The effect of the ultraviolet radiation on the somaclonal variability for

Romanian Biotechnological Letters
Copyright © 2014 University of Bucharest
Vol.19, No3, 2014
Printed in Romania. All rights reserved
ORIGINAL PAPER
The effect of the ultraviolet radiation on the somaclonal variability for
solanum tuberosum
Received for publication, May 05, 2014
Accepted, June 5, 2014
CREŢESCU IULIANA1, PETOLESCU CERASELA*2
1
Banat’s University of Agricultural Sciences and Veterinary Medicine “King
Michael I of Romania” from Timisoara,Faculty of Animal Sciences and
Biotechnologies, 119 Calea Aradului, RO-300654, Timişoara, Romania
2
Banat’s University of Agricultural Sciences and Veterinary Medicine “King
Michael I of Romania” from Timisoara, Faculty of Horticulture and
Forestry, 119 Calea Aradului, RO-300654, Timişoara, Romania
*For correspondence:E-mail:[email protected]
Abstract
Among the non-ionizing radiations, the ultraviolet radiations (UV) are the only ones which
can change the genetic system. The investigations related to the soma clonal variability for
potato (Solanum tuberosum) were focused on the response to the radiation exposure UV-C (λ =
254nm). The biological material used was represented by three types of potato: Cristian, Roclas
and Rustic. For this purpose we used several RAPD markers (Random Amplified Polymorphic
DNA). After the irradiation with UV-C there were some changes at molecular level by the
occurrence in some cases of new DNA bands or the disappearance of DNA bands in other cases
highlighted using three primers (OPF-12, OPW-11 and OPX-01).
Keywords: UV-C, somaclone, potato, genetic variability, RAPD
Introduction
Solanum tuberosum species is part of the Solanaceae family, Solanum type, and has two
subspecies: andigena and tuberosum. The potato, a tubercular plant with high importance in
the human nutrition, animal feeding and industrial processing, cultivated on all continents but
mainly in Europe. In present, due to the demographic explosion (over six billion inhabitants
on the globe), the potato, together with some other plants, represents a hope in insuring the
human food needs competing with the most important food categories: cereals, meat and
fruits.
The ultraviolet radiations (UV) currently represent a very important stress factor for
plants, which can lead to the alteration of the genetic system (EHSANPOUR & al. [1]). Due
to the decrease of the ozone level the negative influence of the UV-B and UV-C radiations
increased.
The main purpose of this research is to study the influence of the ultra violet radiation on
the soma clonal variability from the potato. For this purpose we used several RAPD markers
(Random Amplified Polymorphic DNA).
The part of the electromagnetic spectrum with shorter wave length and higher energy
than the visible light is subdivided in ionized radiations and non-ionized radiations.
The ultraviolet radiations (UV) are non-ionized. The UV spectrum is subdivided in three
bands according to the wave length (λ): UV-C (λ < 280 nm), UV-B (λ = 280-320 nm) and
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UV-A (λ =320-400 nm). On the genetic material act UV radiations with λ = 200-300 nm. The
nucleic acids absorb the radiations with λ = 240-290 nm, the highest mutagen effect have the
radiations with λ = 258-260 nm. Both the DNA and the RNA absorb most intensely the UV at
260 nm (KLUG and CUMMINGS, [2]).
The UV radiations effect (mainly UV-B and UV-C) on the plants vary according to the
species and types of the same species (DANON and GALLOIS, [3]).
The RAPD markers were successfully used also by other researchers to evaluate the
genetic variability of genotypes of the species Solanum tuberosum (DEHGHAN & al., [4];
GHERARDI & al., [5]; CAMPBELL, [6]; CHANDRA, [7]).
RAPD (Random Amplified Polymorphic DNA) was discovered by WILLIAMS & al.
[8] in 1990. The method is fast, relatively low cost and well adapted to obtain a nonradioactive genetic mark (WELSH and McCLELLAND, [9]). It uses a single primer (with
low number of nucleotides) in an amplification reaction (WILLIAMS & al., [8]). Some
problems related to the reproducibility of the method were reported (HANSEN & al., [10];
JONES & al., [11]; VIRK & al., [12]) and the presence of some errors which can occur when
applying the evaluation software of the markers (DEMEKE & al., [13], KARP & al., [14]).
The amplified fragments of DNA are submitted to electrophoresis in a 2% agarose gel,
separating according to the molecular weight. The staining is done with ethidium bromide and
the visualization of the DNA bands is done by exposing the gel which contains the DNA to a
source of UV radiations. We obtain images (photos) which can be scanned and edited by
using specific software.
The RAPD technique is used mainly to identify genetic diversity: Acacia – CASIVA &
al., [15]; Afgekia – PRATHEPHA and BAIMAI, [16]; Astragalus – SANDERSON and
Liston, [17]; Atylosia-Cajanus complex – PARANI & al., [18]; Lotus – CAMPOS & al., [19];
Medicago – BENA & al., [20].
Materials and methods
Biological material
As biological material we used three types of potato procured from the National Institute
for Research and Development of the Patato and Sugar Cane Brasov (I.N.C.D.C.S.Z):
Cristian, Roclas and Rustic. The irradiation experiments with UV-C were made on vitro
plantlets obtained from scalped embryos from sleeping eyes of the mature potato tubercles.
Working methods
Irradiation with UV-C
For the irradiation of the sprouts we used UV lamp in form of tubes which emitted
radiations with wavelength λ= 254nm and approximate intensity 6, 24 µmol photon/m2/s. The
radiation time was 30 minutes.
Molecular techniques for detecting the genetic variability
In order to distinguish the genetic variability we need the following: i) DNA extraction;
ii) its amplification using PCR techniques and iii) migration of the reaction products in the
2% agarose gel by electrophoresis.
The vegetal material collection for the DNA extraction was done after a week of
radiation from the foliar tissue with Maxwell™ 16 Instrument from Promega. This allows
obtaining a quantity of purified vegetal genomic DNA at the proper quality necessary to use it
in the next amplifications. The extracted DNA was distinguished using electrophoresis and
afterwards it was kept at 40C until the amplification with RAPD markers.
PCR amplification with RAPD markers
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The effect of the ultraviolet radiation on the somaclonal variability for solanum tuberosum
For the realization of the PCR reaction, the mix used to obtain the amplification products
was composed by:
1. Kit GoTaq® Green Master Mix 2x (Promega) which consists in: GoTaq® DNA
polymerase; dNTP; MgCl2; reaction buffer, all these components can be found in optimum
concentrations for an efficient amplification of the DNA samples.
2. primer: 10 pmol/µl (from Fermentas company);
3. 50-100 ng genomic DNA;
4. distilled water, free of nucleases.
For the evaluation of the genotypes variability from our study we used 6 RAPD primers
(Table 1.).
Table1. RAPD primers used for the evaluation of the studied genotypes variability
Code
Sequence ( 5’- 3’) Code
Sequence ( 5’-3’)
OPAA-18
TGGTCCAGCC
OPAA-16
GGAACCCACA
OPF-10
GCAAGCTTGG
OPC-16
CACACTCCAG
OPF-12
ACGGTACCAG
OPF-08
GGGATATCGG
OPW-11
CTGATGCGTG
OPW-16
CAGCCTACCA
OPW-13
CACAGCGACA
OPX-03
TTGCGCAGTG
OPX-01
CTGGGCACGA
OPA-14
TCTGTGCTGG
Reaction conditions
The Thermo cycler used was from type Corbett. The amplification was realized in the
following conditions:
i)the initial denaturation of DNA: 4 minutes at 940 C, there were 45 cycles, each cycle
had the following stages:
9 denaturation: 3 minutes, at 940 C;
9 primers attachment:1 minute at 360 C;
9 extension: 2 minutes at 720 C.
ii) DNA synthesis: 3 minutes at 720 C
At the end the samples are cooled till 40 C in the thermo cycler and kept in the
refrigerator until usage.
The analysis of the reaction products through electrophoresis in agarose gel
Results and discussion
After the amplification with the RAPD primers used for the evaluation of the genotypes
variability we obtained many types which presented uniformity. The molecular marker (M)
used determines bands of 1000bp, 750bp, 500bp, 300bp, 150bp and 50bp. Only 3 primers
showed polymorphism: OPF-12, OPW-11 and OPX-01.
The amplification with OPF-12 primer generated bands of 730 bp, 590 bp, and 300bp. It
identified differences between the radiated individuals and the non-radiated ones, fact which
can be noticed in figure 1, next to number 4 individual there is a new band of approximately
400bp.
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Figure 1. Analysis of gel electrophoresis for amplified products using OPF-12primer
Legend: M - molecular marker; 1, 3, 5- non-radiated individuals;2, 4, 6- radiated
individuals;
The amplification with OPW-11 primer generated five bands (of approximately: 1100bp,
755bp, 470bp, 350bp and 220bp Figure 2.) for the non-radiated individuals (1, 3 and 5) while
for the two of the radiated individuals (2 and 4) the biggest one (of 1100bp) is missing. For
the last individual exposed to UV radiation it generated a band of approximately 600bp and
the one of 200bp is missing.
Figure 2. Analysis of gel electrophoresis for amplified products using OPW-11 primer
Legend: M - molecular marker; 1, 3, 5- non-radiated individuals;2, 4, 6- radiated
individuals;
The amplification with OPX-01 primer generated five bands of approximately 750bp,
620bp, 500bp, 340bp and 200bp (Figure 3.). The difference for this primer can be noticed at
individual number 6 where a new band of 800bp appeared.
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The effect of the ultraviolet radiation on the somaclonal variability for solanum tuberosum
Figure 3. Analysis of gel electrophoresis for amplified products using OPX-01 primer
Legend: M - molecular marker; 1, 3, 5- non-radiated individuals;2, 4, 6- radiated
individuals;
SONIYA & al. [21] found that during plant regeneration from tomato plant, more than
90% of regenerated plants had no somaclonal variation using RAPD marker.
Conclusions
After irradiation with UV-C there are changes at molecular level, fact which was
highlighted by the three primers (OPF-12, OPW-11 and OPX-01). We can say that the
polymorphism detected by these primers is not due to the genetic instability or the in vitro
culture methods because there were a low number of subcultures (two).
The ultraviolet mutation can generate mutations at the DNA level; this fact is proved by
the extra DNA bands in some cases and the missing ones in other cases.
References
1. A. A. EHSANPOUR, S. MADANI, and M. HOSEINI, Detection of somaclonal variation in potato callus
induced by uv-c radiation using RAPD-PCR, Gen. Appl. Plant Physiology., 33 (1-2), 3-11(2007).
2. W. S. KLUG and M. R. CUMMINGS, Concepts of genetics, Second edition. Merrill Publishing Company,
1986, pp. 306-312.
3. A. DANON and P. GALLOIS. UV-C radiation induces apoptotic-like changes in Arabidopsis thaliana. FEBS
Letters, 437, 131-136(1998).
4. S. M. DEHGHAN, J. G. HAMPTON, S. E. GARDINER, Genetic analysis among and within populations
forming ecotype and cultivars of lucerne, Medicago sativa (Leguminosae), using RAPD fragments. Plant
Systematics and Evolution, 208 (1-2), 107-119(1997).
5. M. GHERARDI, B. MANGIN, B. GONET, D. BONNET, T. HUGUET, A method to measure genetic
distance between allogamous populations of alfalfa (Medicago sativa L.) using RAPD molecular markers. Theor.
Appl. Genet. 96, 406-412(1998).
6. T. A. CAMPBELL, Molecular analysis of genetic variation among alfalfa (Medicago sativa L.) and Medicago
ruthenica clones. Canadian Journal of Plant Science, 80 (4), 773-779(2000).
7. A. CHANDRA , Bulk Genomic DNA PCR Analysis – A Rapide Method to Estimate Genetic Relatedness
among Heterogeneous Lucerne (Medicago sativa L.) Cultivars. The Japan Mendel Society, Cytologia 72(3), 363368(2007).
8. C. E. WILLIAMS, S. M. WIELGUS, G. T. HABERLACH, C. GUENTHER, H. KIM-LEE and J. P.
HELGESON, RFLP analysis of chrosomal segregation in progeny from an interspecific hexaploid somatic
hybrid between Solanum brevidens x Solanum tuberosum. Genetics, 135, 1167-1173(1993).
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
9343
CREȚESCU IULIANA, PETOLESCU CERASELA
9. J. WELSH and M. MCCLELLAND. Fingerprinting genoms using PCR with arbitrary primers, Nucleic Acids
Res., 18, 7213-7218(1990).
10. M. HANSEN, C. HALLDÉN and T. SÄLL, Error rates and polymorphism frequencies for three RAPD
protocols. Plant Mol Biol Rep., 16, 139-146(1998).
11. C. J. JONES, K. J. EDWARDS, S. CASTAGLIONE, M. O. WINFIELD, F. SALE, C. VAN DE WIEL, G.
BREDEMEIJER, M. BUIATTI, E. MAESTRI, A. MALCEVSHI, N. MARMIROLI, R. AERT, G.
VOLCKAERT, J. RUEDA, R. LINACERO, A. VAZQUEZ and A. KARP, Reproducibility testing of RAPD,
AFLP and SSR markers in plants by a network of European laboratories. Mol Breed., 3, 381-390(1999).
12. P. S. VIRK, J. ZHU, H. J. NEWBURY, G. J. BRYAN, M. T. JACKSON and B.V. FORD-LLOYD.
Effectiveness of different classes of molecular markers for classifying and revealing variations in rice (Oryza
sativa) germplasm. Euphytica, 112, 275-284(2000).
13. T. DEMEKE, R. P. ADAMS, and R. CHIBBAR. Potential taxonomic use of random amplified polymorphic
DNA (RAPD): A case study in Brassica. Theor. Appl. Genet., 84,990-994(1992).
14. A. KARP, K. J. EDWARDS, M. BRUFORD, B. VOSMAN, M. MORGANTE, O. SEBERG, A. KREME, P.
BOURSOT, P. ACTANDER, D. TAUTZ and G. HEWITT Newer molecular technologies for biodiversity
evaluation: opportunities and challenges, Nature Biotechnol., 15, 625-628(1997).
15. P. V. CASIVA, B. O. SAIDMAN, V. C.VILARDI, and A. M. CIALDELLA. First comparative phenetic
studies of Argentinean species of Acacia (Fabaceae) using morphometric, isozymal, and RAPD approaches. Am.
J. Bot., 89, 843-853(2002).
16. P. PRATHEPHA and V. BAIMAI. Molecular characterization of the divergence of rare species of the genus
Afgekia (Papilionoideae, tribe Tephrosieae) by RAPD markers and nucleotide sequence analysis. Sci. Asia., 29,
13-20(2003).
17. M. J. SANDERSON and A. LISTON, Molecular phylogenetic systematics of Galegeae, with special
reference to Astragalus. In: Advances in Legume Systematics, part 7, Phylogeny (Crisp M. and Doyle J. J., eds.).
Royal Botanic Gardens, Kew, U.K., 1995, pp. 331-350
18. M. PARANI, M. LAKSHMI, P. SENTHIL KUMAR and A. PARIDA. Ribosomal DNA variation and
phylogenetic relationships among Cajanus cajan (L.) Millsp., and its wild relatives. Current Science, 78, 12351238(2000).
19. L. P. CAMPOS, J. V. RAELSON and W. F. GRANT. Genome relationship among Lotus species based on
random amplified polymorphic DNA (RAPD). Theor. Appl. Genet., 88, 417-422(1994).
20. G. BENA, M. F. JUBIER, I. OLIVIERI, B. LEJEUNE, Ribosomal external and internal
transcribed spacers: Combined use in the phylogenetic analysis of Medicago (Leguminosae). J. Mol.
Evol. 46, 299-306(1998).
21. E. V. SONIYA, BANERJEE N.S. and M. R. DAS, Genetic analysis of somaclonal variation among callus
derived plants of tomato. Current Science, 80, 1213-1215(2001).
9344
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014