Figure 1.
The development of sticky callus and frog egg-like bodies (FELBs) from the leaf, root, and stem explants of S. nigrum.
A, B and C represent root, stem and leaf explants, respectively; A1, B1 and C1 represent translucent sticky callus induced from root, stem and leaf explants, respectively; A2, B2 and C2 represent FELBs at an early stage of development induced from root, stem and leaf explants, respectively; A3, B3 and C3 represent FELBs at a late stage of development induced from root, stem and leaf explants, respectively; A4, B4 and C4 represent enlarged views of FELBs from parts of A3, B3 and C3.
Figure 2.
Identification and morphological analysis of embryoids and the non-embryogenic callus of frog egg-like bodies (FELBs).
A, B and C represent root, stem and leaf explants, respectively; A1, B1 and C1 represent FELB identification after double staining with acetocarmine and Evans blue for FELB induced from root, stem and leaf explants, respectively; A2, B2 and C2 represent cell morphology and arrangement of FELB embryoids stained with borax-toluidine blue for FELB embryoids induced from root, stem and leaf explants, respectively; A3, B3 and C3 represent cell nuclei of FELB embryoids stained with DAPI and observed in dark-field lighting for FELB embryoids induced from root, stem and leaf explants, respectively; A4, B4 and C4represent enlarged view of A2, B2 and C2, respectively, showing FELB morphology; A5, B5 and C5 represent cell morphology and arrangement of non-embryogenic callus of FELBs stained with borax-toluidine blue, for FELBs induced from root, stem and leaf explants, respectively; A6, B6 and C6 represent cell nuclei of non-embryogenic callus of FELBs stained with DAPI and observed in dark-field lighting, for FELBs induced from root, stem and leaf explants, respectively.
Figure 3.
Microscopic images of different stages of SE by frog egg-like bodies (FELBs) in S. nigrum by using the frozen section technique.
A. Translucent sticky callus induced from a leaf explant; B. FELBs induced from a root explant; C. FELBs induced from a stem explant; D. FELBs induced from a leaf explant; E. The specific localization of FELBs on a leaf explant; F. FELBs containing many embryoids at different developmental stages; G. A spherical, early globular embryo; H-K. Individual FELBs and their embryoids were arranged in different ways, including embryoids in relatively independent FELBs (H and I), two embryoids in one FELB (J), and three embryoids clustered together (K); L-O. Somatic embryos at different developmental stages, including globular shaped embryos (L), heart/torpedo- shaped embryos (M and N), and torpedo- shaped embryos (O).
Figure 4.
Microscopic images showing the development of vascular tissue in the induced embryoids of frog egg-like bodies (FELBs) and somatic embryos of S. nigrum.
A. FELB embryoid without vascular tissue forming; B. The appearance of vascular tissue in the center of a FELB embryoid; C. Elongated FELB embryoid with developing vascular tissue; D. Vascular tissue in FELB embryoids at different developmental stages in one view; E. Longitudinal section of developed vascular tissue in a somatic embryo; F. Transverse section of a somatic embryo with developed vascular tissue; G. Developed vascular tissue in a mature somatic embryo.
Figure 5.
The regeneration process of S. nigrum by frog egg-like bodies (FELBs).
A. Calli induced on a leaf explant; B. Enlarged view of red squares in A, showing FELBs induced in translucent sticky calli; C. Isolated FELBs; D. Isolated FELBs stained with acetocarmine and Evans blue; E. Morphologies of isolated FELBs at different developmental stages stained with acetocarmine and Evans blue; F. Morphologies of the intact individual FELBs at different developmental stages; G. The histological and morphological developmental process of FELBs; H. The regeneration process of FELBs in vitro; I, J, K, and L. The regeneration process of FELBs in vivo; M. A regenerated seedling; N. A flower from a regenerated plant; O. Immature fruits from a regenerated plant; P. Mature S. nigrum fruits from a regenerated plant.
Table 1.
Effect of 2,4-D on the induction of translucent sticky callus in the S. nigrum root, stem, and leaf explants.
Table 2.
Effect of 2,4-D on frog egg-like body (FELB) induction in the S. nigrum root, stem, and leaf explants.
Table 3.
Frequency of plantlet induction from in vivo frog egg-like bodies (FELBs) of S. nigrum root, stem, and leaf explants.