Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.

Somaclonal variation in plants: causes and detection methods

PREFACE REPETITIVE DNA: AN IMPORTANT SOURCE OF VARIATION IN EUKARYOTIC GENOMES Categories of DNA Sequence Mutations Tandem-Repetitive DNA: The Biology of Mini- and Microsatellites Minisatellites Microsatellites Transposable Elements Class I Transposons Class II Transposons Unclassified Transposons Transposons and Genome Evolution Transposons as Molecular Markers DETECTING DNA VARIATION BY MOLECULAR MARKERS Properties of Molecular Markers Traditional Marker Systems Protein Markers and Allozymes DNA Sequencing Restriction Fragment Length Polymorphism (RFLP) Analysis The PCR Generation: Molecular Markers Based on In Vitro DNA Amplification Principle of the PCR Cleaved Amplified Polymorphic Sequences PCR with Arbitrary Primers: RAPD and Its Variants Microsatellites Inter-Repeat PCR DNA Profiling of Genic Regions: Resistance Gene Analog Polymorphism, Sequence-Related Amplified Polymorphism, and Target Region Amplification Polymorphism Hybridization of Microsatellites to RAPD and MP-PCR Products AFLP Analysis and Its Variants Single-Strand Conformation Polymorphism Analysis and Related Techniques Miscellaneous Techniques LABORATORY EQUIPMENT METHODOLOGY Safety Precautions Isolation, Purification, and Quantitation of Plant DNA Collection and Preservation of Plant Tissue in the Field Plant DNA Extraction: General Considerations CTAB Protocol I CTAB Protocol II SDS-Potassium Acetate Protocol DNA Preparation via Nuclei Quantitation of DNA Basic Molecular Techniques Restriction of DNA Polymerase Chain Reaction DNA Sequencing Agarose Gel Electrophoresis PAA Gel Electrophoresis Detection of DNA in Gels Gel Drying Southern Blotting Generation of Radiolabeled Probes, Primers, and PCR Products Blot Hybridization Signal Detection PCR with Arbitrary Primers Standard RAPD Protocol Influence of Reaction Conditions and Components Modifications Microsatellite-Primed PCR Standard Protocol of Microsatellite-Primed PCR Influence of Reaction Conditions and Components Modifications PCR and Hybridization: Combinatory Techniques Assessing the Genomic Copy Number of PCR Amplicons Testing the Homology of Comigrating Bands Random Amplified Polymorphic Microsatellites Amplified Fragment Length Polymorphism Standard AFLP Protocol Using Radioisotopes AFLP Protocol Using Fluorescence-Labeled Primers Selective Amplification of Microsatellite Polymorphic Loci and Microsatellite AFLP Protocols Technical Aspects and Modifications Robustness and Reproducibility Generation and Analysis of Microsatellite Markers Microsatellite Analysis Using Radioisotopes Microsatellite Analysis Using Fluorochromes Technical Aspects and Modifications Generating Microsatellite Markers Without Cloning Microsatellite Cloning CAPS Analysis of cpDNA and mtDNA Standard CAPS Protocol Choice of CAPS Primers EVALUATION OF MOLECULAR MARKER DATA Robustness and Reproducibility Reliability Band Homology Band Linkage and Neutrality Fragment Sizing and Matching General Precautions Equipment Multilocus vs. Single-Locus Approaches Multilocus Markers Single-Locus Markers and Polyploids Band Sharing and Genetic Distances Coefficients of Similarity Dissimilarity Coefficients and Genetic Distances Identity and Uniqueness Clonal Structure Ordination, Clustering, and Dendrograms Ordination Techniques Construction of Dendrograms Population Genetic Analysis Measures of Variation Genetic Differentiation between Populations Genetic Distances between Populations Inbreeding Coefficient and Mating Systems Estimation of Relatedness and Paternity Testing Migration and Hybridization Gene Flow, Isolation-by-Distance, and Spatial Structure Phylogeography and Nested Clade Analysis Statistical Testing of Hypotheses: Analytical and Computational Methods APPLICATIONS OF DNA FINGERPRINTING IN PLANT SCIENCES A Brief History of DNA Fingerprinting Minisatellite and Oligonucleotide DNA Probes Detect Genetic Variation PCR-Based Methods Enter the Stage Microsatellite DNA Analyses Yield Codominant Markers Universal Organellar DNA Primers Produce Uniparental Markers Genotype Identification Individual-Specific DNA Fingerprints Cultivar Identification In Vitro-Propagated Plant Material and Somaclonal Variation Sports and Other Mutants Genetic Diversity Variation and Relatedness among Cultivars Analysis of Population Genetic Diversity and Its Distribution Hybridization and Introgression Plant Conservation Germplasm Characterization and Preservation Plant Taxonomy and Systematics Taxonomic Relationships Revealed by Multilocus DNA Methods Microsatellite Markers in Taxonomic Studies Taxonomic Consequences from DNA Profiling Data Phylogeography Phylogeography Based on cpDNA Phylogeography Based on Nuclear Genes LINKAGE ANALYSIS AND GENETIC MAPS Generating High-Density Genetic Maps Selection of Parent Plants Mapping Population Linkage Analysis The Genetic Map Cytogenetic Maps Genetic vs. Physical Maps Synteny: The Comparative Analysis of Genomes Marker-Assisted Selection Molecular Markers and Positional Cloning WHICH MARKER FOR WHAT PURPOSE: A COMPARISON Morphological Characters and Allozymes vs. DNA Markers Different Kinds of DNA Markers Discriminatory Power Genetic Distances Within- and Among-Population Variation Gene Tagging and Genetic Linkage Mapping Costs Conclusions FUTURE PROSPECTS: SNIPS AND CHIPS FOR DNA AND RNA PROFILING Single-Nucleotide Polymorphisms What Is a SNiP SNP Discovery DNA Microarrays Expression Profiling and Expression Markers APPENDIX 1: PLANT DNA ISOLATION PROTOCOLS APPENDIX 2: SUPPLIERS AND SELLERS OF REAGENTS AND EQUIPMENT APPENDIX 3: COMPUTER PROGRAMS DEALING WITH THE EVALUATION OF DNA SEQUENCE VARIATION AND MOLECULAR MARKER DATA APPENDIX 4: WEB PAGES OF INTEREST REFERENCES INDEX

DNA fingerprinting in plants: principles, methods, and applications

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis. Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail. Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.

Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh

DNA Fingerprinting in Plants: Principles, Methods, and Applications, Second Edition

As part of the development of a molecular toolkit for the study of diversity within large plant germplasm collections, RAPD technology has been applied to accessions of rice (Oryza sativa) obtained from the major world collection held at IRRI (the International Rice Research Institute) which supplies germplasm to breeders. Methods for the speedy extraction of DNA representative of a rice accession, its amplification by PCR to reveal reproducible products, and the analysis of the banding data using numerical techniques have been established. The biological meaningfulness of RAPD data has also been demonstrated by reference to previous work on classification and crossability.

/pdf/use-of-rapd-for-the-study-of-diversity-within-plant-36q5webb1e.pdf

Use of RAPD for the study of diversity within plant germplasm collections

School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom Molecular markers based upon DNA sequence variation have proved extremely effective tools for distinguishing between closely related genotypes. With the advent of PCR technology, methods have been developed that use amplified DNA sequences as molecular markers. These methods require very little template DNA, which can usually be obtained using a simple minipreparation protocol; the production of marker bands is very fast and far less labor intensive than when using restriction fragment length polymorphism (RFLP) technology. PCR-based techniques have already been used in the analysis of relationships between plants. (1-1~ In this paper, we describe methods for producing plant molecular markers using randomly amplified polymorphic DNA (RAPD) technology. In particular, we describe the effect of varying a range of factors during the procedure and define the protocol that we have found most useful. DNA was extracted from 0.1-0.5 grams of fresh leaf material using a rapid minipreparation method based upon the protocols of Sgai-Maroof et al. (11) and Murray and Thompson.02) Yields of DNA were measured using the Hoechst dye assay method with a TKO 100 Minifluorometer according to the manufacturer's instructions. For PCR, all reaction mixtures had a total volume of 50 ~l. Unless otherwise indicated, the mixture contained 1 unit of Taq polymerase (Cetus), 0.2 nmole (= 4 ~M) of a single 10-mer oligonucleotide, 200 ~M with respect to dATP, dCTP, dTTP, and dGTP, 2.5 mM magnesium chloride, the appropriate dilution of the reaction buffer prepared by the company supplying the polymerase, and not more than 0.1 ~g of plant DNA (see below). All reaction mixtures were overlayed with mineral oil before being placed in a Hybaid Thermal Reactor HBTR1. Unless otherwise indicated, all programs had an initial cycle with 94~ for 7 min, the annealing temperature was 36~ (for 1 rain), and the extension time was 4 min (at 72~ The subsequent 35 cycles were the same except that the 94~ denaturation step was applied for only

The random amplification of polymorphic DNA for fingerprinting plants.

RAPD data have been analysed using multivatiate techniques in order to discriminate between sugar beet varieties. While it was not possible to do so using small numbers of plants per variety, with a larger number discrimination was possible.

The Use of RAPD for the Identification of Sugar Beet Varieties

http://www.assbt-jsbr.org/JSBR/Vol30/JSBRVol30No4P291to298TheUseofRAPDfortheidentificationofSugarBeetVarieties.pdf

The use of RAPD for the identification of sugar beet varieties

plants.random amplification of polymorphic DNA for fingerprinting

Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.

The detection of somaclonal variants of beet using RAPD.

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