MX2008010165A - Non-destructive procedure for the isolation of dna from plants - Google Patents

Abstract

The present invention relates to a method for obtaining DNA from a plant comprising collecting the root border cells from a growing root;and extracting DNA from the root border cells. The root border cells are contained in the root exudate of the growing root, which is growing in a medium, such as water, tissue culture medium or soil. Suitably, the root is part of a germinating seed, or the root of a seedling or the adventitious root of a tissue culture plant or plant part.

Description

NON-DESTRUCTIVE PROCEDURE FOR THE INSULATION OF DNA FROM PLANTS FIELD OF THE INVENTION The present invention relates to a non-destructive method for the isolation of plant DNA and the application of the procedures in the genetic analysis of plants or plant populations.

BACKGROUND OF THE INVENTION Plant breeding depends on the efficient use of genetic variation, which resides in the germinal plasma of a particular crop species which determines the phenotype of a plant within a specific environment. Although this has traditionally been done by selecting a combination of desirable traits observed at the phenotypic level, this can be carried out increasingly by selection based on molecular markers which are closely related in a genetic manner to the allelic form of a gene. which contributes to the expression of a specific feature. The selection of traits based on molecular markers is independent of the stage of development of a plant and independent of the environment, which significantly increases the selection process. The number of Ref. : 194715 traits that include complex traits controlled by multiple genes which can be selected by using molecular markers has increased strongly and it is considered that this development will continue at ever larger steps. Another trend in the field of plant breeding arises from inverse genetics. Reverse genetics is related to an approach in which genes are isolated and determined to work by modifying their primary structure or expression. With the current increase in knowledge about function in genes, especially in model systems such as Arabidopsis thaliana, inverse genetics approaches in crop systems are currently gaining effectiveness. In order to determine allelic variability of candidate genes, a large number of DNA diagnostic tools are available which are known to those skilled in the art. Large populations of plants containing natural or induced allelic variations for DNA polymorphisms at the locus of interest need to be screened to acquire a saturated collection of allelic variants. The allelic forms of the genes found in this way can be determined for their contribution to the plant phenotype by association study. The cost of breeding screening or mutant populations is determined to a large extent by the work that it is required to grow and take samples from individual plants of the population under investigation and to prepare DNA from these samples. In the event that a population is available as seed samples representing the genetic variation that resides within individual plants of the population under investigation, a significant amount of labor is invested in harvesting plant-by-plant seeds as related individuals in families. In addition, this exercise requires reiteration of each additional population that is produced and determined for allelic variants at specific genetic loci.

SUMMARY OF THE INVENTION The objective of the present invention is to provide an efficient method for the isolation of plant DNA. A further objective of the invention is to provide an efficient method of DNA isolation that allows the screening of population of plants to determine the presence of allelic variants at specific genetic loci which renders superfluous the requirement of manual digestion of tissue samples or the requirement to harvest seeds from each individual plant of the population under investigation. According to the invention, it has been found that such method may be based on the use of exudate released from the roots, preferably from the roots of very young plants such as seedlings or roots arising from germinating seeds or adventitious roots of plants growing in tissue culture to isolate DNA from the same. More particularly, the method uses the so-called root boundary cells, which are separated from the tip of the root, as the primary source of DNA. Root boundary cells are living cells that surround the apices of the roots of most plant species. A plant naturally detaches the root boundary cells of the roots when it grows in the soil as well as when it grows in a liquid or solid medium. In this way, the method of the present invention is considered to be non-destructive. The cells are separated in advance from the plant in a natural manner and can be harvested by light agitation and separation of the medium, usually fluid surrounding the roots and comprising the root boundary cells. The growing roots maintain producing root-bound cells that can then be harvested again at a later stage. In this way, the invention relates to a method for isolating DNA from a plant, comprising: a) collecting the root boundary cells of a growing root; Y b) extract DNA from the root boundary cells. In principle, DNA can be obtained from all root boundary cells. However, it is very practical to collect the root boundary cells of a root which is part of a germinating seed. The seeds can be embedded in a liquid medium such as water after which they will germinate. Plants in this way can be analyzed at a very early stage of plant development, that is, during germination of the seed. There is no need to wait until the leaves have grown on the plant. However, the method can also be carried out with root boundary cells that come from the root of a seedling. It has also been found that adventitious roots that grow on plant material in tissue culture produce root boundary cells. In accordance with the invention, these root boundary cells can also be used to isolate DNA from them. Various methods for extracting DNA are available and are known to those skilled in the art such as C (Doyle JJ and Doyle JL (1990) Focus 12, 13-15) KingFisher96MR (Thermo Labsystems), etc. The DNA obtained in this way can be of nuclear and cytoplasmic origin and can be analyzed using different nucleic acid analysis technologies. Said Analytical technologies are well known to a person skilled in the art and include but are not limited to polymerase chain reaction (PCR), Sanger sequencing, minisequencing, pyrosequencing, GS20 sequencing, amplified fragment length polymorphism (AFLP), polymorphism Restriction fragment length (RFLP), polymorphic DNA random amplification (RAPD), Invader, Oligonucleotide ligation analysis (OLA) and single characteristic polymorphism (SFP). The invention is further related to the use of a novel non-destructive DNA isolation method to analyze large populations of plants with a very high efficiency for genetic variation at particular loci. This genetic variation can be induced naturally or artificially. The method of isolating plant DNA provided in this manner is an efficient tool for obtaining DNA that can be used to detect genetic variants of specific genes in populations of plants generated through chemical or physical mutagenesis. Alternatively, genetic variants may reside in natural populations. The use of the method of the invention eliminates the need to establish M2 families derived from mutagenized MI plants. The accumulated M2 populations can be use instead of which it is allowed to analyze the M2 populations in a more efficient and flexible way to determine the presence of different allelic forms of specific genes. The method for isolating DNA from plants of the invention is also suitable for use in the genetic typing of plant populations which are useful for quality control purposes of commercial seed lots in order to determine genetic purity and identity. In addition, the DNA isolation procedure can be used for the identification of plants which reside in a genetically distinct population of plants based on the detection of an allelic form of a polymorphic molecular mr which is linked to an allelic form of a gene which determines the expression of a certain phenotypic trait. Large-scale sequencing efforts have produced complete genomic sequences of model plant species such as Arabidopsis thaliana and crop species such as rice. In addition, large quantities of cDNA or EST fragments originating from different tissue samples derived from a large number of crop species such as tomato, lettuce, cucumber, Brassica, melon, corn, etc., have been sequenced. The current challenge is to elucidate the function of individual genes, the regulation of its expression and its genetic interaction. In order to meet this challenge, it will be important to have a large number of allelic variants of each individual gene. This will provide clues as to which biochemical role a gene product plays at a cellular, organism or higher order level and how gene products can interact. In order to take advantage of information regarding the function of the gene in the breeding of plants, the availability of a very efficient and profitable reverse genetic technology is desirable. Reverse genetics refers to an approach in which we start with gene sequence information for which allelic or expression variants are produced which are subsequently analyzed functionally. This terminology is opposed to diverse genetics in which a phenotypic variant is used as the initial material to identify the underlying allelic form of a gene. In order to apply reverse genetics in plant breeding, knowledge of gene function is a requirement. Currently, Arabidopsis thaliana is the plant system studied most widely in relation to the function of genes and the results that come from research with Arabidopsis provide a rich source of information in this regard. Based on homology at the sequence level of One amino acid can predict the function of a homologous protein from a crop species, although direct experimental tests are those that are ultimately required to demonstrate the function of the gene. The genes of the crop species identified based on the gene homology of model species can therefore only be considered candidate genes for specific functions. Genes with similar or identical functions across species can show, although not necessarily do so, high levels of homology and gene functions that reside in a particular model system and can only partially overlap with those that reside in a species of given harvest. Allelic variability, which can be exploited by inverse genetics, occurs either naturally in adapted populations or can be obtained by random mutagenesis using chemical or physical mutagenic agents, for example, ethyl methanesulfonate (ems) or x-rays, respectively. By treating the organs, cells, pollen or seeds of the plant with said mutagenic agents, modifications in random positions in the genomic DNA will be induced which can generate a change in the function of the gene. Given the rapidly increasing knowledge regarding the function of the gene, the wide availability of genomic and cDNA sequences of many plant species and the Availability of populations that present natural and induced genetic variation, reverse genetics technology is of increasing significance as a research document to establish the functions of genes in a model or crop species. In addition, inverse genetics can also be considered as a powerful technology for crop improvement in which allelic variants of genes known to be functionally involved in specific traits can be effectively identified. In order to perform reverse genetics in a crop species, in addition to an objective gene, plant populations are obviously required which contain genetic variants of the genetic loci of the given crop species. Within such populations, genetic variation may have occurred spontaneously or may be induced by mutagenesis as ems. In order to obtain a population with induced mutations, one can incubate, for example, seeds in a solution containing different concentrations of a mutagen like ems. Ems rents mainly G residues from a DNA strand which, during DNA replication, causes pairing with T instead of C. Therefore, the base pairs GC change to base pairs AT at a frequency which is determined by the effective dose of ems and the activity of the repair system of bad pairing of the plant.

The effective dose of ems depends on the concentration used, the size of the seed and other physical properties as well as the time of incubation of the seeds in the ems solution. The seeds which have been treated with ems are typically called MI. As a consequence of the treatment, the tissues of the MI seeds contain random point mutations in the genomes of their cells and those present in the subpopulation of cells which will form the germline tissue will be transferred to the next generation which is called M2. Mutations or combinations thereof which are insufficient haplo will therefore cause sterility or which will induce embryo mortality and will not be transferred to the M2 generation. A procedure similar to that described above for the use of ems applies to other mutagenic agents as well. In order to determine the mutant M2 populations for the presence of desirable allelic variables of the specific genetic loci using reverse genetics, one can carry out different approaches which can be distinguished based on the selected way to harvest and store the population of M2 seeds. On the one hand, one can harvest and store the M2 seeds as a single bulk sample while, on the other hand, one You can harvest and store the M2 seeds as families which means that the M2 seeds are harvested plant by plant and stored separately. When harvesting bulk M2 seeds requires much less work when compared to a situation in which the M2 seeds are harvested separately as families. On the other hand, the harvesting of M2 seeds as families allows the preparation of DNA extracts from a subset of the M2 seeds of each family of the population, which will be used in a diagnostic manner to analyze the population. Once the mutation is identified, only the seed of the family that corresponds to the positively diagnosed sample needs to be grown in a greenhouse or field in order to obtain the mutant. In case an accumulated M2 population is available, plants 2 need to be grown and sampled for DNA extraction and analysis separately, which requires a relatively large resource input for each screening performed. Therefore, both approaches have their specific disadvantages and there is a clear need in the art to provide solutions that overcome these disadvantages of both approaches. When bulk M2 seeds have been harvested, a non-destructive sample is required to identify the individual plant containing the desired allelic variant of a specific gene. Usually, this is done by growing young plants from the M2 population and by sampling these plants individually by taking leaf samples and preparing DNA from them and which are used for analysis. These leaf samples can be accumulated prior to DNA extraction to a degree determined by the dynamic range of the mutation detection platform used. The appropriate marking allows the tracking of the subpopulations that contain the desired mutation and finally the corresponding individual plant. The number of M2 plants which need to be screened depends on the frequency of mutation in the MI plants and the number of independent cells that contribute to the germ lines in the plant species under investigation. In a typical experiment one should care for approximately 10,000 M2 plants when starting from a population of 5000 MI plants to capture the genetic variation induced in the case of 2 independent germ cells / plants. In case the M2 seeds are harvested plant by plant, the induced mutations found in the cells contribute to one or more of the germ lines of the MI plants on which these seeds are harvested and segregate in the M2 family. When preparing a DNA sample of several individuals from an M2 family, one can diagnose by the presence of mutations in a target gene without the need to demonstrate the population each time. In other words, a single DNA sample can be used diagnostically for the M2 family which it represents. Individual plants containing the desired allelic variants of a specific gene can be obtained by generating individuals from only those M2 families which are positively diagnosed for the desired mutations. Although this approach is relatively effective once the M2 families have been established, the establishment of such a family-based system requires work since it also needs individual harvested and processed seeds that have grown in MI plants as well as the establishment of a DNA library as a representative reflection of the genetic variation of the population. It is estimated that the processing of 5000 M2 families requires the entry of at least 250 man-hours. Importantly, in the event that the desired mutation is not present in the population, one should repeat the entire exercise. In order to detect mutations in the isolated DNA of the (accumulated) plant samples, the person skilled in the art has numerous established technologies. The slope (directed local lesions induced in genomes) is based on the specific separation of Cell from the labeled heteroduplex DNA fragments generated by PCR in the position of the bad pairing. The digested samples are analyzed by denaturing gel electrophoresis (for example in a Liquor system) and the presence of the digested PCR fragments indicate the presence of DNA polymorphisms in the accumulated original DNA (Colbert, T. et al., 2001). ) Plant Physiology 126, 480-484). Denaturing high resolution liquid chromatography (dHPLC) can also be applied to accumulated DNA samples. Similar to the tilt procedure, the presence of a mutation generates the formation of heteroduplex molecules which run faster through a dHPLC column compared to homoduplex molecules, which allows the detection of accumulated sample mutations (McCallum, C. et al. al. (2000) Nature Biotechnology 18, 455-457). When fast neutrons are used as mutagenic agents, small deletions are generated in the genomes at random positions. This allows to amplify the mutated loci which as a consequence of the suppression have had a reduced size, by PCR. Since this PCR reaction is specific for the mutated loci, a very high level of sample accumulation becomes feasible. On the other hand, this method only applies to those mutations which allow specific PCR reactions to be designed similar to loci containing deletions (Song, X. et al (2001) The Plant Journal 27, 235-242). It is obvious to a person skilled in the art that basically any available nucleic acid analysis technology can be applied to detect polymorphisms between DNA samples. It can even be applied in direct sequencing of individual samples. In an industrial environment, the platform of choice will be largely determined by its robustness and costs per data point. With the availability of a variety of induced mutation detection technologies and advances in automation and miniaturization, the cost per data point will be reduced to relatively low levels when compared to the costs involved in preparing representative populations and DNA templates. Therefore, improvements in reverse genetics of plants are carried out better in the area of preparation of mutant populations and their representative DNA samples rather than in the detection of induced polymorphisms. Said improvements are provided by the present invention. In any case, that is, the application of reverse genetics on the basis of M2 bulk populations or M2 families, substantial work will be required either during screening or the upper front, respectively. This severely limits the cost-effectiveness of the applicability of inverse genetics approaches in crop species. The method of DNA isolation of the invention can also be used for the selection of plants during the breeding of plants. Progress in plant breeding is obtained through crosses and plant selection of a descendant population. Traditionally, selection occurs at the level of the plant phenotype that manifests under specific growth conditions. For example, plants are selected based on their fruit and color of leaves or forms, their resistance to pathogens, their productivity or other traits or combinations thereof. With the advent of molecular marker technology, the selection of specific traits can be carried out at the DNA level. Technologies have been developed which allow the identification and detection of marker alleles which are genetically strongly related to an allelic form of a gene which is the cause of the expression of a certain phenotype. In the ideal situation, the marker allele and the allelic form of the gene responsible for a certain trait are identical. In this case, the marker allele and the trait allele can not be decoupled by genetic recombination. The use of marker alleles for indirect selection of traits has numerous clear advantages which improve the efficiency of the selection procedure. The selection based on markers independent of the stage of development of a plant and the environment. This allows you to select, for example, fruit characteristics in the seedling stage or select cold tolerance at room temperature. Currently, indirect selection is widely applied using marker alleles in the breeding of modern plants. The most advanced is the application of markers to detect qualitative traits, that is, traits for which the phenotypic variation within the germ plasm is determined by the allelic forms of a single gene. For example, resistances to specific strains of a pathogen are often determined by the presence of dominant R genes which code for receptors which detect the presence or activity of a virulence factor of the pathogen. Although the expression of the resistance phenotype involves many loci, the genetic variation which resides in the germ plasm and which explains the phenotypic value is usually determined by a single locus for the R gene. However, many features are quantitative or continuous which means that many genes can contribute to the phenotypic value of the trait which is often affected by environmental conditions. Such features are, for example, the height of the plant, the flowering time or the yield potential. In addition, the individual genes underlying the complex feature can interact epistatically, which complicates the analysis of their inheritance. With the availability of high density genetic maps and powerful statistical tools, the detection of the loci involved in the expression of these quantitative traits (QTL) is currently feasible. Therefore, it can be anticipated that the number of (complex) traits that can be detected using molecular markers will increase significantly in the near future. In order to apply indirect selection, the seeds of the population from which individual plants need to be selected in a greenhouse must now be germinated to a stage at which a tissue sample can be taken for DNA extraction. Depending on the crop species this can be carried out in the seedling stage or in the young plant stage. After carrying out the DNA analysis, the selection of the plant can be carried out. Obviously, this procedure requires time, space and work which consumes a relatively large part of the total resources needed to carry out indirect selection based on molecular marker. The technology which increases the efficiency of the procedure to obtain an extract of DNA of sufficient quality and quantity to perform DNA analysis can significantly reduce the costs involved in indirect selection based on molecular marker. The present invention provides a novel method for isolating DNA from plants in a highly efficient and non-destructive manner. This method can be applied, for example, to significantly improve the overall efficiency of reverse genetics as well as indirect selection technology. The DNA isolation method can be effectively applied at a very early stage in the development of the plant, that is, in the radicle germination stage of the imbibida seed and, more importantly, does not require sampling of any tissue by perforation or cut and similar. When the seeds are placed under the right conditions of humidity, light and temperature, they will drink water and germination will begin. Currently, the first visible sign of germination is the emergence of the radicle or the tip of the root. As it grows, the area behind the root tip discards viable cells called root boundary cells to the environment (Hawes, M. et al (1998) Annu, Rev. Phytopathol, 36, 311-327).

Although the function of the root boundary cells is not completely clear, the current hypothesis is that these cells protect the plant against toxic elements such as aluminum or pathogenic microorganisms by forming a diffuse boundary between the body of the plant in the soil in which it grows. In addition, root boundary cells can have a function by attracting beneficial microorganisms or by establishing microrhiza. The root boundary cells are therefore very important in controlling the microenvironment of the root system. When the seeds germinate in vitro, for example in water, the root boundary cells are produced and remain equally attached loosely to the root surface. By light agitation, the root boundary cells are released from the root surface, dispersed in the liquid and can be harvested. In accordance with the invention, it has surprisingly been found that these root seed cells can serve as a source of DNA for diagnostic purposes. Most, if not all crop species are known to produce root boundary cells which implies the general applicability of the present invention. When the seeds germinate in water to a stage in which the root has emerged in approximately 1-2 cm, the water contains enough cellular material to carry out a standard DNA extraction procedure by providing enough DNA for further analysis by PCR or other DNA analysis technologies. In accordance with the invention it has also been found that plant roots in vitro produce root boundary cells. Said root boundary cells that do not originate from the root plant of a germinating seed or seedling but from adventitious roots in the tissue culture material and therefore can also be used to isolate DNA from them . When a suspension of root boundary cells is treated for one hour with an effective concentration of deoxyribonuclease, a signal can still be obtained after inactivation of deoxyribonuclease and subsequent DNA extraction (FIG. 15). This corroborates the notion that the DNA which is obtained from the root exudate is in fact derived from deoxyribonuclease resistant structure such as root boundary cells. The amount of DNA which can be obtained is sufficient to carry out many analyzes, especially when using sensitive fluorescence-based detection technologies such as Invader ™ or Invader Plus ™ (Third Wave Technologies). The seeds of most, if not all, crop species which are of physical quality enough germinate after a few days in water. It has been observed that during this very early stage, root boundary cells are formed. The extraction and DNA analysis can therefore surprisingly be carried out within a few days of the beginning of the dried seeds. As the germinated seeds remain viable for at least two weeks in vitro, the time and place normally necessary to grow the plants in a greenhouse as well as the work that is required to harvest tissue samples can be suppressed to a large extent. The same applies to tissue culture roots. The root boundary cells can be isolated from the medium and the plant in vitro can continue to grow. In this way the analysis can be carried out at a very early stage of plant development. It has further been shown according to the invention that the seeds of the accumulated M2 populations can germinate in small sets of 2-5 seeds which provides accumulated DNA samples which can be analyzed on detection platforms which have a dynamic range of detection of a mutant allele in a pool containing 3-9 times the number of wild-type alleles. The accumulated can also be done at the level of root boundary cells by harvesting DNA extracts or the PCR product. The actual accumulation strategy that is taken will depend on the technical conditions of the different procedures that include germination behavior of the seeds in liquid and production of root boundary cells which may differ from one crop species to another as well as the interval dynamic of the detection platform in which an optimum can be found in terms of cost per plant. With respect to efficiency in terms of introduction of inverse genetics resources or indirect selection approaches using accumulated M2 seeds or M2 families or breeding populations, the method according to the present invention has numerous important implications. As the non-destructive DNA extraction procedure using root boundary cells can be carried out very early after germination of the seeds in vitro and given the fact that the seedlings remain viable for at least several weeks in In vitro, the reverse genetics approach in which accumulated M2 populations are used or the indirect selection approach, no more time and greenhouse space is required as well as preparation that requires intense work of plant samples such as leaf discs. This means that the alternative to work with M2 bulk populations, that is, working with M2 families which it requires significant front-loading of resources, it is no longer required. In addition, the indirect selection procedure during aging does not require growing the plant material in a greenhouse which can not be selected. Therefore, with the present invention, any population at hand that needs to be determined in terms of allelic variability at specific loci can be screened with unprecedented efficiency and flexibility when compared to inverse genetics or indirectly known selection procedures currently. in the technique. The DNA isolation method for plants of this invention is applicable in its broadest sense. In the present application, reference is made to several situations in which an effective non-destructive method of DNA isolation is advantageous. These examples are not intended to be limiting. It will be apparent to a person skilled in the art that the method can be used for any DNA isolation and is equally applicable in other situations not mentioned here.

BRIEF DESCRIPTION OF THE FIGURES The present invention will be further illustrated in the non-limiting examples that follow. These examples are referred to in the following figures.

Figures 1A-1B are the root tip of root boundary cells of a cucumber shear in a liquid medium. The left panel shows the image under white light, the panel on the right shows the nuclei of root boundary cells under fluorescent light after staining with DAPI. Figure 2 shows agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis followed by digestion with MspI of the kom20 marker locus of different cucumber DNA samples generated by the DNA extraction method according to this invention . Lane 1: size marker, lanes 2 to 11: 10 single cucumber root boundary cell preparations, lane 12: negative control: lettuce root boundary cell preparation, lane 13: negative control: water lane 14: Positive control: cucumber leaf disc. Figure 3 shows FAM and RED scores expressed as pure multiplet over zero (FOZ) that are obtained after analyzing extracts of cucumber root boundary cell DNA using the kom20 probe set. For each DNA sample, the RED signal is plotted in the the abscissa axis while the FAM signal is plotted on the ordinate axis. Figures 4A-4B in the left panel shows germinated melon seeds are their root tips in liquid medium. The right panel shows the sheared root boundary cells that come from the root. Figure 5 shows an agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis of the mlllkl9 marker locus of different melon DNA samples generated by the DNA extraction method according to this invention. Lane 1: size marker, lanes 2 to 11: 10 single melon root boundary cell preparations, lane 12: melon leaf disk DNA, lane 13: negative control, lettuce root boundary cell preparation , Lane 14: negative control, water. Figure 6 are FAM and RED scores expressed as pure multiplet over zero (FOZ) that are obtained after analyzing DNA extracts from melon root boundary cells using the mlllkl9 probe set. For each DNA sample, the RED signal is plotted on the abscissa axis while the FAM signal is plotted on the ordinate axis.

Figure 7 is the root tip of tomato shear root boundary cells in liquid medium. Figure 8 is an agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis of the lateral suppressor gene from DNA samples other than tomato generated by the DNA extraction method according to this invention. Lane 1: size marker, Lanes 2 to 11: 10 single tomato root boundary cell preparations, Lane 12: tomato leaf disk DNA, Lane 13: negative control, water. Figures 9A-9B are the root tip of Brassica oleracea with bound boundary root cells (left). Detailed view of root limit cells of Brassica olerácea (right). Figure 10 is an agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis of the BoAC02 gene fragment from DNA samples different from Brassica olerácea generated by the DNA extraction procedure according to this invention . Lane 1: size marker, Lanes 2 through 11: Preparations of single root Brassica oleracea boundary cells, Lane 13: negative control, water. Figure 11: agarose gel stained in ethidium bromide showing the bands obtained by PCR analysis of the B region of mitochondrial ORF of the mitochondrial genome of Brassica olerácea. Lane 1: size marker, Lanes 2 to 7: 6 single root Brassica oleracea boundary cell preparations, Lane 8: DNA from a leaf of a Brassica oleracea plant, Lane 9: negative control: water Figure 12: is optical microscopy of root boundary cells sheared from lettuce root after staining with toluidine blue. Figure 13 is agarose gel stained with ethidium bromide showing the bands that are obtained by PCR analysis of the molecular marker attached to the Nasonovia resistance gene, called AS2, from DNA samples other than lettuce generated by the extraction method of DNA, according to this invention. Lane 1: size marker, lanes 2 to 14: 12 individual lettuce root boundary cell preparations, lane 14: size marker, lane 15: negative control; Water, Lane 16: positive control: DNA from a lettuce leaf disc ("pons sla"), Lane 17: negative control: cucumber DNA. Figure 14 are FAM and RED scores expressed as untreated values obtained after analyzing lettuce root boundary cell extracts using the NAS2 probe set. For each DNA sample, the RED signal is plotted on the abscissa axis while the FAM signal is plotted on the ordinate axis. Figures 15A-15B are an agarose gel stained with ethidium bromide showing PCR analysis of DNA extracts from cucumber root exudate treated with deoxyribonuclease. Figure 15A: PCR analysis using the primer combination specific for cucumber kom24. Figure 15B: PCR analysis using RP primer combination specific for lettuce. Lanes 1-18 contain isolated DNA from cucumber root exudate which undergo the following treatments: To the root exudates analyzed in lanes 1-3, 7-9 and 13-15 genomic lettuce DNA is added. No deoxyrubonuclease is added to the samples in lanes 1-6. Deoxyrubonuclease is added to the samples in lanes 7-12 and is immediately inactivated by incubation for 5 minutes at 65 ° C. To the lanes 13-18 deoxyribonuclease is added followed by incubation for 30 minutes at 37 ° C and subsequent inactivation. As controls in figure 15A, water is used (lane 19), lettuce DNA (lane 20) and cucumber DNA (lane 21). As controls in Figure 15B, water is used (lane 19), lettuce DNA (lanes 20 and 21) and cucumber DNA (Lane 22). Figure 16 is the root tip of an adventitious root of a cucumber plant regenerated in vitro after incubation for 24 hours in water at 26 ° C. The shearing of root boundary cells is clearly visible at this stage. Figure 17 is an agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis of the kom24 marker locus of different cucumber DNA samples generated by the DNA extraction method, according to this invention. Lane 1: Preparations of cucumber root boundary cells derived from adventitious roots (BC). Lane 2: DNA from a cucumber leaf disk, lane 3: negative control, water. Figure 18 is the root tip of pepper shear root boundary cells in the liquid medium. Figure 19 are FAM and RED ratings expressed as pure multiples on zero (FOZ) obtained after analyzing DNA extracts from pepper root boundary cells using the GMS probe set. For each DNA sample, the RED signal is plotted on the abscissa axis while the FAM signal is plotted on the ordinate axis. The heterozygous signals are graphed in blue while the homozygous signals of the control samples are plotted in red and green symbols in the graph. Figure 20 is the root tip of corn shear root boundary cells within the liquid medium. Figure 21 is agarose gel stained with ethidium bromide showing the bands obtained by PCR analysis of the invertase gene of wall cells Incwl from different samples' of maize DNA generated by the DNA extraction procedure, in accordance with this invention. Lane 1: size marker. Lanes 2 to 7: Maize DNA of corn root boundary cells. Lane 8: negative control, water. Lanes 9 to 10: positive control, DNA extracted from corn leaf discs. Figure 22: The root tip of limit cells of endive shear root in the liquid medium. Figure 23 is the cell root pulp of carrot shear root limit in the liquid medium.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES EXAMPLE 1 Extraction and DNA analysis of root boundary cells of PePino Cucumber seeds are germinated in 100 μ? of water (milliQ) at 26 ° C. Depending on the objective, different formats can be used such as a 12 x 8 MicronicMR microtube format which allows easy transfer of samples to a 96-well microtiter plate for additional treatments. In a stage where the germinating root has a length of about 1.5 cm, which depends on the variety and quality of the seed, happens after about 18 hours, the tubes containing the germinated seeds are shaken slightly when swirling them during 15 seconds to release the cells from the root limits of the root. The evaluation by optical microscopy clearly shows the presence of cucumber root boundary cells in the medium, as shown in figure 1. The main root and root hairs are not damaged by this procedure. The liquid that contains cells Root limit is used to carry out DNA extraction (plant DNA isolation equipment, Agowa GmbH in combination with King FisherMR robotics, Thermo Labsystems) when adding 100 μ? of lysis buffer (Agowa). The mixture is incubated for 10 minutes at 55 ° C. Subsequently, 300 μ? of DNA binding buffer (Agowa) and the mixture is centrifuged for 5 minutes at 3000 rpm. Subsequently, 15 μ? Are added to the supernatant. of a King Fisher particle suspension (Agowa Magnetic Particles (BL suspension)). After the bound DNA is eluted from the particles using 120 μ? of elution buffer (10 mM Tris-HCl buffer, pH = 7.6), the DNA is ready for analysis. For the cucumber, a random molecular marker residing in the genome, called "kom20", is selected to analyze the DNA obtained from DNA extracts based on root boundary cells. The population used in this example secretes heterozygotes or homozygotes from one of the alleles of the kom20 marker. PCR is carried out using 5 μ? of the total amount of DNA extract that is obtained. The PCR reaction is anticipated to result in a 372 bp fragment when analyzed on an agarose gel. Digestion of the kom20 PCR fragment using the restriction enzyme MspI differentiates between the two alleles of this marker locus in the population used for the experiment in this example. When the Recognition site for the restriction enzyme is present in the PCR product, digestion with MspI results in fragments of 279 and 93 bp. The result of the analysis is shown in figure 2. To confirm this result, the preparations of DNA are analyzed using the fluorescent probe set specific for kom20 (Invader ™) which generates a specific fluorescent signal (which is expressed as pure multiples above zero of FOZ) for each of the kom20 alleles (FAM or RED). Therefore it is expected that the DNA preparations obtained according to the present invention from the individuals of the segregating population analyzed using the kom20 probe generate diagnostic fluorescent signals either for the heterozygous condition of the marker alleles (graphitized on the diagonal, signal RED + FAM) or diagnostic fluorescent signals for the homozygous condition of one of the marker alleles (for this particular case marked RED and plotted on the abscissa axis). The result of the analysis is shown in Figure 3 and confirms what is expected. The genotypic kom20 scores obtained using either PCR / MspI or the fluorescence-based analysis of the population were found to agree for each plant analyzed. Therefore, the results show that the amount of DNA isolated and according to the The process which is the object of this invention is suitable for carrying out DNA marker analysis using PCR in combination with agarose gel electrophoresis or a fluorescence-based probe system as detection platforms. It can be further concluded that the marker cells are derived from DNA from the hybrid tissues that reside in the seeds and not from the maternal tissue since the markers used are segregant, which is not the case in the maternal line used to create the hybrid seeds used for this analysis. To confirm the data obtained using the DNA isolation method according to this invention, the germinated seeds are grown in the greenhouse and samples of the kom20 marker sheets are taken and analyzed using PCR / MspI. The data obtained from the DNA of the leaves are shown to be consistent with the data obtained using DNA from cell extracts of the root boundary cell. This demonstrates that the marker data which is generated through the root boundary cell DNA extracts are representative for an established plant which is grown from the germinating seedlings. To determine the amount of analysis which can be carried out by DNA extract, a series of dilutions of the DNA extract is analyzed using 5 analyzes based on fluorescence in duplicate which detect 5 different randomized markers. It is found that each DNA extract can be diluted at least 20 times without losing any of the signals from the different assays. Since each extract is produced in a volume of 100 μ? 5 μ are used? by analysis, a total of 400 analyzes can be carried out by DNA extract by isolation. In addition, for cucumber, at least 2 rounds of root-boundary cell harvests and DNA isolation can be performed per plantlet, which means that a total of 800 analyzes per seedling can be carried out using DNA extracts from cell lines. root limit. When the germinated seeds which have been used as a source of root-bound cell DNA are stored at 4 ° C, the cucumber seedlings remain viable for at least 3 weeks. This implies that the seedlings have a sufficient level of viability to be transferred to a greenhouse after the DNA analysis has been completed. Therefore, only those plants which have the desired molecular marker qualifications need to be transferred to a greenhouse and plants which do not have the desired molecular marker qualifications can be discarded in a very early in vitro phase. This can result in cost savings considerable.

EXAMPLE 2 Extraction and Analysis of Melon Root Limit Cell DNAs Melon seeds of a self-pollinated offspring of a hybrid variety called Danube are germinated at 100 μ? of water (milliQ) at 26 ° C. The procedure used to isolate melon DNA is comparable to the procedure described for cucumber in example 1 of this application. Figures 4A-4B show the presence of root boundary cells in liquid medium containing melon seedlings. To investigate whether the procedure results in DNA sufficient to detect a marker allele using PCR, 5 μ? of DNA extract to carry out a PCR reaction using primer combination specific for the mlllkl9 marker allele. The PCR reaction is anticipated to result in a 342 bp fragment. The result of this analysis is shown in Figure 5. The result shown in Figure 5 demonstrates that sufficient DNA has been obtained in order to generate the expected DNA fragment by PCR. To demonstrate that the fragment is actually derived from the embryo, an analysis of mlllkl9 based on fluorescence is carried out which it detects both alleles present in the original hybrid and which are anticipated to be segregated in the seeds used in this analysis. Figure 6 shows the results of this experiment. The result clearly demonstrates segregation of the mlllkl9 marker allele in three classes: homozygous A (FA signal), homozygous B (RED signal) and heterozygous, which shows that the DNA is of embryonic origin.

EXAMPLE 3 Extraction and DNA analysis of tomato root boundary cells Tomato seeds are germinated in 50 μ? of water (milliQ) at 26 ° C and when the roots that germinate have an average length of 1.0 cm, DNA is extracted according to example 1 of this application. The analysis by optical microscopy clearly shows the presence of limit cells of tomato root in the medium, as shown in figure 7. PCR is carried out using 5 μ? of a total amount of DNA using a primer combination specific for a known tomato gene called a lateral suppressor which is anticipated to generate a 360 bp band. The result of this experiment is shown in Figure 8. The result clearly demonstrates that the amount of DNA isolated by this procedure is sufficient to generate a PCR fragment of the expected size which can be detected on an agarose gel. It should be noted that in those cases in which a band is not observed (lanes marked with asterisks in Figure 8), the seed has not germinated. This shows that the detection of the PCR fragment using this procedure depends on the germination of the seeds.

EXAMPLE 4 Extraction and analysis of DNA from root boundary cells of Brassica olerácea An experiment is carried out using germinated seeds of Brassica olerácea. The procedure that is carried out is comparable to that described in example 1. The germination temperature is 21 ° C. Figures 9A-9B show cells of the root boundary at the root tip of Brassica olerácea seedlings. In order to demonstrate that the DNA extract obtained from the root boundary cells can be used to detect nucleic acids found in the nucleus, a primer combination is designed which amplifies a fragment of a gene involved in the biosynthesis. of ethylene called BoAC02. It is anticipated that the PCR reaction will result in a 344 bp fragment. The result of this analysis is shown in Figure 10. The result shown in Figure 10 demonstrates that sufficient DNA has been obtained in order to generate the expected nuclear DNA fragment by PCR. Another experiment is carried out in order to demonstrate that in addition to the nuclear sequences, sequences found in the cytoplasmic genomes can be detected in the DNA derived from the root boundary cell. A PCR reaction is carried out which amplifies the region of the ORF B gene that is located in the mitochondrial genome. The fragment of the ORF B region has an expected size of 1180 bp. The result of this experiment is given in Figure 11. The result shows the generation of a specific band for the region of the ORF B gene and demonstrates the cytoplasmic DNA sequences that can be detected using the described DNA isolation procedure.

EXAMPLE 5 Extraction and analysis of DNA from limit cells of lettuce root Lettuce seeds are germinated at 21 ° C in 50 μ? of water (milliQ). Root germination occurs in the following 2 days and the root boundary cells are separated from the root by light agitation. The cells of Limits of lettuce root are visualized by microscopy and are shown in figure 12. DNA extraction is carried out in a stage when the roots have an average length of approximately 1.5 cm. The method of extraction of applied DNA is similar to that described for cucumber in example 1. In order to determine if sufficient DNA has been obtained, a PCR reaction is carried out using a genomic marker, called NAS2, linked to the Nasonovia resistance gene. . The result is given in Figure 13. The result shows the generation of a specific band of the expected size for the applied molecular marker when used in the 5 μ? PCR reaction. of the obtained DNA extract. It is concluded that the DNA isolation procedure according to this invention satisfies the requirements of carrying out DNA analysis similar to PCR. The result shown in Figure 13 demonstrates that sufficient DNA has been obtained in order to generate the expected DNA fragment by PCR. To demonstrate that the fragment is actually of embryonic origin, a NAS2 analysis based on fluorescence is carried out which detects both NAS2 alleles present in the original hybrid and which are expected to segregate in the seeds used in this analysis. The results of this The results show clearly the segregation of the NAS2 marker allele in three classes: homozygous A (FAM signal), homozygous B (RED signal) and heterozygous, which shows that the DNA is of embryonic origin.

EXAMPLE 6 Root exudate DNA is found in the deoxyribonuclease-resistant fraction In order to demonstrate that the DNA obtained from root exudate according to the method described in this invention originates from root boundary cells are carried out Deoxyribonuclease sensitivity experiments. The root boundary cells are produced from cucumber seedlings as described in Example 1 and the isolated DNA is analyzed by PCR using a primer combination specific for the kom24 marker locus. The root exudate is treated with deoxyribonuclease before the DNA extraction is performed. In case the DNA is present in the root exudate as such it will be degraded by deoxyribonuclease. In case the DNA is present within the root boundary cells, deoxyribonuclease treatment will have no effect on the generation of a PCR signal after DNA extraction.

The result of this experiment is shown in Figures 15A-15B. The results show that exogenously added DNA from lettuce is sensitive to deoxyribonuclease treatment when added to cucumber root exudate while the cucumber DNA PCR signal is not lost as a consequence of deoxyribonuclease treatment. This shows that the cucumber root exudate contains cucumber DNA which is protected from the added deoxyribonuclease and therefore derived from the root cell present in the exudate.

EXAMPLE 7 DNA extraction using root exudate from plants that have grown in vitro Cucumber in vitro root shoots are taken for analysis when transferring the seedling from the culture medium in vitro into a macro-well plate containing water which allows to generate root limit cells. After incubation for 24 hours at 26 ° C the formation of root boundary cells is clearly visible by light microscopy, as shown in Figure 16. In this step, the cells of the root boundary are harvested, the DNA is extracted and analyzed by specific PCR for the kom24 marker locus. A clear band diagnoses the presence of cucumber genomic DNA is obtained using the DNA extract from the root boundary cells as shown in Figure 17. The result shown in Figure 17 demonstrates that sufficient DNA has been obtained in order to generate the expected DNA fragment by PCR and therefore it can be concluded that the DNA isolation technology based on root boundary cells is applicable to root exudate derived from adventitious roots of seedlings growing in vitro.

EXAMPLE 8 Extraction and DNA analysis of pepper root boundary cells Pepper seeds are germinated in 40 μ? of water (milliQ) at 26 ° C and when the roots that germinate have a length of an average of 1.0 cm, DNA is extracted according to example 1 of this application. Inspection by optical microscopy clearly shows the presence of pepper root boundary cells in the medium, as shown in Figure 18. The DNA preparations are analyzed using a GMS-specific fluorescent probe set (Invader ™) which generates a specific fluorescent signal (expressed as pure multiples of zero FOZ) for each of the GMS alleles (FAM or RED). In this example, the seeds Hybrid Fl are analyzed knowing that they are heterozygous uniformly for the GMS marker allele. Therefore it is expected that the DNA preparations obtained according to the present invention from individuals of the hybrid population Fl analyzed using GMS probes generate diagnostic fluorescent signals for the heterozygous condition of the marker allele (plotted on the diagonal, RED + FAM signal) . The result of the analysis is shown in Figure 19. The genotypic GMS scores obtained using the fluorescence-based analysis of the population are found to be in agreement with the known genotypic values for this marker locus for each plant analyzed. Therefore, the results demonstrate that the amount of DNA isolated according to the method of this invention is adequate to carry out a DNA marker analysis using a fluorescence-based probe system as a detection platform.

EXAMPLE 9 Formation of corn root boundary cells Corn seeds are germinated in 500 μ? of water (milliQ) at 21 ° C and when the roots that germinate have an average length of 2.0 cm the inspection by optical microscopy clearly shows the presence of limit cells of corn root in the medium, as shown in the figure 20. In this stage, the root boundary cells are collected, the DNA is extracted and analyzed by specific PCR for the cell wall invertase (Incwl, accession number AF050129). A clear band of 620 bp is diagnostic for the presence of maize genomic DNA and is obtained using the DNA extract from the root boundary cells as shown in figure 21.

EXAMPLE 10 Formation of escarole root boundary cells Endive seeds are germinated in 40 μ? of water (milliQ) at 21 ° C and when the roots that germinate have an average length of 1.0 cm the inspection by optical microscopy clearly shows the presence of limit cells of endive root in the medium, as shown in the figure 22 EXAMPLE 11 Formation of carrot root boundary cells Carrot seeds are germinated at 40 μ? of water (milliQ) at 21 ° C and, when the roots that germinate have an average length of 1.0 cm, inspection by optical microscopy clearly shows the presence of carrot root boundary cells in the medium, as shown in figure 23. It is noted that in relation to this date, the best method known by the application to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for obtaining DNA from a plant, characterized in that it comprises: a) collecting the root boundary cells of a growing root; and b) extract DNA from the root boundary cells.
2. 2. The method according to claim 1, characterized in that the root boundary cells are contained in the root exudate of the growing root.
3. 3. The method according to claim 1 or 2, characterized in that the root is growing in a medium.
4. 4. The method according to claim 3, characterized in that the medium is water.
5. 5. The method according to claim 3, characterized in that the medium is a tissue culture medium.
6. 6. The method according to claim 3, characterized in that the medium is ground.
7. 7. The method according to any of claims 1 to 6, characterized in that the root is part of a seed in germination.
8. 8. The method according to any of claims 1 to 6, characterized in that the root is the root of a seedling.
9. The method according to any of claims 1 to 6, characterized in that the root is the adventitia root of a tissue culture plant or a part of a plant.
10. 10. The method according to claim 7, characterized in that the seeds are germinated until the radicle or the tip of the root emerges.
11. The method according to any of claims 1 to 10, characterized in that the root boundary cells are harvested from a root that has germinated at about 1 to 2 cm.
12. 12. The use of the method according to any of claims 1 to 11, for the detection of genetic variants from populations of bulk M2 mutant plants.
13. 13. The use of the method according to any of claims 1 to 11 for the detection of genetic variants of natural plant populations in bulk.
14. 14. The use of the method of compliance with any of claims 1 to 11 for the detection of genetic variants in breeding populations. The use of the method according to any of claims 1 to 11 for the detection of genetic variants in commercial seed batches.