Analysis of Genetic Stability in Some Cyclamen Species
Endemic to Turkey Following Cryopreservation By Vitrification
1
2
2
2
Tolga Izgu , Basar Sevindik , Ozhan Simsek , Pembe Curuk , Yildiz
2
2
Aka Kacar and Yesim Yalcin Mendi
1Department
of Plant Sciences, University of California, Davis, CA, USA
2Department of Horticulture, Faculty of Agriculture, University of Cukurova, Balcalı, Adana, Turkey
INTRODUCTION
The origin of 20 Cyclamen taxons, belonging to Primulaceae and growing under trees and
bushes, is Mediterranean region. Ten Cyclamen species grown naturally in our country, five of
which are endemic to Turkey Cyclamens are under extinction due to destruction of natural
habitats, unconscious usage of agricultural areas and taking out the tubers continuously from
nature for export. For this reason, conservation studies and biotechnological researches on this
important genetic resource are quite significant.
In vitro preservation possibilities of Cyclamen (C. cilicium, C. mirabile, C. parviflorum and C.
pseudibericum) which is an important genetic resource in our country were investigated in this
study. In cryopreservation period, different osmotic reagents (mannitol, sorbitol and sucrose)
and different temperatures were treated with PVS-2 and genetic stability of obtained plantlets
was tested using RAPD primers. The best cryopreservation result was obtained from the
treatment; 0,5 M sucrose+MS basal medium (hormone free), 48 hours, +4°C, darkness and PVS2 solution at 0 C°, 60 min. for all species. This study showed that cryopreservation protocol
worked effectivelly because the plantlets from for Cyclamen species had same genetic
structure after cryopreservation. It was determined that there was no somaclonal variation
between the plantlets obtained after cryopreservation and control plants using RAPD markers.
OBJECTIVES
A. Plant material
Whole endemic cyclamen plants (C. cilicium, C. mirabile, C. parviflorum and C.
pseudibericum) were collected with their tubers intact from locations where they were known
to grow naturally in winter, spring and autumn in natural flora of Turkey (Fig 1, 2, 3 and 4).
C. Regrowth of Explants After Cryopreservation and Assessment of Genetic Stability by
RAPD Markers
The plantlets after cryopreservation and control plants were compared using RAPD marker.
It was found that there was no somaclonal variation in cropreserved plants (Fig 5, 6 and 7).
30 RAPD primers were used for these plants and the gel bands belongs to these primers
were shown in Figure 6. All of the plants had same band profiles. The researchers
mentioned that there was no differences between control and cryopreserved plants
morphologically, sitologically and genetically (Ai et al., 2012; Martin et al., 2012). After
subculturing for a long period, regenerant plants obtained from embryogenic calluses
showed genetically differences (Harding, 2004).
It was shown that cryopreserved plants were genetically same with the controlled plants.
This proved that cryopreservation protocol was optimized. If calluses continued
subculturing in vitro for a long period after cryopreservation, somaclonal variation could
be observed. Some researchers worked on cryopreservation of Citrus sinensis (Marin et al.,
1993), Picea glauca (Park et al., 1994), Picea mariana (Isabel et al., 1993) and Pinus
sylvestris (Haggman et al., 1998 ). They found that no genetic differences was observed
between control and cryopreserved plants. De Verno et al. (1999) mentioned that
somaclonal variation was observed in subcultured embryogenic calluses (2-12 months) after
cryopreservation in Picea glauca. There was no somaclonal variation in directly
regenerated plants after cryopresevation.
Vitrification Based on PVS2
After preculture treatment
Fig 1. C. pseudibericum
Fig 2. C. mirabile
Immersion LN (-196°C)
Transfering of
embryogenic callus to
cryotubes
Adding LS
solution
0°C, 1 hour
Adding PVS-2 solution
TTC viability test
Thawing (37°C, 3min)
Transfering on Regeneration Medium
Fig 3. C. parviflorum
Fig 4. C. cilicium
B. Cryopreservation of Embryogenic Callus Cultures
Embryogenic callus cultures of C. cilicium, C. mirabile, C. parviflorum and C. pseudibericum
were cold hardened for 1 month at 4°C, in darkness. They were then precultured for 48 h at
4°C, in darkness on MS basal media (Murashige and Skoog, 1962) (Fig 5) , containing sorbitol
(2%, 4%), mannitol (2%, 4%), sucrose (0.5 M) or a combination of sorbitol (2%) and mannitol
(2%). Preconditioning of the callus cultures were followed by their transfer to 2-ml Nalgene®
cryovials and incubation in loading solution (LS, 2 M glycerol + 0.4 M sucrose, Matsumoto et al.,
2004) for 30 min at 25°C. Vitrification was performed by discarding the LS and application of
Plant Vitrification Solution 2 (PVS2, 30% glycerol (w/v) + 15% ethylene glycol (w/v) + 15% DMSO
(w/v) + 0.4 M sucrose, prepared in MS medium, Sakai et al., 1990) for 30, 60 or 90 min at 0°C.
After PVS2 treatment, a group of explants was suspended in fresh 0.6 ml of PVS2 solution and
rapidly plunged into liquid nitrogen (LN). After at least 1 h of storage in LN, cultures were
thawed for 2 min in 38°C water bath, rinsed with liquid MS medium, containing 1.2M sucrose
(washing solution) for 20 min at 25°C, and plated on sterile filter paper placed on recovery
medium. After 3 days of culture at 25°C, in darkness, filter paper was removed, calli were
transferred to direct contact with a fresh recovery medium and maintained at 25°C, in
darkness. Second group of explants (control group, LN-) were rinsed in washing solution
immediately after PVS2 treatment, and plated on recovery medium as the LN group.
Fig 5. Callus induction and diferentiation
medium content
Macro elements
(1/2 MS)
Callus induction Differentiation
medium
medium
(mg/L)
(mg/L)
NH4NO3
KNO3
CaCl2 x 2H2O
MgSO4 x 7H2O
KH2PO4
Micro elements
(1/2 MS)
825
950
220
185 MgSO4
85 KH2PO4
(mg/L)
825
950
220
185 MgSO4
85 KH2PO4
(mg/L)
H3BO3
MnSO4 x 1H2O
ZnSO4 x 7H2O
KJ
Na2MoO4 x 2H2O
CuSO4 x 5H2O
CoCl2 x 6H2O
3,1
8,45
5,3
0,415
0,125
0,0125
0,0125
(mg/L)
34,51
250
100
2
0,1
0,5
0,5
(g/L)
30
2
3,7
3,1
8,45
5,3
0,415
0,125
0,0125
0,0125
(mg/L)
34,51
250
100
2
0,1
0,5
0,5
(g/L)
30
2
3,7
(mg/L)
2
0,8
5,5-5,6
(mg/L)
------5,5-5,6
Fe EDTA
Peptone
myo-Inositol
Glysine
Nicotinic acid
Thiamine HCl
Pyridoxine HCl
Sucrose
D(+)-glucose
monohydrate
Gelrite
2,4-D
2iP
pH
Plantlets
Fig 6. RAPD analysis of cyclamens before and after
cryopreservation (UBC-54 RAPD primer), M: Marker (bp), 1: Control
(-LN plantlets) C. persicum, 2, 3: C. persicum (+LN plantlets), 4:
Control (-LN plantlets) C. cilicium, 5, 6: C. cilicium (+LN plantlets), 7:
Control (-LN plantlets) C. pseudibericum, 8,9: C. pseudibericum
(+LN plantlets), 10: Control (-LN plantlets) C. mirabile, 11, 12: C.
mirabile (+LN plantlets), 13: Control (-LN plantlets), C. parviflorum,
14, 15: C. parviflorum (+LN plantlets).
Somatic embryos
(Survival 100%)
10 weeks later
Fig 7. RAPD analysis of cyclamens before and after
cryopreservation (OPB-19 RAPD primer), M: Marker (bp), 1: Control
(-LN plantlets) C. persicum, 2, 3: C. persicum (+LN plantlets), 4:
Control (-LN plantlets) C. cilicium, 5, 6: C. cilicium (+LN plantlets),
7: Control (-LN plantlets) C. pseudibericum, 8,9: C. pseudibericum
(+LN plantlets), 10: Control (-LN plantlets) C. mirabile, 11, 12: C.
mirabile (+LN plantlets), 13: Control (-LN plantlets), C. parviflorum,
14, 15: C. parviflorum (+LN plantlets).
Fig 8. RAPD analysis of cyclamens before and after
cryopreservation (OPB-20 RAPD primer), M: Marker (bp), 1: Control
(-LN plantlets) C. persicum, 2, 3: C. persicum (+LN plantlets), 4:
Control (-LN plantlets) C. cilicium, 5, 6: C. cilicium (+LN plantlets),
7: Control (-LN plantlets) C. pseudibericum, 8,9: C. pseudibericum
(+LN plantlets), 10: Control (-LN plantlets) C. mirabile, 11, 12: C.
mirabile (+LN plantlets), 13: Control (-LN plantlets), C. parviflorum,
14, 15: C. parviflorum (+LN plantlets).
CONCLUSIONS
In this report, a simple, efficient and cost effective protocol for cryopreservation-vitrification
of endemic cyclamen species from embryogenic callus has been presented. In conclusion, the
RAPD techniques used for the genetic stability analysis of plantlets regenerated from frozen
cyclamens did not to detect any polymorphism or somaclonal variation. This result suggested
that cryopreservation could be used for long-term conservation of Cyclamen germplasm.
REFERENCES
1- Aİ, Peng-fei, Li-Ping Lu, and Jian-Jun Songi, 2012. Cryopreservation of in vitro-grown shoot-tips of Rabdosia rubescens by
encapsulation-dehydration and evaluation of their genetic stability. Plant Cell, Tissue and Organ Culture (PCTOC) 108.3:
381-387.
2- De Verno, LL, Park YS, Bonga JM, Barrett, JD., 1999. Somaclonal variation in cryopreserved embryogenic clones of white
spruce [Picea glauca (Moench) Voss.]. Plant Cell Rep 18: 948–953.
3- Haggman, H., Ryyänen, L., Aronen, T., Krajnakova, J., 1998. Cryopreservation of embryogenic cultures of Scots pine.
Plant Cell Tissue Organ Cult 54: 45–53.
4- Harding, K., 2004. Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25, 3–22.
5- Martin, M.L., Gorgorcena. Y., Ortiz, J., Duran-Vila, M., 1993. Recovery of whole plants of sweet orange from somatic
embryos subjected to freezing thawing treatments. Plant Cell Tissue Organ Cult 34, 27–33.
6- Martín, C., 2011. María Teresa Cervera, and María Elena González-Benito, Genetic stability analysis of chrysanthemum
(Chrysanthemum x morifolium Ramat) after different stages of an encapsulation–dehydration cryopreservation
protocol." Journal of plant physiology 168.2: 158-166.
7- Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol.
Plant 15, 473–497.
8- Isabel, N., Tremblay, L., Michaud, M., Tremblay, F.M., Bousquet, J., 1993. RAPDs as an aid to evaluate the genetic
integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B. S. P. Theor Appl Genet 86, 81–87.
9- Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis).
ACKNOWLEDGEMENTS
This research was supported by TUBITAK (The Scientific and Technical Research Council of Turkey)
(Project no.: TOVAG 110O102) project. The authors thank Prof. Dr. Maurizio LAMBARDI (Senior
Researcher of Ivalsa, Trees and Timber Institute of National Research Council (CNR), Florence/Italy)
and Assoc. Prof. Dr. Elif Aylin OZUDOGRU (Researcher of Ivalsa, Trees and Timber Institute of National
Research Council (CNR), Florence/Italy).
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com