Figure 3

Morphological and histological appearance of the different callus phenotypes observed in maintained embryogenic callus cultures assayed for coffee genetic transformation. Whitish type embryogenic callus: morphology, scale bar = 2 mm (A1) and histological aspect (A2), (A3). Note the very loose structure of the callus and the presence of proembryos (pe), isolated embryogenic cells (ec) along with abundant degenerating cells and proembryos (arrow), scale bar = 73 μM (A2); note the segmentation and degenerating process of proembryos (arrow) and the low-density cytoplasm of all cells, scale bar = 36.5 μM (A3). Yellow type embryogenic callus: morphology, scale bar = 2 mm (B1) and histological appearance (B2), (B3); embryogenic callus comprising small cell aggregates similar to proembryogenic masses (PEMs); note the high homogeneity of the tissues, scale bar = 73 μM (B2); note the high nucleus cytoplasm ratio of all cells, the voluminous central nucleus (n) and the numerous small starch grains (s) around the nucleus; note the very dense cytoplasm rich in soluble and reserve proteins (blue staining) in all the cells, scale bar = 36.5 μM (B3). Gray type embryogenic callus: morphology, scale bar = 2 mm (C1) and histological appearance (C2), (C3); note the heterogeneous callus structure comprising a mix of degenerating tissues (on the left) and active (on the right) and intermediate areas, scale bar = 73 μM (C2); most of the cells are vacuolated with a non-central nucleus (n), a small nucleolus (nl) and less abundant but bigger starch grains (s), scale bar = 30 μM (C3).