Introduction

Sugarcane (Saccharum officinarum L.), Poaceae, is a monocotyledonous species grown as a crop in tropical and subtropical areas, which provides the world with approximately 80% of its sugar (sucrose), and 35% of the total ethanol production (Redae and Ambaye 2018). Sugarcane is one of the most efficient plants in the utilization and conversion of solar energy into sugar and other renewable forms of energy and is primarily cultivated for its ability to store easily extractable, high sucrose concentrations in the stem internodes (Tolera et al.2014).

The high level of genetic diversity in sugarcane can be used to develop improved cultivars. Plant tissue culture is a common technique used as a tool in genetic improvement programs. Thus, in vitro cultivation represents an important strategy for large-scale mass clonal propagation of sugarcane, whereby mass production of clonal superior genotypes can be achieved with minimal space and time required. The improvement of physiological and health characteristics of in vitro-derived plants is another benefit, such as an increase in crop yield from 10 to 30% and cane longevity by 30% (Lee et al.2007).

The optimization of the microenvironment inside in vitro culture vessels can also provide additional physiological benefits. Modifications, such as an increase in gas exchange, can contribute to the plant metabolism and help maintain an optimal CO2 concentration, which may also stimulate photoautotrophism, prevent elevated ethylene concentrations and moderate the relative humidity inside culture vessels (Kozai 2010).

Light availability is another key abiotic factor for performance of in vitro plants (Shin et al.2013). Light acts as a source of energy, but is also involved in plant growth modulation. Generally, in in vitro environments, the photon flux density (PFD) used is close to or even below the light compensation point for photosynthesis (Desjardins 1994). Under such conditions, the carbon fixation is lower than the losses due to respiration and photorespiration. In most in vitro propagation systems, the usual PFD is around 40 μmol m−2 s−1 (Singh et al.2017), which is ten times lower than the light levels in ex vitro environments, and a level of light that correlates to natural shading. In a lower energy environment, which includes lower ATP synthesis capacity and lower reducing power (NADPH), CO2 fixation and lower trioses-phosphate reduction in the Calvin Cycle, the biomass accumulation is reduced. The low capacity for carbon fixation tends to be reflected during acclimatization, with an additional likelihood towards photoinhibition when the photoprotection mechanisms are underdeveloped (Sáez et al.2016).

Sugarcane is a plant with a C4 metabolic cycle, which has inherently high compensation and saturation points. In ex vitro conditions, the photosynthetic rate increases until 2000 μmol m−2 s−1 (Sage et al.2013). By providing adequate light and inorganic CO2 levels, there is the potential to increase carbon fixation efficiency in the in vitro environment. The objectives of this study were to evaluate biometric, anatomical and physiological conditions of in vitro propagated sugarcane plantlets using different light intensities and culture vessel types, to stimulate desired photoautotrophic and/or photomixotrophic traits, and consequently increase survival and ex vitro vigor.

Materials and methods

Plant material

Seven-d-old in vitro regenerated shoots isolated from sugarcane meristems (cv CP 96-1252) were transferred to RA40 Microbox plastic containers (SacO2®, Nevele, Belgium) containing Murashige and Skoog (MS) medium (Murashige and Skoog 1962), supplemented with 2% (w/v) sucrose, 0.3 mg L−1 BAP (Benzylaminopurine), and 50 mg L−1 citric acid, and solidified with 3 g L−1 of Phytagel™ (multiplication medium). Approximately 10 mL of culture medium was added to each of the containers, and the explants were cultured for 21 d at the Florida Crystals Biofactory (Belle Glade, Fl) in January 2018. Next, the explants were transported to the University of Florida’s Tropical Research and Education Center (Homestead, Fl) to conduct the experiments.

Root induction using different light intensities and culture vessel types

To optimize the explant rooting and the desired photomixotrophic traits under in vitro conditions, explants with two shoots each were cut into approximately 2-cm-long pieces after 21 d in the multiplication medium and transferred to rooting medium composed of MS medium supplemented with 4% (w/v) sucrose, 1.3 mg L−1 IAA (Indole-3-acetic acid) and 50 mg L−1 citric acid, solidified with 3 g L−1 Phytagel™. Three different types of culture vessels were evaluated, including the RDA60 Microbox, standard model type with no filter, the OV80/OVD80 Microbox, model type with an air exchange filter (7.44 gas exchanges per d) and baby food jars with non-vented polypropylene covers (Sigma-Adrich®, St. Louis MO). Approximately 10 mL of culture media were dispensed per container. The culture vessels were maintained at either 70, 120, or 200 ± 25 μmol m−2 s−1 light level in a growth room with a 12-h photoperiod at 26 ± 2°C. The light sources selected included light-emitting diodes (Philips GreenPower Red/Blue 150 43W and Philips GP DR/W 150 33W, Pila, Poland, respectively), for 70 and 120 μmol m−2 s−1 levels, and fluorescent lamps (OSRAM Sylvania Inc., Wilmington, MA), for the 200 μmol m−2 s−1 level.

Growth assessments

The final data collection was made after 28 d of culture using the growth conditions described. The recorded data included shoot height (cm), leaf number, yellow leaf percentage, shoot and root number and root length. In addition, fresh and dry weight were evaluated. To determine shoot (SDW) and root dry weight (RDW), the plant material was oven dried at 60°C for 72 h. Leaf yellowing was evaluated, and when at least 75% or more of the leaf surface area was yellow, those leaves were considered as a yellow leaf.

Histological and anatomical analyses

Growth and anatomical measurements were evaluated after 28-d cultivation. Samples of leaf tissues from ten plants were fixed in FAA 70% (v/v) (formaldehyde-glacial acetic acid-70% ethyl alcohol), for 72 h and later preserved in 70% ethanol (v/v). Leaf cross sections were obtained by hand using a steel blade and were subjected to clarification with sodium hypochlorite (1.00 to 1.25%, v/v active chlorine), followed by three washes in distilled water, and staining with safrablau solution (0.1% w/v, astra blue and 1% w/v, safranin) on glass slides. For paradermal and cross leaf sections, after safrablau staining, all glass slides were mounted on semi-permanent slides with glycerinated water. The slides were observed under a light microscope (Leica DMLB, Wetzlar, Germany) and photographed using the SPOT 4.7 version software. The images were analyzed in Image Pro Plus image analysis software, with the measurement of six fields per repetition for each analyzed variable.

The parenchyma thickness and the adaxial and abaxial epidermis were determined in cross sections of the leaf. For stomata density (number of stomata per mm2) on adaxial and abaxial faces, the polar diameter (PD), the equatorial diameter (ED) and the PD/ED ratio were evaluated, using the printing technique on the second youngest leaf, as suggested by Ferreira et al. (2017).

Chlorophyll content

Expanded leaves were used to evaluate the chlorophyll content. Leaves were cut into squares measuring 1 cm2, which were then placed into test tubes with lids containing 10 mL of 80% acetone (v/v), and stored for 24 h in a refrigerator at 4°C, protected from light. Extracts were then filtered, and 2 μL of the solution was placed in a ND-2000 NanoDrop spectrophotometer (Thermo Fisher Scientific®, Waltham, MA). The reference sample (blank) consisted of 80% acetone (v/v) solution. The absorbance readings were performed in the ND-2000 NanoDrop at 645, 652, and 663 nm, and chlorophyll a, b and total chlorophyll content were calculated using the obtained readings (Witham and Blaydes 1971; Costa et al.2018). All results were expressed as μg per gram fresh weight of leaf tissue (μg g−1). Relative chlorophyll index (RCI) was determined as suggested by Costa et al. (2018), using a portable SPAD-502 chlorophyll meter (Minolta Co., Osaka, Japan). The device was calibrated using the standard before analysis, and readings were performed on the second youngest leaf.

Plantlet acclimatization

After 28 d under in vitro conditions, the plantlets were thoroughly washed in running tap water to remove any media fragments and moved to ex vitro conditions using 75-mm (three inch) square plastic pots with Pro-Mix BX Biofungicide + Mycorrhizae, covered with a clear 1-L plastic bag for 21 d, and grown under a shade net in a greenhouse with manual irrigation. After 7 d, a 45-degree cut was made 2.5 cm from an upper corner of the plastic bag, and after 14 d, another cut was made across the opposing corner of the bag, and finally after 21 d, the plastic bag was removed. Survival was evaluated after a total of 28 d. From each container, five plantlets were acclimatized to ambient humidity and light intensity conditions. After 60 d, the biometric and chlorophyll analysis was repeated.

Experimental design and statistical analysis

The experimental design was completely randomized, in a factorial arrangement using three light intensities × three culture vessel types, with 20 explants per condition, ten explants per microbox and two explants per baby food jar with 10 mL of medium per culture vessel. The data were normalized by Shapiro-Wilk test at 5% probability and submitted to analysis of variance at 5% probability. Percentage data were transformed to Arc Sin\( \sqrt{x} \). Means were separated by Tukey’s test at 5% probability.

Results

Growth characteristics of in vitro sugarcane plantlets

There was non-factor interaction (p value > 0.05) verified for growth assessments. Multiplication and rooting success were found to be independent in sugarcane plantlets (CP 96-1252), at the tested light intensities and culture vessels used in this study. No differences between the studied factors of light intensity and culture vessel were observed for shoot height and root length. An isolated effect of light intensity was verified for leaf and shoot number in addition to leaf yellowing percentage. Ambiguous behavior at 120 and 70 μmol m−2 s−1 was verified for these variables (leaf and shoot number and leaf yellowing).

Sugarcane plantlets cultured in a microbox with the air filter lid at 200 μmol m−2 s−1 showed the lowest leaf yellowing percentage. There were two shoots that measured less than 2 cm per explant in all treatments. At the end of the study, the explants continued to produce new shoots, which produced six or more leaves each (Table 1).

Table 1. Growth assessments of Saccharum officinarum L. plantlets (CP 96-1252) in different light intensities and culture vessels after 28 d in the rooting phase
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Factor interaction was not observed for SDW (p value > 0.05). However, an isolated effect on culture vessels (p value < 0.05) was observed. The microbox with or without an air filter lid resulted in plants with higher SDW, as shown in Table 2. This result indicates that microbox culture vessels with/or without air filter lids should be preferred over traditional baby food jars for the micropropagation of sugarcane. In this study, although the same amount of medium was used for all culture vessels, the time required for the manipulation of the cultures, such as pouring media and transferring material both in and out of the vessel are extremely different, and much greater for baby food jars. This shows an advantage of the use of the microbox plastic culture vessel. For RDW, an interaction between the factors was observed (p value < 0.05), and therefore, the factors were studied together (Table 2). No significant differences in RDW were observed for light intensities for cultures in baby food jars. In microboxes, however, light intensity below 120 μmol m−2 s−1 increased RDW.

Table 2. Shoot (SDW) and root (RDW) dry weight in Saccharum officinarum L. plantlets (CP 96-1252), under different light intensities and culture vessels after 28 d in the rooting phase
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For chlorophyll content evaluation, interactions between factors were observed (p value < 0.05), and therefore, both factors were studied together. For chlorophyll-a content, the highest content was verified when explants were grown in a microbox under both 70 and 200 μmol m−2 s−1. For the microbox with the air filter lid and baby food jar, the highest light intensity promoted the highest chlorophyll content. For chlorophyll b content analysis, the highest value was found using baby food jars under low intensity light followed by microbox vessels. For total chlorophyll, the highest values were found in leaves from plants grown in microbox vessels using a light intensity of 120 and 200 μmol m−2 s−1 (Table 3).

Table 3. Chlorophyll content in Saccharum officinarum L. plantlets (CP 96-1252), in different light intensities and culture vessels after 28 d in the rooting phase
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Anatomical characteristics of in vitro sugarcane plantlets

All anatomic evaluations showed interactions among the studied factors (p value < 0.05). High light intensity affected the anatomic structure in sugarcane leaves, including both adaxial and abaxial sides, and also in the mesophyll and bulliform cell number (Table 4). Increased values for the epidermis thickness on the adaxial and abaxial leaf surfaces were observed in plants grown in microbox vessels with an air exchange filter under 120 μmol m−2 s−1.

Table 4. Anatomical assessments in Saccharum officinarum L. plantlets (CP 96-1252), in different light intensities and culture vessels after 28 d in the rooting phase
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A strong and significant positive correlation was observed between adaxial epidermis, parenchyma, and mesophyll length in this study, which was similar to bulliform cell number and parenchyma (Table 5). The increased number of bulliform cells resulted in increased leaf thickness (Fig. 1). These correlation analyses helped to gain a better understanding of leaf thickness in sugarcane leaves propagated using different light intensities and culture vessels, such as water stress mitigation.

Table 5. Pearson’s correlation between anatomical assessment in Saccharum officinarum L. leaves under 120 μM m2 s−1 using microbox vessels with air exchange filters after 28 d in rooting media
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Figure 1.
figure 1

Leaf cross section photomicrograph in Saccharum officinarum L. cv 96-1252, cultivated under different light intensities and culture vessels. Microbox without filter, microbox with filter, baby food jar under 70 μmol m−2 s−1 (a, b and c, respectively); 120 μmol m−2 s−1 (d, e and f, respectively), and 200 μmol m−2 s−1 (g, h and i, respectively). Adaxial epidermis (de), abaxial epidermis (be), bulliform cells (bc) and parenchyma (p) (bar = 50 μm).

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Sugarcane is considered to be amphestomatic and shows stomata both in the epidermis of the abaxial and adaxial surfaces, but in greater density in the abaxial surface (Castro et al.2009). For stomatal analysis, an interaction (p value < 0.05) among the factors studied was observed for equatorial diameter (Fig. 2b), ratio between polar and equatorial diameter (Fig. 2c) and stomatal density (Fig. 2e). For stomatal functionality, based upon the ratio between stomatal diameters (polar diameter/equatorial diameter), a higher ratio between stomatal diameters was correlated with an elliptic stomata shape (small stomatal gap). In contrast, a smaller ratio suggests a spherical stomata shape.

Figure 2.
figure 2

Abaxial stomatal analysis of in vitro Saccharum officinarum L. plantlets under different light intensities and culture flasks. Bar = standard error. Means followed by the same letter lowercase in the light intensity, and uppercase in culture flasks indicates no difference according to Tukey’s test at 5% probability. PD = polar diameter. ED = equatorial diameter.

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In this study, similar values obtained for stomatal functionality were observed in leaves at both 70 and 200 μmol m−2 s−1, when grown in microbox vessels with or without air exchange filters. Plants in baby food jars showed a higher stomatal ratio at 120 and 200 μmol m−2 s−1 (Fig. 2). For polar diameter and stomatal number, there were no significant differences between light intensity and culture flasks (p value > 0.05, Fig. 2a, d, respectively). Sugarcane cultivated in vitro using high light intensity conditions and culture vessels designed to allow a high rate of gas exchange between the flask interior and exterior can optimize the desired anatomic features in young plants.

After 28 d of in vitro culture under different light intensities and culture vessels, all plants were acclimatized in plastic containers covered with 75-mm3 clear plastic. All sugarcane in vitro plantlets produced were successfully transferred for acclimatization without any abnormalities or mortality. Growth assessments, particularly the determination of shoot fresh and dry weight after 60 d in greenhouse conditions, showed that in vitro-derived plantlets that originated from microbox vessels with or without air exchange filters had the highest growth values in both in vitro and ex vitro conditions. This was particularly evident for in vitro plantlets cultivated under high light intensities of 120 and 200 μmol m−2 s−1 (Table 6).

Table 6. Growth assessments in Saccharum officinarum L. plantlets (CP 96-1252) at 60 d in ex vitro conditions derived from different in vitro light intensities and culture vessels in the rooting phase
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For improved survival and vigor in ex vitro sugarcane plantlets, it is essential that the entire photosynthetic apparatus is well developed and working properly, which can be observed and confirmed by an increase in total dry weight during in vitro conditions. In this study, plants grown in microbox vessels with air exchange filter lids were taller with functional stomata when cultivated under high light intensity. Consequently, these plants had the highest values of SFW and SDW (Table 7). These results have practical implications, as the sugarcane industry can implement such in vitro production protocols to produce healthier and more vigorous plants with improved anatomical and physiological qualities.

Table 7. Shoot fresh (SFW) and dry weight (SDW) in Saccharum officinarum L. plantlets (CP 96-1252) at 60 d in ex vitro conditions derived from different in vitro light intensities and culture vessels in the rooting phase
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Discussion

Light can modulate plant growth and interact with physiological and morphological processes, depending on the species (Simlat et al.2016). In all in vitro conditions, sugarcane plantlets showed aggressive growth, which included sprouting new shoots, leaves, and roots. Therefore, the leaf yellowing percentage was very important and essential to detect the most effective light intensity and culture vessel type for sugarcane plantlet growth. Yellow leaves can be indicative of an ethylene accumulation, which can increase plantlet mortality during acclimatization, and therefore should be avoided (Catada 2015). Although a high percentage of leaf yellowing was observed, hyperhydricity was not observed in the experimental treatments tested. Hyperhydricity is an anomaly which can occur in closed systems, due to the presence of high humidity and excessive plant growth regulator levels (Silva et al.2013). Lack of hyperhydricity is a positive indication of growth. Although no significant differences in RDW were observed for light intensities for cultures in baby food jars, in the microboxes, light intensity below 120 μmol m−2 s−1 increased RDW. Similarly, Ferreira et al. (2017) showed that lower RDW was observed for sugarcane rooting when fluorescent lamps (50 μmol m−2 s−1) were used compared to LED lamps (70 μmol m−2 s−1).

Different light intensities provided variations in the thickness of the foliar tissues, improved the capacity to use the light for photosynthesis, and result in higher survival during acclimatization. Chlorophyll levels in this study were lower than those observed in studies by Maluta et al. (2013) and Ferreira et al. (2017). It is possible that thylakoid membranes were destroyed under high light intensity, as described in similar study by Maluta et al. (2013). The thylakoid membrane destruction is believed to occur when there is an excessive level of light that exceeds the plant’s photosynthetic capacity (Maluta et al.2013). It would appear that there is not a correlation between thylakoid membrane destruction and ex vitro plant vigor. For example, Ferreira et al. (2017) did not see any significant differences in their acclimatized plants, which is similar to the observations in this study. Although a difference in the photosynthetic pigment level was observed between light intensity and culture vessels, a high survival percentage was obtained from all tested in vitro conditions. Additional studies that correlate plant physiology conditions with actual field conditions should be conducted in the future, as a decrease in yield under field conditions could occur, despite the lack of differences in acclimatized plantlets observed in this study.

The induction of photoautotrophic and/or photomixotrophic behavior of in vitro plants to improve their survival ex vitro has been investigated, including techniques such as flask sealing methods and sucrose levels (Iarema et al.2012), CO2 enrichment (Saldanha et al.2013), and the use of bioreactors to improve gas exchange (Martínez-Estrada et al.2017). In this study, the three different culture vessels were found to promote varying amounts of gas exchange with impacts on ethylene accumulation, and the corresponding damage observed in leaf anatomy.

The increase in light intensity proportionally increases leaf thickness, leaf mass, epidermis and parenchyma thickness, and total leaf cell number (Dickison 2000). However, Gondim et al. (2008) observed an increase in epidermis thickness on both sides of the leaf of Colocasia esculenta (L.) Schott, as the shade intensity increased, in contrast to other tissues analyzed. The plasticity of epidermis physiology is related to the level of light (or shade) intensity, which can cause the epidermis thickness to either increase (Morais et al.2004) or decrease (Gratani et al.2006). The thicker epidermis as observed in this study offers advantages for in vitro-propagated plants, which includes a higher chance of survival during transfer to an ex vitro environment, an increased tolerance to stresses resulting from a shifting cultivation environment, and improvement in acclimatization (Asmar et al.2015). In addition, increased leaf thickness can help prevent water loss under high radiation conditions (Figueiredo et al.2015).

Souza et al. (2013) reported that there are many different cell types or tissues in sugarcane leaves, and more than those found in sugarcane culm. In this study, large amounts of bulliform cells in sugarcane leaves were observed in plants grown under 120 μmol m−2 s−1 in the microbox vessels with an air exchange filter, with an estimate of approximately 13 bulliform cells in the area measured. This is the first report of bulliform cell numbers in sugarcane leaves using different light intensities. The number of bulliform cells changed significantly using different light intensities. In sugarcane leaves, the bulliform cell area can represent almost 30% in cross sections (Ferreira et al.2007), as showed in this study.

In this study, sugarcane leaves displayed a monostratified epidermis, whereby the mesophyll is undifferentiated in palisade and spongy parenchyma, and bulliform cells were observed in all treatments (Fig. 1). Bulliform cells are fan-shaped, different sizes, and observed only on the adaxial side of the epidermis. In addition, vascular bundles are surrounded by a single vascular parenchyma, as reported by Castro et al. (2009).

Bulliform cells are highly vacuolated large cells that are present in monocotyledons (except in the order Helobiae). They are also known as “motor cells” because in drought conditions, they lose turgor pressure, and decrease in size, which causes winding leaves. When water levels become sufficient for the plants, these cells expand, and the leaves open again (Alvarez et al.2008).

A higher PD/ED ratio was observed in this study when sugarcane plantlets were cultivated in the microbox vessels with an air exchange filter under 70 and 120 μmol m−2 s−1, and as a consequence, the stomata were elliptical shaped. This elliptical shape enhances its functionality (Sha Valli Khan et al. 2002), which includes higher CO2 uptake, increased photosynthetic potential, and therefore, a reduced transpiration rate and increased water use efficiency (Castro et al.2009). The greater ratio between stomatal diameters suggests that the most efficient water use was obtained.

Conclusions

By increasing the traditional in vitro cultivation light intensity from around 70 to 200 μmol m−2 s−1, plant height and leaf number per plantlet increased without any negative effects on plant quality and vigor. Consequently, the most efficient culture vessel for sugarcane in vitro production was the microbox type of vessel, with or without an air exchange filter lid. This ensured the production of healthy in vitro plants with desired anatomical and physiological qualities and ex vitro plantlets with improved growth characteristics. The change from a traditional culture vessel (baby food jars), to an alternative vessel design (microbox vessels), associated with increased light intensity allowed the efficient propagation of sugarcane in vitro with improved photoautotrophic and photomixotrophic traits and improved ex vitro plantlet characteristics.