Journal of Applied Botany and Food Quality 92, 1 - 6 (2019), DOI:10.5073/JABFQ.2019.092.001 1 Laboratory for Plant Breeding, Bioplant Center, University of Ciego de Avila, Ciego de Ávila, Cuba 2 Laboratorio de CarboCat, Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Concepción, Región del Bio Bio, Chile 3 Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAMMFL), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador 4 School of Life Sciences, University of Kwazulu-Natal, Durban, South Africa Mutagenic effects of sodium azide on pineapple micropropagant growth and biochemical profile within temporary immersion bioreactors Daviel Gómez1, 2, Lázaro Hernández1, Julia Martínez1, Janet Quiñones1, Byron E. Zevallos3, Sershen4, Lourdes Yabor1, José Carlos Lorenzo1 (Submitted: October 4, 2018; Accepted: November 22, 2018) Summary Sodium azide (NaN3) is widely used to induce mutagenesis within in vitro plant systems. However, since this mutagenesis is un- directed, its unintended effects demand characterization. This study investigated the mutagenic effects of sodium azide (0 - 0.45 mM) on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants within tempo- rary immersion bioreactors (TIBs). The content of soluble phenolics in the culture medium was also evaluated. Irrespective of the con- centration NaN3 decreased shoot multiplication rate (by 87% rela- tive to the control at 0.45 mM) and fresh weight (by 66% relative to the control at 0.45 mM). Levels of chlorophyll a and b, and soluble phenolics in the culture medium were also negatively correlated with NaN3 concentration. Interestingly, NaN3 application increased shoot carotenoid and soluble phenolic levels but had no significant effect on a range of established plant stress biomarkers: cell wall-linked phenolic levels, malondialdehyde and other aldehydes. Given that 0.19 mM NaN3 decreased shoot multiplication rate by 50% and re- sulted in propagants that displayed no morphologically abnormali- ties, increased levels of photoprotective pigments (relative to the control) and no significant increase in lipid peroxidation products, the mutagen can be used at this concentration to induce pineapple mutagenesis in TIB based studies aimed at producing agriculturally- useful mutants. Keywords: In vitro stress; in vitro mutagenesis; plant metabolites; Ananas comosus (L.) Merr.; plant breeding Abbreviations: Sodium azide (NaN3); temporary immersion bio- reactor (TIB). Introduction Plant breeders have generated new varieties using chemical muta- gens for many decades (Dubey et al., 2017). Sodium azide (NaN3) is widely regarded as a relatively safe to handle and very efficient chemical mutagen that is both inexpensive and non-carcinogenic (Salvi et al., 2014). This common laboratory chemical is used widely in organic synthesis, agricultural and medical research (most often as bactericide, pesticide and nitrogen gas generator) but it is most famous for its mutagenic effects in plants and animals (MenDionDo et al., 2016). This mutagenic capacity is based on NaN3’s produc- tion of an organic metabolite, β-azidoalanine, which is valued for its ability to induce chromosomal aberrations at a rate much lower than other mutagens (Dubey et al., 2017). The concentrations of NaN3 that are used for the treatment of plant tissues vary widely across species: e.g. 3 - 10 mM (CaStillo et al., 2001) or 0.5 - 4.0 mM in Hordeum vulgare (Szarejko et al., 2017); 1.0 - 3.0 mM in Pisum sativum (Divanli-türkan et al., 2006); 0.04 - 1.1 mM in Phaseolus vulgaris (Chen et al., 2011); 15.5 - 77.6 mM in Trigonella foenum-graecum (SiDDiquia et al., 2007); and 31.1- 124.3 mM in Brassica napus (huSSain et al., 2017). However, we have not found any report on the use of NaN3 during in vitro production of axillary buds. Despite the wide range of concentra- tions at which NaN3 is used, the mutagen generally acts by crea- ting point mutations in the genome, disturbing metabolic activity, growth and development, and inhibiting protein and DNA replica- tion (ragunathan and PanneerSelvaM, 2007). These effects, the severity of which are concentration dependent (vainStein, 2002), change the balance between growth promoters and their antago- nists (gruSzka et al., 2012). Nevertheless, a number of traits have been improved in a variety of species using NaN3, including days to germination, flowering and silique maturation in Brassica napus (huSSain et al., 2017); related to quality, yield and disease resistance in wheat (Dubey et al., 2017); germination, seedling survival, root length and height, height at maturity, number of leaves, and fruit yield in tomato (aDaMu and aliyu, 2007); and groundnut yield (MenSah and obaDoni, 2007). The mutagen has also been applied to a varie- ty of explant types: e.g. cultures of Pisum sativum seeds (Divanli- türkan et al., 2006), tomato seeds (el kaaby et al., 2015), Brassi- ca napus cotyledons (he et al., 2011), and Hordeum vulgare anthers and microspores (CaStillo et al., 2001). Although the TIB tech- nique is known to increase multiplication rates in many crop species (jiMénez et al., 1999), the use of this culture technique for the NaN3 application has not been reported to date. The increased multiplication rates in TIBs might be caused by the combined advantages of using a solid and liquid culture medium. Micropropagation on solid culture medium allows plant air exchange but nutrient uptake is limited to the explant basal surface (eSCalona et al., 1999). On the other hand, whilst micropropagation in liquid culture medium increases nutrient uptake, it often leads to hyper- hydricity (ziv, 1995). Hyperhydricity is characterized by various degrees of morphological and physiological disorders which can in- clude a glassy, waterlogged-tissue appearance and disordered shoot growth (more specifically in the leaves). Unlike the classic liquid culture procedure (alvarD et al., 1993), in a TIB explants are co- vered with the culture medium only for a few minutes and immer- sion allows nutrient uptake through the entire explant surface. The plant air exchange is restored after removal of the culture medium. Irrespective of the in vitro culture method, explant type or species, the mutations underlying the changes induced by chemical mutagens like NaN3 are undirected and as alluded to above can have nega- tive effects on plant growth and performance. This necessitates the characterization of the unintended effects of NaN3 exposure and identification of the optimum concentration for application within specific species. 2 D. Gómez, L. Hernández, J. Martínez, J. Quiñones, B.E. Zevallos, Sershen, L. Yabor, J.C. Lorenzo Pineapple is the main commercial species of the Bromeliaceae glo- bally (Martín et al., 2015). Fruit production in pineapple reached 25.4 million tons in 2013 (FaoStat, 2016) but to maintain and/ or improve the quality of pineapple production in many parts of the world, researchers are now developing new varieties (yabor et al., 2016). Given that pineapple breeding through conventional tech- niques is extremely costly (loiSon-Cabot and laCoeuilhe, 1989), biotechnological approaches (e.g. induced mutagenesis) provide great potential for improving selected clones more affordably. In vitro-induced mutagenesis has been employed to produce im- proved genetic variants in numerous crop species, including pine- apple (ibrahiM et al., 2009). Research on the efficacy of chemical mutagens, specifically NaN3, in inducing mutagenesis in pineapple is limited and hence, forms the focus of the present research. The study investigated the mutagenic effects of NaN3, applied at a range of concentrations (0 - 0.45 mM), on selected growth (shoot multiplica- tion rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants TIBs. Soluble phenolic content in the culture me- dium was also assessed. The selection of TIBs for the study is based on the high multiplication rate of pineapple (1:66) using this tech- nique, compared with conventional micropropagation containers (1:8) (eSCalona et al., 1999). Materials and methods Plant material, in vitro methods and growth measurements Feld-grown pineapple plants (cv. MD2) served as the source of ex- plants for initiating in vitro cultured buds (Daquinta and benegaS, 1997). These axillary buds, excised after removal of the crown leaves, were decontaminated (with 1% (w:v) Ca(ClO)2 for 10 min) followed by rinsing with tap water. The buds were excised with a por- tion of basal tissue and established in 300 ml glass containers with 5 ml of liquid culture medium per explant. MS salts (MuraShige and Skoog, 1962), 100 mg l-1 myo-inositol, 0.1 mg l-1 thiamine-HCl, 30 g l-1 sucrose, 4.4 μM 6-benzyladenine (BA), and 5.3 μM naph- thaleneacetic acid (NAA) were included in the medium. At 45 d of culture, shoots were transferred to the multiplication medium (original medium supplemented with 9.3 μM BA and 1.6 μM NAA). The shoots were subcultured and multiplied for 6 months, at 45-d intervals, and then placed in TIBs with 3.0 μM paclobutrazol (after eSCalona et al. (1999)). Immersions (2 min in duration) occurred every 3 h for 30 d. Free shoots were located in the bottom of glass vessels (300 mL volume) filled with 200 mL of liquid medium with 5 explants within each of three containers per treatment (40 mL medium / explant). At the beginning of the 30-d-long subculture, different levels of NaN3 (0, 0.15, 0.30 and 0.45 mM) were supplemented in the culture medium. Culture conditions were as follows: 25±1 oC and 80 μmol m-2 s-1 cool fluorescent light for an 8 h photoperiod. Shoot multiplica- tion rate, shoot cluster fresh weight and all biochemical parameters described below were measured after 30 d of culture. Biochemical parameters Plant tissues (100 mg per replicate) were sampled from each of three bioreactors. Phenolics were quantified according to gurr et al. (1992), chlorophylls following Porra (2002) and malondialdehyde and other aldehydes after heath and PaCker (1968). For chlorophyll pigments a and b, the tissue was extracted in 5.0 ml acetone (80%, v:v), centrifuged (14086.8 g, 4 °C, 15 min) and the subsequent super- natants read for absorbance at 646.6 and 663.6 nm using a spectro- photometer (RAYLEIGH, VIS-723G). Similarly, carotenoids levels were measured by reading the absorbance of acetone (80%, v:v) ex- tracts at 470 nm (liChtenthaler, 1987). Phenolic compounds were extracted and quantified (mg gallic acid equivalents per g fresh weight) using a colorimetric assay which involves the reaction of phenols with Folin Ciocalteu reagent (gurr et al., 1992). The reaction of malondialdehyde and other aldehydes with thiobarbituric acid formed the basis of the colorimetric method used to quantify the products of lipid peroxidation (molar extinc- tion coefficient: 1.57 . 105M-1 cm-1) (albro et al., 1986; heath and PaCker, 1968). Phenolic exudation was quantified using a modifica- tion of the hoaglanD (1990) procedure. This involved mixing the culture medium (0.5 ml; one sample per TIB) with 4.5 ml distilled water, after which 0.5 ml Folin Ciocalteau reagent (50% v/v) was added. This mixture was shaken and then left to stand for 5 min before saturated sodium carbonate (1 mL) was added. The mixture was then shaken again, left to stand for 60 min, and then read for absorbance at 725 nm. Phenolic concentration was calculated based on a gallic acid standard curve. Statistical analysis All statistical analyses were performed using SPSS (Version 8.0 for Windows, SPSS Inc., New York, NY). Where data was parametric, tested for normality using a Kolmogorov-Smirnov test, means were compared using a One-Way analysis of variance (ANOVA) in combi- nation with a Tukey post-hoc test (p≤0.05). Additionally, the overall coefficients of variation (OCV) were calculated as follows: (standard deviation/average) * 100. In this formula, the average values of the four NaN3 levels were compared (treatments) to calculate the stan- dard deviation and average. The higher the difference between the four treatments compared, the higher the OCV (lorenzo et al., 2015). OCVs from 2.94 to 30.00% were classified as Low, from 30.00 to 57.06% as Medium and from 57.06 to 84.12% as High. Results Exposure to NaN3 decreased pineapple shoot multiplication rate and fresh weight in a concentration-dependent manner (Fig. 1A, 1B, 1C). At the maximum concentration (0.45 mM) shoot multiplication rate was 12.65% of that obtained in the control, while fresh weight was reduced by 66.42% relative to the control. Despite this reduction in growth and multiplication rate, shoot production was observed across all NaN3 treatment concentrations and the shoots displayed no morphological abnormalities when compared to the control (Fig. 1A). It should also be noted that the inhibitory effects of in- creasing NaN3 concentration were more severe on shoot multiplica- tion rate than cluster fresh weight. In terms of plant pigment contents, NaN3 decreased chlorophyll a (Fig. 2A) levels at the maximum concentration and chlorophyll b (Fig. 2B) at the two highest concentrations, relative to the control. In contrast, NaN3 increased carotenoid levels, compared with the con- trol, at all concentrations tested (Fig. 2C). OCV values were, how- ever, Low (13.65-24.13%) across all three plant pigments and the shoots produced on NaN3 supplemented media showed no signs of chlorosis (Fig. 1A) throughout the growth period. While tissue soluble phenolics (Fig. 2D) increased relative to the control, soluble phenolics levels in the culture medium (Fig. 2F) decreased within increasing NaN3 concentrations, but OCVs were again Low (18.15 and 28.22%, respectively). Interestingly, NaN3 had no significant effects on the levels of cell wall-linked phenolics (Fig. 2E), malondialdehyde (Fig. 2G) or other aldehydes (Fig. 2H). Discussion In light of a predicted reduction in agricultural productivity in many parts of the world due to climate change, induced mutation techno- logy for crop improvement has become an increasing important area of research over the last few decades (Dubey et al., 2017). In this Effects of sodium azide on pineapple 3 regard, chemical mutagens are now useful tools in crop improvement and have been used to produce abiotic stress tolerance and disease resistance in various susceptible crops, improving their yield and quality traits (olawuyi and okoli, 2017). There are several muta- gens available for crop improvement and like NaN3, the mutagen in- vestigated here, each has its own positive or negative effect on plants (heSlot, 1977). Mutagenesis has been employed to introduce many useful traits, including plant size, fruit ripening and resistance to pathogens in a wide range of fruit crops (reviewed by laMo et al. (2017)). How- ever, studies on the effects of chemical mutagens on pineapple are scarce (Paull et al., 2017), while no published reports on the ef- fects of NaN3 on the in vitro growth and biochemistry of pineapple explants were available at the time of this study. In the present study NaN3 decreased shoot multiplication rate and cluster fresh weight (Fig. 1B, 1C). NaN3 has been reported to have similar inhibitory ef- fects on in vitro explant growth in other species (Dubey et al., 2017; huSSain et al., 2017; olawuyi and okoli, 2017; Szarejko et al., 2017). However, at 0.19 mM NaN3, the multiplication rate (1:19) in the TIBs used was lower than the control but still higher to that achieved for pineapple grown in the absence of NaN3 using conventional cul- ture methods (1:8) (eSCalona et al., 1999). Furthermore the pro- pagants generated displayed no morphological abnormalities, when compared to the control (Fig. 1A). Sodium azide did, however, induce significant changes in a number of biochemical parameters relative to the control. Most plants grown in vitro have a different metabolism than in vivo. In the former, they are provided with a carbon source, growth regulators, high humidity, and less carbon dioxide and light, which promote proliferation but also change the autotrophic metabolism to heterotrophic or mixotrophic (ChanDra et al., 2010). Once transferred to the ex vitro environment, micropropagated plantlets must then adapt their metabolism to the new conditions, which can represent a significant stress (teixeira et al., 2017). The biochemical profile of micropropagated plantlets can therefore influence, either negatively or positively, their survival and performance during acclimatization and subsequent ex vitro growth (hazarika et al., 2006). In the present study while in vitro exposure to NaN3 decreased chlo- rophyll levels (a and b) in pineapple shoots, it increased carotenoid levels. Carotenoids are important components of the antioxidant sys- tem in photosynthetic organisms and their absence can increase the extent of photoinhibition (Cabrera, 2002). Both the decreased chlo- rophyll and increased carotenoid levels suggest that NaN3 exposure may reflect a NaN3-stress-related photosynthetic down-regulation Fig. 1: Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/ Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Maximum multiplication rate = 32.93 (0 mM NaN3) Minimum multiplication rate = 4.17 (0.45 mM NaN3) 50%-reduction in multiplication rate = Min. + ((Max. – Min.)/2) = 4.17 + ((32.93 – 4.17)/2) = 18.55 (~0.19 mM) Fig. 1: 4 D. Gómez, L. Hernández, J. Martínez, J. Quiñones, B.E. Zevallos, Sershen, L. Yabor, J.C. Lorenzo capacity and enhanced photoprotection. Increased carotenoid-based photoprotection is a commonly reported stress response (PoMPelli et al., 2010). This may have in turn decreased the potential for photo- oxidation, which can arise as a consequence of stress induced dam- age to the photosynthetic machinery. This suggestion is supported by the fact that NaN3 exposure did not result an increase in malon- dialdehyde and other aldehyde levels (Fig. 2G, 2H) which are com- mon products of cellular damage caused by (photo) oxidative stress (DuMet and benSon, 2000). Under normal growth conditions, basal levels of aldehydes remain low in plant tissues but with exposure to abiotic stress they can accumulate to much higher levels (SerShen et al., 2016). Stress-induced aldehydes can act as generic signal mo- lecules for plants under adverse environmental conditions, inhibi- ting developmental and metabolic processes (MoStoFa et al., 2015). yaDav et al. (2005) for example, showed that the accumulation of aldehydes can inhibit shoot growth. Phenolic compounds are some of the most common products of secondary metabolism in plants and some are essential for plant sur- vival, given their involvement in defense mechanisms under stress situations (SharMa et al., 2012). The regulation of the biosynthesis of phenolics is generally brought about by biotic and abiotic stimuli (bettaieb et al., 2011) and generally accumulate in plant tissues during a stress (joSePh et al., 2015). In the present study, though NaN3 exposure induced a reduction in growth, an unambiguous in- dication of stress, it did not increase phenolic levels in shoot tissues (Fig. 2D, 2E) and or the culture medium (Fig. 2F). This is encourag- ing, since cell wall-linked or insoluble phenolics, in particular, can make cell walls more rigid and less porous inhibiting nutrient uptake and growth (kabera et al., 2014). Their accumulation in in vitro cultures is therefore not beneficial for growth (MaChakova et al., 2008). The results suggest that though NaN3 decreased pineapple shoot multiplication rate within TIBs, propagants displayed no morpho- logically abnormalities, and when produced using 0.19 mM NaN3 exhibited enhanced levels photoprotective pigments and no obvious signs of enhanced lipid peroxidation. The mutagen can therefore be used at this concentration to induce pineapple mutagenesis in TIB based studies aimed at producing agriculturally-useful mutants. Fig. 2: Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/ Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Fig. 2: Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Fig. 2: Effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. Values represent means ± SE (n=3) and are significantly different when labelled with different letters (ANOVA, Tukey, p≤0.05). Overall coefficient of variation (OCV) = (Standard deviation/Average)*100. To calculate this coefficient, the four average values were considered. The higher the difference among results, the higher is the overall coefficient of variation: Low = 2.94 to 30.00%, Medium = 30.00 to 57.06% and High = 57.06 to 84.12%. Fig. 2: Effects of sodium azide on pineapple 5 Author contribution DG, LH, JM, JQ, BEZ, S, LY and JCL designed the research; DG, LH and JM conducted the experiments; DG, LH, JM, JQ, BEZ, S, LY and JCL analyzed the data and wrote the paper; and JCL had primary re- sponsibility for the final content. All authors have read and approved the final manuscript. Acknowledgements This research was supported by the Bioplant Centre (University of Ciego de Ávila, Cuba), the Escuela Superior Politécnica Agropecu- aria de Manabí Manuel Félix López (Ecuador), and the University of Kwazulu-Nathal (South Africa). Compliance with ethical standards Conflict of interest Authors do not have any conflict of interests. Human and animal rights This research did not involve experi- ments with human or animal participants. Informed consent: Informed consent was obtained from all individ- ual participants included in the study. 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Automation and environmental control in plant tissue culture. Springer. Address of the authors: Daviel Gómez, Lázaro Hernández, Julia Martínez, Janet Quiñones, Lourdes Yabor, José Carlos Lorenzo, Laboratory for Plant Breeding, Bioplant Center, University of Ciego de Avila, Ciego de Ávila, 69450, Cuba E-mail: jclorenzo@bioplantas.cu Daviel Gómez, Laboratorio de CarboCat, Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Concepción, Región del Bio Bio, Chile Byron E. Zevallos, Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAMMFL), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador Sershen, School of Life Sciences, University of Kwazulu-Natal, Durban, 4001, South Africa © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en).