Primavera otoño 2020 (Año LXIII Núms. 122-123) Año LXIV Núm. 124-125 horizontes PRIMAVERA / OTOÑO 2021 PUCPR 78 distilled water at the end of the process. This protocol had alcohol that caused tissue damage, but by maintaining it at a concentration of 70% alcohol for a short time, they could sterilize the axillary segment without presenting anomalies. Pandey and Tamta (2016) worked with nodal segments grown in vitro, which allowed them to pass up the sterilization process, providing healthy tissue. Organogenesis Organogenesis is the development of roots, stems, flowers, and leaves from a cell grown in a medium with growth-regulating hormones. In vitro organogenesis was achieved directly and indirectly. Direct organogenesis was obtained directly from cotyledonary tissue collected from the plant, while indirect organogenesis originated from callus, a disorganized cell growth (Pandey & Tamta, 2016). Researchers used mature leaves and placed them in a 2,4-D Murashige and Skoog basal medium in solution with benzyl adenine and α-naphthalene acetic acid to form a callus. They placed the meristematic tissues in basal Murashige and Skoog medium with benzyl adenine, α-naphthalene acetic acid, and gibberellic acid for direct organogenesis. The callus provided the highest number of shoots, but its lineages were more variable than shoots originating from meristematic tissues. Direct organogenesis, where shoots were obtained directly from the explant with slight soma-clonal variation, guaranteed the copy of the genetics of the plant. It is essential to define the symmetry of the explant and depth of the medium. Vibhute et al. (2016) cut nodal segments to 1 cm long and were placed vertically in the in-vitro media, with the nodal base inserted a few millimeters within the culture medium. Another way to sow the tissue was horizontal, leaving a 1 cm long node. The explant produced 4 to 6 fresh shoots, being more efficient than sowing the explants vertically. The vertical seeding in the in-vitro media had a single point of absorption inside the growth medium, while the horizontal seedling had two points of contact at each end. Phytohormones regulate direct organogenesis such as cytokinin, gibberellins, and auxins. Vibhute et al. (2016) investigated the stability of citrus seedlings generated from nodal segments taken from the stems. They evaluated in vitro seedling growth and root and leaf morphogenesis. The new citrus seedlings were exposed to field conditions and grew satisfactorily. Devi et al. (2021) developed a high-efficiency organogenesis protocol for Citrus jambhiri using cytokinins and gibberellins to induce leaf and root organogenesis. They completed experiments to confirm cytokinin and gibberellin signaling by KOI and GA2OXI genes and compared bandwidths on agarose gel expression of GRFI and GRF5 growth genes. Salis et al. (2017) compared the micropropagation of various citrus rootstocks for genetic variation and shoot development. They compared various Citrus cultivars Volkameriana , Citrumelo , Citrange , and Poncirus trifoliata, to study the multiplication of the shoots. They experiment with the basal medium Murashige and Skoog with N6-benzylamine and naphthalene acetic acid for root development. They used inter-simple sequence repeat with molecular primers to increase the genetic profile to analyze variations produced by hereditary variation. The somaclonal variation has proven to be a quick and inexpensive way to get disease-tolerant mutations. Culture media During in vitro propagation, the culture medium regulated tissue growth. Every nutrient medium contained growth hormones, a carbon source, vitamins, a hardening agent, and macro-