Development, chlorophyll content, and nutrient accumulation in shoots of Melaleuca alternifolia in vitro under different light wavelengths and benzylaminopurine (BAP)

Development, chlorophyll content, and nutrient accumulation in shoots of Melaleuca alternifolia in vitro under different light wavelengths and benzylaminopurine (BAP) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development, chlorophyll content, and nutrient accumulation in shoots of Melaleuca alternifolia in vitro under different light wavelengths and benzylaminopurine (BAP) Jean Carlos Cardoso, Antony Cristhian Gonzales-Alvarado This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4249082/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In vitro growing of Melaleuca can contribute to the cloning of superior genotypes. Studies on factors affecting micropropagation are necessary, especially those using recent technologies, such as light-emitting diodes (LEDs). This study aimed at better understanding the effects of wavelengths on the development and physiology of Melaleuca cultivated in vitro, as well as the interaction of LED with N6-Benzylaminopurine (BAP), the main cytokinin used in micropropagation. The BAP and wavelengths, and their interaction, had a strong influence on most variables analyzed, altering the in vitro development and chlorophyll concentrations in the plants and changing the different variables of the culture medium, such as pH, EC, levels of Ca2+, Mg2+ and P in culture media and the nutrient accumulation in the shoots. The results demonstrated that the main effects of adding BAP in the in vitro cultivation of Melaleuca are the increase in the number of shoots, which resulted in a greater increase in fresh and dry mass, reduction in height and chlorophyll contents, complete inhibition of adventitious rooting, higher consumption of Mg and lower consumption of Ca and P from the culture media, higher contents of Fe and lower contents of P, S, Mn, Cu and B in the in vitro tissues. micropropagation tea tree light emitting diodes cytokinin development nutrition chlorophyll Figures Figure 1 Figure 2 Figure 3 Key Message This study evidenced the important interaction of lighting and cytokinin BAP in the culture media, which affects the in vitro development, the metabolism of nutrients, and the chlorophyll contents. Introduction The genus Melaleuca belongs to the family Myrtaceae with around 150 species (Martinho and Rodrigues, 2009) and is used for the production of essential oils on an industrial scale, particularly the species Melaleuca alternifolia Cheel. This species has different properties, such as anti-inflammatory (Caldefie-Chezet et al. 2006; Lahkar et al. 2013), antimicrobial (Carson et al. 2006; Lahkar et al. 2013; Da Silva et al. 2002), fungicide (Lahkar et al. 2013; Tong et al. 1992), and acaricide (Lori et al. 2005), and used in the treatment of insect bites and skin infections (Budhiraja et al. 1999; Lahkar et al. 2013). Melaleuca seedlings result in high heterogeneity and stem cutting shows difficulties such as low rooting percentage (Chen et al. 2016; Iiyama and Cardoso, 2021). Therefore, there is great interest in developing micropropagation protocols to evaluate the feasibility of this technique from the commercial point of view and the performance of plantlets from the micropropagation system. Despite the limited number of studies with Melaleuca micropropagation, they have revealed the substantial potential of using micropropagation as a clonal system (de Oliveira, 2010; Iiyama and Cardoso, 2021). In these studies, the use of the cytokinin N6-Benzylaminopurine (BAP) has proven essential in achieving high rates of multiplication and proliferation of shoots under in vitro conditions (List et al. 1995; de Oliveira, 2010; Iiyama and Cardoso, 2021) by promoting the proliferation of axillary and adventitious buds in tissues cultured in vitro (da Silva et al. 2022). Advances in in vitro conditions, are achieved by using light-emitting diodes (LEDs), which replaced cold or fluorescent lamps, increasing energy efficiency and expanding the types and combinations of LED spectra according to each growing system or plant species cultivated (Sena et al. 2024). Also, the use of LED systems emerges as an extremely promising strategy to modulate the physiological response of plants (Rabara et al., 2017). This approach allows for the generation of a specific wavelength source, which has been shown to induce particular responses in plants, notably an increase in photosynthetic rate and significantly enhanced plant growth (Kozai, 2016). Currently, there are many studies demonstrating the effects of LEDs with different wavelengths (colors) on the in vitro development of Vitis riparia (Poudel et al. 2008), cotton plant (Li and Tang, 2010), Cedrela fissilis (Tadeu, 2017), Helianthus tuberosus (Seedapalee et al. 2021), Ficus carica var. Black Jack (Parab et al. 2021), Pyrus communis (Lupo et al. 2022), Pinus pseudostrobus (Marín-Martinez and Iglesias-Andreu, 2022). Gupta and Jatothu (2013) reported the best development of crops without the induction of damage or photo stress. Despite this, the studies on the in vitro micropropagation of Melaleuca used conventional cold lamps or LED at specific wavelengths (Oliveira, 2010; Amelia et al. 2020; Iiyama and Cardoso, 2021). Therefore, there are still no studies demonstrating the effects of different LED wavelengths on the in vitro development of Melaleuca . In addition, there is little known on the interaction of the different light wavelengths with phytoregulators, such as BAP, as this cytokinin is widely used for micropropagation of several commercial and non-commercial species under in vitro conditions. Finally, few studies have been dedicated to better understanding the chemical changes in culture media in response to the development and cultivation of plants in vitro. This information can help to improve the plant performance under in vitro conditions and to determine key factors that improve the performance of culture media. Thus, the main objectives of this study were to better understand the interaction of cytokinin BAP and different LED wavelengths on the in vitro development of Melaleuca shoots and plantlets, as well as the main changes in culture media related to nutritional characteristics. At the same time, this study also aimed at identifying the best wavelengths associated with the proliferation/multiplication of Melaleuca under in vitro conditions. Materials And Methods Plant material and in vitro growing conditions The plant material used for the experiments consisted of in vitro stem nodal segments (shoots) 1.0 - 1.5 in length, previously established in vitro from the stem apices taken from greenhouse mother plants. These explants were established and propagated in vitrofor approximately nine months to obtain enough stem nodal segments for the experimental design. These nodal segments were grown in 15 mL of Murashige & Skoog (MS) culture medium (1962) containing half the macronutrient concentration (MS ½), 6.5 g L -1 of agar (Agargel, João Pessoa-PB, Brazil), 2% sucrose, 0.1 g L -1 inositol, and pH adjusted to 5.8 (Iiyama and Cardoso, 2021); with or without the presence of BAP (at 0.25 mg L -1 ). The BAP used was added before pH adjustment and was provided from a concentrate solution containing 10 mg BAP (Sigma-Aldrich), previously dissolved in 1 mL NaOH (1M) and 9 mL of distilled water. Test tubes containing one nodal segment each (replicates) were kept in a growth room with a photoperiod set to 14 hours, temperature of 25°C ± 2°C, illuminated with six different wavelengths provided by LED lamps. The photosynthetic photon flux density (PPFD) was measured for each wavelength tested using the Quantum SQ-520 meter (Apogee Instruments, USA). The LED lamps used as treatments were 100% white diffuser module (W: 64.09 μmol/m 2 s -1 ), 2) dark conditions (D: 0.58 μmol/m 2 s – diffuse light), 3) 100% blue LED light (B: 55.58 μmol/m 2 s), 4) 100% red LED light (R: 106.22 μmol/m 2 s), 5) 50%: 50% red and white LED light (RW: 104.51 μmol/m 2 s), and 6) 40%: 60% blue and red LED light (BR: 71.48 μmol/m 2 s). In vitro characteristics of Melaleuca grown under LED spectra and BAP Melaleuca development was analyzed weekly for 30 days of in vitro cultivation. The variables evaluated were the number and length of shoots (cm), shoot height (cm), total fresh and dry masses (g), and shoot mass (g). For the fresh and dry mass determination, plantlets were separated into shoots and roots (when present) or only shoots (no roots) and weighed on a digital analytical scale accurate to 0.1 mg (ML201, Mettler, Switzerland). The dry mass of each part was obtained by drying in an oven at a temperature of 70 °C for 72 h, followed by weighing on the same scale. Chlorophyll a, b, and total chlorophyll contents were also analyzed by spectrophotometry. Chlorophyll a, b, and total chlorophyll concentrations were obtained by macerating 100 mg of the in vitro Melaleuca leaves in a crucible containing 2-3 mL of pure acetone (99.5%) and adjusted to a final volume of 10 mL for spectrophotometer reading. Samples were poured into the manufacturer’s glass vial to measure their absorbance in the IRIS-HI801 spectrophotometer (Hanna Instruments). Absorbance readings were taken at 664 nm and 647 nm according to the equations by Lichtentaler (1987) and against a blank of pure acetone. Chlorophyll concentrations were determined in mg/L using the following equations: chlorophyll a=12.70 (A 664 ) - 2.97 (A 647 ); chlorophyll b=20.70 (A 647 ) - 4.62 (A 664 ), as proposed by Lichtenthaler et al. (1983). Total chlorophyll was obtained by summing up the values of chlorophyll a and b. Stomatal size and density were evaluated in in vitro leaves of Melaleuca . For counting and measuring the polar and equatorial diameters of the stomata, we used leaves from the fourth to the sixth node of the in vitroplantlets (Iiyama and Cardoso, 2021). Leaves were first fixed in Carnoy solution composed of three volumes of ethanol and one of glacial acetic acid (v/v) for 48h. Subsequently, samples were stored in 70% ethanol solution at 8°C until slide mounting. For the visualization of stomata, the leaves were previously immersed in 70% alcohol for one minute and soon after in potassium hydroxide solution 5 M at 45°C for 20-30 s. The digestion reaction was stopped by adding distilled water, followed by mounting the slide with a coverslip and analysis under a light microscope (Nikon Eclipse 201) at a total magnification of 100×, coupled to a high-resolution camera (5 Mp Opticam) and software for stomatal measurement. The variables evaluated were the stomatal density and diameter (µm) in 10 observed fields per leaf. Chemical characteristics of culture media used for Melaleuca in vitro cultivation The purpose was to observe the differences between the initial and final values of different characteristics of the culture medium, such as pH, Electrical conductivity (EC), and some nutrients, to determine the effects of the treatments (LED and BAP) on the chemical changes of culture media after Melaleuca development. The initial (after autoclaving and before cultivation) and final (in vitrocultivated for 30 days) pH and electrical conductivity of the culture media were measured using a pHmeter (SevenCompact, Mettler) and a portable conductivity meter (Hanna Instruments, Portugal). The final and initial pH and EC values were used to obtain the pH variation throughout the in vitro cultivation of Melaleuca . For the analysis of the uptake of the macronutrients Ca, P, and Mg from the culture media, the initial and final values of these nutrients were analyzed to determine how much of these nutrients are absorbed from the culture media. The concentrations (mg/L) of these nutrients in the culture media were obtained using an IRIS-HI801 Spectrophotometer (Hanna Instruments), using the methodologies and solutions provided by the manufacturer. Nutrient analysis in Melaleuca shoots Shoot tissue samples were dried in an oven at 60°C for 48 hours. Afterward, they were ground to a fine powder and sent to the Soil and Plant Analysis Laboratory at Instituto Agronômico de Campinas (IAC), Campinas, Brazil. The nutrients phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), and boron (B) were analyzed by nitric-perchloric digestion and read by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Cinto et al 2022). Experimental design and data analysis. The experiment was conducted in a factorial arrangement of two concentrations of BAP (0.0 mg L -1 and 0.25 mg L -1 ), and six light spectra (W, D, B, R, WR, and BR), with ten repetitions per treatment, and the experimental unit (repetition) was a test tube containing one nodal segment each (1.0-1.5 cm). The experiment was repeated twice. All the results obtained were tested by two-way analysis of variance (ANOVA); when they presented normality (Shapiro-Wilk), the means were compared by Duncan's new multiple range test at p ≤ 0.05 significance. When the data were not normal, and it was not possible to normalize through data transformation, they were tested using the Games-Howel test, also at p ≤ 0.05 significance. For these analyses, the R Studio software (Hill et al. 2022) was used. At the end of the evaluations, a correlation analysis and a Principal component analysis were run for all the variables analyzed. Additionally, the data was added for Correlation and Principal Component Analysis, where one of the factors was modified, and the dark conditions were removed to evaluate the fifteen variables of Melaleuca development since in the dark, Melaleuca does not grow, thus using a 2 x 5 factorial. The correlation was made by the Spearman test at the level of 5% probability. Results Effects of BAP and wavelength on the cultivation and development of Melaleuca in vitro. The effect of BAP and wavelength and their interaction were significant for the number and length of new shoots, shoot height, chlorophyll a, chlorophyll b, and total chlorophyll (CLT) content in leaves. Regarding the fresh and dry mass, the effect of BAP and LED wavelength were significant, but no interaction was observed (Table 1). Our results demonstrated the strong effect of BAP in shifting the type of development of Melaleuca under in vitro conditions (Figure 1). The highest proliferation of shoots (8.1 shoots/nodal segment) was obtained using white LEDs in the presence of BAP, and only 2.5 shoots/nodal segment in the absence of BAP. The opposite was observed for new shoot length and shoot height, in which the addition of BAP resulted in a drastic reduction, from 1.17 cm (-BAP) to 0.54 cm (+BAP), when cultivated under the BR light. Similarly, using white LEDs, shoot height was drastically reduced from 7.3 cm to 1.8 cm in the presence of BAP. Interestingly, the highest fresh and dry mass of plants (0.18 g/plant and 0.015 g/plant, respectively) were obtained using white light in the presence of BAP, with low mass values in the absence of BAP. Except for blue light and cultivation in dark conditions, white light did not differ statistically from red, red+white, and red+blue for most characteristics associated with in vitro Melaleuca development. Levels of chlorophyll ‘a’ and ‘a+b’ were higher (Chla, 39.17 to 49.27; Chl a+b, 57.21 to 73.08 mg L -1 ) in culture media without BAP, and in those added with BAP, the reported values were 16.12 to 26.17 (Chla) and 28.26 to 44.27 mg L -1 (Chla+b), respectively (Table 1). The light spectra (except for dark x light conditions) had a lower influence on chlorophyll contents than BAP but white light resulted in higher contents of Chla and Chla+b compared to the Blue+Red light. Effects of BAP and wavelength on the stomatal density and diameter (DEE and DIE) in Melaleuca leaves. The effect of BAP on the stomatal density and diameter was dependent on the wavelength used for the Melaleuca cultivation (Table 2). The highest density of stomata (235/mm 2 ) was obtained when Melaleuca shoots were cultivated under Red + White light in the presence of BAP, but in the same light source and absence of BAP, the observed density of stomata was only 173.3. In white and red+white lights, stomatal density was higher when BAP was added to culture media; under monochromatic Red light, the stomatal density was higher in the absence of BAP. Except for white light, where stomatal density was higher in the presence of BAP, in most other lights, the absence of BAP resulted in higher (B, R, W+R) or equal (B+R) density of stomata (Table 2). Effects of BAP and wavelength on some chemical characteristics of the culture medium All chemical characteristics of culture media were affected by BAP and the light wavelength used for in vitro cultivation. Apparently, the presence of BAP increased the chemical dynamics between the culture media and the Melaleuca shoots cultivated since in five of six light wavelengths tested, the reduction of pH was more intense in the presence of BAP, with -0.8 to -1.2 point reduction compared to the initial pH. The smallest variations in pH during the in vitro cultivation were verified under dark conditions and the red or white LEDs, in the absence of BAP, with -0.3 to -0.4 point reduction compared to the initial pH (Table 3). Differently from pH, the more intense changes in electrical conductivity (EC) were attributed to light wavelengths than to BAP. For example, the highest ranges in EC were found in culture media incubated under white light and in the absence of BAP (1.13 mS/cm) and blue light (-0.73 to -0.91 mS/cm), independently of the BAP. The BAP had influence only when shoots were cultivated under white or red LED, and in both cases, the greatest variation in EC occurred in the absence of BAP (Table 3). Regarding the nutrient absorption from the media, the greater uptake of calcium from culture media and after in vitro development of Melaleuca was observed in the cultivation under monochromatic red (63.9%) and red+white LED (68.9%) in the absence of BAP. This cytokinin had a strong influence on calcium uptake, generally reducing Ca uptake when shoots were cultivated under White, Red, and red+white LED (Table 3). Similar results were reported for P uptake, in which there was a higher consumption of P (42.9 to 51.0%) in the absence of BAP in the culture medium. The highest percentage of consumption of Mg (20.6-23.2%) occurred under red light, regardless of the presence of BAP, and under the red+white LED in the presence of BAP. The principal component analysis (PCA) showed some new and interesting information about how factors such as BAP and light wavelength affected several variables analyzed (Figure 2). Among these, the increase in stomatal density, number of shoots, and fresh and dry mass in the presence of BAP, while the increase in chlorophyll contents, shoot length, and absorption of Ca and P was observed in the absence of BAP in the culture medium. Effects of BAP and wavelength on nutrient contents in Melaleuca shoot tissues The presence of BAP in the culture medium had a significant effect on seven out of the ten nutrients analyzed in plant tissues cultivated in vitro (Table 4). The concentrations of P, S, Mn, Cu, and B were lower in tissues cultivated in the presence of BAP in the culture medium, and the Fe concentration was higher in the presence of BAP. Regarding the wavelengths tested, the dark condition was the only treatment in which the presence of BAP promoted greater uptake of all analyzed nutrients than the medium in the absence of BAP. Under the light that resulted in better growth of Melaleuca (white and white+red), except Fe, all other nutrients had lower contents in the tissues when BAP was added to culture media. Discussion Effects of BAP and wavelengths onin vitro development of Melaleuca In the present study, a multiplication factor of 5.6 - 8.1 shoots/nodal segment was reported with the addition of a low concentration of BAP (0.25 mg L -1 ) to the culture medium. According to Iiyama and Cardoso (2021), the addition of BAP at a low concentration (0.55 μM = 0.124 mg L -1 ) causes a considerable increase in shoot proliferation, up to 6.54 shoots/explant. Similar results were reported by Oliveira et al. (2010), with the highest proliferation of shoots (5.6 shoots/nodal segment) obtained with the addition of 0.55 μM of BAP to the MS culture medium. The main physiological effect of BAP on the in vitro cultivation of Melaleuca was the promotion of multiple shoots in vitro (Iiyama and Cardoso, 2021) by increasing the development of lateral buds and the proliferation of new shoots (Muleo and Morini, 2006). However, BAP also caused other negative effects on the in vitro development of Melaleuca , such as the strong inhibition of root formation and a considerable decrease in the length and height of shoots (Figure 1). These data are similar to those reported by other authors on the in vitro cultivation of Melaleuca (Oliveira et al. 2010) and other woody species (Bennett et al. 1994; Lin et al. 2013). In fact, cytokinins like BAP are negative regulators of root growth (Nehnevajova et al. 2019). However, it is important to note that more recent studies have shown that although this inhibition occurs in the presence of BAP, shoots from a medium with BAP, when transferred to a rooting medium (absence of BAP), have exhibited better performance and development of roots than those coming from the culture medium without BAP (Iiyama and Cardoso, 2021). Fresh and dry mass showed a strong correlation with the increases in the number of shoots promoted by BAP addition to the media (8.1 shoots/explant) (Figure 2). In different species, this same correlation was detected between fresh and dry mass (FM and DM) with the increased number of shoots/explants (Bello-Bello et al. 2016; Gupta and Jatothu, 2013; Kim et al.,2004; Sarropoulou et al. 2022). Another interesting effect of BAP in culture media is the maintenance of live and growth of shoots since under dark conditions, while in the absence of this cytokinin, the plantlets died before completing 30-d of cultivation. Chlorophyll analysis showed a higher level of chlorophyll a and total (a+b) in the shoots obtained from plantlets cultivated in the absence of BAP, regardless of the light wavelength. As for the effects of light wavelength, there were no differences in chlorophyll concentrations in the absence of BAP. According to Lotfi et al. (2019), blue light is relevant for chlorophyll biosynthesis, chloroplast development, and the stomatal opening (Wu et al. 2007), and similar results were obtained by other authors with different plant species (Hernández and Kubota, 2016; Marín-Martinez and Iglesias-Andreu, 2022). However, there are contrasting results with other species such as bananas, in which the use of white + red and red LED resulted in higher concentrations of chlorophylls than with the use of fluorescent light (Vieira et al. 2015). Conversely, using the same light source, the presence of BAP in the culture medium resulted in a significant reduction in concentrations of chlorophyll a and total but did not influence chlorophyll b. These results are similar to those observed for other species. In Nidularium (Bromeliaceae) in vitro cultivation, increasing concentrations of BAP and other cytokinins in the culture medium resulted in a significant reduction in chlorophyll concentrations in plant tissues (Ördögh, 2015). In transgenic plants of Arabidopsis thaliana , small contents of cytokinins increased the contents of chlorophyll in their shoots (Nehnevajova et al. 2019). The highest stomatal density was obtained with the red+white LED combination, followed by red, blue+red, and white LEDs, while in monochromatic blue light, there was a significant reduction in stomatal density. However, He et al. (2020) observed the highest average density of stomata (10.9) in growing Camellia oleifera under white LED light, and Mihovilović et al. (2020) reported the highest stomatal density (17.3) of Amelanchier alnifolia when cultivated under blue + red LED. Interestingly, Melaleuca cultivated in vitro under blue LED light presented the lowest density of stomata, regardless of the presence of BAP. The opposite was observed in Cucumber seedlings, in which the relative reduction in red light and the increase in blue light resulted in the highest stomatal density (Li et al. 2023). Izzo et al. (2021) reported reduced stomatal density both under red or blue monochromatic light, while their combination (red + blue light) increased stomatal density. Under in vitroconditions, the stomata are normally circular and open (Tichá et al. 1999), and these characteristics were also observed in the present study in Melaleuca shoots. The largest diameter of leaf stomata (26.5 μm and 24.8 μm) was observed under blue or red monochromatic LED lighting, both in the presence of BAP and under white light in the absence of BAP. Similar results were obtained for three species ( Cordyline australis, Ficus benjamina, and Sinningia speciosa ) by Zheng and Van (2017), who reported that the use of blue or red LED resulted in the largest stomatal diameter. Cioć and Pawlowska (2020) reported that the addition of BAP to the culture medium resulted in longer stomata and, consequently, larger diameters. The present study on Melaleuca demonstrated that this increase in stomatal diameter in response to the addition of BAP to the culture medium depended on the wavelength, being higher in the blue, red, and red+white LED. These differential responses to light according to the species were the result of several factors that affected stomata development, such as the species group, light intensity, developmental stage, photosynthetic metabolism (Matthews et al. 2020), and other non-related to light such as the osmotic potential of culture media (Iiyama and Cardoso, 2021). A major obstacle with Melaleuca micropropagation is the acclimatization phase, where Melaleuca plantlets’ rapid dehydration is partially attributed to stomatal density, development and functionality (Iiyama and Cardoso, 2021). Thus, the LED lighting’s influence on the stomata could be used to diminish the stomatal density and diameter for better adaptation of plantlets for acclimatization. Changes in the in vitro environmental conditions are strongly associated with improvements in the acclimatization stage, and stomata characteristics and functionality are some of the main factors affecting acclimatization success, especially in tree species (Asayesh et al. 2017). Effects of BAP and wavelength on the culture medium characteristics and changes Although in vitro studies have extensively explored the influence of culture media, additives, and plant growth regulators on plant development and propagation, little attention has been dedicated to changes in culture media in response to the development of in vitrotissues. Changes in culture media in response to in vitro cultivation systems are influenced by several factors, such as the system used for in vitro cultivation. Kozai et al. (1991) reported that photoautotrophic conditions (no sugar in culture media and CO 2 enriched environment) promoted the highest uptake of PO 4 3- , NO 3 - , Ca 2+ , Mg 2+ , and K + . In the present study with Melaleuca , the highest uptake of nutrient ions PO 4 3- and Ca 2+ also seems to have occurred in the absence of BAP in the culture medium, especially when grown under red and red+white LED. The opposite was registered for Mg 2+ , i.e., the presence of BAP resulted in higher uptake of this nutrient under most spectra used. Gerovac et al. (2016) and Schroeter-Zakrzewska and Kleiber (2014) reported the highest calcium and magnesium uptake using red LED. The widest pH range of culture media after growing Melaleuca in vitro occurred with the presence of BAP, especially with Melaleuca shoots cultivated under blue or red monochromatic LED (-1.1 to -1.2) and in the presence of this cytokinin when shoots were cultivated under blue+red LEDs. For electrical conductivity, the response was contrary, and the greatest variations occurred in the absence of BAP added to the culture medium. The smallest variations in pH and EC, compared to the initial medium, were observed in dark conditions and without BAP, demonstrating that the absence of light for Melaleuca in vitro resulted in limited development of shoots/plantlets and few variations in culture media. Perez-Vazquez et al. (2021) reported that the EC in culture media is negatively correlated with the development of the morrón pepper, showing increased shoot growth (fresh and dry mass) with decreasing EC values. However, our findings with Melaleuca did not demonstrate this correlation between a reduction in EC and a greater accumulation of fresh and dry mass. In other words, other factors related to the type of plant development, such as the formation of plantlets (shoots + roots: absence of BAP) or only shoots (presence of BAP), the higher growth in height of plantlets in vitro (absence of BAP) were associated with increasing uptake of specific nutrients, such as Ca and P, and other components of the culture medium that major contribute to reduction of EC values. The correlation analysis evidenced that a limited number of variables analyzed in Melaleuca demonstrated significant correlations. Among these, the increase in the number of shoots induced by BAP was highly correlated with the increase in fresh (r=0.81**) and dry (r=0.85**) mass of shoots and the reduction in shoot height Also, the higher shoot height promoted by the absence of BAP was correlated with increased chlorophyll a (r=0.77*) and total (r=0.77*) concentrations in the leaves. The presence of BAP is often correlated with the reduction of photosynthetic pigments in different species cultivated in vitro (Martins et al. 2020; 2022). For culture media, we observed a correlation between higher values of final pH (small variations in relation to initial pH), the higher uptake of P by the shoots (r=0.71*), and shoot height (r=0.71*), indicating that higher P uptake increased shoot height in Melaleuca . However, it is not possible to know if this effect is direct or indirect because BAP promotes only shoot induction, and in its absence, shoots and roots were observed in Melaleuca in vitro (Figure 1). The Principal Component Analysis (PCA) revealed that the LED-wavelength component (PC1) explained 50.29% of data variance, and the BAP (PC2) component explained 14.56% of data variation (Figure 3). The PCA analysis showed that the number of shoots, and the fresh and dry mass were highly correlated with the presence of BAP in the culture medium, especially under white or red LED. Effects of BAP and wavelength on the concentration of nutrients in Melaleuca tissues The presence of cytokinin BAP substantially increased the contents of all nutrients in tissues of Melaleuca in vitro under dark conditions (from +62.9% to +341.6% compared to BAP-free media). Cytokinins like BAP are well-known to delay tissue senescence in the dark by continuing higher transcript levels of Photosystem II-related genes and the integrity of chlorophyll pigment-protein, maintaining the Chl a/b ratios (Talla et al. 2016). The addition of BAP and other cytokinins induces photomorphogenesis under dark conditions (Spiro et al. 2004). Thus, cytokinins applied exogenously can maintain active metabolism and morphogenesis, including nutrient uptake of in vitro plantlets under dark conditions. Indeed, cytokinis can promote nutrient accumulation in tissues under adverse conditions (Prasad 2022). However, under light conditions, the addition of BAP resulted in decreased accumulation of nutrients in tissues for most nutrients analyzed, especially phosphorus (P), magnesium (Mg), sulfur (S), manganese (Mn), copper (Cu), and boron (B), compared to tissues cultivated in BAP-free culture media. This is a surprising result since a high amount of fresh and dry mass was observed in the presence of the BAP. Interestingly, other authors report that the increased activity of the cytokinin dehydrogenase enzyme, which leads to cytokinin degradation, resulted in an increment of some nutrients in leaves caused by the enhancement of root system and better scavenging of macro- and micronutrients since cytokinin negatively regulates root growth (Nehnevajova et al. 2019, Prasad 2022). In fact, in our results with Melaleuca , roots in culture media were only observed in the absence of BAP, which explains the higher accumulation of these nutrients in tissues cultivated without this cytokinin. On the other hand, the addition of BAP resulted in higher concentrations of iron (Fe) in all samples, regardless of the light wavelength tested. This is the opposite of that observed by Séguéla et al. (2008), who reported that cytokinins negatively regulate the root iron uptake in Arabidopsis . Nevertheless, Nehnevajova et al. (2019) reported that only iron, among all other nutrients analyzed, was reduced (11-18%) in leaves of transgenic lines of oilseed rape plants overexpressing the CKX2 gene, with lower cytokinin content, compared to the wild-type. Thus, these studies and our results with Melaleuca suggested a more complex mechanism of regulation of iron uptake in plants, in which cytokinins can play an important role in iron uptake and accumulation in tissues. Conclusions There is a strong interaction between the addition of the 6-BAP cytokinin to the culture medium and the LED wavelengths used for the in vitro cultivation of Melaleuca . The addition of 6-BAP had important effects, increasing the number of shoots/explants and, consequently, fresh and dry mass, reducing shoot height, and significantly decreasing the chlorophyll contents of the plants in vitro. White or White+Red LED is recommended in the growing of Melaleuca , aiming at its multiplication in vitro with BAP-supplemented media or in rooting and elongation of shoots without BAP supplementation in media. BAP also negatively affected nutrient concentration in plant tissues, except Fe, which substantially increases independently of light wavelength. Declarations Funding This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Authors contribution statement ACGA contribute with the experimental conduction, data collection and analysis and writing of the manuscript. JCC contribute with the main idea, experimental design, writing, revision and editing of this manuscript. Competing interests The authors have no relevant financial or non-financial interests to disclose. 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BAP (mg L -1 ) Light wavelength ( spectra ) White Dark conditions Blue Red Red+White Blue+Red N umber of shoots 0 2.5 Ba 0.01 Bc 1.88 Bab 2.0 Bab 1.5 Bb 1.7 Bab 0.25 8.1 Aa 5.8 Aa 5.6 Aa 6.9 Aa 6.2 Aa 7 Aa New shoots Length (cm) 0 0.93 Aa 0.02 Bb 1.08 Aa 1.15 Aa 0.9 Aa 1.17 Aa 0.25 0.48 Ba 0.79 Aa 0.85 Aa 0.63 Ba 0.64 Aa 0.54 Ba Shoot height (cm) 0 7.3 Aa 2.2 Ab 5.6 Aab 5.4 Aab 5.7 Aab 4.9 Aab 0.25 1.8 Ba 2.2 Aa 2.5 Ba 2.2 Aa 1.7 Ba 1.8 Ba Fresh mass of shoots (g/plant) 0 0.08 Ba 0.03 Bb 0.05 Ba 0.06 Aa 0.06 Ba 0.06 Ba 0.25 0.18 Aa 0.09 Ab 0.11 Aab 0.08 Ab 0.10 Ab 0.09 Ab Dry mass of shoots (g/plant) 0 0.01 Ba 0.004 Bb 0.007 Bab 0.009 Ba 0.008 Ba 0.009 Ba 0.25 0.015 Aa 0.007 Ab 0.013 Aa 0.012 Aa 0.013 Aa 0.012 Aa Chlorophyll a (mg/L) 0 49.17 Aa - 49.27 Aa 48.33 Aa 39.17 Aa 40.17 Aa 0.25 26.17 Ba - 16.12 Aab 21.3 Bab 17.62 Bab 12.74 Bb Chlorophyll b (mg/L) 0 20.7 Aa - 23.8 Aa 20.61 Aa 18.04 Aa 18.8 Aa 0.25 18.1 Aa - 12.14 Ba 19.04 Aa 12.47 Aa 10.17 Aa Total chlorophyll (a+b) (mg/L) 0 69.87 Aa - 73.08 Aa 68.95 Aa 57.21 Aa 58.97 Aa 0.25 44.27 Ba - 28.26 Bab 40.34 Bab 30.09 Bab 22.91 Bb Different capital letters (colunms) demonstrate a significant difference for the factor absence or presence of BAP and different lowercase letters (lines) shown the differences between the spectrum of light, according to the Duncan test, P <0,05. Table 2: Effect of BAP and wavelength on the density and diameter of the stomata. BAP (mg L -1 ) Light wavelengths White Blue Red Red+White Blue+Red Stomatal density (number of stomata/mm 2 ) 0 116.6 Bb 80 Ac 140 Ab 173.3 Ba 135 Ab 0.25 155 Ab 81.6 Ac 115 Bc 235 Aa 163.3 Ab Stomatal diameter ( μ m) 0 22.5 Aa 18.4 Bc 20.2 Bb 15.1 Bd 18.3 Ac 0.25 18.4 Bb 26.5 Aa 24.8 Aa 18.7 Ab 17 Ab Different capital letters (colunms) demonstrate a significant difference for the factor absence or presence of BAP and different lowercase letters (lines) shown the differences between the spectrum of light, according to the Duncan test, P <0,05. Table 3: Effects of BAP and wavelength on the final culture media pH, EC and the macronutrients (Ca, Mg and P) uptake from the culture media. BAP (mg L -1 ) Light wavelengths White Dark condition Blue Red Red+White Blue+Red pH final (pH final – initial) 0 4.9(-0.4) AaB 5.0(-0.3) Aa 4.5(-0.8) Abc 5.0(-0.4) Aab 4.7(-0.7) Aab 4.2(-1.2) Bc 0.25 4.6(-0.8) Aab 4.7(-0.7) Aa 4.3(-1.1) Aab 4.2(-1,2) Bc 4.4(-1.0) Aab 4.7(-0.8) Aab EC final - initial (mS/cm) 0 -1.13 Aa -0.25 Ac -0.73 Ab -0.43 Ac -0.28 Ac -0.25 Ac 0.25 -0.26 Bb -0.21 Ab -0.91 Aa -0.14 Bb -0.30 Ab -0.26 Ab Ca (% consumed from the media) 0 44.5 Ac 40.2 Ad 39.5 Bd 63.9 Ab 68.9 Aa 40.5 Bd 0.25 21.4 Be 44.9 Ac 55.9 Aa 33.6 Bd 44.9 Bc 50.4 Ab P (% consumed from the media) 0 46.9 Ab 44.9 Ac 30.6 Be 42.9 Ad 51.0 Aa 24.5 Af 0.25 41.3 Ba 13.0 Bc 37.0 Ab 5.4 Be 10.9 Bd 13.0 Bc Mg (% consumed from the media) 0 17.5 Ab 14.3 Acd 8.3 Be 23.2 Aa 12.3 Bd 15.4 Abc 0.25 12.4 Bc 0.0 Bd 16.4 Ab 21.7 Aa 20.6 Aa 16.8 Ab Different capital letters demonstrate a significant difference for the factor, presence and absence of BAP in the culture medium. Lowercase letters show the differences between the spectrum of light, according to the Duncan test, P <0,05. Table 4: Percentage of increase or decrease - in response to the addition of BAP - of nutrient contents in in vitro tissues of shoots of Melaleuca alternifolia cultivated under different LED wavelengths LED wavelength Nutrients in the shoot tissues (%) P K Ca Mg S Fe Mn Cu Zn B White -35.8 -25.9 +7.4 -19.9 -26.9 +21.5 -34.6 -33.8 -2.2 -29.8 Dark conditions +97.3 +106.9 +328.1 +181.9 +392.3 +341.6 +285.2 +62.5 +244.7 +319.5 Blue -21.6 +29.4 -26.3 -3.3 -13.5 +20.3 -19.3 -13.0 +17.6 -24.0 Red -33.3 +4.5 +12.4 +1.6 -17.7 +14.0 -25.1 -30.2 +12.7 +1.3 White+Red -48.2 -12.4 -17.2 -17.0 -39.2 +66.8 -37.2 -57.4 -15.9 -28.7 Red+Blue -36.7 +23.6 +5.1 0.0 +7.6 +3.4 -15.1 -34.5 +18.3 -39.4 Negative number means the decrease and positive number means the increase in percentage of nutrient contents in response to addition of BAP to the culture media, compared with the BAP-free culture media. 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Mg, P, Ca, EC, PH, DIE, DEE, CLT, CLB, CLA, MD, MF, AS, LS and NS (Magnesium, phosphorus, calcium, electrical conductivity, Ph, Diameter of stomata, Density of stomata, Total chlorophyll, Chlorophyll b, Chlorophyll a, Dry mass, Fresh mass, Aerial height, Length of shoots, Number of shoots).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4249082/v1/2b7d9365ec84481255e43154.jpg"},{"id":61897529,"identity":"0d3cb971-4f3e-4ba9-876b-458332494c93","added_by":"auto","created_at":"2024-08-06 21:13:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1202779,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4249082/v1/7bfd0a2d-32d4-4ec1-a545-7fdc0682b17e.pdf"}],"financialInterests":"","formattedTitle":"Development, chlorophyll content, and nutrient accumulation in shoots of Melaleuca alternifolia in vitro under different light wavelengths and benzylaminopurine (BAP)","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThis study evidenced the important interaction of lighting and cytokinin BAP in the culture media, which affects the in vitro development, the metabolism of nutrients, and the chlorophyll contents.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eMelaleuca\u003c/em\u003e belongs to the\u0026nbsp;family\u0026nbsp;Myrtaceae with around 150 species (Martinho\u0026nbsp;and Rodrigues, 2009) and is used for the production of essential oils on an industrial scale, particularly the species \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e Cheel. This species has different properties, such as anti-inflammatory (Caldefie-Chezet et al. 2006; Lahkar et al. 2013), antimicrobial (Carson et al. 2006; Lahkar et al. 2013; Da Silva et al. 2002), fungicide (Lahkar et al. 2013; Tong et al. 1992), and acaricide (Lori et al. 2005), and used in the treatment of insect bites and skin infections (Budhiraja et al. 1999;\u0026nbsp;Lahkar\u0026nbsp;et al. 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMelaleuca\u003c/em\u003e seedlings result in high heterogeneity and stem cutting shows difficulties such as low rooting percentage (Chen et al. \u0026nbsp;2016; Iiyama and Cardoso, 2021). Therefore, there is great interest in developing micropropagation protocols to evaluate the feasibility of this technique from the commercial point of view and the performance of plantlets from the micropropagation system. Despite the limited number of studies with\u003cem\u003e\u0026nbsp;Melaleuca\u003c/em\u003e micropropagation, they have revealed the substantial potential of using micropropagation as a clonal system (de Oliveira, 2010; Iiyama and Cardoso, 2021). In these studies, the use of the cytokinin N6-Benzylaminopurine (BAP) has proven essential in achieving high rates of multiplication and proliferation of shoots under in vitro conditions (List et al. 1995; de Oliveira, 2010; Iiyama and Cardoso, 2021) by promoting the proliferation of axillary and adventitious buds in tissues cultured in vitro\u0026nbsp;(da Silva et al. 2022).\u003c/p\u003e\n\u003cp\u003eAdvances in in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003econditions,\u0026nbsp;are achieved by using light-emitting diodes (LEDs), which replaced cold or fluorescent lamps, increasing energy efficiency and expanding the types and combinations of LED spectra according to each growing system or plant species cultivated (Sena et al. 2024). Also, the use of LED systems emerges as an extremely promising strategy to modulate the physiological response of plants (Rabara et al., 2017). This approach allows for the generation of a specific wavelength source, which has been shown to induce particular responses in plants, notably an increase in photosynthetic rate and significantly enhanced plant growth (Kozai, 2016).\u003c/p\u003e\n\u003cp\u003eCurrently, there are many studies demonstrating the effects of LEDs with different wavelengths (colors) on the\u0026nbsp;in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003edevelopment of \u003cem\u003eVitis riparia\u003c/em\u003e (Poudel et al. \u0026nbsp;2008), cotton plant (Li\u0026nbsp;and Tang, 2010), \u003cem\u003eCedrela fissilis\u003c/em\u003e (Tadeu, 2017), \u003cem\u003eHelianthus tuberosus\u003c/em\u003e (Seedapalee et al. 2021),\u003cem\u003e\u0026nbsp;Ficus carica\u003c/em\u003e var. Black Jack\u0026nbsp;(Parab et al. 2021),\u0026nbsp;\u003cem\u003ePyrus communis\u003c/em\u003e (Lupo et al. 2022),\u0026nbsp;\u003cem\u003ePinus pseudostrobus\u003c/em\u003e (Mar\u0026iacute;n-Martinez and Iglesias-Andreu, 2022).\u0026nbsp;Gupta and Jatothu (2013) reported the best development of crops without the induction of damage or photo stress. Despite this, the studies on the in vitro micropropagation of \u003cem\u003eMelaleuca\u003c/em\u003e used conventional cold lamps or LED at specific wavelengths (Oliveira, 2010; Amelia et al. 2020;\u0026nbsp;Iiyama and Cardoso, 2021).\u0026nbsp;Therefore, there are still no studies demonstrating the effects of different LED wavelengths on the in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003edevelopment of \u003cem\u003eMelaleuca\u003c/em\u003e. In addition, there is little known on the interaction of the different light wavelengths with phytoregulators, such as BAP, as this cytokinin is widely used for micropropagation of several commercial and non-commercial species under in vitro conditions.\u0026nbsp;Finally, few studies have been dedicated to better understanding the chemical changes in culture media in response to the development and cultivation of plants in vitro. This information can help to improve the plant performance under in vitro conditions and to determine key factors that improve the performance of culture media.\u003c/p\u003e\n\u003cp\u003eThus, the main objectives of this study were to better understand the interaction of cytokinin BAP and different LED wavelengths\u0026nbsp;on the in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003edevelopment of \u003cem\u003eMelaleuca\u003c/em\u003e shoots and\u0026nbsp;plantlets, as well as the main changes in culture media related to nutritional characteristics. At the same time, this study also aimed at identifying the best wavelengths associated with the proliferation/multiplication of \u003cem\u003eMelaleuca\u003c/em\u003e under in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003econditions.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material and in vitro growing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant material used for the experiments consisted of in vitro stem nodal segments (shoots) 1.0 - 1.5 in length, previously established in vitro from the stem\u0026nbsp;apices\u0026nbsp;taken from greenhouse mother plants.\u0026nbsp;These explants were established and propagated in vitrofor approximately nine months to obtain enough stem nodal segments for the experimental design.\u0026nbsp;These nodal segments were grown in 15 mL\u0026nbsp;of Murashige \u0026amp; Skoog\u0026nbsp;(MS)\u0026nbsp;culture medium\u0026nbsp;(1962) containing half the macronutrient concentration (MS \u0026frac12;), 6.5\u0026nbsp;g L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eof agar (Agargel, Jo\u0026atilde;o Pessoa-PB, Brazil), 2% sucrose, 0.1 g L\u003csup\u003e-1\u003c/sup\u003e inositol, and pH adjusted to 5.8 (Iiyama and Cardoso, 2021); with or without the presence of BAP (at 0.25 mg L\u003csup\u003e-1\u003c/sup\u003e).\u0026nbsp;The BAP used was added before pH adjustment and was provided from a concentrate solution containing 10 mg BAP (Sigma-Aldrich), previously dissolved in 1 mL NaOH (1M) and 9 mL of distilled water.\u003c/p\u003e\n\u003cp\u003eTest tubes containing\u0026nbsp;one nodal segment each (replicates)\u0026nbsp;were kept in a growth room with a photoperiod set to 14 hours, temperature of 25\u0026deg;C \u0026plusmn; 2\u0026deg;C, illuminated with six different wavelengths provided by LED lamps. The photosynthetic photon flux density (PPFD) was measured for each wavelength tested using the Quantum SQ-520 meter (Apogee Instruments, USA). The LED lamps used as treatments were\u0026nbsp;100% white diffuser module (W: 64.09 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e), 2) dark conditions\u0026nbsp;(D: 0.58 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es \u0026ndash; diffuse light), 3) 100% blue LED light (B: 55.58 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es), 4) 100% red LED light (R: 106.22 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es), 5) 50%: 50% red and white LED light (RW: 104.51 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es), and 6) 40%: 60% blue and red LED light (BR: 71.48 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003es).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003cstrong\u003echaracteristics of \u003cem\u003eMelaleuca\u003c/em\u003e grown under LED spectra and BAP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMelaleuca\u003c/em\u003e development was analyzed weekly for 30 days of in vitro cultivation. The variables evaluated were the number and length of shoots (cm), shoot height (cm), total fresh and dry masses (g), and shoot mass (g). For the fresh and dry mass determination, plantlets were separated into shoots and roots (when present) or only shoots (no roots) and weighed on a digital analytical scale accurate to 0.1 mg (ML201, Mettler, Switzerland). The dry mass of each part was obtained by drying in an oven at a temperature of 70 \u0026deg;C for 72 h, followed by weighing on the same scale.\u0026nbsp;Chlorophyll a, b, and total chlorophyll contents were also analyzed by spectrophotometry. Chlorophyll a, b, and total chlorophyll concentrations were obtained by macerating 100 mg of the in vitro \u003cem\u003eMelaleuca\u003c/em\u003e leaves\u0026nbsp;in a crucible containing 2-3 mL of pure acetone (99.5%) and adjusted to a final volume of 10 mL for spectrophotometer reading. Samples were poured into the manufacturer\u0026rsquo;s glass vial to measure their absorbance in the IRIS-HI801 spectrophotometer (Hanna Instruments). Absorbance readings were taken at 664\u0026nbsp;nm and 647 nm according to the equations by Lichtentaler (1987) and\u0026nbsp;against a blank of pure acetone. Chlorophyll concentrations were determined in mg/L using the following equations: chlorophyll a=12.70 (A \u003csub\u003e664\u003c/sub\u003e) - 2.97 (A\u003csub\u003e647\u003c/sub\u003e); chlorophyll b=20.70 (A\u003csub\u003e647\u003c/sub\u003e) - 4.62 (A\u003csub\u003e664\u003c/sub\u003e), as proposed by Lichtenthaler et al. (1983). Total chlorophyll was obtained by summing up the values of chlorophyll a and b.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStomatal size and density were evaluated in in vitro leaves of \u003cem\u003eMelaleuca\u003c/em\u003e. For counting and measuring the polar and equatorial diameters of the stomata, we used leaves from the fourth to the sixth node of the in vitroplantlets (Iiyama and Cardoso, 2021). Leaves were first fixed\u0026nbsp;in Carnoy solution composed of three volumes of ethanol and one of glacial acetic acid (v/v) for 48h. Subsequently, samples were stored in 70% ethanol solution at 8\u0026deg;C until slide mounting. For the visualization of stomata, the leaves were previously immersed in 70% alcohol for one minute and soon after in potassium hydroxide solution 5 M at 45\u0026deg;C for 20-30 s. The digestion reaction was\u0026nbsp;stopped\u0026nbsp;by adding distilled water, followed by mounting the slide with a coverslip and analysis under a light microscope (Nikon Eclipse 201) at a total magnification of 100\u0026times;, coupled to a high-resolution camera (5 Mp Opticam) and software\u0026nbsp;for stomatal measurement. The variables evaluated were the stomatal density and diameter (\u0026micro;m) in 10 observed fields per leaf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical characteristics of culture media used for \u003cem\u003eMelaleuca\u003c/em\u003e in vitro cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe purpose was to observe the differences between the initial and final values of different characteristics of the culture medium, such as pH, Electrical conductivity (EC), and some nutrients, to determine the effects of the treatments (LED and BAP) on the chemical changes of culture media after \u003cem\u003eMelaleuca\u003c/em\u003e development.\u0026nbsp;The initial (after autoclaving and before cultivation) and final (in vitrocultivated for 30 days) pH and electrical conductivity of the culture media were measured using a pHmeter (SevenCompact, Mettler) and\u0026nbsp;a portable conductivity meter (Hanna Instruments, Portugal). The final and initial pH and EC values were used to obtain the pH variation throughout the in vitro cultivation of\u003cem\u003e\u0026nbsp;Melaleuca\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor the analysis of the uptake of the macronutrients Ca, P, and Mg from the culture media, the initial and final values of these nutrients were analyzed\u0026nbsp;to determine how much of these nutrients are absorbed from the culture media.\u0026nbsp;The concentrations (mg/L) of these nutrients in the culture media were obtained using an IRIS-HI801 Spectrophotometer (Hanna Instruments), using the methodologies and solutions provided by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrient analysis in \u003cem\u003eMelaleuca\u003c/em\u003e shoots\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShoot tissue samples were dried in an oven at 60\u0026deg;C for 48 hours. Afterward, they were ground to a fine powder and sent to the Soil and Plant Analysis Laboratory at Instituto Agron\u0026ocirc;mico de Campinas (IAC), Campinas, Brazil. The nutrients phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), and boron (B) were analyzed by nitric-perchloric digestion and read by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Cinto et al 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design and data analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted in a factorial arrangement of\u0026nbsp;two\u0026nbsp;concentrations of BAP (0.0 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 0.25 mg\u0026nbsp;L\u003csup\u003e-1\u003c/sup\u003e),\u0026nbsp;and\u0026nbsp;six light\u0026nbsp;spectra\u0026nbsp;(W, D, B, R, WR, and BR),\u0026nbsp;with ten repetitions per treatment, and the experimental unit (repetition) was a test tube containing one nodal segment each (1.0-1.5 cm). The experiment was repeated twice.\u003c/p\u003e\n\u003cp\u003eAll the results obtained were\u0026nbsp;tested by two-way\u0026nbsp;analysis of variance (ANOVA); when they presented normality (Shapiro-Wilk), the means were compared by Duncan\u0026apos;s new multiple range test at p \u0026le; 0.05 significance. When the data were not normal, and it was not possible to normalize through data transformation, they were tested using the Games-Howel test, also at p \u0026le; 0.05 significance. For these analyses, the R Studio software (Hill et al. \u0026nbsp; 2022) was used. At the end of the evaluations, a correlation analysis and a Principal component analysis were run for all the variables analyzed.\u003c/p\u003e\n\u003cp\u003eAdditionally, the data was added for Correlation and Principal Component Analysis, where one of the factors was modified, and the dark conditions were removed to evaluate the fifteen variables of \u003cem\u003eMelaleuca\u003c/em\u003e development since in the dark, \u003cem\u003eMelaleuca\u003c/em\u003e does not grow, thus using a 2 x 5 factorial. The correlation was made by the Spearman test at the level of 5% probability.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of BAP and wavelength on the cultivation and development of \u003cem\u003eMelaleuca\u003c/em\u003e in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of BAP and wavelength and their interaction were significant for the number and length of new shoots, shoot height, chlorophyll a, chlorophyll b, and total chlorophyll (CLT) content in leaves. Regarding the fresh and dry mass, the effect of BAP and LED wavelength were significant, but no interaction was observed (Table 1). Our results demonstrated the strong effect of BAP\u0026nbsp;in shifting\u0026nbsp;the type of development of \u003cem\u003eMelaleuca\u003c/em\u003e under in vitro conditions (Figure 1).\u003c/p\u003e\n\u003cp\u003eThe highest proliferation of shoots (8.1 shoots/nodal segment) was obtained using white LEDs in the presence of BAP, and only 2.5 shoots/nodal segment in the absence of BAP. The opposite was observed for new shoot length and shoot height, in which the addition of BAP resulted in a drastic reduction, from 1.17 cm (-BAP) to 0.54 cm (+BAP), when cultivated under the BR light. Similarly, using white LEDs, shoot height was drastically reduced from 7.3 cm to 1.8 cm in the presence of BAP.\u0026nbsp;Interestingly, the highest fresh and dry mass of plants\u0026nbsp;(0.18 g/plant and 0.015 g/plant, respectively) were obtained using white light in the presence of BAP,\u0026nbsp;with low mass values in the absence of BAP. Except for blue light and cultivation in dark conditions, white light did not differ statistically from red, red+white, and red+blue\u0026nbsp;for most characteristics associated with in vitro \u003cem\u003eMelaleuca\u003c/em\u003e development.\u003c/p\u003e\n\u003cp\u003eLevels of chlorophyll \u0026lsquo;a\u0026rsquo; and \u0026lsquo;a+b\u0026rsquo; were higher (Chla, 39.17 to 49.27; Chl a+b, 57.21 to 73.08 mg L\u003csup\u003e-1\u003c/sup\u003e) in culture media without BAP, and in those added with BAP, the reported values were 16.12 to 26.17 (Chla) and 28.26 to 44.27 mg L\u003csup\u003e-1\u003c/sup\u003e (Chla+b), respectively (Table 1). The light spectra (except for dark x light conditions) had a lower influence on chlorophyll contents than BAP but white light resulted in higher contents of Chla and Chla+b compared to the Blue+Red light.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of BAP and wavelength on the stomatal density and diameter (DEE and DIE) in \u003cem\u003eMelaleuca\u003c/em\u003e leaves.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of BAP on the stomatal density and diameter was dependent on the wavelength used for the \u003cem\u003eMelaleuca\u003c/em\u003e cultivation (Table 2).\u0026nbsp;The highest density of stomata (235/mm\u003csup\u003e2\u003c/sup\u003e) was obtained when \u003cem\u003eMelaleuca\u003c/em\u003e shoots were cultivated under Red + White light in the presence of BAP, but in the same light source and absence of BAP, the observed density of stomata was only 173.3. In white and red+white lights, stomatal density was higher when BAP was added to culture media; under monochromatic Red light, the stomatal density was higher in the absence of BAP. Except for white light, where stomatal density was higher in the presence of BAP, in most other lights, the absence of BAP resulted in higher (B, R, W+R) or equal (B+R) density of stomata (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of BAP and\u003c/strong\u003e \u003cstrong\u003ewavelength\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon some chemical characteristics of the culture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemical characteristics of culture media were\u0026nbsp;affected by BAP and the light wavelength used for in vitro cultivation. Apparently, the presence of BAP increased the chemical dynamics between the culture media and the \u003cem\u003eMelaleuca\u0026nbsp;\u003c/em\u003eshoots cultivated since in five of six light wavelengths tested, the reduction of pH was more intense in the presence of BAP, with -0.8 to -1.2 point reduction compared to the initial pH. The smallest variations in pH during the in vitro cultivation were verified under dark conditions and the red or white LEDs, in the absence of BAP, with -0.3 to -0.4 point reduction compared to the initial pH (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferently from pH, the more intense changes in electrical conductivity (EC) were attributed to light wavelengths than to BAP. For example, the highest ranges in EC were\u0026nbsp;found in culture media incubated under\u0026nbsp;white light and\u0026nbsp;in the absence\u0026nbsp;of BAP (1.13 mS/cm) and blue light (-0.73 to -0.91 mS/cm), independently of the BAP.\u0026nbsp;The BAP had influence only when shoots were cultivated under white or red LED, and in both cases, the greatest variation in EC occurred in the absence of BAP (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the nutrient absorption from the media, the greater uptake of calcium from culture media and after in vitro development of \u003cem\u003eMelaleuca\u003c/em\u003e was observed in the cultivation under monochromatic red (63.9%) and red+white LED (68.9%)\u0026nbsp;in the absence\u0026nbsp;of BAP.\u0026nbsp;This cytokinin had a strong influence on calcium uptake, generally reducing Ca uptake when shoots were cultivated under White, Red, and red+white LED\u0026nbsp;(Table 3). Similar results\u0026nbsp;were reported\u0026nbsp;for P uptake, in which there was a higher consumption of P (42.9 to 51.0%) in the absence of BAP in the culture medium.\u0026nbsp;The highest percentage of consumption of Mg (20.6-23.2%) occurred under red light, regardless of the presence of BAP, and under the red+white LED in the presence of BAP.\u003c/p\u003e\n\u003cp\u003eThe principal component analysis (PCA) showed\u0026nbsp;some new and interesting information about how factors such as BAP and light wavelength affected several variables analyzed (Figure 2). Among these, the increase in stomatal density, number of shoots, and fresh and dry mass in the presence of BAP, while the increase in chlorophyll contents, shoot length, and absorption of Ca and P was observed in\u0026nbsp;the absence of BAP in the culture medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of BAP and\u003c/strong\u003e \u003cstrong\u003ewavelength on nutrient contents in\u0026nbsp;\u003cem\u003eMelaleuca\u003c/em\u003e shoot tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of BAP in the culture medium had a significant effect on seven out of the ten nutrients analyzed in plant tissues cultivated in vitro (Table 4). The concentrations of P, S, Mn, Cu, and B were lower in tissues cultivated in the presence of BAP in the culture medium, and the Fe concentration was higher in the presence of BAP. Regarding the wavelengths tested,\u0026nbsp;the dark condition\u0026nbsp;was the only treatment in which the presence of BAP promoted greater uptake of all analyzed nutrients than the medium\u0026nbsp;in the absence of\u0026nbsp;BAP. Under the light that resulted in better growth of \u003cem\u003eMelaleuca\u003c/em\u003e (white and white+red), except Fe, all other nutrients had lower contents in the tissues when BAP was added to culture media.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eEffects of BAP and wavelengths onin vitro\u003c/strong\u003e\u003cstrong\u003edevelopment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof \u003cem\u003eMelaleuca\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, a multiplication factor of 5.6\u0026nbsp;- 8.1 shoots/nodal segment was reported with the addition of a\u0026nbsp;low concentration of BAP (0.25 mg L\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;to the culture medium. According\u0026nbsp;to Iiyama and Cardoso (2021), the addition of BAP at a low concentration (0.55 \u0026mu;M = 0.124 mg L\u003csup\u003e-1\u003c/sup\u003e) causes a considerable increase in shoot proliferation, up to 6.54 shoots/explant. Similar results were reported by Oliveira et al. (2010), with the highest proliferation of shoots (5.6 shoots/nodal segment) obtained with the addition of 0.55 \u0026mu;M of BAP to the\u0026nbsp;MS culture medium.\u0026nbsp;The main physiological effect of BAP on the in vitro cultivation of \u003cem\u003eMelaleuca\u003c/em\u003e was the promotion of multiple shoots in vitro (Iiyama and Cardoso, 2021)\u0026nbsp;by increasing the development of lateral buds and the proliferation of new shoots (Muleo and Morini, 2006). However, BAP also\u0026nbsp;caused other negative\u0026nbsp;effects on the in vitro\u0026nbsp;development of \u003cem\u003eMelaleuca\u003c/em\u003e, such as the strong inhibition of root formation and a considerable decrease in the\u0026nbsp;length and height\u0026nbsp;of shoots (Figure 1). These data are similar to those reported by other authors on the in vitro\u0026nbsp;cultivation of \u003cem\u003eMelaleuca\u003c/em\u003e (Oliveira et al. 2010) and other woody species (Bennett et al. \u0026nbsp;1994; Lin et al. 2013). In fact, cytokinins\u0026nbsp;like BAP\u0026nbsp;are negative regulators of root growth (Nehnevajova et al. 2019). However, it is important to note that more recent studies have shown that although this inhibition occurs in the presence of BAP, shoots from a medium with BAP, when transferred to a rooting medium (absence of BAP), have exhibited better performance and development of roots than those coming from the culture medium without BAP (Iiyama and Cardoso, 2021). Fresh and dry mass showed a strong correlation with the increases in the number of shoots promoted by BAP addition to the media (8.1 shoots/explant) (Figure 2). In different species, this same correlation was detected between fresh and dry mass (FM and DM) with\u0026nbsp;the increased number of shoots/explants (Bello-Bello et al. 2016; Gupta and Jatothu, 2013; Kim et al.,2004; Sarropoulou et al. 2022).\u0026nbsp;Another interesting effect of BAP in culture media is the maintenance of live and growth of shoots since under dark conditions, while in the absence of this cytokinin, the plantlets died before completing 30-d of cultivation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChlorophyll analysis showed a higher level of chlorophyll a and total (a+b) in the shoots obtained from plantlets cultivated in the absence of BAP, regardless of the light wavelength.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs for the effects of light wavelength, there were no differences in chlorophyll concentrations in the absence of BAP.\u0026nbsp;According to Lotfi et al. (2019), blue light is relevant for chlorophyll biosynthesis, chloroplast development, and the stomatal opening (Wu et al. 2007), and similar results were obtained by other authors with different plant species (Hern\u0026aacute;ndez and Kubota, 2016; Mar\u0026iacute;n-Martinez and Iglesias-Andreu, 2022). However, there are contrasting results with other species such as bananas, in which the use of white + red and red LED resulted in higher concentrations of chlorophylls than with the use of fluorescent light (Vieira et al. 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConversely, using the same\u0026nbsp;light source, the presence of BAP in the culture medium resulted in a significant reduction in concentrations of chlorophyll a and total\u0026nbsp;but did not influence chlorophyll b.\u0026nbsp;These results are similar to those observed for other species. In \u003cem\u003eNidularium\u003c/em\u003e (Bromeliaceae) in vitro cultivation, increasing concentrations of BAP and other cytokinins in the culture medium resulted in a significant reduction in chlorophyll concentrations in plant tissues (\u0026Ouml;rd\u0026ouml;gh, 2015). In transgenic plants of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, small contents of cytokinins increased the contents of chlorophyll in their shoots (Nehnevajova et al. 2019).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest stomatal density was obtained with the red+white LED combination, followed by red, blue+red, and white LEDs, while in monochromatic blue light, there was a significant reduction in stomatal density. However,\u0026nbsp;He et al.\u0026nbsp;(2020) observed the highest average density of stomata (10.9) in growing\u0026nbsp;\u003cem\u003eCamellia oleifera\u003c/em\u003e under white LED light, and\u0026nbsp;Mihovilović\u0026nbsp;et al. (2020) reported the highest stomatal density\u0026nbsp;(17.3) of\u0026nbsp;\u003cem\u003eAmelanchier alnifolia\u003c/em\u003e when cultivated\u0026nbsp;under blue + red LED. Interestingly, \u003cem\u003eMelaleuca\u003c/em\u003e cultivated in vitro under blue LED light presented the lowest density of stomata, regardless of the presence of BAP. The opposite was observed in \u003cem\u003eCucumber\u0026nbsp;\u003c/em\u003eseedlings, in which the relative reduction in red light and the increase in blue light resulted in the highest stomatal density\u0026nbsp;(Li et al. 2023). Izzo et al. (2021) reported reduced stomatal density both under red or blue monochromatic light, while their combination (red + blue light) increased stomatal density.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder in vitroconditions, the stomata are normally circular and open (Tich\u0026aacute; et al. \u0026nbsp;1999), and these characteristics were also observed\u0026nbsp;in the present\u0026nbsp;study\u0026nbsp;in \u003cem\u003eMelaleuca\u003c/em\u003e shoots. The largest diameter of leaf stomata (26.5 \u0026mu;m and 24.8 \u0026mu;m) was observed under blue or red monochromatic\u0026nbsp;LED lighting, both in the presence of BAP\u0026nbsp;and under white light in the absence of BAP. Similar results were obtained for three species (\u003cem\u003eCordyline australis, Ficus benjamina,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Sinningia speciosa\u003c/em\u003e) by Zheng and Van (2017), who reported that the use of blue or red LED resulted in the largest stomatal diameter. Cioć and Pawlowska (2020) reported that the addition of BAP to the culture medium resulted in longer stomata and, consequently, larger diameters. The present study on\u0026nbsp;\u003cem\u003eMelaleuca\u003c/em\u003e demonstrated that this increase in stomatal diameter in response to the addition of BAP to the culture medium depended on the wavelength, being higher in the blue, red, and red+white LED.\u0026nbsp;These differential responses to light according to the species were the result of several factors that affected stomata development, such as the species group, light intensity, developmental stage, photosynthetic metabolism (Matthews et al. 2020), and other non-related to light such as the osmotic potential of culture media (Iiyama and Cardoso, 2021).\u003c/p\u003e\n\u003cp\u003eA major obstacle with \u003cem\u003eMelaleuca\u003c/em\u003e micropropagation is the acclimatization phase, where \u003cem\u003eMelaleuca\u003c/em\u003e plantlets\u0026rsquo; rapid dehydration is partially attributed to stomatal density, development and functionality (Iiyama and Cardoso, 2021). Thus, the LED lighting\u0026rsquo;s influence on the stomata could be used to diminish the stomatal density and diameter for better adaptation of plantlets for acclimatization. Changes in the in vitro environmental conditions are strongly associated with improvements in the acclimatization stage, and stomata characteristics and functionality are some of the main factors affecting acclimatization success, especially in tree species (Asayesh et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of BAP and wavelength on the culture medium characteristics and changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough in vitro\u0026nbsp;studies have extensively explored the influence of culture media, additives, and plant growth regulators on plant development and propagation, little attention has been dedicated to changes in culture media in response to the development of in vitrotissues. Changes in culture media in response to in vitro cultivation systems are influenced by several factors, such as the system used for in vitro cultivation. Kozai et al. (1991) reported that photoautotrophic conditions (no sugar in culture media and CO\u003csub\u003e2\u003c/sub\u003e enriched environment) promoted the highest uptake of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, and K\u003csup\u003e+\u003c/sup\u003e. In the present study with \u003cem\u003eMelaleuca\u003c/em\u003e, the highest uptake of nutrient ions PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e also\u0026nbsp;seems to have occurred in the absence of BAP in the culture medium, especially when grown under red and red+white LED. The opposite was registered for Mg\u003csup\u003e2+\u003c/sup\u003e, i.e., the presence of BAP resulted in higher uptake of this nutrient\u0026nbsp;under most spectra used. Gerovac\u0026nbsp;et al. (2016) and Schroeter-Zakrzewska and Kleiber (2014) reported the highest calcium and magnesium uptake using red LED.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe widest pH range of culture media after growing \u003cem\u003eMelaleuca\u003c/em\u003e in vitro\u0026nbsp;occurred with the presence of BAP, especially with \u003cem\u003eMelaleuca\u003c/em\u003e shoots cultivated under blue or red monochromatic LED (-1.1 to -1.2)\u0026nbsp;and in the presence of this cytokinin when shoots were cultivated under blue+red LEDs.\u0026nbsp;For electrical conductivity, the response was contrary, and the greatest variations occurred in the absence of BAP added to the culture medium. The smallest variations in pH and EC, compared to the initial medium, were observed in\u0026nbsp;dark conditions\u0026nbsp;and without BAP, demonstrating that the absence of light for \u003cem\u003eMelaleuca\u003c/em\u003e in vitro\u0026nbsp;resulted in limited development of shoots/plantlets and few variations in culture media.\u0026nbsp;Perez-Vazquez et al. (2021) reported that the EC in culture media is negatively correlated with the development of the morr\u0026oacute;n pepper, showing increased shoot growth (fresh and dry mass) with decreasing EC values.\u0026nbsp;However, our findings with \u003cem\u003eMelaleuca\u003c/em\u003e did not demonstrate this correlation between a reduction in EC and a greater accumulation of fresh and dry mass. In other words, other factors related to the type of plant development, such as the formation of plantlets (shoots + roots: absence of BAP) or only shoots (presence of BAP), the higher growth in height of plantlets in vitro (absence of BAP) were associated with increasing uptake of specific nutrients, such as Ca and P, and other components of the culture medium that major contribute to reduction of EC values.\u003c/p\u003e\n\u003cp\u003eThe correlation analysis evidenced that a limited number of variables analyzed in \u003cem\u003eMelaleuca\u003c/em\u003e demonstrated significant correlations. Among these, the increase in the number of shoots induced by BAP was highly correlated with the increase in fresh (r=0.81**) and dry (r=0.85**) mass of shoots and the reduction in shoot height Also, the higher shoot height promoted by the absence of BAP was correlated with increased chlorophyll a (r=0.77*) and total (r=0.77*) concentrations in the leaves. The presence of BAP is often correlated with the reduction of photosynthetic pigments in different species cultivated in vitro (Martins et al. 2020; 2022). For culture media, we observed a correlation between higher values of final pH (small variations in relation to initial pH), the higher uptake of P by the shoots (r=0.71*), and shoot height (r=0.71*), indicating that higher P uptake increased shoot height in \u003cem\u003eMelaleuca\u003c/em\u003e.\u0026nbsp;However, it is not possible to know if this effect is direct or indirect because BAP promotes only shoot induction, and in its absence, shoots and roots were observed in\u0026nbsp;\u003cem\u003eMelaleuca\u003c/em\u003e in vitro (Figure 1).\u003c/p\u003e\n\u003cp\u003eThe Principal Component Analysis (PCA) revealed that the LED-wavelength component (PC1) explained 50.29% of data variance, and the BAP (PC2) component explained 14.56% of data variation (Figure 3). The PCA analysis showed that the number of shoots, and the fresh and dry mass were highly correlated with the presence of BAP in the culture medium, especially under white or red LED.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of BAP and wavelength on the concentration of nutrients in \u003cem\u003eMelaleuca\u003c/em\u003e tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of cytokinin BAP substantially increased the contents of all nutrients in tissues of \u003cem\u003eMelaleuca\u003c/em\u003e in vitro under dark conditions (from +62.9% to +341.6% compared to BAP-free media).\u0026nbsp;Cytokinins like BAP are well-known to delay tissue senescence in the dark\u0026nbsp;by continuing higher transcript levels of Photosystem II-related genes and the integrity of chlorophyll pigment-protein, maintaining the Chl a/b ratios (Talla et al. 2016). The addition\u0026nbsp;of BAP and other cytokinins induces photomorphogenesis under dark\u0026nbsp;conditions (Spiro et al. 2004). Thus, cytokinins applied exogenously can maintain active metabolism and morphogenesis, including nutrient uptake of in vitro plantlets under dark\u0026nbsp;conditions. Indeed, cytokinis can promote nutrient accumulation in tissues under adverse conditions (Prasad 2022).\u003c/p\u003e\n\u003cp\u003eHowever, under light conditions, the addition of BAP resulted in decreased accumulation of nutrients in tissues for most nutrients analyzed, especially phosphorus (P), magnesium (Mg), sulfur (S), manganese (Mn), copper (Cu), and boron (B), compared to tissues cultivated in BAP-free culture media. This is a surprising result since a high amount of fresh and dry mass was observed in the presence of the BAP. Interestingly, other authors report that the increased activity of the cytokinin\u0026nbsp;dehydrogenase enzyme, which leads to cytokinin degradation, resulted in an increment of some nutrients in leaves caused by the enhancement of root system and better scavenging of macro- and micronutrients since cytokinin negatively regulates root growth (Nehnevajova et al. 2019, Prasad 2022). In fact, in\u0026nbsp;our results with \u003cem\u003eMelaleuca\u003c/em\u003e, roots in culture media were only observed in the absence of BAP, which explains the higher accumulation of these nutrients in tissues cultivated without this cytokinin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, the addition of BAP resulted in higher concentrations of iron (Fe) in all samples, regardless of the light wavelength tested. This is the opposite of that observed by S\u0026eacute;gu\u0026eacute;la et al. (2008), who\u0026nbsp;reported that cytokinins negatively regulate the root iron uptake in \u003cem\u003eArabidopsis\u003c/em\u003e. Nevertheless, Nehnevajova et al. (2019) reported that only iron, among all other nutrients analyzed, was reduced (11-18%) in leaves of transgenic lines of oilseed rape plants overexpressing the CKX2 gene, with lower cytokinin content, compared to the wild-type. Thus, these studies and our results with \u003cem\u003eMelaleuca\u003c/em\u003e suggested a more complex mechanism of regulation of iron uptake in plants, in which cytokinins can play an important role in iron uptake and accumulation in tissues.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThere is a\u0026nbsp;strong interaction between the addition of the 6-BAP cytokinin to the culture medium and the LED wavelengths used for the in vitro cultivation of \u003cem\u003eMelaleuca\u003c/em\u003e. The addition of 6-BAP had important effects, increasing the number of shoots/explants and, consequently, fresh and dry mass, reducing shoot height, and significantly decreasing the chlorophyll contents of the plants in vitro. White or White+Red LED is recommended in the growing of \u003cem\u003eMelaleuca\u003c/em\u003e, aiming at its multiplication in vitro with BAP-supplemented media or in rooting and elongation of shoots without BAP supplementation in media. BAP also negatively affected nutrient concentration in plant tissues, except Fe, which substantially increases independently of light wavelength.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACGA contribute with the experimental conduction, data collection and analysis and writing of the manuscript. JCC contribute with the main idea, experimental design, writing, revision and editing of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmelia ZR, Supriyanto, Wulandari AS (2020) Effect of 6-BAP application on shoot production of \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e. IOP Conf. Series: Earth and Environ Sci 528:012063. https://doi.org/10.1088/1755-1315/528/1/012063\u003c/li\u003e\n\u003cli\u003eArgentel LM, Fonseca IR, Garatuza JP, Yapez EG, Gonzalez JA (2017) Effect of salinity on calli of wheat varieties during \u003cem\u003ein vitro \u003c/em\u003eculture. Rev Mex de Cienc Agr\u0026iacute; 8(3):477\u0026ndash;488. https://doi.org/10.29312/remexca.v8i3.25\u003c/li\u003e\n\u003cli\u003eAsayesh ZM, Vahdati K, Aliniaeifard S, Askari N (2017) Enhancement of \u003cem\u003eex vitro\u003c/em\u003e acclimation of walnut plantlets through modification of stomatal characteristics \u003cem\u003ein vitro. \u003c/em\u003eSci Hort 220(16):114-121. https://doi.org/10.1016/j.scienta.2017.03.045\u003c/li\u003e\n\u003cli\u003eBello-Bello JJ, Martinez-Estrada E, Caamal-Velazquez JH, Moralesramos V (2016) Effect of LED light quality on \u003cem\u003ein vitro\u003c/em\u003e shoot proliferation and growth of vanilla (\u003cem\u003eVanilla planifolia\u003c/em\u003e Andrews). Afri J Biotec 15(8):272\u0026ndash;277. https://doi./10.5897/AJB2015.14662\u003c/li\u003e\n\u003cli\u003eBennett IJ, Mccomb JA, Tonkin CM, Mcdavid DAJ (1994) Alternating cytokinins in multiplication media stimulates \u003cem\u003ein vitro \u003c/em\u003eshoot growth and rooting of \u003cem\u003eeucalyptus globulus \u003c/em\u003elabill. Ann Bot 74(1):53\u0026ndash;58. https://doi.org/10.1093/aob/74.1.53\u003c/li\u003e\n\u003cli\u003eBudhiraja SS, Cullum ME, Sioutis SS, Evangelista l, Habanova ST (1999) Biological activity of \u003cem\u003emelaleuca alternifolia \u003c/em\u003e(tea tree) oil component, terpinen-4-ol, in human myelocytic cell line hl-60. J Man Physiol Therap.22(7):447\u0026ndash;453. https://doi.org/10.1016/s0161-4754(99)70033-3\u003c/li\u003e\n\u003cli\u003eCaldefie-chezet F, Fusillier C, Jarde T, Laroye H, Damez, MV, Guillot J (2006) Potential anti-inflammatory effects of \u003cem\u003emelaleuca alternifolia\u003c/em\u003e essential oil on human peripheral blood leukocytes. Phyto Res 20(5):364\u0026ndash;370. https://doi.org/10.1002/ptr.1862\u003c/li\u003e\n\u003cli\u003eCarson CF, Hammer KA, Riley TV (2006) \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 19(1):50\u0026ndash;62. https://10.1128/CMR.19.1.50-62.2006\u003c/li\u003e\n\u003cli\u003eChen B, Li J, Zhang J, Fan H, Wu L, Li Q (2016) Improvement of the tissue culture technique for \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e. 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Front in Plant Sci 8:9\u0026ndash;17.https://doi.org/10.3389/fpls.2017.00917\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Effect of BAP and wavelength on \u003cem\u003ein vitro\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003edevelopment of the melaleuca shoots.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eLight wavelength (\u003c/strong\u003e\u003cstrong\u003espectra\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.778705636743215%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWhite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.745302713987474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDark conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.745302713987474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.745302713987474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.745302713987474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed+White\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.24008350730689%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue+Red\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003cstrong\u003eumber of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e2.5 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.01 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e1.88 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e2.0 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e1.5 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e1.7 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e8.1 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e5.8 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e5.6 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e6.9 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e6.2 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e7 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eNew shoots\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLength\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.93 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.02 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e1.08 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e1.15 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.9 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e1.17 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.48 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.79 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.85 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.63 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.64 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e0.54 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eShoot height\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e7.3 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e2.2 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e5.6 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e5.4 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e5.7 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e4.9 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e1.8 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e2.2 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e2.5 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e2.2 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e1.7 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e1.8 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eFresh\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emass\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of shoots (g/plant)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.08 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.05 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.06 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.06 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e0.06 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.18 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.09 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.11 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.08 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.10 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e0.09 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eDry mass of shoots (g/plant)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.01 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.004 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.007 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.009 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.008 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e0.009 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e0.015 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.007 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.013 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.012 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e0.013 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e0.012 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eChlorophyll a (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e49.17 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e49.27 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e48.33 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e39.17 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e40.17 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e26.17 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e16.12 Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e21.3 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e17.62 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e12.74 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eChlorophyll b (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e20.7 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e23.8 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e20.61 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e18.04 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e18.8 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e18.1 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e12.14 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e19.04 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e12.47 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e10.17 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.9290780141844%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal chlorophyll (a+b) (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e69.87 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e73.08 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e68.95 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e57.21 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e58.97 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.702127659574469%\" valign=\"top\"\u003e\n \u003cp\u003e44.27 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e28.26 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e40.34 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.070921985815604%\" valign=\"top\"\u003e\n \u003cp\u003e30.09 Bab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.943262411347519%\" valign=\"top\"\u003e\n \u003cp\u003e22.91 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent capital letters (colunms) demonstrate a significant difference for the factor absence or presence of BAP and different lowercase letters (lines) shown the differences between the spectrum of light, according to the Duncan test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;0,05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Effect of BAP and wavelength on the density and diameter of the stomata.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.00881834215167%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLight wavelengths\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.70954356846473%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWhite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.502074688796682%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.70954356846473%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.502074688796682%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed+White\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.57676348547718%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue+Red\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.00881834215167%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStomatal density (number of stomata/mm\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e116.6 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e80 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e140 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e173.3 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.34215167548501%\" valign=\"top\"\u003e\n \u003cp\u003e135 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e155 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e81.6 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e115 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e235 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.34215167548501%\" valign=\"top\"\u003e\n \u003cp\u003e163.3 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.00881834215167%\" colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStomatal diameter (\u003c/strong\u003e\u003cstrong\u003e\u0026mu;\u003c/strong\u003e\u003cstrong\u003em)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e22.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e18.4 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e20.2 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e15.1 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.34215167548501%\" valign=\"top\"\u003e\n \u003cp\u003e18.3 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.991181657848324%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e18.4 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e26.5 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.75485008818342%\" valign=\"top\"\u003e\n \u003cp\u003e24.8 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\"\u003e\n \u003cp\u003e18.7 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.34215167548501%\" valign=\"top\"\u003e\n \u003cp\u003e17 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent capital letters (colunms) demonstrate a significant difference for the factor absence or presence of BAP and different lowercase letters (lines) shown the differences between the spectrum of light, according to the Duncan test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;0,05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e Effects of BAP and wavelength on the final culture media pH, EC and the macronutrients (Ca, Mg and P) uptake from the culture media.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLight wavelengths\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.335766423357665%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWhite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.510948905109489%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDark condition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.153284671532848%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.335766423357665%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.153284671532848%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed+White\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.510948905109489%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlue+Red\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003epH final (pH final \u0026ndash; initial)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e4.9(-0.4) AaB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e5.0(-0.3) Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e4.5(-0.8) Abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e5.0(-0.4) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e4.7(-0.7) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e4.2(-1.2) Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e4.6(-0.8) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e4.7(-0.7) Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e4.3(-1.1) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e4.2(-1,2) Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e4.4(-1.0) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e4.7(-0.8) Aab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEC final - initial (mS/cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e-1.13 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e-0.25 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e-0.73 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e-0.43 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e-0.28 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e-0.25 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e-0.26 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e-0.21 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e-0.91 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e-0.14 Bb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e-0.30 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e-0.26 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCa (% consumed from the media)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e44.5 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e40.2 Ad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e39.5 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e63.9 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e68.9 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e40.5 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e21.4 Be\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e44.9 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e55.9 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e33.6 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e44.9 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e50.4 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP (% consumed from the media)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e46.9 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e44.9 Ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e30.6 Be\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e42.9 Ad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e51.0 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e24.5 Af\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e41.3 Ba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e13.0 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e37.0 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e5.4 Be\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e10.9 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e13.0 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"87.82051282051282%\" colspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg (% consumed from the media)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e17.5 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e14.3 Acd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e8.3 Be\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e23.2 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e12.3 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e15.4 Abc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.179487179487179%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e12.4 Bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e0.0 Bd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e16.4 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.224358974358974%\" valign=\"top\"\u003e\n \u003cp\u003e21.7 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.064102564102564%\" valign=\"top\"\u003e\n \u003cp\u003e20.6 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.621794871794872%\" valign=\"top\"\u003e\n \u003cp\u003e16.8 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent capital letters demonstrate a significant difference for the factor, presence and absence of BAP in the culture medium. Lowercase letters show the differences between the spectrum of light, according to the Duncan test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;0,05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003c/strong\u003e Percentage of increase or decrease - in response to the addition of BAP - of nutrient contents in \u003cem\u003ein vitro\u003c/em\u003e tissues of shoots of \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e cultivated under different LED wavelengths\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"608\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.460526315789474%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eLED wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.53947368421052%\" colspan=\"10\"\u003e\n \u003cp\u003e\u003cstrong\u003eNutrients in the shoot tissues (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.023483365949119%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.784735812133073%\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.784735812133073%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.784735812133073%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.741682974559687%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.154598825831702%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.154598825831702%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.219178082191782%\"\u003e\n 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width=\"9.421487603305785%\"\u003e\n \u003cp\u003e+21.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.421487603305785%\"\u003e\n \u003cp\u003e-34.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.9421487603305785%\"\u003e\n \u003cp\u003e-33.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.59504132231405%\"\u003e\n \u003cp\u003e-2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.59504132231405%\"\u003e\n \u003cp\u003e-29.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.537190082644628%\"\u003e\n \u003cp\u003eDark conditions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.776859504132231%\"\u003e\n \u003cp\u003e+97.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.264462809917354%\"\u003e\n \u003cp\u003e+106.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.264462809917354%\"\u003e\n \u003cp\u003e+328.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.264462809917354%\"\u003e\n 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width=\"8.59504132231405%\"\u003e\n \u003cp\u003e+18.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.59504132231405%\"\u003e\n \u003cp\u003e-39.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNegative number means the decrease and positive number means the increase in percentage of nutrient contents in response to addition of BAP to the culture media, compared with the BAP-free culture media.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"micropropagation, tea tree, light emitting diodes, cytokinin, development, nutrition, chlorophyll","lastPublishedDoi":"10.21203/rs.3.rs-4249082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4249082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In vitro growing of Melaleuca can contribute to the cloning of superior genotypes. Studies on factors affecting micropropagation are necessary, especially those using recent technologies, such as light-emitting diodes (LEDs). This study aimed at better understanding the effects of wavelengths on the development and physiology of Melaleuca cultivated in vitro, as well as the interaction of LED with N6-Benzylaminopurine (BAP), the main cytokinin used in micropropagation. The BAP and wavelengths, and their interaction, had a strong influence on most variables analyzed, altering the in vitro development and chlorophyll concentrations in the plants and changing the different variables of the culture medium, such as pH, EC, levels of Ca2+, Mg2+ and P in culture media and the nutrient accumulation in the shoots. The results demonstrated that the main effects of adding BAP in the in vitro cultivation of Melaleuca are the increase in the number of shoots, which resulted in a greater increase in fresh and dry mass, reduction in height and chlorophyll contents, complete inhibition of adventitious rooting, higher consumption of Mg and lower consumption of Ca and P from the culture media, higher contents of Fe and lower contents of P, S, Mn, Cu and B in the in vitro tissues.","manuscriptTitle":"Development, chlorophyll content, and nutrient accumulation in shoots of Melaleuca alternifolia in vitro under different light wavelengths and benzylaminopurine (BAP)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 18:56:35","doi":"10.21203/rs.3.rs-4249082/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f239bc7-6e73-45f0-85df-f1df52bbb12e","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-06T21:04:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-17 18:56:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4249082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4249082","identity":"rs-4249082","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}