Documente Academic
Documente Profesional
Documente Cultură
DOI 10.1007/s00497-002-0131-y
O R I G I N A L A RT I C L E
Introduction
Since Takebe and co-workers first succeeded in regenerating tobacco mesophyll protoplasts in normal plants
(Takebe et al. 1971), this approach has gained interest
due to its possible application in genetic improvement.
Since the early 1970s, whole plants have been obtained
from protoplasts derived from a number of somatic cell
types in several hundred plant species, and many species
have been genetically modified through either somatic
hybridization or direct gene transfer into the protoplasts
(Puite 1992).
Gametophytic line cells have additional advantages,
since protoplasts of different ploidy and different nuclear
specificity can be obtained depending on the developmental stage of the cells subjected to enzymatic treatment (Tanaka 1994). To date, protoplasts have been
successfully isolated from tetrads (Arnalte et al. 1991),
microspores (Zhou 1989b), megasporocytes and egg
cells (Mouritzen and Holm 1995; Pnya et al. 1999),
binucleate or trinucleate pollen grains (Tanaka et al.
1987; Zhou 1989a, b; Fellner and Havranek 1992), and
also from pollen tubes (Kroh and Knuiman 1988; Rutten
and Derksen 1990, 1992). These protoplasts were further
used in gametic or gametosomatic fusion experiments
(Lee and Power 1988; Ueda et al. 1990; Desprez et al.
1995) leading at least in some cases to the production
of triploid hybrid plants (Pirrie and Power 1986).
In addition to their application in the formation of
cells with a triploid chromosome set, microspore- or pollen-derived protoplasts can also contribute to new transformation techniques that are potentially useful as alternatives to those in which whole microspores or pollen
grains are used (Tanaka 1994). Protoplasts isolated from
sperm cells and egg cells have recently been used in in
vitro fertilization studies (Kranz 1999; Scholten and
Kranz 2001).
The applications summarized above require that
either gametophytic protoplasts or their fusion products
(zygotes) reform the cell wall, an absolutely essential
event that precedes mitotic division and the subsequent
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Results
Isolation of pollen tube-protoplasts
After pollen tubes were placed in enzyme solution, within 30 min of incubation the tube wall became thinner,
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and the cytoplasm underwent plasmolysis and fragmented into several parts of unequal size (Fig. 1A). At the
same time, many pollen tubes displayed characteristic
displacements of the cytoplasm that formed spherical
zones at the apical end, which in the next step apparently
gave rise to separate protoplasts floating in enzyme solution (Fig. 1A). The number of protoplasts increased rapidly between 60 and 90 min of incubation, as the pollen
tubes disintegrated. The isolated protoplasts were spherical varied in size, and ranged in diameter from 4 to
23 m (Fig. 1B). Staining with DAPI clearly showed that
enzymatic digestion of the pollen tubes resulted in the
release of cytoplasts, which lack nuclei, and karyoplasts
that contained one, two or three nuclei (Fig. 1C).
Electron microscope observation of the morphology
of freshly isolated protoplasts revealed that they were
surrounded by plasma membrane and lacked remnants of
the cell wall (Fig. 3A). As was found for the pollen tubes
(Fig. 2A), the protoplast cytoplasm was relatively dense,
rich in ribosomes, dictyosomes, mitochondria, plastids,
short cisterns of endoplasmic reticulum, and sometimes
in electron-dense spherical bodies (Fig. 3A).
Wall components of pollen tubes
Discussion
Pollen tube wall
The structure of the olive pollen tube wall described here
confirms previous reports on the two-layered structure
observable in subapical regions of tube walls in other
species (Taylor and Hepler 1997). At the proximal tube
end (i.e., close to the grain), the mature tube wall of
some species is composed of three layers: internal callosic, intermediate cellulosic, and the outermost layer composed of pectins (Li et al. 1993). This arrangement was
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Gametophytic protoplasts also provide a suitable model for studies of pollen or pollen tube biology, in particular the molecular characteristics of the plasma membrane
(Fan et al. 2001) and cell wall ontogenesis (Rutten et al.
1991). Available information about the nature of wall
components deposited by regenerating gametophytic protoplasts is compared to somatic protoplasts scarce and
based only on the results of cytochemical staining (MikiHirosige et al. 1988; Zhou 1989a). Detailed information
on the chemical nature and subcellular distribution of
wall components is still lacking.
The results presented in this paper show that callose
is abundantly synthesized at the olive protoplast surface
and that within 12 h of culture it forms a thick continuous layer. This process has been previously described for
somatic protoplasts of several species (Caumont et al.
1997; Mnster 2001), pollen-derived protoplasts of
Lilium longiflorum and Nicotiana tabacum, and megasporocyte protoplasts of Hordeum vulgare (MikiHirosige et al. 1988; Rutten et al. 1991; Mouritzen and
Holm 1995) on the basis of aniline blue staining. The
precise distribution of this polymer in pollen-derived
protoplasts is, to our knowledge, revealed here for the
first time by electron microscopy and immunocytochemistry. The deposition of callose may reflect at least two
phenomena: (1) the translation of mRNA(s) encoding
enzyme(s) involved in callose synthesis, which previously existed in growing pollen tubes and contributed to the
formation of the callose layer during tube growth, and
(2) de novo transcription and translation of mRNA activated by plasma membrane wounding in the course of
enzymatic treatment or mechanical damage to the protoplast. Currently, we have no direct evidence to state
whether callose also appears around the cytoplasts,
which do not possess the nucleus necessary for the transcriptional processes. For this reason, neither of the phenomena proposed above can be ruled out. In some other
species the callose wall is, however, also synthesized by
cytoplasts, a fact that gives support to the first hypothesis (Kroh and Knuiman 1988; Rutten and Derksen 1992).
On the other hand, support for the second hypothesis
comes from studies of somatic protoplasts isolated from
cells which do not synthesize callose under in planta
conditions. In these protoplasts, secretion of -(13)glucan, the event that typifies the initial phases of cell
wall reformation and precedes cellulose deposition was
found in a number of species, such as soybean, carrot
and sunflower (Klein et al. 1981; Mock et al. 1990;
Caumont et al. 1997). Callose secretion is most frequently ascribed to the cell-wounding-response (Shea
et al. 1989). The fibrillar material deposited on the olive
protoplast surface was identified as PTA-positive, and
may reflect cellulose deposits, but confirmation will
require more specific procedures.
Along with the synthesis of -glucans at the surface
of pollen tube protoplasts of olive, pectic epitopes recognized by the JIM7 antibody start to be secreted. The
structure of these epitopes has not yet been fully characterized, but optimal antibody binding is known to occur
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