LIGHT PULSES IN THE MICROPROPAGATION OF Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson x Corymbia citriodora (Hook.) K.D.Hill & L.A.S.Johnson CLONE: in vitro ELONGATION AND ROOTING

LED lamps emit light in specific spectrums, significantly enhancing the quantum efficiency of photosynthesis (QFP) and, consequently, plant growth and development. Corymbia citriodora , Corymbia torelliana , and some of their interspecific hybrids are being incorporated into clonal forestry programs. However, a major bottleneck in propagation is related to rooting. This study aimed to analyze the effects of light pulses with different spectral qualities on the in vitro elongation and rooting stages, using micropropagation techniques in a hybrid clone of Corymbia torelliana x Corymbia citriodora . Four light sources were utilized: (2F) fluorescent, (LED V+/A) red (99%) + blue (1%), (LED V/A+) red (1%) + blue (99%), and (LED V/A) red (72%) + blue (28%). The treatments compositions consisted of combining these light sources with different exposure durations of 7, 14, 21, and 28 days (without pulse), totaling 13 treatments. After 28 days of cultivation, the explants were evaluated based on the following parameters: survival (%), contamination (%), callogenesis (%), rooting (%), culture medium oxidation (%), shoot length (cm), and plant vigor (scores from 1 to 3). The results indicated that the lowest oxidation rate of the culture medium occurred with the V/A+ LED light source after 28 days. However, this result did not directly affect vigor or in vitro development. Additionally, under the tested conditions, using V+/A LEDs (a higher proportion of red) for 7 days stimulated shoot growth and root emission, leading to the best in vitro development.


INTRODUCTION
The success of productivity in plantations of exotic forest species in Brazil is intrinsically linked to several factors, with clonal silviculture being a highly relevant component.A set of techniques has allowed the development of plantations, improving activities from the vegetative propagation of selected materials to the establishment and management of clonal forests (Xavier et al., 2021).
In this context, species of Corymbia, such as Corymbia torelliana (F.Muell.)K.D. Hill & L.A.S. Johnson and Corymbia citriodora (Hook.)K.D. Hill & L.A.S. Johnson as well as their interspecific hybrids have been incorporated into clonal forestry cultivation initiatives.These species are significant in the forestry sector due to the quality and density of their wood and their resilience to harsh and adverse conditions (Assis, 2014).However, due to low rooting levels -less than 5% -companies in the forestry sector face difficulties in the vegetative propagation of the species.Therefore, rooting has been the main obstacle in the propagation of seedlings of the genus Corymbia (Reis et al., 2014).
Micropropagation is a widely used technique in agriculture that involves in vitro propagation from plant parts, enabling faithful reproduction of selected genotypes (Chen et al., 2002).The technique has been applied to multiply virus-free plants in less time and physical space (Abiri et al., 2020), and to enhance the efficiency of various processes within Corymbia seedling production (Brondani et al., 2018;Souza et al., 2019;Trueman et al., 2018).However, despite these advances, inducing rhizogenesis and managing environmental conditions remain significant challenges in the forestry sector.
The elongation process in micropropagation of stumps is essential to ensure that the shoots reach a desirable shoot length, given the need to maintain the plant architecture for the rooting stage (Xavier et al., 2021).Several factors impact the rooting of the stumps, including genetics, physiological conditions, mineral nutrition of the mother plant, storage and health of the stumps, environmental conditions, and carbohydrate levels in the vegetative propagules (Hartmann et al., 2011;Xavier et al., 2021).However, the development of in vitro environmental control systems has been proposed as a solution to overcome challenges in the large-scale production of high-quality plants (Gago et al., 2014;Arencibia et al., 2017;Shukla et al., 2017).
Environmental control can be facilitated in commercial micropropagation since the light factor can be easily managed.LED (Light Emitting Diode) lamps can replace fluorescent lamps emitting a broad spectrum, including unnecessary and low-quality wavelengths for promoting plant growth (Ramírez-Mosqueda et al., 2017).The use of LED lamps has been a solution to overcome challenges associated with inefficient lighting, heat dissipation, high energy consumption and inadequate spectrum of traditional sources such as high-pressure sodium lamps, fluorescent lamps, metal halide lamps, and incandescent bulbs (Álvarez et al., 2014).
In in vitro environments, lamps can be managed to alter the photoperiod (light hours), light intensity (photon flux), and spectral quality (different wavelengths), influencing the morphology and growth of plant cells, tissues, and organs (Higuchi and Hisamatsu, 2016;Rehman et al., 2017;Zakurin et al., 2020).Therefore, LED lights have desirable characteristics such as spectral composition control, long durability, emission of specific wavelengths, and reduced heat emission and they are compact, easily manageable, and installable in growth chambers (Li et al., 2010;Muneer et al., 2014).
Adjustments to the wavelengths of LEDs create combinations of red and blue light that can generate specific physiological responses in plant photoreceptors, such as phytochromes (red light) and cryptochromes (blue light) (Muneer et al., 2014).Recent research has demonstrated how different light qualities affect plant metabolism (Batista et al., 2018), demonstrating that the absorption of blue and red light emitted by LED lamps exerts a substantial influence on plant development and physiology, representing about 90% of the emitted light (Lazzarini et al., 2018).
Considering the above, we hypothesize that pulses from different light sources enhance in vitro growth and development by stimulating stem elongation and rooting.Therefore, the objective of this study was to analyze the effects of using light pulses with different spectral qualities on the in vitro elongation and rooting stages through micropropagation techniques, in a hybrid clone of Corymbia torelliana × Corymbia citriodora.

Study location and experimental material
The experiment was conducted at the Tissue Culture Laboratory II of the Institute of Applied Biotechnology for Agriculture (BIOAGRO) at the Federal University of Viçosa, in Viçosa, Minas Gerais, Brazil.
The plant material used for the explants was sourced from a clone of Corymbia torelliana (F.Explants were multiplied in 55 mL test tubes containing 10 mL of JADS culture medium (Correia et al., 1995).The medium was composed of JADS salts with the addition of 30 g L -1 of sucrose, 100 mg L -1 of myoinositol, 800 mg L -1 of Polyvinylpyrrolidone (PVP), and 5,5 g L -1 of agar for solidification, and the pH was adjusted to 5.8 ± 0.1.The culture medium was supplemented with 0,5 mg L -1 of 6-benzylamiporurine (BAP) and 0,01 mg L -1 of α-naphtaleneacetic acid (NAA) for the multiplication phase.The test tubes were capped with polypropylene lids, and the the culture medium was autoclaved at 1.5 atm and 121°C for 20 minutes.
The explants were subcultured on a culture medium containing the hormonal balance of the multiplication stage until the desired number of plants was obtained.After the multiplication stage, the explants were transferred to the JADS culture medium, but with 0,3 mg L -1 of BAP and 0,01 mg L -1 of NAA, for 15 days for the stabilization stage before the experiment.
For the experimental setup, the explants were standardized to 1,0 cm of aerial part and two leaf pairs (Figure 1), individualized and free of callogenesis at the base of the stump, and cultivated on JADS culture medium supplemented with 0,02 mg L -1 of BAP and 0,2 mg L -1 of IBA.The hormonal balance was adjusted to promote the elongation and rooting stages (Figure 1).

Cultivation conditions and light source quality
After introducing the explants, the tubes were sealed with polypropylene caps and secured with micropore tape.The material was maintained in a growth room with a photoperiod of 16 hours, a constant temperature of 25 ± 2 ºC, a relative humidity of 60 ± 5%, and an irradiance of 60 μmol m -2 s -1 mas measured by a LI-COR® LI-250A Light Meter.
Four light sources, each with different spectral qualities, were used in the treatments: fluorescent light (2F); red+/blue LED with 99% red and 1% blue, with wavelengths of 650 nm and 450 nm, respectively; red/blue+ LED with 1% red and 99% blue, with wavelengths of 650 nm and 450 nm, respectively; and red/blue LED with 72% red and 28% blue, with wavelengths of 650 nm and 450 nm, respectively.
At the end of 28 days of culture, the explants were assessed for survival (%), contamination (%), rooting (%), callogenesis at the base of the stump (%), shoot length (cm), vigor (rated from 1 to 3), and medium oxidation (%).The experiment was replicated twice under identical conditions.To ensure standardization during the evaluation, the assessors were trained and participated in both experimental trials.The vigor assessment methodology involved assigning scores ranging from 1 to 3, considering undesirable plant characteristics such as explant oxidation, leaf abscission, chlorosis, and hyperhydricity.Plants with a score of 1 exhibited at least two undesirable characteristics; plants with a score of 2 exhibited at least one undesirable characteristic; and plants with a score of 3 did not exhibit any undesirable characteristics.The final score for each treatment was calculated as the arithmetic mean of the scores assigned to the repetitions.Consequently, a score closer to 3 indicates higher vigor of the explant.
The percentage data that did not exhibit a normal distribution according to the Kolmogorov-Smirnov test were transformed using the equation: arc sin(√(x/100)).The data were subjected to analysis of variance (ANOVA), and the means were compared using Tukey's test at a 5% significance level.

Survival, callogenesis, contamination, and explant vigor
The results indicated that the characteristics of contamination, survival, callogenesis at the base of the stump and vigor showed no significant differences in relation to the light source and duration of exposure, meaning they acted independently.All treatments achieved a high average survival rate of 99.4% ± 1.10% and high callogenesis at the base of the stump with 99.23% ± 1.20%.The maximum contamination observed was 10% ± 13.7% (Table 1).
However, after 28 days of the experiment, it is worth noting that the average scores assigned for vigor were above 2.6 (± 0.1949), demonstrating that few undesirable characteristics were observed in the explants.Therefore, none of the treatments hindered in vitro development.

Elongation and Rooting
After 28 days of cultivation, bud elongation was significantly (5%) influenced by light pulses with different spectral qualities and duration.The greatest shoot length (cm) (Figure 3) was achieved in treatment T5, which involved 7 days of pulsing with R+/B LED light followed by 21 days under 2F light, with an average length of 2.7 cm ± 0.241.This treatment promoted nearly threefold growth of the explant compared to its initial length, whereas treatment T12 resulted in a shoot length of less than 0.5 cm.
The rooting of the stumps was also significantly influenced by light pulses with different spectral qualities and duration at a 5% significance level (Figure 4).The treatment with 7 days of pulsing with R+/B light followed by 21 days under 2F light (T5) achieved the highest rooting rate, reaching 55% (± 26%) of explants with root emission.This is a noteworthy average from the perspective of propagating hybrid clones of the genus Corymbia, given the recalcitrance of these materials to rooting.

DISCUSSION
Our results confirmed the hypothesis that both aspects of the treatments (light spectrum quality and duration of exposure) promoted the in vitro development of the analyzed clone.It appears that the optimal time for inducing accelerated growth is 7 days under the R+/B LED light (with a higher red-to-blue light ratio), which provides the ideal combination of wavelengths in the red region at 650 nm (99%) and blue at 450 nm (1%).Therefore, the timely regulation of the light spectrum during the elongation and rooting phases highlighted the importance of spectral quality in the in vitro development of Corymbia, potentially yielding better results than fluorescent white light.
Elongation of the explants was essential for producing mini-stumps and facilitating the handling and architecture of the seedlings.Generally, higher photosynthetic quantum efficiency (PQE) results in increased dry matter, root system expansion, and stem elongation in many plant species (Bilodeau et al., 2019).The light source with a higher proportion of red light may have promoted Light pulses in the micropropagation of... Superbi et al, 2024 greater PQE when comparing the parameters (shoot length and rooting percentage) with treatment T0, which used only white light.
It is important to highlight that even a small addition of blue light was sufficient to produce a superior effect on in vitro growth and development (Brown et al., 1995;Yorio et al., 1998).
Phytochromes play a fundamental role in acquiring information perceived by the aerial part of the plant.A low red ratio, for example, can influence root behavior, affecting both growth and morphology.The role of light quality in controlling root function may have been overlooked over the years, but some studies report results with woody species (Santos Junior et al., 2022), including Eucalyptus (Souza et al., 2019;Frade et al., 2023).Red light has been shown to stimulate root growth in Enhalus acoroides seedlings (Heemboo et al., 2023).Additionally, the addition of far-red light only during the initial growth phase of medicinal cannabis improved rooting without promoting stem elongation (Sae-Tang et al., 2024).
The lowest values for culture medium oxidation occurred under the R/B+ LED (with a higher proportion of blue light) with no pulsing.Although this was a significant result, this characteristic did not directly affect vigor or overall plant development.The influence of the light source with a higher proportion of blue light (R/B+ LED) may have been limited because not all absorbed blue photons are utilized in photosynthesis.Therefore, photosynthetic pigments did not fully realize the absorption of photons from the R/B+ LED.As a result, they did not contribute significantly to the growth and production of the explants.Of the total blue light absorbed, 20% might have been used by non-photosynthetic pigments or dissipated as heat or fluorescence (Bilodeau et al., 2019).In contrast, red LEDs emit a narrow light spectrum (620-750 nm), highly absorbed by chlorophyll and phytochromes (Muneer et al., 2014).
Light sources with specific wavelengths induce photomorphogenic responses in explants cultivated in controlled environments.This technology is beneficial for eucalyptus plants by optimizing in vitro development and productivity (Frade et al., 2023).In each stage of development, an optimal behavior will be desired for the explant.The wavelength was considered an essential factor in the in vitro multiplication of E. grandis × E. urophylla, as it reduced hyperhydricity, a higher number of shoots per explant, and greater shoot length (Souza et al., 2020a;Souza et al., 2020b).
Controlling plant morphology in vitro requires constant protocol updates due to the innovations emerging in the market.Artificial lighting technologies offer numerous possibilities for light manipulation and management.The light sources used in the experiments act in precise ways and do not always come with detailed descriptions of the spectral quality employed.However, the lack of reproducibility in protocols results in outcomes that are difficult to compare (Batista et al., 2018).The distinguishing feature of this work was achieving high rooting percentages for Corymbia compared to other studies with the genus (Reis et al., 2014), as well as specifying the proportions of each wavelength used, making the experiments comparable within the genus (Reis et al., 2014), as well as specifying the proportions of each wavelength used, allowing for experiment comparability.
The high maintenance cost of in vitro cultivation systems is justified by the expenses related to system setup, electricity, and the challenge of providing the irradiance necessary to reach the light saturation point (LSP) (Xavier et al., 2021).However, it is important to highlight the precedents that this work sets for testing growth acceleration strategies.This is especially relevant for regions with low solar intensity, where these methods could be explored as a supplementary light source.It is important to consider that in vitro cultivation conditions may differ from ex vitro conditions and should be adjusted according to the goals of seedling production.

CONCLUSION
Light pulses with different spectral qualities promoted both elongation and rooting.The R+/B LED light for 7 days, followed by a switch to 2F light for 21 days, was identified as the best combination of duration and spectral quality for the in vitro development of a Corymbia torelliana × Corymbia citriodora clone.This work contributed to advancing vegetative propagation techniques for the Corymbia genus.In addition, it opened new opportunities for the use of LEDs with specific spectral qualities, suggesting light pulses as a method for accelerating growth during different phases of in vitro development.