Figure 1.
Histological analysis of NECs in banana cultivar ‘Baxijiao’ (A, B) and ECs and somatic embryos in cultivar ‘Yueyoukang 1’ after periodic acid-Schiff reagent (PAS) staining.
Starch and surface mucilage were stained pink while nuclei were stained blue. A. An overview of NECs; B. A detail of NECs showing very few starch granules (pink staining, arrows) around nuclei (blue staining). C. An overview of ECs partially surrounded by “starch-rich cells” (arrows); D. A detail of ECs showing many starch granules (pink staining, arrows), especially around nuclei (blue staining); E. A median histological section through pre-globular embryo; F. A median histological section through globular embryo with well-developed epidermis (arrowheads); G. A median histological section through pear-shaped embryo showing epidermis (arrowheads), subepidermal and cortical cells (CC) with starch (arrow); H. A median histological section through cotyledonary embryo showing epidermis (arrowheads), cortical cells (CC), starch-rich cells in the basal (arrow) as well as in the lateral parts of the embryo and vascular tissue in the middle part of the embryo (star); Bars represent 20 µm in B and D; 50 µm in A, C, E and F; 100 µm in G and 200 µm in H.
Figure 2.
Whole-mount histochemical staining of surface-localized pectins in banana NECs of cultivar ‘Baxijiao’ (A, B) as well as in ECs and somatic embryos of cultivar ‘Yueyoukang 1’ (C–F) with ruthenium red.
A. and B. Histochemical staining of clumps composed of NECs. Please, note very weak pink labeling only in few peripherally localized cells and small cell groups; C. and D. Histochemical staining of clumps composed of ECs. Please, note stronger pink/red staining of EC clusters; E. Cluster of embryos at different embryogenic stages from pre-globular to globular and pear-shaped. Please, note an intense pink/red staining at the surface of these somatic embryos. F. Cluster of late-stage cotyledonary embryos. Please, note weaker staining of these embryos and a net-like structure at their outer surface; Bars represent 1 mm in A–D, and 500 µm in E and F.
Figure 3.
Semi-quantitative developmental analysis by immunodot of the relative abundance of pectin epitopes in extracts prepared from NECs (cultivar ‘Baxijiao’) and from ECs and somatic embryos (cultivar ‘Yueyoukang 1’) during somatic embryogenesis of banana (Musa spp. AAA).
N: non-embryogenic cells; E: embryogenic cells; P: proembryos; G: globular embryos; L: late embryos.
Table 1.
Antibodies used to study distribution of the pectic epitopes in the cell walls of banana embryogenic cultures.
Figure 4.
Immunofluorescence localization of pectin epitopes in non-embryogenic cells (NECs) of banana (Musa spp. AAA, cultivar ‘Baxijiao’) and the effect of NaOH de-esterification on the immunofluorescence labeling of these epitopes.
A and I. The 2F4 epitope was immunolocalized only in few cell walls (A), while base treatment increased this immunolocalization (I); B and J. The LM18 epitope was immunolocalized to the surface of some small cell groups (B) and this immunolabeling slightly increased after base treatment (J); C and K. The CCRC-M38 epitope was immunolocalized all over the section through NECs (C) with no obvious changes after base treatment (K); D and L. The JIM5 epitope was mainly immunolocalized to the surface of some small cell groups (D) but this JIM5 immunolabeling disappeared after base treatment (L); E and M. The CCRC-M34 epitope was weakly immunolocalized in NECs (E) but immunolabeling disappeared after base treatment (M); F and N. The JIM7 epitope showing a very weak immunolocalization in NECs (F) and no visible immunolabeling after base treatment (N); G and O. The LM20 epitope showing a very weak immunolocalization in NECs (G) but no visible immunolabeling after base treatment (O); H and P. The LM5 epitope showing immunolocalization in NECs (H) and slight decrease of immunolabeling after base treatment (P). Bars represent 50 µm.
Figure 5.
Immunofluorescence localization of pectin epitopes in embryogenic cells (ECs) of banana (Musa spp. AAA, cultivar ‘Yueyoukang 1’) and the effect of NaOH de-esterification on the immunofluorescence labeling of these epitopes.
A and I. The 2F4 epitope was almost non-dectable by immunolabeling in banana ECs before (A) and also after base treatment (I); B and J. The LM18 epitope was immunolocalizated in ECs (B) with slight increase of immunolabeling after base treatment (J); C and K. The CCRC-M38 epitope showing strong, evenly distributed immunolocalization in ECs (C), with no obvious changes after base treatment (K); D and L. The JIM5 epitope was immunolocalized in ECs (D) but no visible immunolabeling was detected after base treatment (L); E and M. The CCRC-M34 epitope showing a weak immunolocalization in ECs (E) with no visible immunolabelling after base treatment (M); F and N. The JIM7 epitope immunolocalized in ECs (F), with weaker immunolabeling after HG de-esterification by base treatment (N); G and O. The LM20 epitope showing weak immunolocalization in ECs (G) which disappeared after base treatment (O); H and P. The LM5 epitope immunolocalized in starch-rich cells (arrows in H) showing less immunolabeling after base treatment (P). Bars represent 50 µm.
Figure 6.
Immunofluorescence localization of pectin components in proembryos to early globular embryos of banana (Musa spp. AAA, cultivar ‘Yueyoukang 1’) and the effect of NaOH de-esterification on the immunofluorescence labeling of these epitopes.
A and I. The 2F4 epitope was immunolocalized to the ECM at the surface of epidermal cells (arrow in A) while slight increase of immunolabeling was detected after base treatment (arrow in I); B and J. The LM18 epitope showing strong immunolocalization in epidermis and weaker in cortical cells (B) with increased immunolabeling after base treatment (arrow in J); C and K. The CCRC-M38 epitope showing strong and evenly distributed immunolocalization in the proembryo (C) with no visible changes after base treatment (K); D and L. The JIM5 epitope was immunolocalized in the ECM of epidermal cells (arrow in D) and this immunolabeling disappeared after base treatment (L); E and M. The CCRC-M34 epitope was weakly immunolocalized in the proembryo (E) and immunolabeling disappeared after base treatment (M); F and N. The JIM7 epitope was immunolocalized in the epidermis (arrow in F) and in the cortical cells (F), and these imunolabelings decreased after base treatment (N); G and O. The LM20 epitope showing moderate immunolabeling in the proembryo (G) but no visible immunolabeling after base treatment (O); H and P. The LM5 epitope showing evenly distributed immunolocalization in the proembryo (H) with immunolabeling decrease after base treatment (P). Bars represent 50 µm, except A and I where they represent 100 µm.
Figure 7.
Immunofluorescence localization of pectin components in late embryos of banana (Musa spp. AAA, cultivar ‘Yueyoukang 1’) and the effect of NaOH de-esterification on the immunofluorescence labeling of these epitopes.
A and I. The 2F4 epitope was immunolocalized in the ECM covering epidermal cells (arrow in A) but also in the cells in the middle parts of the embryos (A) with very slight increase of the immunolabeling after base treatment (I); B and J. The LM18 epitope was immunolocalized in epidermal (arrow in B) and cortical cells (B) with slight increase of the immunolabeling after base treatment (arrow in J); C and K. The CCRC-M38 epitope was immunolocalized to outer cell layers as well as to the cells in the middle part of embryos (C) with no changes after base treatment (K); D and L. The JIM5 epitope showing weak immunolocalization in epidermal and cortical cells but stronger one in parenchyma cells surrounding vascular tissue (arrow in D), and depletion of these immunolabelings after base treatment (L); E and M. The CCRC-M34 epitope showing weak immunolocalization in epidermal and cortical cells of late embryos (arrow in E) which disappeared after base treatment (M); F and N. The JIM7 epitope was immunolocalized to the cells of the middle part of late embryos (arrow in F), and this immuolabeling decreased after base treatment (N); G and O. The LM20 epitope showing relatively strong immunolocalization to the cells of the middle part of late embryos (G) with decrease of this immunolabeling after base treatment (O); H and P. The LM5 epitope was immunolocalized to cell-cell junctions (arrow) of cortical parenchyma cells (H), and this immunolabeling decreased after base treatment (P). Bars represent 50 µm in B, E, F and J; and 100 µm in other images.
Table 2.
The intensity of immunolabeling with different pectin antibodies in diverse cell types during somatic embryogenesis of banana.
Table 3.
The degree of pectin methyl-esterification (DM) during somatic embryogenesis of banana.