In vitro banana plantlets measuring about 5 cm in height

A volume of 50 mL of
medium was dispensed into baby food jars, and the medium was autoclaved at 121 ◦C
and 20 psi for 20 min. In vitro banana plantlets measuring about 5 cm in height had
their leaves and roots trimmed to 2 cm shoot-tips, which were used as explants. Baby
food jars with polypropylene lids were sealed with parafilm, and cultures were placed
in a growth chamber under controlled environmental conditions of 27 ± 2

◦C and a 16 h

photoperiod at 100 μmol m−2
s
−1 PPFD. In vitro shoots were subcultured to fresh MS

medium at four-week intervals.
2.2. Light Sources
Three different light sources were evaluated: two provided by LED lighting, LED-1 at
116 μmol m−2
s
−1
(Philips GreenPower DR/B 3:1 150 43W) and LED-2 at 90 μmol m−2
s
−1
(Philips GreenPower DR/W 3:1 150 33W); and one provided by fluorescent lighting (Philips
9A fluorescent bulbs 40W) at 100 μmol m−2
s
−1
(FL). Photoperiod was 16/8 h (light/dark).
The spectral energy distribution for the lighting used in this study is shown in Figure 1:
LED-1 showed peak emissions at 440 nm and 650 nm (Figure 1A), LED-2 showed peak
emissions at 440 nm and 670 nm (Figure 1B); and fluorescent lighting (FL) showed a broader

Horticulturae 2022, 8, 92 3 of 12

spectrum with peaks in the green (550 nm), blue (440 nm), and some additional peaks in
between (490 nm, 590 nm, 610 nm, and 710 nm) (Figure 1C). The intensity and composition
of all light sources were measured using an LI-180 Li-Cor spectrometer (Li-Cor, Lincoln,
NE, USA).

Figure 1. Intensity and composition of lighting as photosynthetically active radiation (PAR) for the
different light treatments in this study: (A) LED-1 = 116 μmol m−2
s
−1
; (B) LED-2 = 90 μmol m−2
s
−1
;

and (C) fluorescent light (FL) = 100 μmol m−2
s
−1
. While both LED lights peak in the red range
(660–670 nm) with some blue peaks (440–470 nm), fluorescent light has a broader distribution with
peaks in the green (550 nm), blue (440 nm), and some additional peaks in between (490 nm, 590 nm,
610 nm, and 710 nm). Maximum irradiance (mW m−2

) is shown for each spectrum. Measurements

were obtained with an LI-180 Li-Cor spectrometer.
2.3. In Vitro Growth and Development
Explants from all treatments were evaluated four weeks after in vitro establishment
for shoot length, root length and number, plantlet fresh and dry weight, shoot fresh and
dry weight, root fresh and dry weight, root length, and number determined. Dry weight
was determined by oven-drying plantlets at 70 ◦C until they reached constant weight. Five
random plants were selected per treatment.

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2.4. Relative Chlorophyll Content

Relative chlorophyll content was evaluated as SPAD value by placing the third ex-
panded leaf of each plantlet, counted from top downwards, in a portable SPAD-502 chloro-
phyll meter (SPAD-502, Minolta Co., Ltd., Tokyo, Japan). Five random plantlets were

selected per treatment.
2.5. Stomata Analysis
The middle third portion of the third and fourth fully expanded leaves were cut into
approximately 1 cm × 1 cm sections. Impressions of the leaves were obtained by placing
leaf sections on top of a thin layer of super glue (Elmer’s Products, Inc., Westerville, OH,
USA), spread over microscope glass slides, and removing the leaf after drying. Impressions
were obtained for both the adaxial and abaxial surfaces of the leaves. Stomata observations
were performed under an optical Leica DMLB microscope (Leica microsystems, Buffalo,
NY, USA) at 200× magnification. Images were recorded using a SPOT 4.7 digital camera
coupled to the microscope and analyzed using the SPOT basic software (SPOT Imaging,
Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The number of stomata was
counted under the microscope for both the abaxial and adaxial surfaces of leaves. Three
different areas of 5 mm in diameter were selected as replicates for observations for each
light treatment. Results were expressed as a means of counts of the three areas per mm2
.