Primavera otoño 2020 (Año LXIII Núms. 122-123) Año LXIV Núm. 124-125 horizontes PRIMAVERA / OTOÑO 2021 PUCPR 79 and micronutrients. The macronutrients were nitrogen, calcium, magnesium, and potassium; the micronutrients were boron, cobalt, copper, iron, manganese, and zinc. Murashige and Skoog MS inorganic salt medium provided the best nutrition compared to the woody plant culture medium (Mahmoud et al., 2020). An essential element was the hardening agent. When added to the mixture, it limited the movement of nutrients within the medium. Tissue media culture used polymers such as agar and gels within tissue culture media to solidify the reagent mix. Solidifying agents provided anchoring to protect tissue from an aqueous environment. An aqueous solution reduced oxygen by increasing the tissue’s hydration, leading to a growth abnormality called hyperhydricity (Niedz & Marutani-Hert, 2018). Researchers worked with a paper-like cellulose polymer to provide the anchoring tissue with a liquid medium and oxygen. They stacked several cellulose paper sheets and moistened them with a liquid medium of either N6-benzyl amino or zeatin riboside to increase shoot regeneration. Tzatzani et al. (2018) experimented with direct organogenesis by treating the tissue with Murashige and Skoog medium combined with N6-benzyl amino and N6benzylaminopurine to improve the protocol. Using cytokinin such as N6-benzylamine combined with gibberellin and adding vitamins at the correct concentration provided better multiplication (Pandey & Tamta, 2106). Within the different combinations, using 0.1 mg / L of α-naphthalene acetic acid auxin and 0.5 mg / L of N6-benzylamine reported the best results, yielding 2 to 2.5 shoots per explant. The N6- benzylamine used alone had better results than in combination with gibberellins, yielding 4 to 6 shoots with the stems down horizontally (Bulbarela-Marini et al., 2019). Shoots were separated from the explant and placed in individual media. When the shoots reached 100 to 155 mm in height, they were transferred to a rooting Murashige, and Skoog medium made plus the α-naphthalene acetic acid auxins. Among the most used auxins were acetic acid 1H-indole-3-butanoic acid and indole-3-acetic acid auxin. The best results were reported for root organogenesis when 2.0 mg / L of 1H- indole-3-butyric acid were combined with 0.1 mg / L indole-3-acetic acid auxin (Tzatzani et al., 2018). They obtained 1.45 roots per shoot with a maximum growth of 3.22 cm. For in vitro propagation, rooting was complex organogenesis. Chromatography and spectrometry helped determine how 6-benzylamine-like compounds increased root production (Aremu et al., 2017). Researchers quantified and identified new metabolic routes for meta- topolin and its derivatives to develop fewer toxic compounds than auxin phytohormones. Low toxicity compounds were more resistant to sterilization processes and improved root organogenesis processes. Successful roots demonstrated continuous growth and white color when removed from in-vitro for hardening. During the in vitro, culture media presented unique problems caused by the growth parameters. Long et al. (2017) studied nutrient solutions at various pH values to test the physiological response of Citrus sinensis . There was insufficient adsorption of minerals at alkaline pH values, while acidic pH values inhibited leaf and root development. Increased acidity reduced metabolic processes, including photosynthesis and biological respiration. Production of ethylene gases caused tissue senescence and leaf loss. Most citrus tissue culture experiments failed because of the high frequency of leaf abscission. Researchers used a tissue culture media vessel to control temperature, moisture, and light intensity.