2.5. Stomatal Measurements
The length and width of three randomly chosen stomata were measured on both the adaxial and abaxial surfaces of each leaf, across three distinct sections of the leaf. A total of five randomly selected plantlets were used per treatment.
2.6. Anatomical Observations
Anatomical analysis was performed on the middle-third section of the second fully developed leaf. Cross-sections were made using a steel blade for freehand sectioning, followed by fixation in 70% FAA (formaldehyde-acetic acid-ethyl alcohol 70%) for 48 hours. The samples were then preserved in 70% ethanol (v/v). After decolorization using sodium hypochlorite (1–1.25% active chlorine), followed by triple rinsing in distilled water and staining with toluidine blue (0.05% w/v), the leaf sections were mounted on semi-permanent slides with glycerinated water. The slides were examined and photographed using a Leica DMLB microscope with an SPOT 4.7 digital camera and software. The analysis involved evaluating five fields per replicate for each measured variable. The following epidermal and parenchymal tissues were assessed for thickness: abaxial surface epidermis (ASE), adaxial epidermis (ABE), abaxial hypodermis (AH), adaxial hypodermis (AbH), palisade parenchyma (PP), and spongy parenchyma (SP). A total of five randomly selected plants were used per treatment.
2.7. Experimental Design and Statistical Analysis
A completely randomized experimental design was employed, consisting of six treatments (three light sources × two banana varieties), with five replications per treatment. Each replication included 3 baby food flasks, each containing a single in vitro plantlet, resulting in a total of 90 experimental units. Data were analyzed using analysis of variance (ANOVA) in the R statistical program, with means compared using the Least Significant Difference (LSD) test at the 5% significance level.
3.1. In Vitro Growth and Development
The light source did not significantly affect most of the in vitro growth and development parameters for both banana varieties, including stem diameter, shoot and root fresh weight, leaf number, shoot and root length, and shoot and root dry weight (Table 1). However, plantlets of ‘Little Prince’ grown under LED-1 and LED-2 exhibited greater overall fresh weight and shoot length compared to those grown under fluorescent lights. Conversely, plantlets of ‘Truly Tiny’ showed increased fresh weight only under LED-1, with no significant differences in shoot length.
3.2. Relative Chlorophyll Content
There were significant differences in relative chlorophyll content between the light sources and banana varieties (Figure 2). Generally, plantlets grown under LED lighting displayed higher SPAD values compared to those under fluorescent lamps. SPAD values were 36.84 for ‘Little Prince’ and 41.54 for ‘Truly Tiny’ under LED-1, 31.62 for ‘Truly Tiny’ and 40.92 for ‘Little Prince’ under LED-2, and 24.12 for ‘Little Prince’ and 24.56 for ‘Truly Tiny’ under fluorescent lamps. While no significant difference in relative chlorophyll content was observed between plantlets of both varieties under LED-1 and LED-2, values under these LED conditions were consistently higher than those under fluorescent lamps. However, ‘Truly Tiny’ under LED-2 exhibited similar values to both varieties grown under fluorescent lights. Leaves of plantlets under LED-1 and LED-2 appeared dark green, while those under fluorescent lighting showed a yellowish-green hue.
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