A comparative structural analysis of direct and indirect shoot ...

Shoot regeneration in opium poppy
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Results
Shoot regeneration was initiated from hypocotyls of primary
somatic embryos (Fig. 1 a). The emerging shoot primordia
appeared on the hypocotyl surface of the somatic embryo
hypocotyl, but they did not disrupt the surface tissues (Fig.
1 b). In addition, no general tissue reorganisation was observed
(Fig. 1b). Due to these features, this kind of regeneration
was termed direct shoot organogenesis in this study.
Indirect shoot regeneration involved tissue dedifferentiation,
callus formation, and cell re-differentiation leading to de novo
shoot regeneration (Fig. 1 c). Tissue organisation and cell
morphogenesis of indirectly regenerated shoot primordia were
completely different in comparison to underlying callus tissue
(Fig.1d).
Direct shoot regeneration
Surface integrity of the embryo hypocotyl suggested that adventitious
shoots arose from superficial cell layers (Fig. 1 b).
Initially, sub-epidermal cells were activated and divided. The
plane of the first cell division seemed to be anticlinal (Fig.
2 a), but subsequent cell divisions were observed in both anti-
and periclinal planes (Fig. 2 b). Afterwards, dividing subepidermal
and epidermal cells created organogenic nodules,
the first recognisable structures on the hypocotyl surface
(Figs. 2 c, d). The two most important morphogenetic features
of these cells were dense non-vacuolated cytoplasm (Figs.
2 a, b, c) and reduced cell size (Fig. 2 d).
The first apparent indication of cell specification was observed
in the phase of apical meristem formation. The cells
within nodule apices adopted tunica-corpus zonation (Figs.
2 e, f). The original epidermal cells participated in differentiation
of the tunica layer (Fig. 2 c). This explains why the tunica
and protomeristem maintained morphological and histological
continuity between the forthcoming buds and the remaining
hypocotyl surface (Figs. 1b, 2 c, d, g). Cell specialisation
continued by both histodifferentiation of the shoot apical meristem
(Figs. 2 e, f) and transformation of organogenic nodules
into buds (Figs. 2 g, h). Adventitious buds were visible on
the hypocotyl surface (Figs. 2 g, h) and regular activity of the
apical meristem was detected, including formation and growth
of leaf primordia (Figs. 2 h, i).
Indirect shoot regeneration
Distinct mode of indirect induction of organogenesis was
based on callus production. Cell activation triggered a morphogenetic
switch in some cells within rarely dividing callus
tissue, resulting in the establishment of dividing, meristemlike,
and shoot-forming tissue (Fig. 3 a). Typical features of
meristemoids (representing population of competent cells)
were small cell size, cytoplasmic density, minimal vacuolation
(Figs. 3 a, b), and cell adhesion after cell division (Figs. 1 d,
3 a, b, c). The first cell differentiation events within the multicellular
meristemoids resulted in the formation of both peripheral
and central cell layers, with only peripheral cells continuing
their divisions (Fig. 3 b). Reserves were stored in central
cells in the form of starch (Figs. 3 b, d) and lipids (Figs. 3 e, f).
These cells were non-dividing but their cytological parameters
indicated high metabolic activity. Frequent endo- and
exocytosis in these cells (Fig. 3 e) and cell wall ingrowth for-
Figure 1. Shoot buds regenerated in the callus culture of P.
somniferum L. a. Somatic embryo (SE) in the culture where
the shoot (SH) regeneration from the hypocotyl was
induced. Bar = 1 mm. b. Directly regenerated shoots with
leaf primordia, visible as protuberances on the hypocotyl
surface of somatic embryo. Bar = 100 µm. c. Shoot bud
regenerated in the organogenic callus culture. The white
compact tissue represents meristemoids. Bar = 1 mm. d.
Shoot primordium on the surface of callus tissue. Bar =
100 µm.