Effect of different phytohormones on in vitro multiplication of Solanum tuberosum L. var. Cecilia

Effect of different phytohormones on in vitro multiplication of Solanum tuberosum L. var. Cecilia

Catherine Lizzeth Silva Agurto ¹, Michel Leiva Mora ²*, Fredy Santiago Córdova Frías ³,
Willian Ernesto Tipán Chinachi ³, Tannia Elizabeth Gómez Pinto ⁴, Alfredo Jiménez González ⁵, Miguel Angel Osejos Merino ⁶, Rene Nazareno Ortiz ⁷, Yosbel Lazo Roger ⁸ ,
Justo Antonio Rojas Rojas⁹

¹Docente investigador (Instituto Superior Tecnológico Pelileo/ Pelileo/ Ecuador);

²*Laboratorio de Biotecnología. Departamento de Agronomía, Facultad de Ciencias Agropecuarias (Universidad Técnica de Ambato/ Ambato, Ecuador);

³Docente investigador (Instituto Superior Tecnológico Pelileo/ Pelileo/ Ecuador);

⁴Docente investigador (Instituto Superior Tecnológico Pelileo/ Pelileo/ Ecuador);

⁵Carrera de Ingeniería Forestal. Facultad de Ciencias Naturales y de la Agricultura. Universidad Estatal del Sur de Manabí/Ecuador;

⁶Carrera de Ingeniería Ambiental. Facultad de Ciencias Naturales y de la Agricultura. Universidad Estatal del Sur de Manabí/Ecuador;

⁷Universidad Técnica Luis Vargas Torres de Esmeraldas, Ecuador;

⁸Centro de Gestión Internacional de Capacitación y posgrado.

⁹Instituto Superior Tecnológico Consulting Group Ecuador. Sede Santo Domingo;

* Correspondencia: m.leiva@uta.edu.ec; Tel.: +593 999937691

RESUMEN

Las fitohormonas se utilizan ampliamente en plantas de Solanum tuberosum con la finalidad de acelerar el proceso de multiplicación in vitro. El objetivo principal de esta investigación fue evaluar el efecto de diferentes concentraciones de fitohormonas: auxinas (AIA, AIB, ANA), citoquininas (6-BAP, TDZ, Zeatina) y giberelinas (AG3) sobre características morfológicas de Solanum tuberosum L. var. Cecilia. Se utilizaron plantas establecidas in vitro, la multiplicación in vitro se realizó mediante segmentos nodales cultivados en MS + 20 g. L⁻¹ de sacarosa + 7 g.L⁻¹ de agar + fitohormonas y se mantuvieron bajo condiones de luz blanca fluorescente total. Se evaluó el número de nudos, número de hojas, altura de la planta y número de brotes. A los 21 días al utilizar AIB (0.05 mg.L^-1 y 0.25 mg.L^-1) se incrementó el número de nudos y el número de hojas; mientras que, las concentraciones de Zeatina de 0.05 mg.L^-1, 0.1 mg.L^-1 incrementaron el número de hojas, altura de la planta y número de brotes, adicionalmente la concentración de 0.15 mg.L^-1 incrementó la altura de planta, con respecto al AG3 la concentración de 0.15 mg.L^-1 incrementó el número de nudos de plantas de S. tuberosum var. Cecilia. En base a los resultados del presente trabajo se concluyó que el AIB, Zeatina y AG3 favorecieron la multiplicación in vitro de plantas de S. tuberosum var. Cecilia.

Palabras clave: cultivo de tejidos; papa; reguladores de crecimiento; yemas.

ABSTRACT

Phytohormones are widely used in Solanum tuberosum plants to accelerate the in vitro multiplication process. The main objective of this research was to evaluate the effect of different concentrations of phytohormones: auxins (IAA, IBA, NAA), cytokinins (6-BAP, TDZ, Zeatin), and gibberellins (GA3) on the morphological characteristics of Solanum tuberosum L. var. Cecilia. In vitro established plants were used, and the in vitro multiplication was performed using nodal segments cultured in MS medium supplemented with 20 g.L⁻¹ sucrose, 7 g.L⁻¹ agar, and phytohormones. The plants were maintained under total fluorescent white light conditions. The number of nodes, number of leaves, plant height, and number of shoots were evaluated. At 21 days, the use of IBA (0.05 mg. L⁻¹ and 0.25 mg. L⁻¹) increased the number of nodes and the number of leaves. Meanwhile, Zeatin concentrations of 0.05 mg. L⁻¹ and 0.1 mg. L⁻¹ increased the number of leaves, plant height, and number of shoots. Additionally, the concentration of 0.15 mg. L⁻¹ increased the plant height compared to GA3, and the concentration of 0.15 mg. L⁻¹ increased the number of nodes in S. tuberosum var. Cecilia plants. Based on the results of this study, it was concluded that IBA, Zeatin, and GA3 promoted the in vitro multiplication of S. tuberosum var. Cecilia plants.

Keywords: buds, growth regulators, potato, tissue culture.

INTRODUCTION

Solanum tuberosum L., commonly known as potato and belonging to the Solanaceae family¹, stands out as the world's fourth most important food crop due to its high productivity and nutritional value for human consumption². In 2017, global production reached 388 million tons cultivated over 19.5 million hectares³.

The primary global challenge in potato cultivation lies in reducing yield losses caused by the degeneration of planting material associated with various diseases ⁴. The most economically significant losses are caused by late blight (caused by Phytophthora infestans de Bary)^5-7. This disease can devastate crops within two weeks under high humidity conditions ⁸. Moreover, it can survive even in adverse conditions and quickly spread through soil, water, rain, and wind⁹. Similarly, viruses pose a significant threat to potato crops worldwide. Viral infections are concerning not only due to the noticeable decline in production but also because the disease often lacks visible symptoms¹⁰.

The known susceptibility of this crop to disease, primarily viral infections, implies a scarcity of high-quality seed, significantly restricting potato production in several countries^12,13. Acquiring certified seed can be difficult for farmers due to its high cost or limited availability¹⁴. However, enhancing seed quality through healthy and virus-free potato plantlets and microtubers15 is crucial. This improvement can lead to a yield increase of 15% to 20%¹⁶.

Micropropagation technology represents an alternative to conventional propagation and has proven to be effective in systematically eliminating viral infection¹⁹, ensuring the acquisition of high-quality and disease-free planting material^20-22. This technology offers a solution to the challenge mentioned above in potato production²³. It is based on cultivating plant cells, tissues, organs, seeds, protoplasts, or embryos in a sterile environment within a nutrient medium^24,25. Factors such as temperature, photoperiod, humidity, light, and medium components must be meticulously controlled to provide an optimal growth environment^26,27.

The culture medium is one of the most critical aspects that directly influences the success of the micropropagation technique^28,29. The addition of plant hormones in appropriate quantities is crucial for regulating plant growth and development^30-32. Among the growth hormones, cytokinins and auxins are commonly employed^33,34. Among cytokinins, 6-benzylaminopurine (6-BAP) and kinetin (KIN) stand out for their ability to stimulate cell division and shoot development. On the other hand, key auxins include 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA), which play a crucial role in the overall development of plants^35,36.

Although phytohormones significantly regulate the growth and development of in vitro-cultured plant tissues³⁷, their effects can vary considerably depending on factors such as plant species, genotype, and culture conditions. This variability can pose challenges in successfully reproducing plants through micropropagation ^38,39. Another critical aspect is the loss of tissue regeneration capacity over time. Moreover, excessive or inappropriate use of phytohormones can lead to undesired effects, including shoot deformations, hyperhydration (excessive tissue moisture)^41-44, necrosis, or explant oxidation⁴⁵.

Based on the issues mentioned earlier, the present study aimed to evaluate the effect of different concentrations of phytohormones, including auxins (IAA, IBA, NAA), cytokinins (6-BAP, TDZ, Zeatin), and gibberellins (GA3), on the number of nodes, number of leaves, number of shoots, and plant height of Solanum tuberosum L. var. Cecilia.

MATERIAL AND METHODS

The experiment was conducted at the Plant Biotechnology Laboratory of the Faculty of Agricultural Sciences at the Technical University of Ambato, located in the Cevallos canton, Tungurahua province, from April to September 2022.

Material vegetal

In vitro, established potato plants were utilized for this study. The in vitro multiplication stage involved culturing nodal segments on MS medium supplemented with 20 g.L⁻¹ sucrose, 7 g.L⁻¹ agar, and varying concentrations of phytohormones (auxins, cytokinins, and gibberellins). The cultures were maintained under full-spectrum fluorescent white light conditions.

Experiment 1: Influence of auxin concentrations (NAA, IBA, IAA) on in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

Table 1. Treatments corresponding to different concentrations of naphthaleneacetic acid (NAA) for the in vitro multiplication stage.

Table 2. Treatments corresponding to different concentrations of indole-3-butyric acid (IBA) for the in vitro multiplication stage.

Table 3. Treatments corresponding to different concentrations of indole-3-acetic acid (IAA) for the in vitro multiplication stage.

Experiment 2: Influence of cytokinin concentrations (6-BAP, TDZ, Z) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

Table 4. Treatments corresponding to different concentrations of 6-benzylaminopurine (6-BAP) for the in vitro multiplication stage.

Table 5. Treatments corresponding to different concentrations of thidiazuron (TDZ) for the in vitro multiplication stage.

Tabla 6. Tratamientos correspondientes a diferentes concentraciones de Zeatina (Z) para la etapa de multiplicación in vitro.

Experiment 3: Influence of gibberellin concentrations (GA3) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

Table 7. Treatments corresponding to different concentrations of gibberellic acid (GA3) for the in vitro multiplication stage.

Evaluations

The plant length (cm), number of leaves, number of nodes, and number of shoots per plant were determined at 21 days for each experiment.

Experimental design and statistic analysis

A completely randomized design was employed for each experiment. A control group (without phytohormone application) was included for each type of phytohormone. The data obtained for the variables, including the number of leaves, number of nodes, and number of shoots per plant, were recorded using SPSS version 26.0. Normal distribution was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was examined using Levene's test. For variables that did not meet these requirements, the Kruskal-Wallis test was used, followed by the Mann-Whitney U test. The significance level was set at 95%.

RESULTS

Experiment 1. Influence of auxin concentrations (NAA, IBA, IAA) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

At the 21-day evaluation, the naphthaleneacetic acid (NAA) at the utilized concentrations did not affect the number of nodes, leaves, shoots, and plant height of S. tuberosum (Table 8 and Figure 1).

Table 8. Effect of naphthaleneacetic acid (NAA) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Mean ranks that do not share common letters within the same column significantly differ according to the Kruskal-Wallis test, complemented by the Mann-Whitney U test at a significance level of p<0.05, with a sample size n=10

Figure 1. In vitro plants of S. tuberosum var. Cecilia at the multiplication stage on MS medium with NAA concentrations: 0.1 mg.L-1 (A), 0.25 mg.L-1 (B), Control (C) at 14 and 21 days.

At 21 days, when using 0.05 mg.L^-1 and 0.25 mg.L-1 of IBA, there was an increase in the number of nodes, number of leaves, and number of shoots per plant, while the plant height variable did not show any differences (Table 9 and Figure 2).

Table 9. Effect of indole-3-butyric acid (IBA) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Figura 2. Plantas in vitro de S. tuberosum var. Cecilia en etapa de multiplicación en MS + concentraciones de AIB: 0.05 mg.L^-1 (A), 0.25 mg.L^-1 (B), Control (C) a los 14 y 21 días.

At the 21 day evaluation of the effect of IAA on the in vitro multiplication of S. tuberosum var. Cecilia, no statistically significant differences were observed among the treatments when evaluating the number of nodes, leaves, shoots, and plant height (Table 10 and Figure 3).

Table 10. Effect of indole-3-acetic acid (IAA) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Figura 3. Plantas in vitro de S. tuberosum var. Cecilia en etapa de multiplicación en MS + concentraciones de AIA: 2 mg.L^-1 (A) y 2.5 mg.L^-1 (B), Control (C) a los 14 y 21 días.

Experiment 2: Influence of cytokinin concentrations (6-BAP, TDZ, Z) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

At the 21-day evaluation of the effect of 6-BAP on the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia, no statistically significant differences were observed among the treatments (Table 11 and Figure 4).

Table 11. Effect of 6-benzylaminopurine (6-BAP) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Figura 4. Plantas in vitro de S. tuberosum var. Cecilia en etapa de multiplicación en MS + concentraciones de 6-BAP: 0.3 mg.L^-1 (A), 0.4 mg.L^-1 (B), Control (C) a los 14 y 21 días.

At the 21-day evaluation of the effect of TDZ, it was determined that there were no statistically significant differences when evaluating the number of nodes per plant, number of leaves per plant, number of shoots, and plant height (Table 12 and Figure 5).

Table 12. Effect of Thidiazuron (TDZ) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Figure 5. In vitro plants of S. tuberosum var. Cecilia at the multiplication stage on MS medium with Thidiazuron concentrations: 1 mg.L-1 (A), 2 mg.L-1 (B), Control (C) at 14 and 21 days.

At 21 days, the concentrations of Zeatin at 0.05 mg.L^-1 and 0.1 mg.L^-1increased the number of leaves and number of shoots per plant, except for the concentration of 0.25 mg.L^-1. The remaining treatments and the control increased the plant height (Table 13 and Figure 6).

Tabla 13. Efecto de las concentraciones Zeatina (Z) sobre el número de nudos, número de hojas, número de brotes y altura de planta en la multiplicación in vitro de segmentos nodales de S. tuberosum var. Cecilia a los 21 días.

Figure 6. In vitro plants of S. tuberosum var. Cecilia at the multiplication stage on MS medium with Zeatin concentrations: 0.05 mg.L-1 (A), 0.1 mg.L-1 (B), 0.15 mg.L-1 (C), and Control (D) at 14 and 21 days.

Experiment 3: Influence of gibberellic acid (GA3) concentrations on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

At the 21-day evaluation of the effect of GA3, the concentration of 0.15 mg.L^-1 increased the number of nodes per plant; however, no statistically significant differences were observed for the number of leaves, shoots, and plant height (Table 14 and Figure 7).

Table 14. Effect of gibberellic acid (GA3) concentrations on the number of nodes, leaves, shoots, and plant height in the in vitro multiplication of nodal segments of S. tuberosum var. Cecilia at 21 days.

Figure 7. In vitro plants of S. tuberosum var. Cecilia is at the multiplication stage on an MS medium with GA3 concentrations of 0.15 mg.L^-1 (A), Control (B) at 14 and 21 days.

DISCUSSION

Influencia de concentraciones de auxinas (ANA, AIB, AIA) sobre la multiplicación in vitro de plantas de papa var. Cecilia.

Auxins are plant hormones primarily known for their role in plant root development ⁴⁶. In the in vitro cultivation of S. tuberosum, adding auxins to the culture medium can enhance the formation of roots in the explants. The concentration and type of auxin used can influence the degree of stimulation of root formation ⁴⁷. For instance, naphthaleneacetic acid (NAA) is a commonly used auxin in plant micropropagation and has been shown to induce root formation in various plant species ⁴⁸, including potatoes.

Auxins can regulate cellular growth by influencing gene expression in cell division and elongation ⁴⁹. It has been demonstrated that they can activate the expression of genes responsible for synthesizing structural cellular proteins and enzymes involved in cellular expansion and growth ⁵⁰. This regulation directly impacts the proper development of tissues during in vitro multiplication ⁵¹.

In addition to these direct effects, auxins can interact with other phytohormones, such as cytokinins and gibberellins, to coordinate tissue growth and in vitro culture development ⁵². The relationship between different phytohormones and their balance in the culture medium are critical aspects to consider in optimizing the in vitro multiplication process of S. tuberosum ⁵³.

The concentrations of naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) used in the study may have been insufficient or excessive to promote an increase in the number of leaves, nodes, shoots, and plant height ^54,55. If the concentrations were not within the optimal range, it is possible that no effect on in vitro multiplication was observed ^56,57. Research has determined that NAA did not influence in vitro multiplication of S. tuberosum when shoot height and multiplication rate were evaluated ⁵⁸. Regarding the effect of indole-3-butyric acid (IBA), low concentrations positively impacted in vitro multiplication.

Influence of cytokinin concentrations (6-BAP, TDZ, Z) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

Cytokinins are phytohormones that play a crucial role in plant growth and development ⁵⁹, including in vitro multiplication of crops such as S. tuberosum. They are commonly used in plant micropropagation, where plant tissue is cultured under controlled conditions ⁶⁰.

These phytohormones promote cell division and tissue expansion, significantly contributing to in vitro multiplication by enabling rapid cell proliferation ⁶¹. They can induce shoot formation and can develop into complete plants when properly cultivated ⁶². They are responsible for promoting meristem activity, which are regions of active plant growth, stimulating the production of new buds and shoots ⁶³.

In addition to stimulating vegetative growth and development, cytokinins can delay cellular aging and promote longevity of plant cells ⁶⁴, which is beneficial for maintaining the health and viability of in vitro cultured tissues over extended periods. Research indicates that supplementation of the medium with concentrations of 6-benzylaminopurine (6-BAP) ranging from 0.5 to 1 mg.L⁻¹ did not show an apparent effect on in vitro multiplication when evaluating the number of nodes and plant height in S. tuberosum group Phureja ⁶⁵.

Some authors have noted that S. tuberosum cv. Almera and Diamante explants measuring 1 to 2 cm with 2 nodes, cultured in MS medium supplemented with TDZ and auxin concentrations showed positive effects by exhibiting more shoots per explant when using 3.0 mg.L⁻¹ of TDZ combined with 0.1 mg. L⁻¹ of NAA ⁶⁷. However, in this study, high concentrations of Zeatin had no effect when used on the Cecilia variety. This could be attributed to the fact that each variety responds differently to a specific phytohormone.

Research conducted to evaluate the effect of Zeatin on the in vitro multiplication of S. tuberosum indicated that a medium supplemented with 0.1 mg.L⁻¹ of Zeatin + 0.5 mg.L⁻¹ of 2,3,5-triiodobenzoic acid (TIBA) promoted an increase in the number of shoots ⁶⁸. Furthermore, they noted that higher concentrations inhibited shoot development. The obtained results are consistent with the findings reported by the authors.

Influence of gibberellin concentrations (GA3) on the in vitro multiplication of Solanum tuberosum L. var. Cecilia plants.

Gibberellic acid (GA₃) can stimulate both cell elongation and division, resulting in increased growth of in vitro cultured plant tissues ⁶⁹. It is a crucial factor in plant micropropagation, as greater cell growth leads to more shoots and, consequently, a higher multiplication rate ⁷⁰. The use of GA₃ in the micropropagation of S. tuberosum activates the expression of genes involved in the initiation and development of shoots, leading to increased shoot formation on in vitro multiplied plants ⁷¹.

GA₃ can influence plant tissue morphogenesis, encompassing the formation of roots, stems, nodes, and leaves. This aspect is crucial for producing healthy seedlings that have a higher chance of surviving during acclimatization ⁷². Additionally, GA₃ can affect various metabolic processes such as protein synthesis, photosynthesis, and nutrient uptake, directly contributing to a higher propagation rate under in vitro conditions by providing optimal conditions for the growth and development of plant cells ⁷³.

Research conducted for the micropropagation of S. tuberosum using a liquid culture medium with different concentrations of phytohormones indicates that MS medium supplemented with 0.25 mg. L⁻¹ of GA3, 5 mg. L⁻¹ of pantothenic acid, 1 mg. L⁻¹ of thiamine and 20 g.L⁻¹ of sucrose, under constant agitation, yielded better results at in vitro multiplication stage ⁵⁸. Similarly, some authors mentioned that a concentration of 0.1 mg. L⁻¹ of GA₃ significantly increases plant height and the number of nodes and leaves. It was also observed that high concentrations of GA₃ lead to a decrease in leaves number and number of nodes per explant ⁷⁴. These findings are consistent with the results obtained in the present study.

CONCLUSIONS

The present study concluded that specific phytohormone treatments positively affected the in vitro multiplication of Solanum tuberosum L. var. Cecilia. The auxin IBA at concentrations of 0.05 mg.L⁻¹ and 0.25 mg.L⁻¹ increased the number of nodes and leaves. The cytokinin Zeatin at concentrations of 0.05 mg.L⁻¹ and 0.1 mg.L⁻¹ significantly improved leaf number, plant height, and shoot proliferation, while 0.15 mg.L⁻¹ Zeatin promoted increased plant height. The gibberellin GA3 at a concentration of 0.15 mg.L⁻¹ notably increased the number of nodes. These findings offer valuable insights for optimizing in vitro multiplication protocols for Solanum tuberosum L. var. Cecilia contributes to the efficient production of disease-free potato plantlets and promotes sustainable agricultural practices.

Supplementary Materials: The following are available online at www.revistabionatura.com/xxx/s1, Figure S1: title, Table S1: title, Video S1: title.

Author Contributions: Conceptualization, Michel Leiva Mora, Justo Antonio Rojas Rojas and Catherine Lizzeth Silva Agurto; methodology, Catherine Lizzeth Silva Agurto, Yosbel Lazo Roger and Michel Leiva Mora; software, Catherine Lizzeth Silva Agurto and Freddy Santiago Córdova Frías; validation, Catherine Lizzeth Silva Agurto, Michel Leiva Mora, and Willian Ernesto Tipán Chinachi; formal analysis, Catherine Lizzeth Silva Agurto and Willian Ernesto Tipán Chinachi; investigation, Catherine Lizzeth Silva Agurto, Alfredo Jiménez González, Miguel Angel Osejos Merino, Rene Nazareno Ortiz and Michel Leiva Mora; resources, Catherine Lizzeth Silva Agurto, Miguel Angel Osejos Merino and Michel Leiva Mora; data curation, Freddy Santiago Córdova Frías, Justo Antonio Rojas Rojas and Tannia Elizabeth Gómez Pinto; writing – original draft preparation, Catherine Lizzeth Silva Agurto and Michel Leiva Mora; writing – review and editing, Catherine Lizzeth Silva Agurto and Michel Leiva Mora; visualization, Willian Ernesto Tipán Chinachi, Rene Nazareno Ortiz and Freddy Santiago Córdova Frías; supervision, Catherine Lizzeth Silva Agurto; project administration, Michel Leiva Mora; funding acquisition, Catherine Lizzeth Silva Agurto and Michel Leiva Mora. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Acknowledgments. Se thank the support provided by the research coordinator, Deysi Alexandra Guevara Freire, and the authorities of the Faculty of Agricultural Sciences and Research and Development Directorate (DIDE-FCAGP) at the Technical University of Ambato. We also acknowledge the recognizing and supporting the efforts made in this project, which was self-funded by Engineer Catherine Silva Agurto and Doctor Michel Leiva Mora.

Conflicts of Interest: The authors declare no conflicts of interest.

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Received: April 14, 2024 / Accepted: July 18, 2024 / Published: 15 September 2024

Citation: Silva Agurto C L, Leiva Mora M, Córdova Frías F S, Tipán Chinachi W E, Gómez Pinto T E, Jiménez González A, Osejos Merino M A, Nazareno Ortiz R, Lazo Roger Y, Rojas Rojas J A. Effect of different phytohormones on in vitro multiplication of Solanum tuberosum L. var. Cecilia.

Bionatura journal. 2024;1(3):22. doi: 10.70099/BJ/2024.01.03.22

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