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Comparative studies between

ferns gametophyte and


sporophyte by bidimensional
electrophoresis
Florentina Aldea*, Florenta Elena Helepciuc*, Cristian Banciu*, Aurelia Brezeanu*,
Liliana-Cristina Soare^
*Institutul de Biologie Bucuresti al Academiei Romane
^Universitatea din Pitesti, Facultatea de Stiinte
 Athyrium filix-femina, Polypodium vulgare and Asplenium trichomanes are three
valuable ferns species due to their potential as ornamental plants and secondary
metabolite production originate to Valsan Valley protected area.
 Our primary experiments were focused on developing an optimal in vitro
multiplication method and medium term conservation for these species. The
reactivity of each species has been evaluated on the basis of biometric
(morfometric and gravimetric) and biochemical determinations.
 Current study shows the differences between gametophytes and sporophytes of the
mentioned species by comparing the proteomic profiles and determination of
ploidy levels of two types of cultures.
Sporophytes of ferns arising from homogenates of gametophytes

spores  gametophytes cultures formation of


homogenates of sporophytes
gametophytes

sporophytes Polypodium Asplenium Athyrium filix-


vulgare trichomanes femina
Materials and Methods

Biological material was


represented by gametophytes and
sporophytes from Athyrium filix-
femina, Polypodium vulgare and
Asplenium trichomanes obtained
by in vitro culture on MS 1/2
medium.

 The protein samples were separated by two-dimensional gel electrophoresis and stained
with Coomassie Brilliant Blue 250. Proteins were isolated using to a phenol-based
extraction procedure (Hurkman and Tanaka 1986). Ploidy level was determined by flow
cytometry using two step procedure with Otto I+II buffers and the fluorochrome DAPI.
Results
 Although in previous studies using PAGE technique we have not
observed significant differences between gametophyte and sporophyte,
the patterns obtained by two-dimensional gel electrophoresis showed
the existence of distinct spots for both gametophyte and sporophyte
extracts. The differences in protein expression suggest that gametophyte
and sporophyte have different metabolic pathways of protein synthesis.

G1 G2 G3 G4 G5 S1 S2 S3 S4 S5

Total protein spectrum from


Polypodium vulgare (left,
G1-G5 gametophytes, right,
S1-S5 sporophytes)
Results
 Determination of ploidy level
showed in case of Athyrium
filix-femina and Asplenium
trichomanes both sporophyte
and gametophyte were
diploid, while for Polypodium
vulgare we detected that the
gametophyte was haploid and
sporophyte was diploid. It is
possible that the diploides
origin of Athyrium filix-
femina and Asplenium
trichomanes gametophytes to
appeare by a false meiosys.
Results

gametophyte sporophyte
Athyrium filix-femina
Results

gametophyte sporophyte
Asplenium trichomanes
Results

gametophyte sporophyte

Polypodium vulgare
Conclusions

 This study presents a preliminary analysis of proteomics profiles and compares the ploidy
level for detection of further changes that can occur at the molecular level during the
cryopreservation process. Proteomic analysis allows the observation of translationally
manifested cell physiological response.