Plant Regeneration from Seed Cotyledon Tissues of Avocado (Persea americana Mill.)

We focused on establishing an efficient protocol for the micropropagation of avocados from seed cotyledons. In this study, we conducted two experiments: 1) initiation of tissue culture from avocado seed cotyledons, and 2) shoot and callus induction from in vitro avocado seedlings. In experiment 1, avocado seed cotyledons were cut into four small slices of 5–10 mm in thickness (B, M1, M2, and T) and transferred to MS medium (Murashige and Skoog 1962) without a plant growth regulator (MS0) and MS medium supplemented with 2, 4- dichlorophenoxyacetic acid and benzyl adenine (MS10/3). Fluorescence microscopy with two fluorescent dyes, Cellstain DAPI solution for nuclear staining and calcofluor white stain for cell wall staining, was used to verify the pattern of histological differentiation. Overall, the M1 slice, containing the mitotically active embryonic region, was the best for establishing an in vitro avocado seedling culture. The best site-specific response value, specifically 82.3% shooting, was identified with M1 slices. In experiment 2, fresh node segments, of approximately 1.5 cm were collected from in vitro shoots and cultured on MS0 to determine the effect of illumination on rooting, shooting, and callusing. The light condition resulted in a high capacity for target tissue greening and enhanced shoot formation with moderate calluses. In contrast, the explants in the dark condition mainly induced roots within 2 weeks. Overall, the node segments of avocado plants grown in vitro had better potential for shoot bud induction under dark conditions in a short time compared to those under light conditions. Once the cultured shoots were transferred from dark to light conditions, they continued to grow with healthy leaf flashing and shoot elongation. Moreover, after transplanting the regenerated avocado plants into the soil, they safely developed pot seedlings in a greenhouse.