In vitro propagation of avocado faces several limitations. To optimize the establishment phase, we evaluated three plant material types: etiolated shoots, 30-day covered field shoots, and uncovered field shoots, collected at two time points. Biochemical and anatomical analyses were conducted to understand material performance during establishment. Across both collection times, etiolated shoots exhibited minimal oxidation, enhanced bud sprouting, reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels, increased peroxidase (POD) activity, and improved xylem development, consistently outperforming field-derived materials. Using etiolated shoots, we optimized disinfection and in vitro multiplication protocols. Pre-disinfection with 3 mL L−1 Phyton 27® and 2% sodium hypochlorite yielded the highest survival rates. In multiplication experiments, varying concentrations of 6-benzylaminopurine (BAP) and meta-topolin (MT), supplemented with gibberellic acid (GA3), did not significantly affect growth variation. However, 8.88 µM BAP with 0.29 µM GA3 resulted in the greatest number of sprouted buds.