In Vitro Growth and Development

The length and width of three randomly selected stomata were measured for both the
adaxial and abaxial surfaces of each leaf in three different sections of each selected leaf.
Five random plantlets were selected per treatment.
2.6. Anatomical Observations
Anatomical studies were conducted using the middle-third section of the second
completely developed leaf. Leaf cross-sections were obtained by freehand sectioning using
a steel blade, fixed in 70% FAA (formaldehyde-acetic acid-ethyl alcohol 70%) for 48 h,
and then preserved in ethanol 70% (v/v). Following discoloration in sodium hypochlorite
(1–1.25% active chlorine), triple-rinsing in distilled water, and staining in toluidine blue

(0.05% w/v), leaf sections were subsequently fixed on semi-permanent slides with glyceri-
nated water [25]. The slides were examined and photographed under the same light Leica

DMLB microscope and the SPOT 4.7 idea digital camera and software, as described earlier.
The images were evaluated by assessing five fields per repetition for each variable analyzed.
The thickness of the abaxial surface epidermis (ASE), adaxial epidermis (ABE), abaxial
hypodermis (AH), adaxial hypodermis (AbH), palisade parenchyma (PP), and spongy
parenchyma (SP) were determined. Five random plants were selected per treatment.
2.7. Experimental Design and Statistical Analysis
The experimental design was completely randomized and consisted of six treatments
(three light sources × two banana varieties), with five replications per treatment. Each
replication consisted of 3 baby food flasks containing 1 in vitro plantlet per flask for a
total of 90 experimental units. Data were collected and submitted to analysis of variance
(ANOVA) using the R statistical analysis program, with means compared by LSD (Least
Significant Difference) test at the 5% level of significance.
3. Results
3.1. In Vitro Growth and Development
The light source had no significant effect on most in vitro growth and development
parameters evaluated 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, ‘Little Prince’ plantlets produced under LED-1 and LED-2 showed larger overall
plantlet fresh weight and shoot length compared to plantlets under fluorescent lights, while
‘Truly Tiny’ plantlets showed higher fresh weight only under LED-1, but no differences in
shoot length.

Banana
Variety

Light
Source

Stem
Diameter *
(mm)

Plant Fresh
Weight
(g)
Shoot Fresh
Weight
(g)
Root Fresh
Weight
(g)

Number
of Leaves

Shoot Length
(cm)

Root Length
(cm)

Number of
Roots

Shoot Dry
Weight
(g)

Root Dry
Weight
(g)

3.2. Relative Chlorophyll Content
Significant differences were shown in relative chlorophyll content among the different
light sources and banana varieties (Figure 2). In general, the relative chlorophyll content
of plants under LED lighting showed greater SPAD values compared to plantlets 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 (Figure 2). However, relative
chlorophyll content was not significantly different between plantlets of both varieties under
LED-1 and LED-2, but higher than FL, with the exception of ‘Truly Tiny’ under LED-2,

which had similar values to both varieties under FL. The leaves of plantlets under LED-
1 and LED-2 showed a dark green color, while the leaves of plantlets under FL had a

yellowish-green color.