A total of 50 mL of medium was added to baby food jars, which were then autoclaved at 121°C and 20 psi for 20 minutes. In vitro banana plantlets, approximately 5 cm in height, had their leaves and roots trimmed to 2 cm, with the remaining shoot-tips used as explants. The jars, equipped with polypropylene lids, were sealed with parafilm and placed in a growth chamber under controlled conditions: a temperature of 27 ± 2°C and a 16-hour light period with 100 μmol m⁻²s⁻¹ photosynthetic photon flux density (PPFD). Every four weeks, in vitro shoots were subcultured onto fresh MS medium.
2.2. Light Sources
Three light sources were tested: two types of LED lighting—LED-1 at 116 μmol m⁻²s⁻¹ (Philips GreenPower DR/B 3:1 150 43W) and LED-2 at 90 μmol m⁻²s⁻¹ (Philips GreenPower DR/W 3:1 150 33W)—and one fluorescent lighting system (Philips 9A fluorescent bulbs 40W) at 100 μmol m⁻²s⁻¹ (FL). The photoperiod for all light treatments was 16 hours of light followed by 8 hours of darkness. The spectral energy distributions of the light sources are presented in Figure 1. LED-1 displayed peak emissions at 440 nm and 650 nm (Figure 1A), while LED-2 peaked at 440 nm and 670 nm (Figure 1B). Fluorescent light (FL) exhibited a broader spectrum, with peaks in the green (550 nm), blue (440 nm), and additional peaks in the 490 nm, 590 nm, 610 nm, and 710 nm ranges (Figure 1C). Light intensity and composition were measured using an LI-180 Li-Cor spectrometer.
2.3. In Vitro Growth and Development
Four weeks after establishment, explants from all treatments were assessed for various growth parameters, including shoot length, root length and number, plantlet fresh and dry weight, shoot fresh and dry weight, and root fresh and dry weight. Dry weight was measured after oven-drying the plantlets at 70°C until they reached a constant weight. Five randomly selected plants per treatment were used for each measurement.
2.4. Relative Chlorophyll Content
The relative chlorophyll content of plantlets was measured using a portable SPAD-502 chlorophyll meter (Minolta Co., Ltd., Tokyo, Japan), with readings taken from the third expanded leaf of each plantlet, counting from the top down. Five random plantlets per treatment were selected for this analysis.
2.5. Stomatal Analysis
The middle third of the third and fourth fully expanded leaves were cut into approximately 1 cm² sections. Leaf impressions were obtained by placing the leaf sections on a thin layer of superglue (Elmer’s Products, Inc., Westerville, OH, USA) spread on microscope slides, and then removing the leaf after the glue dried. Impressions were made for both the adaxial and abaxial surfaces of the leaves. Stomatal observations were conducted under a Leica DMLB microscope (Leica Microsystems, Buffalo, NY, USA) at 200× magnification. Images were captured using a SPOT 4.7 digital camera and analyzed with SPOT basic software (SPOT Imaging, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The number of stomata was counted on both the adaxial and abaxial surfaces, with three 5 mm² areas selected for each treatment. Results were expressed as the average number of stomata per mm² across the three areas.