for Windows).
3.4. Stomatal index, stomatal aperture and acclimatization The stomatal index and percentage of closed stomata showed significant differences among the culture systems. The highest stomatal index was found in the semisolid and partial immersion systems with 7.43 ± 0.21 and 7.13 ± 0.32%, respectively. A lower stomatal index was found in TI, without presenting significant differences among the frequencies evaluated. Regarding the percentage of closed stomata, shoots cultured on liquid medium systems presented the highest percentages (between 23–29%). On the other hand, shoots cultured in semisolid medium had the lowest percentage of closed stomata, with 2% (Fig. 3). Regarding acclimatization, the plantlets obtained from the TIS showed survival percentages higher than 90%.No statistical differences were found for survival regarding frequency treatments (Fig. 4). The lowest survival percentages were found in the plantlets obtained in semisolid medium, with 80.4. Fig. 5 shows plantlets obtained in the TIS at 30 and 90 days of culture under greenhouse conditions.
3. Results 3.1. Evaluation of the different culture systems The results show significant differences among the three culture systems evaluated (Table 1). The highest number of shoots was obtained in the TI treatment with 31.50 ± 0.50 shoots per explant, followed by the partial immersion system and culture in semisolid medium, with 7.25 ± 0.16 and 4.50 ± 0.18 shoots per explant, respectively. Regarding shoot length, the shoots in TI obtained the longest length (1.76 ± 0.05 cm) in comparison with the explants in partial immersion and in semisolid medium (1.39 ± 0.4 and 1.15 ± 0.4 cm, respectively). For the variables fresh weight, dry weight and number of leaves, the shoots obtained in TI had the highest values compared to the semisolid medium and partial immersion (Fig. 1). As for chlorophyll content, the results showed significant differences among the different culture systems evaluated. The highest chlorophyll contents were found in the liquid medium culture systems, while the lowest chlorophyll values were obtained in the semisolid medium culture system (Fig. 2a).
4. Discussion 4.1. Evaluation of the different culture systems The evaluation of the different in vitro culture systems demonstrated the usefulness of TI to increase the quantity and quality of shoots produced with respect to the conventional micropropagation system in semisolid medium. The liquid culture media in partial and TI improved shoot production, which reduces the production costs of the culture medium and labor. According to Dewir et al. (2014), liquid culture systems offer many potential advantages over solid cultures, such as faster growth rates, rapid uptake of nutrients and growth regulators by tissues and dilution of exuded growth inhibitors i.e. phenolics released by explants. These advantages have allowed the use of liquid media to be an alternative for commercial-scale propagation, especially when using TIS. In this study, the Ebb-and-Flow bioreactor proved to be the most efficient system for the micropropagation of A. andreanum. In other reports for this species, Lee-Espinosa et al., 2003 obtained a maximum of 10 and 15 shoots per explant in the cvs Midori and Kalapana, at 75 and 90 d, respectively. Similarly, Gantait et al., 2008 reported for the cv CanCan a production of ten shoots per explant at 50 d of culture. On the other hand, Bejoy et al., 2008 reported in cv Agnihothri a maximum of 9.7 shoots by indirect organogenesis. Gantait et al., 2012 reported that in cv CanCan it is possible to regenerate 17.2 shoots per protocorm like bodies (PLBs). In another study, Gu et al.,
3.2. Evaluation of the immersion frequency Regarding the evaluation of immersion frequencies, no significant differences were found in the morphological variables evaluated (Table 1). Shoot production in the immersion frequencies every 4, 8 and 12 h were 31.50 ± 0.50, 31.62 ± 0.67 and 33.12 ± 0.97, respectively. Chlorophyll content also showed no significant differences (Fig. 2b). However, these results allow us to recommend the frequency of 2 min every 12 h for the micropropagation of A. andreanum due to the lower use of electrical energy. 3.3. Evaluation of culture medium volume per explant When evaluating the initial culture medium volume per explant, the 187
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Fig. 1. Effect of culture system and immersion frequency in TIS on in vitro multiplication of A. andreanum. a) semisolid medium, b) liquid medium in partial immersion, c) temporary immersion for 2 min every 4 h, d) temporary immersion for 2 min every 8 h, and e) temporary immersion for 2 min every 12 h. Bar = 1 cm.
Fig. 3. Effect of culture system and immersion frequency in TIS on stomatal index and percentage of closed stomata during the multiplication stage of A. andreanum cv. Rosa after 45 days of in vitro culture. Bars represent the mean ± standard error. Bars followed by a different letter denote significant statistical differences (Tukey, P ≤ 0.05). TI: Temporary immersion.
production with respect to the indirect organogenesis micropropagation system. Similar to the results obtained in this study, the use of TIS has recently been reported in Stevia rebaudiana (Ramírez-Mosqueda and Iglesias-Andreu, 2016; Vives et al., 2017), Quercus robur (Gatti et al., 2017), Gerbera jamesonii (Mosqueda et al., 2017) and Dianthus caryophyllus (Ahmadian et al., 2017). Roels et al., 2005 reported that in Musa AAB the use of a Temporary Immersion Bioreactor (TIB) increased the multiplication rate with respect to the semisolid medium. The same results were reported by Farahani and Majd (2012) and Vives et al. (2017) in Musa spp and Stevia rebaudiana, respectively. In ornamental plants, Hahn and Paek (2005) reported in Dendranthema grandiflorum that the use of TI resulted in greater fresh weight, dry weight, shoot length and leaf area compared to semisolid culture. In the same way, Daquinta et al. (2007) reported that the multiplication rate of Caladium × hortulanum was more than 12 times higher than under a semisolid culture. Similar results were reported by Ahmadian et al. (2017), who showed that TI
Fig. 2. Chlorophyll contents during the multiplication stage of A. andreanum cv. Rosa after 45 days of in vitro culture. Effect of a) culture system and b) volume of medium per explant. Bars represent the mean ± standard error. Bars followed by a different letter denote significant statistical differences (Tukey, P ≤ 0.05). IT: Temporary immersion.
2012 obtained 24.9 and 24.7 shoots through indirect organogenesis in cvs Alabama and Sierra, respectively. More recently, Bhattacharya et al., 2015 regenerated up to 25.6 shoots per explant in cv Tinora. To date, our micropropagation system in TIS, compared to the previously mentioned reports, suggests the possibility of doubling or tripling the production of shoots per explant compared to direct organogenesis systems from shoots in semi-solid medium and even improving 188
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4.2. Evaluation of the immersion frequency Immersion frequency is an important aspect when using TIS since it plays a vital role in nutrient intake, growth regulators and hyperhydricity control (Etienne and Berthouly, 2002; Watt, 2012). It has been shown that immersion frequency varies among species. Mordocco et al. (2009) reported a higher percentage of regeneration in explants of three cultivars of Saccharum spp. (cv: Q117, Q165 and Q205) when using an immersion frequency of 1 min every 24 h. Akdemir et al. (2014) decreased hyperhydration in Pistacia vera by prolonging the immersion frequencies, from 16 min every 8 h to 16 min every 24 h. This study is the first reported evaluation of different immersion frequencies to optimize the production of shoots in A. andreanum. Although no significant differences were found in the variables evaluated, our results allow us to recommend an immersion frequency of every 12 h due to the fact that less electrical energy is required per day for the operation of the compressor and solenoid valve that make up the immersion system. Similar to our results, Mosqueda et al. (2017) found no significant differences in the multiplication rate of Gerbera jamesonii among the immersion frequencies; however, they reported that the frequencies of 4 min every 8 and 12 h produced longer shoots with fewer hyperhydration symptoms compared to the frequency of 4 min every 6 h.
Fig. 4. Effect of culture system on survival percentage of A. andreanum cv. Rosa after 30 days of acclimatization under greenhouse conditions. Bars followed by a different letter denote significant statistical differences (Tukey, p ≤ 0.05).
produces more than 10 times the shoot production in Dianthus caryophyllus in comparison to semisolid culture. One of the main advantages of using TI is that it promotes ventilation within the culture vessel, allowing the removal of volatile compounds such as ethylene (Roels et al., 2006), recirculating the carbon dioxide necessary for photosynthesis (Aragón et al., 2014) and increasing stomatal functionality in the leaves compared to those obtained in semisolid environments (Afreen, 2008). These factors, together with the loss of apical dominance, could explain the increase in the multiplication rate of A. andreanum. In this sense, our results showed that total chlorophyll content is higher in culture systems in liquid medium than in semisolid medium. Similar results were reported by Arencibia et al. (2013) in the micropropagation of Rubus spp. Probably the increase in the content of chlorophylls in liquid medium is due to a greater availability of nutrients and other compounds, which can be taken practically throughout the explant through leaves and tissues while, in semisolid medium, the explants obtain the components of the culture medium only from the area in contact with the medium.
4.3. Evaluation of culture medium volume per explant The initial medium volume per explant should be a variable to optimize in TI. Although it is a factor that is usually omitted, several authors have shown its importance. Lorenzo et al. (1998) showed that 50 mL of medium per explant resulted in a higher multiplication rate than lower or higher amounts of medium (27.5 or 72.5 mL per explant) during the micropropagation of Saccharum spp. Ramos-Castellá et al. (2014) reported an increase in shoot production by optimizing the medium volume at 25 mL per explant in Vanilla planifolia. According to Etienne and Berthouly (2002), large volumes of medium per explant are usually inefficient because the in vitro cultures produce extracellular chemicals that stimulate shoot formation, which would be diluted when large volumes of medium are used. In addition to this, the initial medium volume determines the quantity of explants inside a bioreactor that, once developed, will compete for the space, light and nutrients of the culture medium. In this sense, the greater the number of explants per bioreactor, the greater the chances the explants could lack the optimal conditions for their development. The results of this study suggest that 25 mL of medium per explant is sufficient to maintain a high multiplication rate in A. andreanum.
Fig. 5. Acclimatized plantlets of A. andreanum cv. Rosa obtained from Ebb-And-Flow Bioreactor at a) 30 days and b) 90 days of ex vitro culture under greenhouse conditions. Bar = 10 cm. 189
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4.4. Stomatal index, stomatal aperture and acclimatization
References
According to Zobayed (2005), by applying ventilation, some anatomical and physiological changes that are common in conventional micropropagation can be prevented, including the production of malfunctioning stomata, which remain permanently widely open. Closed stomata and a low stomatal index generate a low transpiration rate, avoiding the loss of water and dehydration, one of the most frequent causes of plant death, during acclimatization (Aragón et al., 2014; Vieira et al., 2015). The results obtained in this study show that the TIS favors a low stomatal index and a high percentage of closed stomata compared to the system in semisolid medium, suggesting the functionality of the stomata, thereby promoting a lower transpiration rate and probably a higher photosynthetic rate. It also demonstrated a greater accumulation of photosynthetic pigments. The success of a micropropagation protocol not only depends on the number of shoots obtained by the explants but also depends on the morphological quality and vigor present in the plantlets produced (Martre et al., 2001). Therefore, acclimatization is an important stage that determines the success of micropropagation. Reports similar to this study have demonstrated that the TIS environment prepares the plantlets for the stress of acclimatization (Yang and Yeh, 2008; Aragón et al., 2010; Regueira et al., 2018). Escalona et al. (2003) reported that TI bioreactor-derived Ananas comosus plantlets showed remarkable nutrient uptake, indicating a higher photo-mixotrophic metabolism. Ahmadian et al. (2017) reported that TIS have a suitable effect on enhancing the elongation and root induction of Dianthus caryophyllus plantlets during acclimatization in comparison with plants from solid culture. Recently, Zhang et al. (2018) reported that Bletilla striata plants obtained using TIS showed uniform growth, enhanced leaf and stem development and substantially improved pseudobulbs. There are great differences in the physiology of the plants between the semisolid medium culture and TIS. The results show that among the factors that can contribute to improve the acclimatization of the plants obtained in TIS in comparison with those obtained in semisolid medium are a greater accumulation of photosynthetic compounds, a lower stomatal index and a higher percentage of closed stomata. In this regard, Yang and Yeh (2008) reported that, during ex vitro acclimatization, Calathea orbifolia plants produced in TIS had higher photosynthetic rates and subsequently a greater leaf area and fresh and dry weights compared to those obtained in semisolid medium. Likewise, Aragón et al. (2014) showed that TIS-derived plantlets exhibit better growth and stomatal function regulation and can therefore have better water loss control and even accumulate more starch in the leaves, which could be used during the first days of ex vitro acclimatization. Finally, the evaluation of the immersion frequencies and the medium volume per explant showed no significant differences in shoot production; however, they are important because they allow obtaining a high multiplication rate without decreasing quality, reducing production costs with respect to the traditional system in semisolid medium. Therefore, the use of TIS with an immersion frequency of 2 min every 12 h with an initial volume of 25 mL of culture medium per explant is recommended. In conclusion, it is herein reported for the first time that the TI technique is an alternative for the commercial micropropagation of A. andreanum. In addition to increasing the multiplication rate, these systems improve the quality of the shoots without a previous rooting stage and promote an increased survival rate during acclimatization.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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