Ecophysiological Adaptations of Musa haekkinenii to Light Intensity and Water Quality

Abstract

Musa haekkinenii is a compact wild banana species with emerging value in ornamental horticulture, yet its adaptive responses to environmental factors remain underexplored. This study investigated the morpho-physiological and anatomical responses of M. haekkinenii to contrasting light regimes and irrigation water qualities to identify optimal cultivation conditions. A 210-day factorial experiment was conducted under subtropical greenhouse conditions using a split-plot design, with light intensity (full sun vs. shade) and irrigation water quality (reverse osmosis vs. well water) as treatment factors. Plants grown under shaded conditions and irrigated with reverse osmosis water exhibited significant increases in plant height, pseudostem diameter, leaf number, and sucker production, alongside enhanced pigment accumulation and photosynthetic performance. In contrast, full-sun plants irrigated with well water showed reduced growth, lower photosynthetic efficiency, and increased substrate salinity, indicating additive effects of light and osmotic stress. Leaf anatomical analysis revealed greater stomatal size and density under shade, particularly when combined with high-quality irrigation. Multivariate analysis further supported the association of favorable trait expression with shaded conditions and reverse osmosis water. These findings highlight the importance of microenvironmental management in enhancing the physiological stability and ornamental quality of M. haekkinenii, supporting its potential application in sustainable urban landscaping.

1. Introduction

Ornamental horticulture represents a dynamic sector within global agriculture, driven by the increasing demand for aesthetically valuable and adaptable plant species [1,2]. The global floriculture market, valued at $6.69 billion in 2023, underscores the economic significance of optimizing cultivation strategies for high-value ornamental species [3]. The commercial success of an ornamental plant is contingent not only on its visual attributes but also on its physiological adaptability to diverse environmental conditions, which directly impacts marketability and production efficiency [4,5]. Among tropical ornamentals, species that demonstrate resilience to environmental stressors while exhibiting compact growth and distinctive floral characteristics possess a competitive advantage in the global market [1].

Musa haekkinenii N.S.Lý & Haev. (Musaceae), a recently identified wild banana species holds substantial potential for commercial ornamental production [6]. Unlike other Musa species, M. haekkinenii exhibits a compact growth habit, reaching a pseudostem height of only 1.0–1.5 m, making it the smallest known species within the Callimusa section [6,7]. Its unique morphological traits—including an erect inflorescence, vibrant orange-red bracts, and persistent floral structures—distinguish it from commonly cultivated ornamental bananas such as Musa coccinea [6,7]. These attributes make M. haekkinenii particularly suitable for urban landscapes and confined horticultural spaces, where space-efficient species are increasingly favored. However, despite its commercial potential, the physiological responses of M. haekkinenii to key environmental factors remain unexplored. Previous studies on Musa physiology have primarily focused on triploid cultivars grown for fruit production [8,9], with limited attention to wild diploid species such as those characterized in metabolomic or genomic diversity studies among Musa wild relatives [10]. Polyploid banana cultivars, including autotriploid and allotriploid varieties, often display enhanced vigor, broader environmental adaptability, and greater tolerance to abiotic stresses compared with wild diploid progenitors [11,12]. In this context, evaluating the ecophysiological responses of M. haekkinenii provides a baseline for defining optimal acclimatization conditions for propagation in ornamental cultivation.

Light intensity is a fundamental environmental factor regulating plant growth, photosynthetic efficiency, and physiological adaptations [13]. High-light conditions often induce structural modifications, including increased leaf thickness and enhanced cuticle deposition, which contribute to improved water-use efficiency and stress tolerance [14,15]. However, excessive irradiance can cause photoinhibition, leading to a decline in photosynthetic performance due to oxidative damage to the photosynthetic apparatus, particularly photosystem II [16]. To mitigate transpirational water loss under such conditions, plants frequently reduce stomatal aperture, which can result in midday depression of photosynthesis—an extensively documented response among species exposed to high irradiance [17]. In contrast, shade-adapted plants exhibit morphological and physiological adjustments that enhance light capture, including increased leaf surface area, elevated chlorophyll content, and reduced leaf thickness, thereby optimizing carbon assimilation under low-light conditions [14,18]. These responses underscore the importance of elucidating the optimal light environment for M. haekkinenii to maximize its ornamental quality and commercial viability.

Water quality is another critical factor influencing plant physiology and market viability. The chemical composition of irrigation water directly affects nutrient bioavailability, osmotic balance, and overall plant vigor, with significant implications for aesthetic quality and growth performance [19]. Well water, frequently enriched with dissolved minerals, may provide essential nutrients but often introduces salinity stress, which disrupts photosynthetic efficiency, reduces stomatal conductance, and induces nutrient imbalances [20,21]. Salinity-induced osmotic stress leads to excessive ion accumulation, triggering oxidative damage, premature senescence, and reduced ornamental quality [22,23,24,25]. High sodium and chloride concentrations, in particular, compromise cellular homeostasis, leading to irreversible physiological impairments [20,25]. In parallel, secondary metabolites such as glucosinolates have been shown to contribute to osmotic adjustment and stress tolerance under saline conditions, particularly in Brassicaceae, highlighting the broader relevance of metabolite-mediated responses to salinity [26]. In contrast, desalinated water sources, such as reverse osmosis (RO) water, eliminate salinity stress but require precise nutrient supplementation to maintain metabolic balance and sustain optimal growth [27]. The increasing reliance on desalinated irrigation water in horticultural production presents both opportunities and challenges, highlighting the importance of tailored nutrient management strategies to optimize plant health and ornamental appeal [27].

Beyond environmental influences, the commercial feasibility of M. haekkinenii is largely determined by its growth performance under varying cultivation conditions. Morphological traits, including plant height, pseudostem diameter, leaf number, sucker production, and flowering dynamics, serve as critical indicators of market potential [28]. Given its compact growth habit and striking floral attributes, M. haekkinenii represents a high-value ornamental species with broad applicability in both interior and exterior landscaping. Understanding the interactions between light intensity and water quality in shaping these phenotypic traits is fundamental to refining its cultivation protocols and enhancing its commercial appeal. As consumer preferences shift toward compact, visually distinctive tropical ornamentals, optimizing production strategies for M. haekkinenii will be essential for maximizing its economic potential and market penetration.

To our knowledge, this is the first study to evaluate the morpho-physiological and anatomical responses of M. haekkinenii under controlled conditions, thereby filling a critical research gap and providing a basis for optimized cultivation protocols. This study aimed to investigate the ecophysiological responses of M. haekkinenii to varying light intensities and water qualities to establish optimal cultivation strategies for commercial production in subtropical climates. By assessing key morphological and physiological traits, the research provides a comprehensive framework for enhancing productivity and marketability. Specifically, the study sought to (i) evaluate growth parameters, including plant height, pseudostem diameter, leaf number, sucker production, and flowering patterns, to define optimal morphological traits for commercial production; (ii) analyze gas exchange regulation by measuring stomatal anatomy, stomatal conductance, electron transport rate, and photosynthetic efficiency to determine plant acclimation to different environmental conditions; (iii) assess nutrient leaching and substrate salinity dynamics to optimize irrigation management and prevent nutrient imbalances affecting plant vigor; and (iv) identify optimal environmental conditions for large-scale cultivation by integrating morphological and physiological insights to enhance production efficiency and economic viability. By elucidating the interactions between light intensity, water quality, and plant physiological responses, this research provides science-based guidelines to optimize production and promote sustainable ornamental plant cultivation globally. These objectives directly support the ornamental horticulture industry, where optimizing environmental management is essential for producing compact, visually distinctive, and stress-resilient plants with high market value.

2. Materials and Methods

2.1. Experimental Site and Plant Material

This study was conducted at the Fairchild Tropical Botanic Garden Research Center (FTBG) in Coral Gables, Florida, United States (latitude: 25°40′36″ N, longitude: 80°16′19″ W) at 3 m.a.s.l., from January to August 2024.

Six-month-old Musa haekkinenii plants were obtained from mother plants cultivated under natural conditions at FTBG. Individuals were selected based on uniformity in size and physiological condition to ensure experimental consistency at the onset of the study. Each plant was transplanted into 28 cm diameter plastic pots (14 L volume) filled with a standardized Fairchild substrate composed of 40% Canadian peat, 20% coir pith, 20% pine bark, 10% perlite, and 10% dolomite (v/v). A controlled-release fertilizer (Florikan^® CRF 18-6-8 with micronutrients; Florikan ESA LLC, Sarasota, FL, USA) was incorporated at 15 g per pot following the manufacturer’s incorporation rate for containerized ornamentals. This substrate formulation has been previously standardized at Fairchild Tropical Botanic Garden to ensure adequate aeration, drainage, and nutrient retention, thereby providing suitable conditions for sustained root development and vegetative growth of tropical ornamentals.

2.2. Experimental Design and Treatment Application

A 2 × 2 factorial experiment was conducted using a split-plot design to evaluate the effects of light intensity and water quality on the growth and physiological responses of Musa haekkinenii. Light intensity was assigned to the main plots, while water quality treatments were applied to the subplots. A split-plot design was selected because irrigation treatments were applied at the main-plot level, while light treatments were implemented at the subplot level. This arrangement ensured uniform application of treatments and minimized management variability [29]. The light levels applied in this experiment were selected to simulate restricted-light environments typical of greenhouse and indoor ornamental production

The first factor, light intensity treatments, was defined based on the monthly average PAR values recorded throughout the experiment (January to August 2024) inside the greenhouse at 2 m height, using a HOBO MX2308 Temp/RH/PAR Data Logger (Onset Computer Corporation, Bourne, MA, USA). Average values reached 450 µmol m⁻² s⁻¹ under full sun and 100 µmol m⁻² s⁻¹ under shade conditions. The latter (100 µmol m⁻² s⁻¹) was achieved by covering the plots with a polyethylene shade cloth (50% light reduction, black, 70% UV stabilized, Green-Tek^®, Janesville, WI, USA). These measurements provided consistent estimates of the average radiation received by plants across treatments (Figure S1).

The second factor, water quality treatments, included reverse osmosis (RO) water, with low electrical conductivity (EC = 27 µS/cm) due to the removal of dissolved minerals, and well water (WW), with higher EC (330 µS/cm) due to naturally occurring mineral content. Plants were irrigated using automatic overhead sprinklers three times per day (8:00, 12:00, and 16:00 h), delivering 2 mm of water per event, equivalent to 6 mm per day.

Each of the four treatment combinations was replicated five times, (one plant per pot), resulting in a total of 20 experimental units. The choice of five replicates thus reflects a balance between statistical rigor and logistical feasibility, aligning with recommendations in agronomic research that replication levels be sufficient to maintain acceptable error variance relative to the expected treatment effects [30,31]. Baseline measurements were taken at 15 days after treatment (DAT), followed by subsequent evaluations at 30, 60, 90, 120, 150, 180, and 210 DAT to assess long-term treatment effects.

Temperature (°C) and relative humidity (%) were monitored throughout the experimental period using a HOBO MX2308 Temp/RH/PAR Data Logger (Onset Computer Corporation, Bourne, MA, USA) installed 2 m height at both microenvironments. Seasonal trends are presented in Supplementary Figure S2.

2.3. Morphological Traits

Morphological traits were evaluated at each assessment period to monitor plant growth dynamics. Plant height (cm) was determined by measuring from the substrate surface to the insertion point of the first leaf, while pseudostem diameter (cm) was quantified at a height of 5 cm above the substrate [32] using a digital caliper (Mitutoyo Corp., Kawasaki, Japan; accuracy ±0.01 mm) to ensure precise structural measurements. Furthermore, the total number of fully expanded leaves, number of suckers (rhizome), and the occurrence of flowering were also recorded.

2.4. Physiological Measurements

2.4.1. Chlorophyll Content and Vegetation Indices

Chlorophyll content was evaluated from 15 to 210 DAT using two optical sensors to quantify relative leaf pigment indices (Figure 1a,b). Measurements were conducted employing an SPAD-502 chlorophyll meter (Konica Minolta Sensing, Inc., Osaka, Japan) and an atLEAF chlorophyll meter (FT Green LLC, Wilmington, DE, USA). Chlorophyll content readings were collected from the youngest fully expanded leaf, specifically the third leaf from the apical region, with measurements taken at three distinct points on the leaflet to ensure methodological consistency [33]. Additionally, the Normalized Difference Vegetation Index (NDVI) was determined using a GreenSeeker™ Optical Sensor (Trimble Industries, Inc.), positioned 45 cm above the plant canopy (Figure 1c), following the methodological framework outlined by [34]. NDVI measurements were obtained at 15 and 210 DAT to assess variations in canopy greenness and overall plant vigor throughout the study [35]. All measurements were performed using five subsamples per experimental unit.

2.4.2. Stomatal Conductance and Electron Transport Rate Measurements

Stomatal conductance (g_s, mol m⁻²s⁻¹) and electron transport rate (ETR, μmol electrons m⁻²s⁻¹) were measured concurrently using a Li-600 porometer/fluorometer (LI-COR Biosciences, Lincoln, NE, USA) to assess gas exchange dynamics and photosynthetic efficiency (Figure 1d). Data were collected monthly between 9:00–11:30 a.m. under ambient environmental conditions using the same leaf selected for chlorophyll measurements to maintain consistency and capture peak transpiration and photochemical activity [32].

2.5. Nutrient Leachate

Leachate samples were obtained from each experimental unit using individual containers placed beneath each pot during irrigation. Following [34,36], plants were irrigated to saturation, and an additional 350 mL of distilled water was applied to facilitate leachate collection. The leachate was analyzed for calcium (Ca²⁺, ppm), potassium (K⁺, ppm), sodium (Na⁺, ppm), and nitrate (NO₃⁻, ppm) concentrations using a LAQUAtwin ion meter (Horiba Scientific, Kyoto, Japan). Prior to each measurement session, the ion meter was calibrated according to the manufacturer’s specifications, employing two-point calibration with standard solutions specific for each ion (Horiba calibration solutions at known concentrations). Between measurements, electrodes were rinsed with distilled water to prevent cross-contamination. Additionally, electrical conductivity (EC, µS/cm), pH, and total dissolved salts (ppm) were measured with an ExStik EC500 conductivity meter (EXTECH Instruments, FLIR Commercial Systems, Goleta, CA, USA) to evaluate the impact of water quality on substrate salinity and nutrient availability.

2.6. Leaf Anatomical Analysis

Leaf anatomical traits were evaluated at 210 DAT using samples collected from the third fully expanded leaf from the apical region, following the same protocol applied for chlorophyll measurements. The leaves were sampled, and ~1 cm² sections were excised from the central lamina (excluding midrib and margins). Samples were cleared in a 1:1 solution of 30% H₂O₂ and 100% CH₃COOH at 70 °C until pigments were fully removed, rinsed in distilled water, and carefully separated into epidermal and mesophyll layers using fine forceps. To enhance contrast, sections were stained with Safranin O (1% w/v) for 10–15 min, rinsed, and mounted on slides with CytoSeal (Fisher Scientific, Waltham, MA, USA), following the protocol described by [37].

Stomatal measurements were conducted using photomicrographs captured at 40× g magnification. Stomatal length (μm), width (μm), and count were quantified on both adaxial and abaxial leaf surfaces to calculate stomatal density (mm²) and estimate individual stomatal size (μm²). Image analysis was performed using ImageJ software version 1.x [38].

2.7. Maximum Stomatal Conductance

The theoretical maximum stomatal conductance (g_smax) was calculated based on the model proposed by [39], which integrates key anatomical features of stomata. This model calculates g_smax based on stomatal density (D_s), the maximum pore area ($a_{\mathrm{m}\mathrm{a}\mathrm{x}}$), and the estimated depth of the stomatal pore ($d_p$), along with constants for the diffusivity of water vapor in ai and the molar volume of air ($v$) at 25 °C. The diffusivity of water vapor was set at 0.0000249 m² s⁻¹, and the molar volume of air at 0.0224 m³ mol⁻¹. The depth of the pore was assumed to be proportional to guard cell length, approximated as 0.36 times the guard cell length ($l_g$), following the method of [40]. The maximum pore area was estimated by modeling the stomatal aperture as a circle, where the pore diameter was taken as half the guard cell length. Accordingly, $a_{\mathrm{m}\mathrm{a}\mathrm{x}}$ was calculated using the equation $\pi {\left({\displaystyle \frac{p}{2}}\right)}^2$, with $p$ representing half of $l_g$. Using these parameters, g_sₘₐₓ was derived from empirical measurements of guard cell length and stomatal density, following the procedure adopted by [37].

$$g_{s,\mathrm{m}\mathrm{a}\mathrm{x}}={\displaystyle \frac{D_s\hspace{0.17em}{a}_{\mathrm{m}\mathrm{a}\mathrm{x}}\cdot \frac{d_{{\mathrm{H}}_2\mathrm{O}}}{v}}{d_p+\frac{\pi }{2}\sqrt{a_{\mathrm{m}\mathrm{a}\mathrm{x}}/\pi }}}$$

2.8. Statistical Analysis

A linear mixed-effects model was applied to analyze the response variable, incorporating fixed and random effects [41]. The model was specified as follows:

$$Y\sim 1+Time\times Light\times Water+\left(1+Time\mid Block\right)$$

where Y is the response variable, Time is the measurement time, Light is the light intensity treatment, Water is the water quality treatment, and Block represents replicate blocks included as a random effect. The random structure (1 + Time|Block) accounts for variability across experimental replicates and allows for random slopes for Time, capturing potential differences in temporal responses among replicates.

All statistical analyses and graphics were performed using R statistical software version 4.5.0 [42]. A two-way analysis of variance (ANOVA) was applied to evaluate the effects of light intensity and water quality on plant growth and physiological traits, and to determine whether these factors and their interaction exerted statistically significant influences. When significant differences were detected (p < 0.05), Tukey’s multiple comparison tests were applied to determine pairwise differences among treatment means, using the emmeans 1.11.2-8 package [43]. A Principal Component Analysis (PCA) was performed with the FactoMineR 2.12 R package [44] to evaluate correlations between morphological, physiological, and anatomical traits, providing a multivariate assessment of environmental treatment effects. The graphs were made with inti 0.6.8 R package [45].

3. Results

3.1. Morphological Responses to Light Intensity and Water Quality

To evaluate the growth parameters of Musa haekkinenii, plant height, pseudostem diameter, leaf production, sucker formation, and flowering were assessed and compared between conditions (Figure 2). These traits serve as key indicators of plant vigor, reflecting the impact of environmental factors on growth dynamics [28].

Plant height varied significantly across treatments, with shade-grown plants exhibiting greater elongation than those exposed to full sun. Differences between treatments became evident after 90 DAT, when shade-grown plants under reverse osmosis surpassed 75.06 cm, whereas full-sun plants remained below 53 cm (Figure 2a). By 210 DAT, the tallest plants were observed in the shade under reverse osmosis irrigation, reaching 85.92 cm, followed by those under the shade with well water (78.24 cm). In contrast, full-sun conditions limited height expansion, with plants irrigated with reverse osmosis and well water reaching 64.56 cm and 59.48 cm, respectively.

Pseudostem diameter followed a similar pattern, with shade-grown plants developing thicker stems. At 210 DAT, plants under shade with reverse osmosis exhibited the largest pseudostems at 5.65 cm, followed by shade with well water (4.00 cm). Full-sun plants displayed significantly reduced diameters, measuring 3.92 cm with reverse osmosis and 3.20 cm with well water. The divergence in structural development became noticeable after 90 DAT, with shade-grown plants maintaining a steady increase in pseudostem thickness, while full-sun plants showed a slower expansion rate (Figure 2b).

Leaf production was consistently higher in shade-grown plants, indicating enhanced vegetative growth under reduced light conditions. At 210 DAT, shade with reverse osmosis resulted in the highest leaf count (9.4 leaves), followed by shade with well water (8.6 leaves). Full-sun plants exhibited significantly lower leaf production, with 7.6 leaves under reverse osmosis and 4.4 leaves under well water. Treatment differences became evident after 90 DAT, as shade-grown plants exhibited continuous leaf formation, whereas full-sun plants maintained a slower growth rate (Figure 2c).

Sucker formation followed a similar trend, with shade-grown plants producing more suckers than those in full sun. At 210 DAT, shade with reverse osmosis irrigation resulted in the highest sucker count (4.0 suckers), followed by shade with well water (2.2 suckers). In contrast, full-sun plants exhibited limited vegetative propagation, with 1.8 suckers under reverse osmosis and 0.6 suckers under well water. No suckers were recorded before 30 DAT, suggesting that early growth stages were dedicated to primary shoot development (Figure 2d).

Flowering was delayed across all treatments, with no floral emergence before 120 Flowering was delayed across all treatments, with no floral emergence before 120 DAT. By 150 DAT, the first inflorescences appeared in shade-grown plants irrigated with reverse osmosis water and in full-sun plants irrigated with reverse osmosis water, both averaging 0.4 flowers per plant. By 210 DAT, flowering was most pronounced in shade-grown plants irrigated with reverse osmosis water, averaging 1.0 flower per plant, while shade with well water averaged 0.4 flowers per plant. In contrast, flowering remained limited under full-sun conditions, with reverse osmosis plants averaging 0.6 flowers per plant and well-watered plants only 0.2 flowers per plant (Figure 2e).

3.2. Chlorophyll Content, Photosynthetic Efficiency, and Gas Exchange Dynamics

To assess the physiological performance of Musa haekkinenii, parameters including chlorophyll content, stomatal conductance (g_s), electron transport rate (ETR), and stomatal density were evaluated for 210 days. These traits were selected as integrative indicators of photosynthetic capacity and stress response, allowing us to evaluate how plants functionally acclimate under the experimental conditions. These parameters provided direct insights into plant acclimation under varying light intensity and water quality treatments [13,46].

Chlorophyll content, quantified using SPAD and atLEAF indices, exhibited significant variation across treatments. Shade-grown plants consistently maintained higher chlorophyll levels than full-sun plants throughout the experiment. By 210 DAT, SPAD values were highest in shade with reverse osmosis irrigation (51.88), followed by shade with well water (48.22). Full-sun plants exhibited significantly lower SPAD values, with 40.16 under reverse osmosis and 35.84 under well water. A similar trend was observed in the atLEAF index, where shade with reverse osmosis reached 60.09, followed by shade with well water at 53.2. The lowest chlorophyll content was recorded in full sun, with values of 51.42 under reverse osmosis and 45.34 under well water. Differences among treatments became more pronounced after 90 DAT, as shade-grown plants exhibited a steady increase in chlorophyll content, while full-sun plants showed limited accumulation. By 180 DAT, SPAD values peaked at 51.66 in shade with reverse osmosis, in contrast to 37.78 in full sun with reverse osmosis. Concurrently, atLEAF values reached 58.7 in shade with reverse osmosis, whereas full sun with well water resulted in the lowest recorded value (44.49) (Figure 3a,c).

Stomatal conductance (g_s) exhibited a progressive increase over time, with significant treatment effects. By 210 DAT, the highest g_s was recorded in shade with reverse osmosis (0.55 mol m⁻² s⁻¹), followed by shade with well water (0.33 mol m⁻² s⁻¹). Full-sun plants exhibited significantly lower stomatal conductance, with values of 0.17 mol m⁻² s⁻¹ under reverse osmosis and 0.09 mol m⁻² s⁻¹ under well water. Differences between treatments became apparent after 90 DAT, with shade-grown plants maintaining a continuous increase in g_s, while full-sun plants exhibited lower values, likely due to increased evaporative demand and stomatal closure as a protective mechanism (Figure 3b).

Electron transport rate (ETR) followed a similar trend, with higher values in shade-grown plants compared to those in full sun. By 210 DAT, shade with reverse osmosis recorded the highest ETR (278.65 µmol electrons m⁻² s⁻¹), followed by shade with well water (240.36 µmol electrons m⁻² s⁻¹). Full-sun plants exhibited significantly lower ETR, with values of 193.75 µmol electrons m⁻² s⁻¹ under reverse osmosis and 151.44 µmol electrons m⁻² s⁻¹ under well water. The highest ETR values were observed at 90 DAT, where shade and reverse osmosis reached 314.83 µmol electrons m⁻² s⁻¹. Following this peak, values gradually declined across all treatments, suggesting a shift in photosynthetic activity over time (Figure 3d).

Normalized Difference Vegetation Index (NDVI), a proxy for canopy greenness and vigor, was consistently higher in shade-grown plants compared to those in full sun. By 210 DAT, shade with reverse osmosis recorded the highest NDVI (0.89), followed by shade with well water (0.85). Full-sun conditions resulted in significantly lower NDVI values, with 0.78 under reverse osmosis and 0.72 under well water (Figure 3e). For vapor pressure deficit (VPD, Figure 3f) varied significantly across treatments, influenced primarily by light conditions. Full sun with reverse osmosis exhibited the highest VPD (3.99 kPa), followed by full sun with well water (3.26 kPa). Shade treatments showed lower VPD, with 3.19 kPa under reverse osmosis and 2.73 kPa under well water.

To investigate anatomical responses associated with physiological acclimation and gas exchange regulation, stomatal traits were quantified on both the abaxial and adaxial leaf surfaces across all treatments (Figure 4). Specifically, the relationships among stomatal size (S_s), maximum stomatal density (D_s), and theoretical maximum stomatal conductance (g_smax) were examined following the framework established by [37,39,47].

Stomatal size (S_s) varied across treatments and leaf surfaces. On the adaxial surface, the largest stomata were recorded under shade with well water (S_s = 1833.38 µm²), and the smallest under full sun with well water (S_s = 1464.99 µm²). On the abaxial surface, stomatal size ranged from 1504.88 µm² in shade with well water to 1289.22 µm² in full sun with well water.

Stomatal density (D_s) was consistently higher on the abaxial surface compared to the adaxial surface. The highest abaxial D_s was observed in full sun with well water (786.95 mm⁻²), followed by full sun with reverse osmosis (717.29 mm⁻²) and shade with reverse osmosis (691.71 mm⁻²). The lowest abaxial value was recorded under shade with well water (669.40 mm⁻²). On the adaxial surface, D_s values ranged from 704.78 mm⁻² (full sun with well water) to 546.72 mm⁻² (shade with well water).

Theoretical maximum stomatal conductance (g_smax) was generally higher on the abaxial surface and followed trends in stomatal density. The highest abaxial g_smax was observed in full sun with reverse osmosis (1.17 mol m⁻² s⁻¹), followed by full sun with well water (1.05 mol m⁻² s⁻¹), shade with well water (0.97 mol m⁻² s⁻¹), and shade with reverse osmosis (0.91 mol m⁻² s⁻¹). On the adaxial surface, g_smax values were lower overall, with the highest value observed in shade with reverse osmosis water (0.53 mol m⁻² s⁻¹) and the lowest in full sun with well water (0.40 mol m⁻² s⁻¹).

Photomicrographs captured at 40× magnification (Figure 4c,d) corroborated these trends, with visibly more abundant stomata observed in full sun treatments, particularly on the abaxial surface. Quantitatively, the highest stomatal number (Sn) on the abaxial surface was recorded in full sun with well water (8.69), followed closely by full sun with reverse osmosis (8.08). In contrast, shaded treatments exhibited lower stomatal frequencies, with Sn values of 7.00 in shade with well water and 6.44 in shade with reverse osmosis. On the adaxial surface, overall stomatal counts were lower across all treatments, with the highest Sn observed in shade with reverse osmosis (3.46), and the lowest in shade with well water (2.43).

3.3. Nutrient Leaching Dynamics and Substrate Salinity in Response to Irrigation Treatments

Understanding nutrient loss and salinity accumulation under different irrigation regimes is vital for developing sustainable fertilization and irrigation strategies for Musa haekkinenii [48]. Nutrient leaching and substrate salinity critically affect nutrient availability, root zone conditions, and plant health. Significant variations in nitrate (NO₃⁻), sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺) leaching were observed, influenced by light intensity, water quality, and time (Figure 5).

The highest nitrate leaching was observed in full-sun plants irrigated with well water, peaking at 295.0 ppm at 30 DAT. In contrast, the lowest levels were recorded in shade-grown plants under reverse osmosis irrigation, with values of 40.0 ppm at 15 DAT and 76.6 ppm at 210 DAT (Figure 5a). Sodium leaching followed a similar trend, with the highest levels in full sun with well water at 30 and 210 DAT, while shade-grown plants irrigated with reverse osmosis exhibited the lowest sodium concentrations, ranging from 5.8 to 7.8 ppm throughout the experiment (Figure 5b).

Potassium leaching displayed distinct patterns, with the highest losses occurring in full-sun plants irrigated with well water, reaching 155.0 ppm at 30 DAT and remaining elevated throughout the study. In contrast, reverse osmosis irrigation under shaded conditions resulted in significantly lower potassium leaching, with values ranging from 30.8 to 59.6 ppm (Figure 5c). Calcium leaching exhibited a dynamic pattern, with an initial increase observed at 30 DAT, particularly in well-watered treatments, where shade-grown plants under well water irrigation peaked at 336.0 ppm. Reverse osmosis irrigation significantly reduced calcium leaching, with final values as low as 36.8 ppm at 210 DAT (Figure 5d).

Substrate salinity, assessed through salt concentration, electrical conductivity (EC), and pH varied significantly among treatments. Salt accumulation exhibited a similar trend, with full-sun plants irrigated with well water displaying the highest salt concentrations at 827.6 ppm at 30 DAT, followed by a gradual decline. By 210 DAT, salt levels remained high in full-sun plants irrigated with well water at 762.0 ppm, whereas shade-grown plants irrigated with reverse osmosis water exhibited the lowest salt accumulation at 114.0 ppm. These findings indicate that reverse osmosis irrigation effectively reduced salt buildup, whereas well-water irrigation led to sustained salt retention, increasing the potential risk of substrate salinity stress (Figure 5e).

Electrical conductivity (EC) levels were consistently higher in well-watered treatments, particularly under full-sun conditions, while reverse osmosis irrigation resulted in lower EC values. By 30 DAT, full-sun plants irrigated with well water exhibited the highest EC (1656.8 µS/cm), whereas the lowest value was recorded in shade-grown plants irrigated with reverse osmosis water at 249.0 µS/cm (Figure 5f).

Substrate pH fluctuated throughout the experimental period, with full-sun plants irrigated with well water displaying the highest pH at 8.5 by 90 DAT. In contrast, shade-grown plants irrigated with reverse osmosis water presented pH values, ranging from 7.01 to 7.45. By the end of the study, pH values ranged from 7.29 in full sun with well water to 7.45 in shade with reverse osmosis irrigation (Figure 5g).

3.4. Optimizing Environmental Conditions for Large-Scale Cultivation

To determine the optimal environmental conditions for cultivating Musa haekkinenii, a principal component analysis (PCA) was performed 210 days after treatment. This analysis integrated morphological, physiological, and anatomical traits across all light and water quality treatments to identify the traits most strongly differentiating treatments and to assess their combined contribution to overall plant performance (Figure 6).

The PCA biplot of variables (Figure 6a) shows that Dim 1 (62.24%) primarily captures variability related to growth and photosynthetic performance. Traits with strong positive loadings along Dim 1 include plant height, pseudostem diameter, number of leaves, suckers, and flowers, as well as NDVI, SPAD, atLEAF, ETR, and g_s. Stomatal traits—particularly stomatal density (SD_lower, SD_upper) and stomatal size on the adaxial surface (area.wst40x_upper)—also aligned closely with these performance indicators, suggesting their integrated role in gas exchange regulation and physiological vigor.

Dim 2 (15.34%) explained variation largely attributable to anatomical and ionic stress factors. Nutrient and salinity indicators including NO_3⁻, Na⁺, K⁺, Ca²⁺, EC, and salt concentration showed strong negative loadings along Dim 1 and moderate positive influence on Dim 2. These variables were inversely correlated with growth-related traits, suggesting that elevated salinity and nutrient accumulation may impair developmental outcomes. Vapor pressure deficit (VPDleaf) loaded orthogonally, implying an independent effect from both primary principal components.

The PCA of individuals (Figure 6b) effectively distinguished treatment effects. Plants grown under shade and irrigated with reverse osmosis water were tightly clustered in the upper-right quadrant of the PCA space, indicating strong association with favorable growth, photosynthetic efficiency, and anatomical adaptation. In contrast, full-sun plants irrigated with well water occupied the lower-left quadrant, aligning with elevated salinity markers and reduced growth, indicating significant physiological stress. Intermediate responses were observed for shade–well water and full sun–reverse osmosis treatments, highlighting partial mitigation of either light or salinity stress under those regimes.

Collectively, the PCA underscores that integrating moderate light intensity (shade) with high-quality irrigation (reverse osmosis) provides the most favorable conditions for maximizing growth, physiological performance, and anatomical integrity in Musa haekkinenii. This combination minimizes salinity-related constraints while enhancing photosynthetic and morphological traits, offering a strategic framework for sustainable and commercially viable ornamental production.

4. Discussion

Musa haekkinenii is a promising ornamental species with significant commercial potential due to its unique floral characteristics and adaptability to confined urban spaces. However, realizing this potential in large-scale production systems requires a detailed understanding of its ecophysiological responses to different environments. Among these factors, light intensity and water quality are particularly influential, as they govern both growth dynamics and physiological response. This study presents a comprehensive assessment of these abiotic factors, offering insights into the species’ acclimation mechanisms and providing evidence-based recommendations for optimized cultivation in controlled environments.

The results indicate that light intensity plays a fundamental role in shaping plant morphology and physiological acclimation and performance. Shade-grown plants exhibited greater height, increased pseudostem diameter, and higher leaf production (Figure 2a–c), consistent with previous findings that reduced irradiance often promotes cell elongation and chlorophyll accumulation as adaptive mechanisms to enhance light capture, although biomass responses may vary depending on species and environmental conditions [49]. Similarly, the morphological characterization of fifteen ornamental banana accessions in the Godavari zone of Andhra Pradesh revealed significant variability in pseudostem height, girth, leaf area, and petiole length, highlighting the genetic basis of trait differentiation within Musa ornamentals [50]. Such comparative studies emphasize that both ecophysiological responses and morphometric variation are central to defining the horticultural potential of ornamental bananas across diverse environments. Additionally, increased chlorophyll content in shade conditions (Figure 3a,c) has been associated with enhanced light capture efficiency, particularly in shade-tolerant species, as observed in tree seedlings responding to spectral light composition [51]. In contrast, full-sun exposure led to lower chlorophyll levels (Figure 3a,c), likely due to oxidative stress and pigment degradation, which can impair photosynthetic efficiency and overall plant aesthetics, as high light intensity has been shown to induce photoinhibition and damage photosystem II [52].

In addition to light, water quality significantly influenced physiological responses, particularly stomatal conductance (g_s) and electron transport rate (ETR). Plants irrigated with reverse osmosis water exhibited higher g_s and ETR compared to those irrigated with well water (Figure 3b,d). The negative effects of well-water irrigation were associated with increased salinity (Figure 5e), which can lead to osmotic stress, reduced water uptake, and alterations in plant physiological functions, as observed in studies on salinity stress in ornamental species [53]. Increased salinity not only limits growth but also affects flowering patterns, a factor of great relevance in commercial ornamental production. The use of well water irrigation can influence nutrient dynamics in the root zone, potentially leading to ion accumulation and osmotic imbalances that affect plant vigor, a phenomenon previously observed on wastewater irrigation and soil quality [54].

Substrate salinity patterns further supported these findings, with well water irrigation leading to increased salt accumulation, electrical conductivity, and pH fluctuations over time (Figure 5e–g). These results align with previous research demonstrating that saline irrigation exacerbates substrate salinization, reducing nutrient bioavailability and affecting plant performance [55,56,57,58]. In contrast, reverse osmosis irrigation effectively maintained more stable nutrient availability and lower electrical conductivity levels (Figure 5a–f), minimizing the risk of salt-induced stress. The progressive accumulation of salts in well water treatments highlights the long-term challenges associated with high-salinity irrigation, reinforcing the need for precise water management strategies to sustain high-quality ornamental production [59].

In addition to salinity, the higher pH of well water likely reduced micronutrient solubility, particularly Fe, Mn, and Zn, which in turn limited nutrient uptake efficiency and overall growth performance. By contrast, reverse osmosis water, with its near-neutral pH, created a more favorable chemical environment for nutrient absorption, supporting improved g_s, ETR, and morphological development. Similar responses have been documented in Musa cultivars, where elevated salinity and pH were associated with impaired nutrient uptake, reduced photosynthetic efficiency, and growth inhibition [60,61]. These results suggest that pH-driven nutrient availability, together with salinity-induced ionic stress, contributed to the reduced performance of plants irrigated with well water in this study.

With respect to physiological response as instantaneous water use efficiency (WUE), our findings underscore the importance of evaluating both the ratio between ETR and g_s, and the influence of vapor pressure deficit (VPD) in modulating transpirational loss (Figure 3f). Although ETR and g_s both increased in shaded, RO-irrigated treatments, their ratio remained relatively constant, suggesting that intrinsic WUE did not proportionally improve. VPD measurements confirmed consistently lower values in shaded environments (100 µmol m⁻² s⁻¹), particularly under reverse osmosis irrigation (Figure 3f), supporting the hypothesis that reduced atmospheric demand lowers water flux from the leaf despite higher stomatal openness. This finding is consistent with physiological models emphasizing the dependency of transpiration on VPD rather than g_s alone [39,62]. Thus, enhanced WUE in these treatments is likely a result of reduced evaporative pressure rather than anatomical optimization alone. These integrated anatomical and physiological adjustments reflect an acclimation strategy in M. haekkinenii, enabling it to balance carbon gain and water conservation under varying environmental conditions. The results corroborate the broader hypothesis that stomatal size-density scaling and conductance ratios are critical determinants of species’ adaptation to abiotic stress, particularly in tropical ornamentals cultivated under controlled conditions [37,47].

Beyond physiological responses, anatomical traits such as stomatal size (S_s), stomatal density (D_s), and theoretical maximum stomatal conductance (g_smax) offered additional insight into the acclimation strategies of Musa haekkinenii under contrasting light and water conditions. These traits showed treatment-specific variation, with more pronounced effects on the abaxial surface. The highest D_s was observed under full sun with well water, but this treatment also exhibited the smallest stomatal size and a relatively low g_smax—suggesting that elevated density alone does not ensure high conductance. Conversely, shaded plants irrigated with well water developed the largest stomata but had the lowest Dₛ and intermediate g_smax values. These outcomes highlight a complex anatomical trade-off shaped by environmental conditions such as high irradiance and water salinity, both of which appear to constrain stomatal development and reduce anatomical efficiency for gas exchange. These findings partially align with the stomatal size-density trade-off model proposed by [39], which posits that smaller, densely packed stomata maximize conductance. However, the present data and visualizations indicate a generally positive correlation between S_s and D_s (Figure 4a), contradicting the trade-off assumption and instead suggesting coordinated development. As noted by [47] this positive relationship implies that g_smax should also rise in response to increases in both S_s and D_s. Yet, in Figure 4b, this expected relationship is not entirely evident—particularly in full sun treatments where high D_s does not always translate into proportionally high g_smax—indicating that additional environmental constraints may modulate conductance beyond anatomical potential.

Importantly, the relationship between stomatal traits and gas exchange in M. haekkinenii mirrors patterns observed in other ornamental crop systems. For instance, ornamental plants subjected to water limitation show reduced g_s and altered WUE, consistent with acclimation strategies documented in drought-stressed ornamentals [63]. Under salinity stress, ornamental crops employ osmotic adjustment, ROS-scavenging pathways, and stomatal regulation to maintain photosynthetic function [64]. These comparative insights strengthen the interpretation that the stomatal and physiological trade-offs observed in M. haekkinenii are not species-specific anomalies but rather part of a broader set of ornamental crop adaptation mechanisms. Moreover, stomatal density and chlorophyll content in cultivated Musa genotypes have also been shown to vary significantly across genomic groups, with abaxial densities reaching up to 233 e/mm² in M. balbisiana cultivars and strong correlations between stomatal traits, chlorophyll concentration, and resistance to foliar pathogens such as Mycosphaerella fijiensis [65]. In addition, stomatal morphology and conductance have been documented to vary widely across Musa germplasm [66] and water-stressed bananas exhibit reduced g_s and altered osmotic regulation comparable to our findings [67]. These responses align with the broader environmental physiology of bananas, where light intensity, salinity, and water availability are known to strongly regulate stomatal density and gas exchange [68]. Together, these Musa-specific comparisons reinforce the novelty of evaluating M. haekkinenii within the ornamental banana context while situating its responses within established physiological patterns of the genus.

This discrepancy becomes especially apparent when considering the performance of plants grown under shade with reverse osmosis water. Notably, this treatment yielded intermediate values for both stomatal size and density yet achieved a relatively high g_smax. This outcome suggests that optimal gas exchange may not rely on maximizing any single anatomical trait but rather on achieving a balanced configuration that avoids the extremes induced by environmental stressors. These observations align with the findings of [37], who emphasized that stomatal coordination is both genetically regulated and environmentally responsive, especially under saline and high-light conditions—both of which are reflected in the present dataset.

The responses of M. haekkinenii to light intensity and water quality align with previous studies on tropical ornamentals, where moderate shade has been shown to enhance biomass allocation, chlorophyll retention, and overall plant aesthetics [69,70,71,72]. Similarly, the detrimental effects of high-salinity irrigation, including reduced stomatal conductance due increased ion toxicity, have been widely documented in Musa cultivars and other salt-sensitive species [73,74]. These findings suggest that combining controlled light exposure with high-quality irrigation water is a viable strategy to enhance plant quality and commercial viability, as has been proposed for other high-value ornamental species.

While this study provides valuable insights into the optimal cultivation conditions for M. haekkinenii, certain limitations must be acknowledged. The controlled experimental setup may not fully replicate field conditions, where additional abiotic and biotic factors influence plant performance [23,75]. Moreover, the study focused on short-term physiological responses; further research is needed to assess long-term acclimation mechanisms, flowering dynamics, and potential metabolic shifts under extended cultivation. Phenological stages were not recorded in this experiment, which limited the ability to relate growth dynamics to developmental phases. Another important consideration is that the baseline evaluation was established 15 days after the onset of the experiment, which resulted in significant initial differences in several variables and, consequently, influenced the interpretation of early-stage comparisons. It is important to emphasize that this work represents the first study evaluating the physiological responses of M. haekkinenii, and future studies should adopt standardized frameworks such as the BBCH scale to improve comparison across experiments. In addition, total dry matter production was not quantified, since the plants were part of the living collection at Fairchild Tropical Botanic Garden. This limited the ability to assess whole-plant growth and stress responses in terms of total biomass accumulation. Finally, a more in-depth biochemical analysis of secondary metabolites could provide further insights into the species’ resilience and ornamental appeal under different environmental conditions. Despite these limitations, this study represents one of the first evaluations of the ecophysiological responses of M. haekkinenii, offering a framework for optimizing its commercial production and guiding future research on its adaptation to diverse cultivation environments.

Although the conditions applied in this study were different than those experienced in the natural habitats of M. haekkinenii, they were chosen to represent restricted-light environments typical of greenhouse and indoor ornamental production. This distinction is critical, as the results are directly relevant to horticultural management practices rather than ecological field conditions. The integration of morphological, physiological, and anatomical analyses highlights how microenvironmental management can be leveraged to enhance the ornamental quality and commercial cultivation potential of this compact Musa species.

The results of this study underscore the necessity of integrating moderate light conditions with high-quality irrigation water to optimize the growth and physiological stability of M. haekkinenii. These findings offer a foundation for developing sustainable large-scale cultivation practices by mitigating environmental stressors while enhancing desirable morphological traits. Future research should focus on refining fertilization strategies and evaluating temperature interactions under field conditions for commercial production. Additionally, genetic studies on salinity and light tolerance could further support breeding programs aimed at enhancing stress resilience and ornamental quality in M. haekkinenii.

5. Conclusions

This study provides the first experimental evidence of the ecophysiological responses of Musa haekkinenii, wild diploid banana, with high ornamental potential, under contrasting light and water quality regimes. Restricted light conditions enhanced morphological traits such as pseudostem diameter, leaf number, and sucker production while also improving physiological parameters including stomatal conductance, chlorophyll indices, and electron transport rate. Plants irrigated with reverse osmosis water promoted stable growth and physiological performance compared to well water, likely due to its neutral pH and lower salinity, which reduced ionic stress and improved nutrient availability. These findings underscore the importance of optimizing both light and water management in controlled environments to ensure plant vigor, visual quality, and stress resilience. From a practical perspective, maintaining moderate shading (~100 µmol m⁻² s⁻¹ PAR) and low-salinity irrigation water (~30 µS/cm) promotes compact growth and improves overall ornamental performance. For small-scale nurseries and urban landscaping, these results demonstrate that M. haekkinenii can thrive under light-restricted environments such as shaded courtyards, patios, or indoor spaces. These findings underscore the importance of optimizing both light and water management to ensure plant vigor, aesthetic appeal, and stress resilience, thereby expanding the commercial applications of M. haekkinenii in the ornamental horticulture industry.