MISCELLANEOUS REPORTS 83 ISSN 0253-6749

ADVANCEMENTS IN MOLECULAR BIOLOGYANDPOTENTIALAPPLICATIONS IN CYPRUS AGRICULTURE

I.M. Ioannides

AGRICULTURAL RESEARCH INSTITUTEMINISTRY OFAGRICULTURE, NATURAL RESOURCES

AND THE ENVIRONMENT

FEBRUARY 2002

CYPRUSNICOSIA

Editor - in Chief

Dr A.P. Mavrogenis, Agricultural Research Institute, Nicosia, Cyprus.

All responsibility for the information in this publication remains with the author(s). The use oftrade names does not imply endorsement of or discrimination against any product by theAgricultural Research Institute.2

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SUMMARYDuring the last decade, the Agricultural sector worldwide has been transformed by

the numerous applications of the explosive emergence of the current methodologies ofmolecular biology and biotechnology toward the production of improved agriculturalproducts. This article provides a brief account of current molecular biology technolo-gies and describes ways that these can benefit the Cyprus Agricultural Sector.

ΠΕΡΙΛΗΨΗ

Τα τέλη του εικοστού αιώνα βρίσκουν την Γεωργία της Κύπρου σε ένα κρίσιµοσταυροδρόµι. Η Κυπριακή γεωργία αντιµετωπίζει προβλήµατα αναβάθµισης καιαναδιάρθρωσης. Πολλές από τις αιτίες µπορεί να αναζητηθούν στην αλλαγή τωνδεδοµένων στη διεθνή πολιτική και οικονοµική σκηνή, σε συνδυασµό µε την έλλει-ψη και διαρκή υποβάθµιση της ποιότητας των υδάτινων πόρων στο νησί. Σήµερα,διεθνώς η γεωργία είναι ο τοµέας που προσφέρεται για την εφαρµογή της αλµα-τώδους επιστηµονικής επανάστασης στις σύγχρονες µεθοδολογίες µοριακής βιο-λογίας και γενετικής για την παραγωγή βελτιωµένων γεωργικών και κτηνοτροφι-κών προϊόντων. Η εφαρµογή των σύγχρονων αυτών µεθόδων και στην Κύπρο θαέχει ως αποτέλεσµα την αναβάθµιση της κυπριακής γεωργίας, λύνοντας προβλή-µατα που καθηµερινά αντιµετωπίζει ο γεωργοκτηνοτρόφος

ADVANCEMENTS IN MOLECULAR BIOLOGYANDPOTENTIALAPPLICATIONS IN CYPRUS AGRICULTURE

I.M. Ioannides

INTRODUCTIONThe beginning of the new millennium

finds Cyprus agriculture at a critical stage, inimmediate need of reconstruction and reor-ganization. Many of the causes can be tracedto the sweeping changes in the globaleconomies during the last decade that haveprofoundly affected international trading.Other important reasons relate to the continu-ous degradation and depletion of the island’swater resources. Among the less obviouscauses is the low utilization, so far, of the cur-rent methodologies of molecular biology andbiotechnology that would offer substantialbenefits toward the production of improvedagricultural products.During the last decade, the Agricultural

sector worldwide has been transformed by thenumerous applications of the newmethodolo-gies. This article provides a brief account ofcurrent molecular biology technologies anddescribes ways that these can benefit theCyprus Agricultural Sector.

CURRENT METHODOLOGIES OFMOLECULAR BIOLOGYAND

BIOTECHNOLOGYThe structure of DNAThe science of Molecular Biology, as its

name implies, studies the organisms at themolecular level, that is, at the level of DNA(deoxyribonucleic acid). The DNA is thecommon molecule of life among all livingorganisms, as diverse asmicrobes, plants, ani-mals and humans. It is the carrier of all thegenetic information (genes) that are inheritedfrom one generation to the next. Each organ-ism has a distinct set of genes that distin-guishes not only the different species, but alsoindividuals within a species. DNA is a verylong molecule, resembling a long thread. Theseminal discovery (Watson and Crick, 1953)that the DNA molecule has a highly regularshape, and is in fact a double helix, providedclues to the long-sought explanation of how aDNA molecule replicates itself, thus passingthe genetic information to the next genera-tion, and brought the Nobel prize to those whodiscovered it.

The chemical structure of DNA, and thusthe chemical structure of the genes, compris-es a large number of deoxyribonucleotides.Each deoxyribonucleotide consists of a nitro-gen base, a sugar, and a phosphate group.There are only four nitrogen bases, i.e., ade-nine, thymine, cytosine and guanine, but thepossible combinations of them across thelength of a DNA molecule are limitless. Agene may comprise hundreds or thousands ofthese bases, as part of the respectivenucleotides, and what differentiates one genefrom another is the various serial combina-tions of these bases, which is known as thesequence of the DNA.Discovery of PCRIn the mid-eighties, another breakthrough

gave a new impetus in the study of DNAmol-ecules. The discovery of the PolymeraseChain Reaction (PCR) by K.B. Mullis (Saikiet al., 1985) revolutionized molecular biologyand brought a Nobel prize to its inventor in thenineties. The PCR allowed for the first timethe synthesis of practically limitless quanti-ties of a specified DNA sequence withoutresorting to more laborious cloning method-ologies. This was a necessary step to any fur-ther characterization of genes and it paved theway for numerous PCR-based technologieswithout which the current advent in genomicswould not have been possible.Genomics and DNA sequencing technolo-giesThe term “genome” refers to the entire

genetic material of an organism.Genomics is a rapidly emerging area of

research that came into existence during thelast decade of the last century. It embraces thestudy of whole genomes, their molecularorganization, evolution and function of themyriads of constituent genes. The advent ofgenomics is a direct consequence of the dis-covery of methods to sequence nucleic acids(Sanger et al., 1977; Maxam and Gilbert,1977) and of the flexibility provided by PCRmethodologies. These original methods havebeen improved with extensive automationand development of advanced robotic tech-nologies during the last decade. High-through-put (HTP) sequencing is the key con-cept in genomics and it is what permitted thesequencing of whole genomes, like the

human and the Arabidopsis (Maheshwari etal., 2001).BioinformaticsThe sequencing of entire genomes has

been driving the development of many othernovel technologies, including the new field ofbioinformatics (Hagen, 2000). Eukarioticgenomes comprise thousands of genes andmillions of base pairs, so there is a massiveoutput of data that must be stored, retrievedand analyzed. The handling of this enormousamount of information requires new compu-tational tools and new mathematicalapproaches that are under continuous devel-opment.There are certain characteristics of the

DNA sequence that are crucial to the devel-opment of many applications in agriculture.Specifically, the DNA sequence of any indi-vidual is independent of the environmentalconditions and the farming systems. In addi-tion, all tissues from an individual plant oranimal share the same DNA sequence, at anylevel of development. In practice, this meansthat plants or animals can be tested at theDNA level at the seedling stage or at a veryyoung age, circumventing the need to waituntil a particular trait has been fullyexpressed. In the following section, referenceis made to a number of challenges and prob-lems in Cyprus Agriculture that can be suc-cessfully faced with the application of appro-priate technologies of molecular biology,resulting in large economic benefits.APPLICATIONS OF THE TECHNOLO-GIES OFMOLECULAR BIOLOGY TOVARIOUS SECTORS OF CYPRUS

AGRICULTUREGenetic identificationThere is a number of technologies under

the general designation of genetic identifica-tion techniques or identification at the DNAlevel. These technologies, through the charac-terization of specific DNA sequences, enablethe exact identification, as the name implies,of the individual that contributed the DNAspecimen among a group of very similar indi-viduals.At the level of applied agriculture, genetic

identification is of primary importance in cas-es where the phenotypic or morphological

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differentiation is difficult or even impossible.Example of a case that morphological differ-entiation may be impossible is when the traitunder study is controlled by genes with dom-inant action or by genes that their actionbecomes apparent at later stages of develop-ment.Aproblem that has already affected ani-mal production in Cyprus, and in particularsome of ARI’s experimental sheep popula-tions, is the disease scrapie. The timely iden-tification of individual sheep that are carriersof the PrP genotype for scrapie is of primaryimportance.On other occasions, genetic identification

can be used to successfully distinguish amongclones of various species that are importantfor Cyprus agriculture. These species includethe vine (Vitis vinifera), the almond tree(Amygdalus communis), the olive tree (Oleaeuropea), and various citrus species. Clonesare either morphologically indistinguishableor the morphological variation may representnon-genetic (environmental) causes. Geneticidentification can provide evidence for theuniqueness of certain Cyprus clones, espe-cially when the products have desirable qual-ity characteristics that consumers prefer andhelp ensure cultivation, marketing and pro-tection.Diagnostic technologies for the productionof healthy reproductive materialThere is a number of technologies based on

the Polymerase Chain Reaction (PCR) thatcan be used successfully for the timely diag-nosis of plant pathogens in the greenhouse orin the fields. Of particular importance is thecontribution of these methods to the produc-tion of pathogen-free reproductive stocks ofvines, citrus, and other relevant species.Identification of resistant genes in plantsand animalsThe aforementioned technologies can be

applied not only at the diagnostic level, butfor an in-depth investigation of various hostsand parasites, with the aim to identify and iso-late specific genes that are responsible forresistance to various diseases (Michelmore,2000). These efforts are greatly aided bymethodologies that produce genetic maps inplants and animals of agricultural interest. Inthe beginning of the nineties, the first plantgenes for resistance to bacterial pathogens

were isolated in laboratories in the USA.Since then, there is an influx of relevant stud-ies. This is an area that can greatly benefitCyprus Agriculture. The existence of resist-ance genes in a plant cultivar or in an animalbreed, is a prerequisite for the reduction in theuse of pesticides and the promotion of a moresustainable agriculture.Abiotic stress tolerance, including droughttoleranceThe development of cultivars with

enhanced tolerance to various abiotic stressesis of great importance for today’s agriculture.Many genes contributing to this type of toler-ance have been recently identified, using themodel plant Arabidopsis thaliana. Examplesinclude genes involved in drought stress (Ishi-tani et al., 1997), genes involved in salt toler-ance (Zhu, 2001), and genes involved in met-al toxicity (Ma et al., 2001).In addition to the above, there are other

important emerging molecular technologiesthat will prove indispensable in the nearfuture, but are presently not yet developed tothe stage of user-friendliness that permits amore widespread use in applied agriculture.Microarray technology (DNA chips) has aprominent role in this new generation of tech-nologies (Lemieux et al., 1998; Ruan et al.,1998). Microarrays allow studies on genome-wide or global patterns of gene expression,permitting visualization of the activity of hun-dreds and thousands of genes simultaneously.The basic principle involves the immobiliza-tion of multiple genic sequences (probes) ona solid glass or nylon support and the subse-quent hybridization with the chosen RNA ofcDNA, which has been tagged with a fluores-cent dye for visualization purposes. In thefuture, DNA chips accommodating entireplant or animal genomes will have a majorimpact in Agricultural Sciences.In conclusion, given the strong interna-

tional attention on the genomic technologies,it is expected that their development will alsobe viewed as of primary importance forCyprus Agricultural Research.

REFERENCESMa, J.F., P. Ryan, and E. Delhaize. 2001. Alu-

minum tolerance in plants and the com-plexing role of organic acids. Trends inPlant Science 6(6):273-278.

Hagen, J.B. 2000. The origins of bioinformatics.Nature Reviews Genetics 1:231-236.

Lemieux, B., A. Aharoni, and M. Schena. 1998.Overview of DNA chip technology.Molecular Breeding 4:277-289.

Maheshwari, S.C., N. Maheshwari, and S.D.Sopory. 2001. Genomics, DNAchips and arevolution in plant biology. Current Sci-ence 80(2):252-261.

Michelmore, R. 2000. Genomic approaches toplant disease resistance. Current Opinionin Plant Biology 3:125-131.

Ishitani, M., L. Xiong, B. Stevenson, and J. Zhu.1997. Genetic analysis of osmotic and coldstress transduction in Arabidopsis. PlantCell 9:1935-49.

Ruan, Y., J. Gilmore, T. Conner. 1998. TowardsArabidopsis genome analysis: monitoringexpression profiles of 1400 genes usingcDNAmicroarrays. Plant Journal 15:821-833.

Saiki, R.K., S.J. Scharf, F. Faloona, K.B. Mullis,G.T. Horn, H.A. Erlich, and N. Arnheim.1985. Enzymatic amplification of beta-globin sequences and restriction site analy-sis for diagnosis of sickle cell anemia. Sci-ence 230:1350-1354.

Watson, J.D. and F.H.C. Crick. 1953. Molecularstructure of nucleic acids. Nature 171:737-738.

Zhu, J.K. 2001. Plant salt tolerance. Trends inPlant Science 6(2):1360-1385.

Sanger F., S. Nicklen, and A.R. Coulson. 1977.DNA sequencing with chain terminatinginhibitors. PNAS 74:5463-5467.

Maxam, A. M., and W. Gilbert. 1977. A newmethod of sequencing DNA. PNAS74:560-564.

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