Influence of the Hybrid Compound La(NO3)3@Zn-MOF on the In Vitro Growth of Sugarcane (Saccharum spp. L.)

TL;DR

This study shows that a hybrid compound can boost sugarcane growth in lab conditions but high doses are harmful.

Contribution

The novel hybrid compound La(NO3)3@Zn-MOF is shown to stimulate sugarcane growth at low concentrations.

Findings

5 mg L−1 La(NO3)3@Zn-MOF increased shoots and leaves in sugarcane plantlets.

High concentrations (10–20 mg L−1) caused toxicity and inhibited growth.

Fresh and dry matter increased significantly at 5 mg L−1 La(NO3)3@Zn-MOF.

Abstract

In agriculture, the use of Porous Coordination Polymers (PCPs), also known as Metal–Organic Frameworks (MOFs), has emerged as a promising area of research for biological applications, particularly as long-lasting delivery systems for biostimulant chemical compounds. The objective of this study was to evaluate the effect of different concentrations of the hybrid compound La(NO3)3@Zn-MOF and La(NO3)3·6H2O on the in vitro growth of sugarcane cv. Mex 69–290. To assess the effect on sugarcane (Saccharum spp. L.), plantlets were grown in flasks containing Murashige and Skoog (MS) liquid medium without growth regulators. Each treatment consisted of three independent culture flasks, each containing three sugarcane plantlets, and different concentrations (0, 2.5, 5, 10, and 20 mg L−1) of La(NO3)3@Zn-MOF and La(NO3)3·6H2O were added separately. After 30 days of culture, various growth variables were evaluated, including explant length, number of leaves, number and length of shoots, fresh matter, dry matter, and chlorophyll content (a, b, and total). The 5 mg L−1 concentration of La(NO3)3@Zn-MOF increased the number of shoots and leaves in the sugarcane plantlets, and significant increases in fresh and dry matter were observed, while no statistically significant differences were detected in explant length, shoot length, or chlorophyll a, b, and total chlorophyll. However, inhibitory effects were observed at concentrations of 10 and 20 mg L−1 of La(NO3)3@Zn-MOF and La(NO3)3·6H2O, respectively. In conclusion, the hybrid compound La(NO3)3@Zn-MOF exhibited biostimulatory effects on sugarcane growth and physiology under in vitro conditions, whereas high concentrations of the lanthanum(III) salt caused toxicity symptoms in the sugarcane plantlets.

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1. Introduction

Metal–organic frameworks (MOFs) are porous materials composed of self-assembled metal ions and organic ligands that form open crystalline networks [1]. MOFs exhibit several valuable physicochemical properties, including high structural diversity, tunable porosity, large surface area, and remarkable thermal and chemical stability [2]. In addition, they are of particular interest for delivering biologically relevant compounds because their adjustable pores can be functionalized to accommodate specific molecules [3].

Moreover, MOFs have a wide range of promising applications across fields such as chemistry, drug delivery, biomedicine, food safety, and plants. Their unique structural properties can be harnessed to influence metabolic processes by regulating the absorption, storage, and controlled release of various molecules. In agricultural systems, MOFs can directly affect plant growth and development through the release of agronomically important compounds, such as fertilizers, pesticides, herbicides, nucleotides, and proteins [3,4,5], thereby enhancing resistance to biotic and abiotic stresses [5].

For instance, [1] reported that Fe-MOFs promoted the release of essential nutrients (N, P, and Fe) in the soil during the growth of rice (Oryza sativa L.), thereby increasing crop yield. Similarly, [6] demonstrated that oxalate–phosphate amine MOFs (OPA-MOFs), which contain nutrients such as N, P, and Fe, enhanced nitrogen use efficiency in wheat (Triticum aestivum L.) by slowly releasing ammonium into the soil. Furthermore, [7] showed that Fe-MOFs reduced the toxic effects of tebuconazole on T. aestivum plantlets during early growth.

Among MOFs, those composed of lanthanide ions are generally more porous and thus offer a wide range of applications [2], including analyte sorption, gas storage, drug delivery, and environmental pollution control [8]. Lanthanides, as inorganic compounds, are also considered plant biostimulants. At low concentrations, they can stimulate plant growth and enhance yield through hormetic effects [9,10]. Several studies have demonstrated that rare earth elements, such as cerium [11,12], europium [13], neodymium [14], and lanthanum, have been used in agriculture to increase crop yields and improve product quality [15,16,17].

Lanthanum belongs to the group of elements known as the lanthanides, which comprise the 15 elements with atomic numbers 57 to 71, from lanthanum to lutetium. This group includes cerium, europium, promethium, and thulium, among others [17]. It is the most electropositive element among the rare earths, is uniformly trivalent, and exhibits chemical properties like those of the alkaline earth elements [18]. The effects of lanthanum and its compounds on cellular systems are of considerable interest because they promote nutrient absorption, a behaviour attributed to its electropositive character and its chemical similarity to alkaline earth elements such as calcium(II) and magnesium(II) [15]. Several studies have shown that lanthanum induces positive effects on plant growth in rice (Oryza sativa) [19,20], red sage (Salvia miltiorrhiza) [21], and tulip (Tulipa gesneriana L.) [22].

Sugarcane (Saccharum officinarum) is a tropical grass and a significant source of global sugar production, supplying approximately 60% of the world’s sugar and generating a wide range of agro-industrial products, including molasses and ethanol [23,24]. However, climate change has intensified both biotic and abiotic stresses on crops, significantly affecting global agricultural yields through a complex interplay of environmental factors and plant physiological responses [23]. Consequently, improving sugarcane productivity increasingly relies on advanced, innovative strategies to mitigate these stresses and enhance overall plant performance.

Today, in vitro culture is an effective and indispensable technique for producing large numbers of pathogen-free, genetically uniform plants, often leading to higher yields and superior quality than those propagated by traditional methods [25,26]. In this study, a metal–organic framework (MOF) based on zinc(II) formate combined with lanthanum(III) nitrate was used to develop a new hybrid compound. When applied to sugarcane, this compound mitigated toxic effects by enhancing physiological, morphological, and biochemical responses. The objective of this research was to evaluate the effects of different concentrations of the hybrid compound La(NO_3_)3@Zn-MOF and La(NO_3_)3·6H_2_O on the in vitro development of sugarcane cv. Mex 69-290.

2. Results

2.1. Characterization of La(NO3)3@Zn-MOF

The characterization of the La(NO_3_)3@Zn-MOF hybrid composite was carried out by powder X-ray diffraction (PXRD) and Fourier Transform Infrared spectroscopy (FTIR).

A comparison was made between the diffractogram of the reflux-synthesized Zn-MOF and the existing bibliographic data for this procedure, validating the experimental results, as shown in Figure 1A. Comparing intensities, a similarity was observed among the 16, 21, 23, 26, 31, 33, and 34° signals, with a slight shift to the right relative to the experimental Zn-MOF. However, all signals exhibited this shift in the same direction. This demonstrates that a structure like that previously reported was obtained. On the other hand, the resulting diffraction pattern of the La(NO_3_)3@Zn-MOF hybrid composite was the product of a mixture of crystalline phases, as shown in Figure 1B. This mixture consisted of the crystalline phase of the Zn-MOF and the crystalline phase of La(NO_3_)3·6H_2_O, confirming that the samples match, as similarity was observed in the signals at 14, 20, 22, 25, 26, 30, 33, and 34° concerning the undoped Zn-MOF, which retains its crystalline phase. The remaining signals correspond to the crystal lattice generated by the presence of La(NO_3_)3·6H_2_O.

Finally, the La(NO_3_)3@Zn-MOF hybrid composite and its precursors (Zn-MOF and La(NO_3_)3·6H_2_O) were characterized using FTIR spectroscopy to observe the behaviour of the nitrate anion in the hybrid material. Figure 2 shows the spectra obtained from the PerkinElmer^®^ FTIR/FTNIR spectrophotometer model Spectrum 100 with an attenuated total reflection (ATR) accessory, confirming the doping of the Zn-MOF with La(NO_3_)3·6H_2_O. The FT-IR spectrum of La(NO_3_)3·6H_2_O shows stretching bands associated with both the nitrate anion (NO_3_^−^) and the water of hydration: 1380–1450 cm^−1^, ν_3_ (asymetric N–O); 1030–1060 cm^−1^, ν_1_ (symetric N–O); 3200–3600 cm^−1^, ν(O–H); and 400–600 cm^−1^, ν(La–O). The FT-IR spectrum of Zn-MOF exhibits stretching bands attributable to the formate ligand (HCOO^−^) and the small amount of adsorbed water: 1580–1620 cm^−1^ ν(asymetric COO^−^), 1350–1385 cm^−1^ ν(symetric COO^−^), 3200–3600 cm^−1^ ν(O–H), and 400–600 cm^−1^ ν(Zn–O). The FT-IR spectrum of La(NO_3_)3@Zn-MOF shows that the main stretching bands originate from formate, nitrate, and water of hydration. Doping does not create new bands but modifies their intensities and can slightly shift their positions.

In addition, although the crystalline structure of MOFs is usually very regular, mortar grinding causes fragmentation. The SEM micrograph of the Zn-MOF shows an irregular and porous structure (Figure 3A). Once the Zn-MOF was doped with La(NO_3_)3, the SEM micrograph revealed clusters of spherical and individual particles on the surface of the material (Figure 3B). Therefore, mechanochemical grinding led to a surface interaction in which La^3+^ ions can coordinate with the formate oxygens or with structural defects, while NO^3−^ anions coordinate with unsaturated Zn^2+^ sites.

2.2. Effect of La(NO3)3@Zn-MOF and La(NO3)3·6H2O on the In Vitro Growth of Sugarcane

The number of shoots showed statistically significant differences among the treatments evaluated, with the highest value recorded at the 5 mg L^−1^ concentration of La(NO_3_)3@Zn-MOF (14.83 ± 0.70), while the lowest value was observed in the treatment with 20 mg L^−1^ of La(NO_3_)3·6H_2_O (Figure 4A). In contrast, shoot length showed no statistically significant differences among the treatments with La(NO_3_)3@Zn-MOF, the lanthanum salt, and the control (Figure 4B).

Likewise, the number of leaves showed a significant increase in plantlets treated with 5 mg L^−1^ of La(NO_3_)3@Zn-MOF, with an average of 51.33 ± 5.10 leaves, while the lowest value was recorded in the treatment with 20 mg L^−1^ of La(NO_3_)3·6H_2_O, with 3.88 ± 1.46 leaves (Figure 4C). Plant length, on the other hand, did not show statistically significant differences between the treatments evaluated (Figure 4D).

Fresh matter showed statistically significant differences between the treatments evaluated, with the maximum value reached in the treatment with 5 mg L^−1^ of La(NO_3_)3@Zn-MOF (2.06 ± 0.32 g), while the minimum value was recorded in the treatment with 20 mg L^−1^ of La(NO_3_)3·6H_2_O (Figure 5A). Similarly, dry matter also showed statistically significant differences, with the highest value observed in the concentration of 5 mg L^−1^ of La(NO_3_)3@Zn-MOF (0.21 ± 0.002 g) and the lowest in the treatment with 20 mg L^−1^ of La(NO_3_)3@Zn-MOF (0.02 ± 0.005 g) (Figure 5B).

Representative images of plantlets exposed to different concentrations of La(NO_3_)3@Zn-MOF showed greater growth than those exposed to La(NO_3_)3·6H_2_O and the control, as shown in Figure 6.

2.3. Effect of La(NO3)3@Zn-MOF and La(NO3)3·6H2O on the Chlorophyll Content of Sugarcane

The evaluations of chlorophyll a, chlorophyll b, and total chlorophyll do not show statistically significant differences among the treatments evaluated (Figure 7). However, variations in photosynthetic pigment content were observed among treatments. In general, the highest values for chlorophyll a, chlorophyll b, and total chlorophyll were observed in plantlets treated with 2.5 mg L^−1^ of La(NO_3_)3@Zn-MOF, while the lowest values corresponded to the treatment with 20 mg L^−1^ of La(NO_3_)3·6H_2_O. Furthermore, treatments with the hybrid compound tended to have higher chlorophyll content than treatments with the La(NO_3_)3·6H_2_O and the control.

3. Discussion

3.1. Synthesis of La(NO3)3@Zn-MOF

The hybrid compound La(NO_3_)3@Zn-MOF was synthesized via mechanochemical milling. The Zn-MOF employed in this study consists of formate anions used as organic ligands and zinc(II) used as the metal centre. The Zn-MOF was subsequently doped with La(NO_3_)3·6H_2_O and stored, possibly for controlled release. Previous studies have reported the use of zinc-based MOFs due to their biocompatibility [27].

This study evaluated the effect of the hybrid compound La(NO_3_)3@Zn-MOF on the in vitro growth of sugarcane. At a concentration of 5 mg L^−1^, La(NO_3_)3@Zn-MOF promoted significant increases in the number of shoots and leaves, as well as in the fresh and dry matter of the sugarcane plantlets.

According to [14], lanthanides tend to bind to the carboxyl groups of the plant cell wall, particularly those present in pectin, thereby stimulating plant development and contributing to mechanical strength and flexibility. The stimulatory response of plants depends on the concentration of the biostimulant: at low concentrations, lanthanides exert beneficial effects, whereas at high concentrations, they can become toxic, a phenomenon known as the hormetic effect [28,29].

3.2. Evaluation of the Effect of La(NO3)3@Zn-MOF on the In Vitro Growth of Sugarcane

In this study, the application of high concentrations (10 and 20 mg L^−1^) of La(NO_3_)3·6H_2_O resulted in the inhibition of shoot and plantlets length. Plantlets exposed to the highest doses exhibited toxicity symptoms, including reduced colour intensity, leaf yellowing, weak stems, vitrification, and necrosis. In contrast, plantlets treated with La(NO_3_)3@Zn-MOF did not display deficiency or toxicity symptoms; instead, they showed an intense green colouration and vigorous growth. This enhanced performance is attributed to the greater availability of lanthanum(III) ions within the Metal–Organic Framework, which effectively functions as a storage and controlled-release system.

This is likely because the lanthanum(III) salt in the liquid medium was fully available to the plant, with no mechanism for controlled administration. In contrast, the lanthanum(III) incorporated into the hybrid compound exhibited a controlled-release behaviour, ensuring more efficient utilization and increasing the shoot multiplication rate in sugarcane. According to [3], MOFs possess controlled-delivery capabilities for pesticides and other agronomically relevant molecules. Together, these results suggest that La(NO_3_)3@Zn-MOF may serve as a promising biostimulant to enhance the in vitro multiplication of this crop.

This enhanced performance could be attributed to the greater availability of lanthanum(III) ions within the Metal–Organic Framework, which could function effectively as a controlled storage and release system. However, to support this claim, it would be necessary to evaluate the release kinetics of the lanthanum(III) ion in both the hybrid compound and the inorganic salt by quantification using inductively coupled plasma optical emission spectroscopy (ICP-OES) or atomic absorption spectroscopy (AAS).

The impact of lanthanum(III) on plants depends on several factors, including the cultivated species, developmental stage, applied concentration, treatment duration, and environmental growing conditions [30]. Positive effects on nutrient uptake are often reflected in improved plant growth. Lanthanum(III) salts can influence the absorption and accumulation of other elements in plants through synergistic or antagonistic interactions [31]. For example, a previous study [32] reported that applying 2 mg L^−1^ of LaCl_3_ to four varieties of pepper (Capsicum annuum L.) increased the number of leaves.

Similarly, La(NO_3_)3@Zn-MOF exhibited a hormetic effect on the number of leaves, although the optimal concentration depends on the crop species. For sugarcane cv. Mex 69–290, a dose of 5 mg L^−1^ produced the most pronounced effect. These results indicate that the hybrid compound can improve seedling quality by enhancing specific growth parameters and biomolecule concentrations, depending on the genotype and exposure duration [31]. The authors in [33] emphasized that plantlet matter is a key parameter for assessing growth and development, as it reflects increases in overall size and biomass accumulation.

In this study, an increase in shoot production was observed, resulting in a significant rise in biomass and, consequently, in both fresh and dry weight. A related study [22] evaluated the effects of two lanthanum(III) sources—lanthanum(III) chloride and lanthanum(III) nitrate—each at a concentration of 8 mg L^−1^, on the preservative solution of fifteen varieties of cut tulip flowers (Tulipa gesneriana L.). Lanthanum(III) nitrate increased the fresh matter of vase stems and prolonged their lifespan. Moreover, the nitrate form of lanthanum(III) had more pronounced effects on the metabolism of cut tulip flowers than the chloride form, likely due to the influence of the anion.

Other studies have indicated that high chloride concentrations in the growth medium can affect nutrient uptake, including N, P, K, Ca, S, and Zn, through antagonistic interactions and by altering the selective permeability of plant membranes [34]. Chloride can interact with nutrients in the solution, thereby reducing their availability and potentially causing both deficiencies and toxicities in plants [35].

On the other hand, plants can utilize nitrate as a nitrogen source, and many species exhibit improved growth when supplied with this form of nitrogen [36]. Evidence from [37] indicates that the application of 1 and 4 mg L^−1^ of lanthanum(III) nitrate and lanthanum(III) chloride to tulip (Tulipa gesneriana L.) crops resulted in a greater increase in dry matter accumulation. Similarly, researchers in [38] observed that low concentrations of lanthanum(III) salts in Salvia miltiorrhiza plantlets promoted dry matter accumulation, whereas higher concentrations did not enhance biomass production.

However, data from [39] suggest that the application of 100 mg L^−1^ of lanthanum(III) chloride to Chinese cabbage (Brassica chinensis L.) stimulated growth and increased both fresh and dry weight. In the present study, the sustained-release system was designed to deliver La(NO_3_)3·6H_2_O efficiently within the sugarcane plant’s metabolism. The Zn-MOF exhibits considerable thermal and chemical stability, gradually releasing the target molecule—in this case, lanthanum(III) ions—into plant cells. By supplying ions slowly in accordance with the plant’s requirements, the MOF avoids the toxicity associated with high doses while preserving the integrity of plant tissues.

3.3. Effect of La(NO3)3@Zn-MOF on the Quantification of Chlorophyll a, b and Total

Different concentrations of the hybrid compound and lanthanum(III) salt produced variable effects on chlorophyll content. In this study, no significant differences were observed in chlorophyll a, chlorophyll b, or total chlorophyll in sugarcane. However, a decreasing trend was observed for both La(NO_3_)3@Zn-MOF and La(NO_3_)3·6H_2_O at 10 mg L^−1^. Reductions in chlorophyll content may result from altered pigment synthesis or enhanced chlorophyll degradation. For example, the study presented in [32] reported that the application of 2 mg L^−1^ of lanthanum(III) to a sweet pepper variety stimulated chlorophyll biosynthesis.

Similarly, the evidence reported in [40] suggests that the application of 5 mg L^−1^ of lanthanum(III) increased both the photosynthetic rate and the chlorophyll index. In contrast, the analysis conducted in [41] revealed that applying 10 mg L^−1^ of lanthanum(III) oxide to maize (Zea mays) had negative effects on chlorophyll content. Adequate amounts of lanthanum(III) have been shown to enhance photosynthesis in tobacco [42] and rice [43]; however, high concentrations (170–340 mg L^−1^) reduce photosynthetic activity.

Research evidence in [32] demonstrates that the effect of lanthanum(III) nitrate on photosynthesis depends primarily on the applied concentration and the method of application. The findings reported in [44] suggested that the positive effects of lanthanum(III) nitrate on chlorophyll content may result from increased levels of essential macronutrients involved in chlorophyll biosynthesis. Conversely, the results obtained in [45] showed no significant changes in chlorophyll a or b content in barley plants treated with lanthanum(III) nitrate at concentrations ranging from 10 to 200 mg L^−1^.

However, evidence presented in [46] highlights that lanthanum(III) nitrate neither increased nor decreased chlorophyll content in Brassica chinensis L. In the present study, La(NO_3_)3@Zn-MOF was shown to stimulate growth during in vitro culture of sugarcane var. Mex 69–290 without causing toxicity at high concentrations, owing to the controlled and prolonged release of La(NO_3_)3·6H_2_O.

The effects of lanthanum(III) nitrate on plant physiology have been reported in various species. Differences in plant responses are attributed to the applied dose, method of application, and the physical and chemical properties of the growth medium. These responses are further influenced by nutrient interactions, crop type, and developmental stage [33]. In the present study, a three-way interaction was observed between the experimental model, the concentrations of the hybrid compound, and lanthanum(III) nitrate. This finding indicates that plants’ response to lanthanum is both species-specific and concentration-dependent [46].

4. Materials and Methods

4.1. Synthesis of Zn-MOF

A MOF based on the Zn^2+^ cation as the metal center and the formate anion as the organic ligand was designed using reflux heating. To achieve this, the precursors Zn(NO_3_)·6H_2_O (Merck KGaA^®^, Darmstadt, Germany, 2.7760 g, 9.3315 mmol, ≤99.0%) and formic acid (Merck KGaA^®^, 1.2886 mL, 27.9947 mmol, 95–98%) were mixed in a 1:3 ratio (metal:ligand), and 5 mL of N,N-dimethylformamide (DMF) were added. The reaction mixture was then refluxed for 6 h at 120 °C. The crystalline solids obtained from the reaction were collected by vacuum filtration through a 250 mL Büchner funnel fitted with Double-Rings^®^ filter paper, then washed with methanol and placed in a drying oven at 80 °C overnight. Zn-MOF was obtained in 70% stoichiometric yield. All solvents and reagents were of analytical grade and were purchased commercially from Merck KGaA^®^.

4.2. Mechanochemical Synthesis of La(NO3)3@Zn-MOF

The Zn-MOF was doped with the lanthanum(III) salt at 5% (w/w) by mechanical grinding in a mortar utilizing a mixture of Zn-MOF (95 mg, 0.4740 mmol) and La(NO_3_)3·6H_2_O (5 mg, 0.0115 mmol). During the 40 min process, a few drops of acetonitrile were added to promote maceration. Finally, the mixture was dried in an air oven at 80 °C for 2 h [47].

4.3. X-Ray Powder Diffraction Analysis

The diffraction pattern of the synthesized material was recorded on a D2 Phaser benchtop analyzer (Bruker^®^, Billerica, MA, USA). The data were processed employing DIFFRAC.SUITE version 11 software, supplied with the equipment.

4.4. SEM Micrographs

Scanning electron microscopy (SEM) measurements were performed with a JEOL JSM-5900-LV (JEOL Ltd., Tokyo, Japan). Oxford ISIS energy dispersive spectroscopy (EDS) was used to study the lanthanide concentration in Zn-MOF (microscope operating at an accelerating voltage of 3–30 kV).

4.5. In Vitro Establishment and Multiplication

For the in vitro establishment of sugarcane tissue cultures (var. Mex 69–290), 10 cm-long explants were collected from mother plants grown at the Cotaxtla Experimental Field of the National Institute of Forestry, Agricultural and Livestock Research (INIFAP) in Cotaxtla, Veracruz, México (18.93243, −96.19233). The explants were placed under constant agitation for 20 min in a solution containing 1 g L^−1^ of the fungicide Captan 50 plus^®^ (Ingeniería Industrial, S.A. de C.V., México City, Mexico) and the bactericide Intermicin 500^®^ (Internacional Química de Cobre, S.A. de C.V., México City, Mexico).

Three washes were performed with sterile distilled water. Subsequently, in a laminar flow hood, the layers covering the apex were removed and reduced to 3 cm in length. The selected apices were rinsed in a 45% (v/v) (6% a.i.) NaClO solution of CloralexTM (Industrias Alen, S.A de S.V, Santa Catarina, NL, México) and Tween 20^®^ (Merck KGaA^®^, Darmstadt, Germany) under agitation for 20 min. The tips were rinsed four times with sterile distilled water and grown in test tubes (22 mm × 150 mm) containing 25 mL of semi-solid MS medium [48] supplemented with 30 g L^−1^ sucrose and 100 µM methylene blue (Merck KGaA^®^, Darmstadt, Germany) as an antioxidant. The pH of the culture medium was adjusted to 5.8, and 2.9 g L^−1^ of Phytagel^®^ (Merck KGaA^®^, Darmstadt, Germany) was added as a gelling agent. The cultures were transferred to an incubation room at 24 ± 2 °C with low relative humidity (<40%), under fluorescent lighting and a photoperiod of 16 h light and 8 h dark. After four weeks, the shoots were transferred into 500 mL flasks with 30 mL of previously sterilized semi-solid MS medium, supplemented with 0.1 mg L^−1^ of BAP (6-benzylaminopurine, Merck KGaA^®^), 1 mg L^−1^ of KIN (Kinetin, Merck KGaA^®^), 0.5 mg L^−1^ of IAA (indoleacetic acid, Merck KGaA^®^) and 30 g L^−1^ of sucrose and a pH adjusted to 5.8. The cultures were incubated under the temperature and irradiation conditions described above.

After in vitro establishment, the plant material was propagated under the following conditions to obtain sufficient material for the different treatments. The cultures were transferred to an incubation room at 24 ± 2 °C with low relative humidity (<40%), under fluorescent lighting and a photoperiod of 16 h light and 8 h dark. After four weeks, the shoots were transferred to 500 mL flasks containing 30 mL of previously sterilized semi-solid MS medium, supplemented with 0.1 mg L^−1^ BAP (6-benzylaminopurine, Merck KGaA^®^), 1 mg L^−1^ KIN (kinetin, Merck KGaA^®^), 0.5 mg L^−1^ IAA (indole-3-acetic acid, Merck KGaA^®^), and 30 g L^−1^ sucrose, with the pH adjusted to 5.8. The cultures were incubated under the temperature and light conditions described above.

4.6. Treatments with La(NO3)3·6H2O and La(NO3)3@Zn-MOF

After three 30-day subcultures, 2 cm-long, rootless explants of the Mex 69–290 cultivar were used, with three plantlets grown per flask as an experimental unit. Sugarcane plantlets were propagated in liquid MS medium supplemented with 30 g L^−1^ of sucrose, with different concentrations (0, 2.5, 5, 10, and 20 mg L^−1^) of the La(NO_3_)3@Zn-MOF hybrid compound and the La(NO_3_)3·6H_2_O. For each concentration treatment, three culture flasks were used per run, resulting in nine independent experimental units per treatment. Each solution was placed in 500 mL flasks containing 10 mL of culture medium per treatment. The pH of the medium was adjusted to 5.8, and the culture medium was sterilized by autoclaving for 20 min at 120 °C. The shoots were cultured under the irradiation and temperature conditions described previously. After 30 days, the following morphological parameters were evaluated: number and length of shoots, number of leaves, plant length, fresh matter and dry matter, and biochemical parameters: chlorophyll a, b, and total chlorophyll content. Plantlet measurements were averaged within each culture flask, which was treated as the experimental unit for statistical analysis to ensure independence among observations and to avoid pseudoreplication.

4.7. Determination of Chlorophyll

Chlorophyll quantification was performed using the method described by [49]. For each sample, 100 mg of plant tissue was weighed and macerated in liquid nitrogen. The sample was placed in conical tubes (Falcon™; Corning Incorporated, Corning, NY, USA) and diluted to 10 mL with 80% acetone. The samples were refrigerated at −4 °C in the dark for 24 h. The extract was then filtered through Whatman No. 1 filter paper and diluted to 20 mL with 80% acetone. Finally, the sample was mixed using a vortex mixer. Subsequently, 2.5 mL aliquots were transferred to a quartz cell, and their absorbance was measured at 663 and 645 nm using a spectrophotometer (GENESYS™ 10S UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA), with 80% acetone as the blank.

4.8. Experimental Design and Statistical Analysis

All experiments were carried out following a completely randomized design in triplicate. Data was analyzed using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (p ≤ 0.05) using the SAS (version 9.4; Statistical Analysis System) software.

5. Conclusions

In this study, a hybrid compound was developed and employed as a controlled-release system for La(NO_3_)3·6H_2_O in in vitro-grown sugarcane plantlets. It was demonstrated that a concentration of 5 mg L^−1^ of the hybrid compound effectively stimulates sugarcane growth. To date, the synthesis and application of this specific hybrid compound have not been previously reported.

Therefore, the use of MOFs for in vitro propagation of sugarcane represents an innovative strategy to enhance the physiology of this crop. This hybrid compound has the potential to advance plant biotechnology as a biostimulant, thereby increasing productivity and sustainability in sugarcane cultivation. However, it is essential to establish safe dosage limits, as lanthanum has demonstrated both beneficial effects on plant physiology and improvements in certain yield parameters. These findings highlight the potential of the hybrid compound in in vitro propagation, where it exerts a hormetic effect on plant development.