Annals of RSCB
Vol. XVI, Issue 1
COMPARATIVE STUDY OF DIANTHUS GIGANTEUS SUBSP.
BANATICUS LEAVES ANATOMY IN DIFFERENT GROWING
CONDITIONS
II. ULTRASCTRUCTURAL ASPECTS
Liliana Jarda1, Victoria Cristea2, C. Crăciun1, S. Tripon1
1
FACULTY OF BIOLOGY AND GEOLOGY; 2 “AL. BORZA” BOTANICAL GARDEN,
BABES-BOLYAI UNIVERSITY, CLUJ-NAPOCA, ROMANIA
Summary
In the ultrastructural study of Dianthus giganteus subsp. banaticus leaflets grown in
different conditions, we used plants, from wild (i), from in vitro classical culture plants (ii),
and from photoautotrophic culture plants (iii) with some modifications which are detailed in
the material and methods section. The observations in the leaf blade, by means of electron
microscope, show that the ultrastructure of the leaflet blade coming from photoautotrophic in
vitro culture is similar with that of wild leaflets, having well defined cells arranged in tissues,
while leaflets coming from classical in vitro culture have cells of irregular shape and are not
well defined in tissues. Chloroplasts’ structure and position into the cells is also different to
classical in vitro culture unlike the photoautotrophic in vitro culture and wild samples.
Key words: in vitro, ex vitro, Dianthus giganteus subsp. banaticus, chloroplasts, NAB.
[email protected]
high number of chloroplasts and intensify
RUBISCO activity (Lee et al., 1985,
Vargas-Suarez et al., 1996, Serret et al.,
1996, Cristea et al., 1999). In our study, we
try to demonstrate that also in the case of
Dianthus giganteus subsp. banaticus
species the use of a photoautotrophic stage
before vitroplantlets acclimatization may be
useful and may reduce plant material loss
when transitioning plants from aseptic to
greenhouses and in the air cultures.
Introduction
In vitro cultures biotechnology is an
efficient multiplication method of rare and
endangered plants, as well as of the
ornamental plants (Zăpârţan, 2001, Miclăuş
et al., 2006, Liu and Liu, 2010). Plants
obtained by such methods need a period of
acclimatization before planting them in
greenhouses or in the air (Lee et al., 1985).
During this acclimatization period,
many of the plants are being lost because of
the impact of passing from a highly humid
atmosphere to a dryer one and, also, from a
heterotrophic nutrition (as in case of in vitro
cultures) to a photoautotrophic one. Plants
transition through a photoautotrophic stage
before acclimatization may reduce plant
material loss because plants growing onto a
medium without an organic carbon source
will induce the development of the
photosynthetic apparatus, which is less
developed to in vitro plants (Cristea et al.,
1996). Ultrastructural analyses on different
plant species show that inorganic CO2
supplementation of culture vessels induce a
Material and methods
Plant material was sampled from
Dianthus giganteus subsp. banaticus plants,
from wild (i), from in vitro classical culture
plants (ii), and from photoautotrophic
culture plants (iii). See the previous article
for the used plant material and its
preparation for the ultrastructural study. For
these investigations we used the same
samples from which ultrathin sections of 30
– 60 nm were obtained by using the same
Leica UC6 ultramicrotome with a Diatome
35º diamond knife. These sections were put
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Annals of RSCB
Vol. XVI, Issue 1
and the intercellular spaces are small (Fig. 1
G). Each cell has a large central vacuole,
while in the parietal cytoplasm there are
numerous lenticular chloroplasts and the
nucleus with obvious nucleolus (Fig.1 H).
The leaf mesophyll is made up of
large cells, each with a large central vacuole
with vacuolar juice, and the parietal
cytoplasm with chloroplasts and nucleus
(Fig. 1 H). Generally, chloroplasts from the
palisade tissue are lenticular, have classical
structure and lack the plastoglobuli (Fig. 2
A). At the bottom of the cells, right beneath
the palisade tissue, the chloroplasts have
numerous grana and relatively numerous
plastoglobuli in the stroma (Fig. 2 B).
The lacunar mesophyll is made up
of numerous spherical-oval cells, with
relatively uniform walls and arranged in
groups of 3-6 cells attached to one another
or linked in small chains, having aeriferouslacunar spaces of different dimensions (Fig.
2 C). Each cell has a central vacuole, the
nucleus and chloroplasts being grouped,
and tonoplast being tightly attached to
plasmalemma (Fig. 2 C). Other cells nearby
the bottom of the palisade tissue have
numerous chloroplasts, a large amount of
cytoplasm and a relatively high number of
mitochondria of normal structure (Fig. 2
D). It is worthy to mention that in such
cells, in the chloroplasts’ stroma, there is a
high number of plastoglobuli (Fig. 2 D),
probably of carotenoid-phospholipidic
nature. The cells of the lacunar mesophyll
from the bottom of the leaf blade have
deformed cell walls, are smaller and have
few chloroplasts with or without
plastoglobuli, and the lacunar spaces are
larger (Fig. 2 E).
The vessel network is very well
developed, being mainly surrounded by
cells of the lacunar mesophyll (Fig. 2 F). At
the veins level, the phloem cells have a
small diameter and are very tight. The cells
have a finely pellicular cytoplasm arranged
at the periphery along plasmalemma, where
you can find the nucleus and some small
chloroplasts, and in the center the majority
of space is occupied by a vacuole (Fig. 2
onto 200 Mesh network electrolytic grids,
which were double contrasted with uranyl
acetate and lead citrate, and then were
examined by means of a JEM JEOL 1010
electron microscope, the images being
recorded with a Mega View III camera. The
used methods and technique are according
to the international standards (Kay, 1967,
Ploaie and Petre, 1979, Crăciun et al.,
1993-1994, Corneanu et al., 1995, Hayat,
2000, Cachiţă et al., 2003, Pavelka and
Roth, 2005).
Results and discussions
The superior epidermal wall is
smooth towards exterior, has no cuticle, is
almost without undulations and has a
thickness of 4µm (Fig.1 B) in wild plants.
The inferior epidermal wall has
approximately the same thickness, but has
relatively rare major undulations (Fig. 1 C)
and minor undulations represented by a
cuticle, probably waxy, of 0.3 – 0.5 µm
thickness, with undulations which appear in
cross sections like some conic crests (Fig. 1
E). Here and there onto this epidermis there
are stomata with ostioles in the “closed”
position (Fig. 1 F).
Beneath the superior wall the
epidermis is made up of a single row of
relatively large cells, in which the nucleus
and lenticular chloroplasts are arranged
here and there and only onto the cell wall
towards the palisade tissue, as it can be
observed in cross sections (Fig. 1 A). All
these cells have a large vacuole with
vacuolar juice and flocculent material (Fig.
1 A and B).
The cells of the inferior epidermis
are also arranged in a single row, have very
little cytoplasm pellicular-parietal arranged
(Fig. 1 D), where it can be observed
nucleus and rare small chloroplasts (Fig. 1
C). Here and there, between the external
wall and the epidermal cells, it can be
observed accumulations of electrondense
material, which is probably senescent (Fig.
1 C and E).
The palisade tissue is made up of
numerous elongated cells, tightly arranged,
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Vol. XVI, Issue 1
have aberrant shapes, and have large
lacunar spaces between them (Fig. 3 G).
The majority of them have deteriorated
tonoplast and plasmalemma, and the nuclei
and chloroplast are being highly
deteriorated or are missing (Fig. 3 G). In
other cells, the damaged chloroplasts float
in the vacuolar juice, after tonoplast breaks
down (Fig. 3 H and 4 A).
The veins are less developed and
contain a low number of cells, in the
phloem as well as in the xylem (Fig. 4 B).
In case of xylem, the lignin deposits are
incomplete (Fig.4 B and C). The xylem
cells have narrow lumen and a damaged
material inside (Fig. 4 D).
The ultra structural investigations
on
the
samples
coming
from
photoautotrophic in vitro culture confirm
the results obtained in the optic microscopy
study. The obtained images indicate a very
similar situation to the control leaflets ultra
structure, meaning that the alterations
described in leaflets ultra structure coming
from in vitro culture are no longer present
or are minor and insignificant. The same
situation it was observed by Gardenia
jasmoides ( Serret, et al., 1996), and the
authors suggest that, these situation may
affect positively the further acclimation to
the ex vitro condition.
Thus, the superior epidermal wall is
thinner, having 2 - 3µm thickness and a
very thin waxy epicuticular layer (Fig. 5 A).
The superior epidermal cells are arranged in
a single row and a vacuolar juice with
flocculated material is present into the
single large vacuole (Fig. 5 A). The inferior
epidermis (Fig. 5 B and C) is made up of a
single row of cells, covered by a wall with
small undulations, thinner than the control,
having generally a thickness between 1 2µm. (Fig. 5 B), but it is thicker of
approximately 3 - 5µm nearby stomata (Fig.
5 C). As in the case of superior epidermis,
the cytoplasm of these cells is pellicular and
almost lack of cell components (Fig. 5 B
and C). The vacuole occupies the whole cell
volume, and flocculated material is present
into the vacuolar juice. Here and there
G). The xylem is tightly attached by
phloem and is also made up of elongated
cells with thick walls and lignin sediments
(Fig. 2 H).
The analysis of images obtained by
means of the electron microscope for the
leaflets blade coming from plants
maintained in classical in vitro culture
shows that there is not a clear
differentiation of the leaf blade mesophyll
into the palisade tissue and lacunar tissue,
because the cells are similar in shape, have
irregular outline, have undulated walls
because of lack of rigidity, whereas the
intercellular spaces are much larger,
forming lacunae all over the mesophyll.
The wall of the superior epidermis
is smooth, without undulations, of 2.5 µm
thick, so thinner than the normal thickness
(Fig. 3 A). The cells of the superior
epidermis are arranged in a single row, the
vacuole occupies all the cell volume,
because the tonoplast seems destroyed and
the organelles are missing. The vacuoles
content is relatively clear, so mainly
aqueous (Fig. 3 A).
Right beneath the epidermis there
are relatively disparate cells, which belong
to the palisade tissue, because the
longitudinal diameter is larger than the
transverse one, and are tightly attached to
the basal membrane of the epidermal cells
(Fig. 3 B). The cells have a central vacuole
with clear aqueous content, and in the
pellicular cytoplasm there are lenticular
chloroplasts, having stroma with starch
grains, many of them being real
amyloplasts (Fig. 3 B). In such cells the
nucleus
becomes
heterochromatic,
suggesting its functional damage (Fig. 3 D),
a similar situation was observed by Cachiţă
et al., 2003, to chrysanthemum in vitro
culture. In some cells it can be seen the
electrondense cytoplasm, which becomes a
dark mass, metabolically inactive (Fig. 3
C), and also the progressive installation of a
structural deterioration of chloroplasts and
of the whole cells (Fig. 3 F).
The cells from the central part of the
leaflet blade have thin and undulated walls,
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Vol. XVI, Issue 1
unknown, but probably they are of viral
nature. According to some previous
research, this viral material would cause an
intensification of RNAm and RNAr
synthesis, which, by means of NAB
structures, ease their transportation into the
cytoplasm for the intensification of proteic
synthesis processes.
The transition from the palisade
tissue to lacunar mesophyll is gradual,
meaning that cells beneath the palisade
tissue contain few chloroplasts (Fig. 5 H),
or the chloroplasts contain more starch
grains (Fig. 6 A), as well as mitochondria
interspersed between chloroplasts (Fig. 6
B). Towards the center of the leaf blade, the
cells are larger, have undulated walls, few
chloroplasts, and the intercellular spaces are
larger (Fig. 6 C). As it is approached the
inferior epidermis, the cells either have
many chloroplasts (Fig. 6 D), or have
irregular shapes and few chloroplasts (Fig.
6 E).
The veins contain the vascular
system with close structure to that of the
normal leaves (Fig. 6 F). Noted also that in
some phloem cells the cytoplasm occupies
the whole cell volume, having obvious
nucleus and numerous mitochondria,
meaning that these are very young cells
(Fig. 6 F).
stomata with opened ostioles can be seen
(Fig. 5 C). It is worthy to notice that there is
a differentiation of the leaflet mesophyll
into the palisade tissue and lacunar tissue,
even if they are less obvious than in case of
the control.
The palisade tissue, disposed right
beneath the superior epidermal cells, is
made up of elongated cells, as they are seen
in longitudinal sections (Fig. 5 D) and
appear oval-rounded as seen in the cross
sections (Fig. 5 E). The resemblance of
these cells with those of the control leaflet
consist in the fact that they contain
numerous lenticular chloroplasts with
normal structure and are arranged side by
side along the cell walls (Fig. 5 D).
Between chloroplasts there are nuclei and
mitochondria, of normal structure (Fig. 5
E). All the cells have the majority of the
volume occupied by a vacuole delimitated
towards cytoplasm by tonoplast, and the
vacuolar juice has relatively little flocculent
material (Fig. 5 D).
The chloroplasts of the palisade
cells, especially those from the upper part
of the cells, towards the epidermal cells, are
lenticular, with classical normal structure of
grana and inter-grana thylakoids, with a
rich stroma in plastidial ribosomes, with
few plastoglobuli and few and small starch
grains (Fig. 5 F).
Cells nuclei are found between
chloroplasts (Fig. 5 F). They are
euchromatic type, so very active
metabolically for synthesis processes,
which are also confirmed by the presence
nearby nucleoli of a NAB (Nucleolar
Associated Body) structure (Fig. 5 G),
knowing that they are especially present in
nuclei of cells with high metabolic activity,
as in the case of young, mersitematic cells
(Jakab et Crăciun, 2009, Crăciun et al.,
1980, 1984, 1996, Chamberland and
Lafontaine, 1993, Lafontaine, 1965, Recher
et al., 1969, Wergin et al., 1970). Noted
also the presence in the nuclear chromatin,
right
besides
nucleoli,
of
some
paracrystalline
and
parallelepipedic
structures (Fig. 5 G), which signification is
Conclusion
The ultrastructural investigations confirm
the results obtained in optic microscopy.
The images obtained for leaflets coming
from photoautotrophic in vitro culture show
a very similar situation to the ultrastructure
of control leaflets, meaning that the
alterations described to leaflets coming
from in vitro cultures are no more present
or are minor and insignificant. The
photoautotrophic cultures may replace
classical in vitro cultures or may represent a
stage before vitroplantlets acclimatization,
this stage playing the role of preparing
plants for an autotrophic nutrition,
developing the photosynthetic apparatus
which is less developed to plants coming
from classical in vitro culture.
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Acknowledgements
Funding came from programs co-financed by
The
SECTORAL
OPERATIONAL
PROGRAMME HUMAN RESOURCES
DEVELOPMENT,
Contract
POSDRU
6/1.5/S/3 – „Doctoral studies: through science
towards society“, and with the support by a
grant from the Romanian Ministry of
Education and Research on “Parteneriate PN
II” Programme (CNMP), Project 31-008/2007.
The authors are thankful to Dr. Oana RoşcaCasian for her help with translation and to the
Biological Research Institute, Cluj-Napoca,
for technical support.
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Fig. 1 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; control sample; wild plants.
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Fig. 2 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; control sample; wild plants.
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Fig. 3 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; classical in vitro culture plants.
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Fig. 4 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; classical in vitro culture plants.
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Fig. 5 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; photoautotrophic in vitro culture
plants.
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Fig. 6 Ultrastructure of Dianthus giganteus subsp. banaticus leaflet; photoautotrophic in vitro culture
plants.
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