*3.4. Anatomical Observations*

Some parameters of leaf anatomy of in vitro plantlets were significantly affected by the light source, including abaxial epidermis (AbE), and abaxial (AbH) and adaxial (AdH) hypodermis for both varieties (Table 3). However, no significant differences were observed for adaxial epidermis (AdE) and for palisade (PP) and spongy (SP) parenchyma for both varieties (Table 3). The abaxial epidermis (AbE) was significantly thinner than the adaxial epidermis (AdE) (Table 3). As for the organization of mesophyll, the banana is dorsiventral or bifacial, with the palisade parenchyma oriented towards the adaxial epidermis and immediately below the adaxial hypodermis and the spongy parenchyma facing the abaxial epidermis (Figure 4). The palisade parenchyma was comprised of closely packed cylindrical cells, exhibiting one to two layers of cells, while the spongy parenchyma was comprised of two to three layers of spongy mesophyll cells, packed with inconspicuous air spaces (Figure 4). The cells were not well defined and had more or less a round shape under FL (Figure 4). However, no statistical differences were observed between most parameters evaluated.

**Table 3.** Effects of light source on leaf anatomy of two ornamental banana varieties grown in vitro; Musa 'Little Prince' and 'Truly Tiny'. LED-1 = 116 μmol m−<sup>2</sup> s−1; LED-2 = 90 μmol m−<sup>2</sup> s−1; fluorescent light (FL) = 100 μmol m−<sup>2</sup> s<sup>−</sup>1.


\* Means within columns followed by the same letter are not significantly different at *p* ≤ 0.05 according to the LSD (Least Significance Difference) test. The tests were conduct independently within each parameter (adaxial surface epidermis, abaxial surface epidermis, adaxial hypodermis, abaxial hypodermis, palisade parenchyma, spongy parenchyma). AbE = Abaxial Epidermis; AdE = Adaxial Epidermis; AbH = Abaxial Hypodermis, AdH = Adaxial Hypodermis; PP = Palisade Parenchyma; SP = Spongy Parenchyma.

**Figure 4.** Cross-section of in vitro banana leaves (Musa 'Little Prince' and 'Truly Tiny') under different light sources. LED-1 = 116 μmol m−<sup>2</sup> s−1; LED-2 = 90 μmol m−<sup>2</sup> s−1, fluorescent lighting (FL) = 100 μmol m−<sup>2</sup> s<sup>−</sup>1. AdE = adaxial epidermis; AbE = abaxial epidermis; AdH = adaxial hypodermis; AbH = abaxial hypodermis; PP = palisade parenchyma; SP = spongy parenchyma. Bar = 50 μm.

#### **4. Discussion**

The maintenance of the quality of in vitro plantlets requires an environment that provides optimum conditions for growth and development [26]. Light is among some of the main factors influencing the growth and development of in vitro plantlets, including light quality, intensity, and the photoperiod [27]. In this study, our goal was to evaluate the effect of different light sources on in vitro growth and development of two ornamental banana varieties. However, for most parameters evaluated, no significant differences were observed. The stem diameter of in vitro banana plantlets in our study was not significantly affected by the light source. This contrasts with similar studies showing an increase in stem diameter of in vitro banana plantlets under LED lighting (45, 60, and 75 μmol m−<sup>2</sup> s−<sup>1</sup> PPF) compared to fluorescent lights (45 μmol m−<sup>2</sup> s−<sup>1</sup> PPF) [22], although light intensities evaluated were lower as compared to the light intensities evaluated in our study [22]. This could indicate that lower light levels are more desirable to induce a larger stem diameter in in vitro banana plantlets. Another aspect was the spectral distribution evaluated [22], where LED lighting had a 9:1 = red:blue ratio and FL had an 8:2 = red:blue ratio. Our study had similar red:blue ratios for both LEDs compared to FL, which had almost an inverse red:blue ratio and a peak in the green (Figure 1), indicating that the light spectral distribution might have influenced the results observed. Similar results were reported in rapeseed in vitro plantlets under LED lighting [28]. In contrast, the stem diameter of Cymbidium in vitro plantlets was not affected by different light sources [29], thus showing similarities to our study. In our study, however, stem diameter had a correlation with banana varieties, whereas 'Little Prince' showed a larger average diameter than 'Truly Tiny', suggesting a genotype effect. Such differences in responses confirm that the influences and mechanisms related to light quality, intensity, and photoperiod in plants are rather specific to plant species or cultivars. A similar genotype effect has been shown for two annatto (*Bixa orellana*) cultivars grown under different irradiance and light quality [2].

Banana in vitro plantlet fresh weight was slightly greater under LEDs compared to FL, but differences were not statistically significant between LED-1 and LED-2 for both varieties. This trend was observed when banana in vitro plantlets were cultured under LED lighting [22] as well as in rapeseed [28], where no significant differences in fresh weight of plantlets under different LED lighting were observed.

Shoot fresh and dry weight of banana in vitro plantlets were not significantly different under different light sources. Similarly, no significant differences were observed for root fresh and dry weight for both banana varieties. However, similar studies with in vitro banana [30] and Doritaenopsis [31] plantlets under LED reported higher shoot and root fresh weight.

Because the spectral energy distribution of red and blue lights aligns with chlorophyll absorption, it is generally accepted that LED lighting improves plant growth and development by enhancing the net photosynthetic rate [32–34]. However, in our study, the effect of LED lighting on shoot length was significantly different from FL only for one banana variety, 'Little Prince', reinforcing the concept of a genotype effect, as previously indicated.

There were no statistically significant differences among plantlets for root length under the different light sources evaluated in our study. These results contrast with those on banana [23] and rapeseed [28], where root length was significantly different between different light sources. However, these studies showed no significant differences among plantlet stem lengths under different light treatments, thus showing similarities with our study.

In our study, the relative chlorophyll content of banana in vitro plantlets grown under LED lighting was higher compared to fluorescent lighting. Similar results for in vitro banana plantlets were reported [35], showing that higher chlorophyll values were obtained when banana plantlets were cultured under LED lights. Similar behavior has been reported for other species, including chrysanthemum [11], Doritaenopsis [32], and rapeseed [28].

The general morphology of plantlets proved quite homogeneous under all lighting treatments evaluated. No differences were observed for leaf number among the different light treatments, and our results are similar to those reported in bananas [35] and Cymbidium. However, in similar studies with banana and Anacardium othonianum plantlets, a higher number of leaves was observed under fluorescent lamps [15,30,36]. However, strawberry in vitro plantlets showed a higher number of leaves in plantlets cultured under

LED compared to fluorescent lighting [37]. The great diversity in responses in vitro among different species as affected by light intensity and quality illustrates the complexity of the topic and the need for continued studies to narrow down the best parameters for proper growth and development of plants in vitro.

Light sources influenced leaf stomata in our study. The number of stomata was generally higher on the abaxial than the adaxial leaf surface. Stomata of plantlets grown under the LED lighting were ellipsoid and closed, compared to open stomata and guard cells with round shapes in plantlets under FL (Figure 3). In general, banana plantlets grown under FL had larger numbers of stomata in both adaxial and abaxial leaf surfaces and similar higher values for length and width of stomata in adaxial leaf surfaces (Figure 3). However, for the abaxial leaf surfaces, while the length was slightly higher for FL, no differences were observed for width. Comparable results were reported in chrysanthemums [11]. However, another study with bananas [35] indicated the increased formation of stomata on both leaf surfaces (adaxial and abaxial) when banana plantlets were grown under LED compared to FL lighting.

In the present study, the epidermis of the adaxial leaf surface was thicker than the abaxial epidermis. These results corroborate a similar study of bananas in vitro [38], where the adaxial surface was thicker than the abaxial surface in leaves of in vitro-derived bananas under acclimatization. However, most of the anatomical leaf features measured in the current study were similar among the different light sources regardless of the banana variety. Similar results have been reported in pepper, showing a similar thickness in anatomical leaf features [39]. High light intensity also affected the anatomic structure in sugarcane leaves, including both adaxial and abaxial sides, and in the mesophyll and bulliform cell number [40]. However, no differences were observed in anatomic structures for ornamental bananas in our study, except for the thickness of adaxial compared to abaxial leaf surfaces.
