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Bulgarian Journal of Agricultural Science, 19 (2) 2013, 18–21
Agricultural Academy
EFFECT OF ABA PRETREATMENT ON CHLOROPLASTS STRUCTURE IN HYPERICUM
RUMELIACUM BOISS. PLANTS SUBJECTED TO CRYOPRESERVATION
D. KOLEVA*, Ts. GANEVA and M. STEFANOVA
Sofia University “St. Kliment Ohridski”, Faculty of Biology, BG - 1164 Sofia, Bulgaria
Abstract
KOLEVA, D., Ts. GANEVA and M. STEFANOVA, 2013. Effect of ABA pretreatment on chloroplasts structure in Hypericum
rumeliacum Boiss. plants subjected to cryopreservation. Bulg. J. Agric. Sci., Supplement 2, 19: 18–21
Chloroplast ultrastructure in mature leaves’ cells of in vitro cultured Hypericum rumeliacum Boiss. plants regenerated
from cryopreserved shoot tips pretreated with abscisic acid (ABA) for 3, 7 and 10 days periods respectively was analyzed by
transmission electron microscopy (TEM). The chloroplasts in the control-unfrozen plants were lenticular with well-developed
peristromium and thylakoids. The chloroplasts in regenerants passed 3-day period of pretreatment with ABA were structured
almost the same to the control ones while in regenerated after 7 day pretreatment plants, reduced volume of the thylakoids was
observed. In 10 day pretreated regenerants TEM revealed round shaped chloroplasts and both grana and stromal thylakoids
were destroyed. The structural analysis of the plastid apparatus in regrown H. rumeliacum plants pointed out the 3-day ABApretreatment as the most efficient for cryopreservation on sub-cellular level of organization.
Key words: abscisic acid, chloroplasts, cryopreservation, Hypericum rumeliacum
Abbreviations: ABA – abscisic acid; TEM – transmission electron microscopy
Introduction
Preservation of plant material at ultralow temperatures
(cryopreservation) is a promising method for long-term conservation and storage of valuable plant genotypes. Although, it is
known that freezing could cause damages on tissue, cellular and
sub-cellular levels. The undisputable necessity of this method
and the problems related to its application are well-founded
reasons for many studies to direct their attention to optimizing
the cryopreservation protocols (Mycock et al., 2010). There are
still many open questions that refer to the structural and functional status of the treated plants (Kaczmarczyk, 2008). One of
the steps in the cryopreservation process is the plant material
pretreatment that could increase the survival rate and genetic
stability (Yordanova et al., 2010). Abscisic acid is routine applied cryoprotectant against ultrastructural damages in the plant
material subjected to ultralow temperatures (Haisel et al., 2006;
Kaczmarczyk, 2008; Brunakova et al., 2010).
*E-mail: [email protected]
Hypericum rumeliacum Boiss. is a Balkan subendemic
species with conservational value for the Bulgarian flora.
At the same time number of studies reported accumulation
of phenolics and flavonoids with valuable pharmacological
properties (Kitanov, 2001; Galati et al., 2008). Danova et al.
(2010) reported that in vitro cultured H. rumeliacum plants
produce high levels of phytochemical compositions commensurable to the levels in the intact plants.
The aim of the present study was to analyze by TEM the
chloroplast ultrastructure in mature leaves’ cells of in vitro
cultured H. rumeliacum plants regenerated from cryopreserved shoot tips pretreated with ABA for 3, 7 and 10 days
periods respectively.
Material and Methods
The preculture treatment performed to H. rumeliacum was
based on 0.076 μM ABA exposure of shoot tips in RMB0.5 liq-
Effect of ABA Pretreatment on Chloroplasts Structure in Hypericum rumeliacum Boiss. Plants...
uid culture medium for 3, 7 and 10 days periods. Further explants were treated for 20 minutes in LS solution (2M glycerol
and 0.4M sucrose) at room temperature. Plant shoot tips were
dehydrated in PVS3 (50% w/v sucrose and 50% w/v glycerol)
for 90 minutes on ice and finally directly immersed into liquid
nitrogen. After one week of storage, thawing was performed
in water bath at 40ºC for 1 minute. Tips were rinsed in liquid
RMB0.5 containing 1.2 M sucrose. Afterwards shoot tips were
cultivated on semi-solid RMB0.5 for regeneration. The transmission electron microscopy was performed approximately 2
months after thawing. Leaf segments (1 mm2) from the middle
part of fully expanded leaves were fixed in 3 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 12 h at
4ºC. Then the leaf segments were post fixed in 1% KMnO4 in
the same buffer for 2 h at room temperature. After dehydra-
Fig. 1. Control plant chloroplast
Fig. 2. 3-day ABA-pretreated plant chloroplast
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tion by increasing concentrations of ethylalcohol (from 25%
to 100%), the samples were embedded in Durcupan (Fluka,
Buchs, Switzerland) and cross-sectioned with Reichert-Jung
(Wien, Austria) ultramicrotome. Observation was performed
by JEOL 1200 EX (Tokyo, Japan) electron microscope.
Results
The chloroplasts in the control-unfrozen plants were lenticular with well-developed peristromium entirely orientated to the
cell wall (Figure 1). The internal membrane system consisted
of large-areal grana with different height (4–30 thylakoids) and
long stromal thylakoids. Only in few chloroplasts there were
small stromal zones where the thylakoids were orientated perpendicular to the longitudinal axis (Figure 1 – arrow).
Fig. 3. 7-day ABA-pretreated plant chloroplast
Fig. 4. 10-day ABA-pretreated plant chloroplast
D. Koleva, Ts. Ganeva and M. Stefanova
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The chloroplasts in plants passed 3-day pretreatment
with ABA were structured in almost the same way as the
control ones (Figure 2). The internal membrane system
had comparatively large volume. It consisted of well developed grana and stromal thylakoids.
The chloroplasts in regenerated after 7-day pretreatment with ABA plants had reduced volume of the thylakoid system than the control ones (Figure 3). The observed grana were not so large-areal and the number of the
thylakoids did not exceed 10. The amount of the stromal
thylakoids was also small. The thylakoid membranes were
well developed and the peristromium was with comparatively large volume.
The chloroplast structure in regenerated after 10-day
pretreatment with ABA plants differed greatly from the
control ones (Figure 4). They were round shaped. The
observation revealed internal membrane system in state
of destruction. There were no distinguishable grana and
stromal thylakoids in the stroma.
In none of the examined experimental plants, starch
grains in the stroma were found.
Discussion
Chloroplasts are the organelles mainly affected by the
low temperature conditions (Kratsch and Wise, 2000).
There are many records that low temperatures cause
destruction and reorganization of the thylakoid system
in chloroplasts and deviations in the metabolic transport (Taylor and Craig, 1971; Ristic and Ashwort, 1993;
Kratsch and Wise, 2000; Deryabin et al., 2007; Popov
et al., 2007). Other cell organelles such as mitochondria
and nucleus are much more stable in same experimental
conditions (Ishikawa, 1996; Kratsch and Wise, 2000). According to Dellaire et al. (1994) ABA protection ability
does not depend on its concentration. The present study
revealed that if the structure of the chloroplasts is considered as a criterion for the cryoprotective role of ABA then
for H. rumeliacum the duration of the pretreatment is very
important. The longer the duration of the pretreatment is,
the weaker is the protective effect of ABA and the stronger is the negative effect of the low temperatures along
with the other stress factors on the ultra structure of the
plastid apparatus.
The TEM analysis supported and completed the results
from the histological light microscopy observation of the
leaves of H. rumeliacum plants in same experimental conditions (Danova et al., 2009).
Conclusions
The structural analysis of the plastid apparatus in recovered H. rumeliacum plants pointed out the 3-day ABA
pretreatment as the most efficient for cryopreservation on
sub-cellular level of organization and supported the assertion of the great significance of ABA-pretreatment duration for photosynthetic tissue structuring.
Acknowledgements
This work was supported by grants of the Bulgarian
Ministry of Education Youth and Science (DNTC/BGSK-01/2012) and from Slovak Research and Development
Agency (APVV SK-BG 0012-10).
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