MXPA06002799A - Methods and compositions for enhanced plant cell transformation - Google Patents

Abstract

Methods and compositions to increase Agrobacterium transformation efficiency (frequencies) in monocot host plants include increasing histone levels in the host plant, and using histones and L-cysteine at certain stages in monocot transformation.

Description

METHODS AND COMPOSITIONS FOR INTENSIFIED TRANSFORMATION OF VEGETABLE CELLS Field of the Invention The invention relates to intensified frequencies of transformation mediated by Agrobacterium plant due to the addition of histones to the plant to be transformed. Specific methods for enhancing monocot transformation frequencies are also described where both histones and L-cysteine are factors. BACKGROUND OF THE INVENTION Agrobacterium tumefaciens is a gram negative soil bacterium that has been exploited by plant biologists to introduce external DNA into plants. The efficient Agrobacterium-mediated transformation of dicotyledonous plants was first reported in the mid-1980s. Because monocotyledonous plants are not natural hosts for Agrobacterium tumefaciens, the development of transformation systems that use this vector for monocotyledons is behind that one for the dicotyledons. Direct DNA delivery techniques including electroporation, microprojectile bombardment and silicon carbide fiber treatment were developed for the transformation of monocotyledons as alternatives for the delivery of Agrobacterium-based DNA. The production of fertile transgenic corn plants Ref.: 170204 was first reported in 1990 when using a bombardment of microprojectiles. Reports of production of fertile transgenic maize plants using silicon carbide fiber treatment and by electroporation followed a few years later. The first well-documented report of fertile transgenic maize plants by Agrobacterium was published by Ishida et al. in 1996 followed by a second report by Negrotto et al. in 2000. Although the high frequency Agrobacterium-mediated transformation was reported in these studies, and also in a few maize transformation laboratories in a private industry, these frequencies have not been reproduced in public corn processing laboratories. Factors contributing to the lack of reproducibility in the public sector may include: 1) omission of critical details in the protocol and media descriptions in published reports, 2) lack of access to specialized binary vectors by public researchers and 3) reluctance or inability to transfer proprietary information from private industry to the public sector. The advantages of using a transformation system based on Agrobacterium for maize (high frequency transformation, low copy, simple insertion of transgenes, improved stability of transgene expression, low cost in relation to biolistics and the potential to introduce large fragments of DNA in the genome of the plant) makes it imperative that optimized protocols are developed, published and made readily available to maize researchers in the public sector. Although known for this practical application, the current mechanism of DNA transfer from bacteria to plants is not fully understood. Moreover, there are some limitations on the use of this transformation vector, for example, difficulties in transforming monocots and transforming frequencies may be too low to be useful. At present, even some dicots, for example, many Arabidopsis ecotypes and mutants, can not easily or efficiently be transformed by a root transformation method using generally Agrobacterium. It is believed that Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti plasmid, designated T-DNA to the plant, and integrate the T-DNA into the plant genome. Little is known about the integration process of T-DNA, and no plant genes involved in the integration have been previously identified. The DNA that is transferred from Agrobacterium to the plant cell is a segment of the Ti or tumor-inducing plasmid called T-DNA (transferred DNA). The virulence genes (vir) responsible for the processing and transfer of the T-DNA are reported to be somewhere else on the Ti plasmid. The role of vir genes in the processing of T-DNA, the formation of bacterial channels for the export of T-DNA and the placement of bacteria to the plant cells is reported. In contrast, little is known about the role of plant factors in the transfer and integration of T-DNA. Transformations can be transient or stable. Stable transformation is preferred because it is required to produce transgenic plants. Many plant species resist a stable transformation by Agrobacterium. However, these species are transiently transformed in a simple manner to express GUS activity or symptoms of viral infection after agroinoculation. BMS corn cells are easily transiently transformed and can express and process a gzs-A-intron transcript encoded by the pBISN1 of the binary vector. The published results imply that at least in this transformation system, the T-DNA can be directed to the corn kernels and converted to a competent form in double-stranded transcription. However, the lack of a stable detectable transcript suggests that the integration of T-DNA may be deficient. Thus, by making the integration of T-DNA more efficient and stabilizing the expression of T-DNA genes are important factors to improve the transformation of corn. The integration of exogenous DNA is reported to be enhanced by the provision of DNA in plant cells with one or more Agrobacterium genes that can encode proteins within the cells of plants. This technique, referred to as "agrolistic transformation", is just an improvement over the biolistic transformation by which DNA is supplied to plants by a non-biological method such as a "gene gun" (biolistic transformation). In this improvement, the genes encoding the virulence proteins that normally function in Agrobacterium are transferred to the plants together with a T-DNA substrate. Then the substrate acts on the cells of the plant to make a T-DNA molecule. However, the described technique does not include the use of plant genes or other factors discussed herein. The technique was not shown to make a plant more susceptible to transformation. One goal of this method was to increase the prediction of the location of the integration not its frequency. Furthermore, "agrolistic transformation" is an expensive procedure that requires a lot of infrastructure and resources, someone of experience will have to go through the laborious process every time to develop a transgenic plant. The isolation of a supposed plant factor has been reported recently. Bailas and Citovsky demonstrated that a α-caryoferrin plant (AtKAP a) can interact with nuclear localization sequences VirD2 in a 2-hybrid yeast interaction system, and is presumably involved in the nuclear translocation of the T complex. By using a similar method , a type 2C tomato protein phosphatase, DIG3, which can interact with VirD2 NLS, is identified. Unlike AtKAP a, DIG3 plays a negative role in nuclear import. After the T-DNA / T complex enters the nucleus it must be integrated into the chromosome of the plant. The chromosomal DNA of the plant is packed into nucleosomes that consist mainly of histone proteins. The input T-DNA may have to interact with this nucleosome structure during the integration process. However, T-DNA can be preferentially integrated into the transcribed regions of the genome. These regions are believed to be temporarily free of histones. Exactly how the integration of T-DNA takes place is unknown. Recent reports have implicated the involvement of the VirD2 protein in the integration process of T-DNA. Several ecotypes of the dicotyledon Arabidopsis are resistant to transformation by Agrobacterium. Transforming mutant rat5 resistant to Arabidopsis transformation with a wild-type RAT5 gene (histone H2A) is reported by the inventors to be complementary to the mutant phenotype.

In monocots, corn is the most studied plant model that has an important economic value. Although genetic transformation systems for maize have been established in private laboratories, the lack of such systems is still a key limitation for public researchers. This is due to the fact that most public research groups do not have access to the resources and infrastructure necessary for corn processing through currently available procedures. In addition, current technology has serious limitations including low efficiency and production, difficulty with the transformation of endogenous lines, unpredictable transgene copy numbers and integrity, and the silencing of undesirable transgenes during development and over generations. Because fertile transgenic corn (Zea mays) was first produced using a biolistic spray gun, corn transformation technology has served as an important tool in the development of germplasm and its research, addressing fundamental biological issues through the study of corn transgenic Recent reports have shown that the transformation of corn mediated by Agrobacterium tumefaciens may offer a better alternative than the biolistic pistol for the supply of transgenes for corn. This gene delivery system results in a higher proportion of stable transgenic events with a low copy number compared to the biolistic gun, offers the possibility of transferring larger segments of DNA in recipient cells and is highly efficient. Reproduction protocols for maize transformation mediated by A. tumefaciens have used superbinary vectors in which the A. tumefaciens strain carries additional copies of virB, virC, and virG, to infect the immature zygotic embryos of the A188 endogamous line or the Hi II hybrid line. The immature zygotic embryos of Hi II were transformed by the inventor at an average efficiency of 5.8% using the superbinary vector of A. tumefaciens in strain LBA4404. Because the cost of licensing this proprietary technology for use on a broader scale may be prohibitive for a public sector laboratory, the inventors implemented a standard binary vector system of A. tumefaciens (which is not superbinary) to transform the immature zygotic embryos of Hi II of corn. The stable transformation of maize using a standard binary vector to infect the meristems of the shoots was previously reported, but the adoption of this method was hampered by its lack of firmness. The development of an efficient and reproducible method to transform corn using a standard binary vector will not only provide the researchers with the benefits already mentioned, it would also facilitate the construction of the vector when compared with a superbinary vector. The final assembly of a superbinary vector system involves the co-integration of the gene of interest into a large plasmid (pSBl) in a strain of A. tumefaciens LBA4404 by means of homologous recombination. In contrast, the assembly of a standard binary vector does not require this additional step, making a more efficient way to confirm the introduction of a gene of interest in a strain of A. tumefaciens. The transformation of corn (Zea mays) using a standard binary vector system of Agrobacterium tumefaciens (not superbinary) was achieved by the inventors. The immature zygotic embryos of the Hi II hybrid line were infected with the A strain. tumefaciens EHA101 which contains a standard binary vector and is grown together in the presence of 400 mg / L of L cysteine. The inclusion of L-cysteine in the joint culture medium leads to an improvement in the transient expression of glucuronidase observed in the target cells and a significant increase in the efficiency of stable transformation, but is associated with a decrease in the response of the embryo after the culture in parallel. The average stable transformation efficiency (number of bialaphos-resistant events recovered per 100 infected embryos was 5.5%.) Progeny and Southern blot analysis confirmed the integration, expression and inheritance of the transgenes bar and gus in the R0 generations, R and R2 of transgenic events Stable, fertile transgenic maize was routinely produced using a standard binary vector system of A. tumefaciens The level of stable transformation achieved is attributed to the complement of a culture medium combined with 400 mg / L Cys. This antioxidant treatment also increased the supply of T-DNA to the competent embryogenic embryonic scutellum cells of infected embryos.A similar increase in trans gus gene expression, followed by an increase in the efficiency of stable transformation is reported in the nodule explants of cotyledonous soybean infected with A. tumefaciens and grown together on a medium supplemented with Cys. Contrary to expectations, the increase in stable transformation efficiency observed with the 400 mg / L Cys treatment is associated with a decrease in the embryo ratio as it results in an embryogenic callus compared to the 0 mg / L Cys treatment . This reduction in the embryo response is not related to the interaction of the plant pathogen per se, because uninfected embryos also showed a reduced response at 400 mg / L Cys. It is likely that Cys concentrations as high as 400 mg / L are toxic to maize cells. A similar negative impact of 80 mg / L Cys on embryogenesis in Japanese rice explants was reported by Enriquez-Obregon et al. (1999). Comparable stable transformation rates were achieved using Cys concentrations as low as 100 mg / L, and this treatment was associated with better embryo recovery after a joint culture, than that observed using the 400 mg / L Cys treatment. A corn transformation mediated by A. Tumefaciens using a standard binary vector system reproduces although the variation in experimental efficiency persists. By using a joint culture medium with 7 days of preparation this variation is minimized. The average transformation efficiency is around 5.5%. Information on plant factors and other factors that affect the frequencies of transformation of Agrobacterium in plants is needed to improve the performance of this method in both dicotyledonous and monocotyledonous. Brief Description of the Invention Methods and compositions for increasing the efficiency of Agrobacterium transformation in a host plant include adding histones to the host plant and for monocotyledons, also adding L-cysteine and using a standard binary vector (a "binary vector system"). simple ", not super). Histones can be added either transiently or genes encoding histone can be stably incorporated into a host plant genome. A polynucleotide sequence encoding a plant histone protein can be integrated into the genome of the host plant or only introduced transiently to express the polynucleotide sequence encoding a histone protein in the plant. The host plant expressing the polynucleotide sequence encoding a histone protein in the plant to increase histone base levels is transformed with a DNA molecule of interest by means of Agrobacterium. There are 4 types of central histones (H2A, H2B, H3 and H4). A suitable plant histone protein is a member of the family of the Arabidopsis H2A gene, for example RAT5. One aspect of the invention is a transgenic plant with at least one additional copy of a polynucleotide sequence encoding a histone H2A protein in the plant. The polynucleotide sequence can encode a histone H2A protein in the plant that is a member of the H2A gene family of Arabidopsis.

A method to increase the stable transformation efficiency of Agrrsjbacterium in host plants includes the steps of: a) selecting a host plant that expresses a polynucleotide sequence that encodes a protein H2A of plant histone with which histone levels in the host plant are increased; b) introducing a transformation vector with a DNA molecule of interest into an Agrobacterium strain; c) providing at least one antioxidant in a joint co-culture medium; d) infecting the cells of a host plant with the Agrobacterium strain in a joint culture medium e) providing suitable conditions for the recovery of infected cells; and f) selecting the infected cells for the transformants expressing the DNA molecule of interest. The host plant can be a monocot, for example, corn and the antioxidant can be L-cysteine. The concentration of L-cysteine can be between about 100 mg / L and 400 mg / L. The infection of the cells in the joint culture medium can be for about 3 days.

One aspect of the invention is a genetic construct comprising at least one copy of a histone gene in addition to that in the host plant initially which when expressed may increase the frequencies of transformation in a host plant. The histone gene can encode H2A for example a gene of RAT5 Arabidopsis. One aspect of the invention is a host cell transformed by at least one copy of a gene involved in a T cell integration where the gene can effect overexpression of histone to improve the frequencies of plant transformation. A method for increasing the transformation frequencies of AgroJbacterium in a host plant includes the steps of: a) increasing the histone levels in the host plant as compared to the normal levels of histone in the host plant; and b) transforming the host plant with Agrobacterium. Transformation frequencies can be measured by the number of tumors produced in the host plant or by using detectable markers if the transformation has occurred. The histone of H2A can be H2A-1. One aspect of the invention is a plant cell with an overexpression of plant histones sufficient to increase the transformation efficiency of the plant cell by Agrobacterium.

A method to increase the efficiency of transformations in a host plant, the method includes the steps of: (a) transforming a host plant with a transcriptional activator of the histone genes; (b) selecting a transgenic host plant that over expresses at least one history gene; and (c) transforming the transgenic host plant that over expresses at least one histone gene with a DNA molecule of interest. The host plant is a monocotyledonous plant and the transcription factor is a transcription factor of the b-ZIP family. The transcription factor of b-ZIP is HBP-la.

A method to increase the transformation efficiency in a host plant, the method comprises: (a) transforming the host plant with an activator; (b) separating by exclusion a progeny of the transformed host plant to increase expression of the histone gene; and (c) transforming the progeny of the host plant that has been increased with a DNA molecule of interest. The activator can be an enhancer element of CaMv 35S or any other equivalent promoter or enhancer elements.

Definitions Activator: a nucleic acid sequence that activates transcription. Intensifier: a nucleic acid sequence that greatly increases the expression of a gene in its vicinity; the sequence can be located at the 5 'or 3' end and its orientation is not fixed. H2A: a member of the family of H2A gene. The members of the H2A gene are also denoted by HTA. The RAT5 of Arabidopsis is one such member of the H2A / HTA gene family. Infected: Agrobacterium is in the host cell. Retransformation: the transformation of a host plant having at least one additional copy of a polynucleotide encoding histone H2A protein stably integrated into the genome of the host plant. Efficiency of. transformation: (number of successful events / number of infected embryos) x 100. The number of transgenic events is an indication of stable transformation. The calculation of transformation efficiency can also involve influencescence, calluses, seeds or other biological material that can be infected with Agrobacterium to produce a transformant.

Brief Description of the Figures Figures 1A-1C show the rat5 mutant: (fig.lA) stable transformation of the wild-type Arabidopsis ecotype Ws, the mutant rat5 and the progeny Fl; (Fig. IB) sequence of the binding region of rat5 / T-DNA; (fig.lC) pattern of integration of T-DNA into rat5; LB, T-DNA extreme left; RB, T-DNA extreme right; pBR322, pBR322 sequences containing the ß-lactamase gene and the ColEl origin of replication; Tn903, kanamycin resistance gene for the selection of E. Coli; Tn5, kanamycin resistance gene for plant selection. Figures 2A-2D show the complementation of rat5 mutant and the over expression of RAT5 in wild-type Arabidopsis plants; (fig.2A) binary vector maps pKM4 and (fig.2B) pKM5 RB, T-DNA extreme right; LB, T-DNA extreme left; Pnos, polyadenylation signal sequence of nopaline cintasa; Histone H2A, histone H2A gene coding sequence RAT5; pH2A, promoter sequence of histone H2A gene RAT5; Pnos; promoter of nopaline cintasa; hpt, hygromycin resistance gene; pAg7, polyadenylation signal sequence of agropin tape; uidA, gene without promoter gusA; the arrows above the histone H2A, uidA, and the htp genes indicate the direction of transcription; (fig.2C) complementation of the rat5 mutant; (fig.2D) assay of tumorigenesis of Ws transgenic plants that over express histone RA 5A H2A gene. Figures 3A-3B show the T-DNA integration assays of plants rat5 and Ws; (fig.3A) transient and stable GUS expression in Ws and rat5; (fig.3B) T-DNA integration in plants rat5 and Ws. Figure 4 shows the map of the binary corn transformation vector pE2250 to generate the founding lines of the corn and a flow diagram of the construction of the vector. Figure 5 shows a Northern Blot of the transgenic corn lines expressing H2A. Figures 6A-6E show the cDNA sequences from HTA1 to HTA13. The coding region is highlighted in bold letters and the UTR region 5 'and 3' in normal letters. Figures 7A-7B show the amino acid sequences of HTAla to HTA13. Figures 8A-8M show the genomic sequences of HTA1 through HTA13. Figures 9A-9D show that the results of Agrobacterium-mediated transformation in the lower transgene copy number (fig.9A) and in the expression of the superior gene (fig.9B) compared to the transformation of corn biolistic gun (number of copy L: 1-3; M: 4-10; H: 10-20; VH: > 20). (figs.9C-9D) show that the transgene expression is more stable in the transformants derived from Agrobacterium (fig.9C) than in the derivatives of the bombardment (fig.9D). Figure 10 is a schematic illustration of the T-DNA region of a standard binary vector pTF102.LB, left end; RB, far right; bar, phosphinotricin acetyltransferase gene; gus-int, glucuronidase gene that contains an intron; P35S, CaMV 35S promoter, TEV, translational enhancer of tobacco etch virus; Tvsp, soybean vegetative storage protein terminator; T35S, terminator CaMV 35S; H, HindIII.

Detailed Description of the Invention The invention relates to methods and compositions for increasing the transformation frequencies of AgaroJbacteriuiT? in plants due to the addition of histone directly or by incorporation into a host plant of at least one plant gene involved in the integration of the T-DNA host. This differs from some methods in previous publications, because the plant-host genes, not the bacterial genes of Agrobacterium, are used to improve transformation. In one embodiment, the addition of at least one histone H2A gene encoded by the Arabidopsis RAT5 gene enhances the transformation frequency, most likely due to the overexpression of histone compared to the natural expression levels of the host. The gene may be either in transgenic plants or carried by the T-DNA transformation agent for the practice of the invention. To identify the genes of plants involved in the transformation mediated by Agrobacterium, a collection of T-DNA labeled with Arabidopsis is separated by exclusion for mutants that are resistant to the transformation of Agrobacterium (rat mutant). A mutant labeled with Arabidopsis T-DNA, rat5, is characterized in that it is deficient in the integration of T-DNA and is resistant to root transformation mediated by AgrroJbacterium. Both DNA and genetic immunoblot analyzes indicate that there are two copies of T-DNA integrated as a tandem repeat in a single place in rat5. There are no major reconfigurations in the rat5 plant DNA that immediately surrounds the T-DNA insertion site. These data suggest strongly that in rat5 the T-DNA had inserted in a gene necessary for the transformation mediated by Agrobacterium. The sequence of the plant junction at the far left of T-DNA indicates that the T-DNA was inserted into the 3 'untranslated region of a histone H2A gene. This insertion is in the upward direction of the consensus polyadenylation signal. By separating a collection of Ws cDNA from the Arabidopsis ecotype by exclusion, and sequentially forming 20 different histone H2A cDNA clones and performing a computer database search, at least six different histone H2A genes were identified. These genes encode proteins that are 90% more identical at the amino acid sequence level. Thus, the histone H2A genes comprise a family of multigenes in Arabidopsis. The accession number to gene bank AB016879 contains a report of a DNA sequence of some of the clones derived from chromosome 5 of Arabidopsis thaliana. One of these sequences is a histone H2A gene that is identical to the RAT5 gene. however, this report neither teaches nor suggests a role for histone in improving transformation frequencies. The overexpression of the histone genes of the present invention outweighs the poor performance that limits the use of Agrobacterium as a transformation vector. Many plants can be transiently transformed by Agrobacterium in a way that expresses the transformation gene for a period of time, but they are not transformed stably due to integration problems of T-DNA. Therefore, transgenic plants are not produced. The H2A gene (RAT5) plays an important role in the illegitimate recombination of T-DNA within the genome of the plant and the overexpression of the gene intensifies the transformation.

The transient and stable expression data of GUS (ß-glucuronidase) and the evaluation of the amount of T-DNA integrated into the genomes of the rat5 plants of Arabidopsis and wild-type indicates that the mutant rat5 is deficient in the integration of T-DNA necessary for transformation. The complement of the rat5 mutation is carried out by expressing the wild-type histone RAT5 H2A gene in the mutant plant. Surprisingly, overexpression of RAT5 in wild-type plants increases the transformation efficiency of Agrobacterium. Additionally, the transient expression of a RAT5 gene from the input T-DNA was sufficient to complement the rat5 mutant and also not to increase the transformation efficiency of the wild-type Arabidopsis plants. By adding the histone directly to the host plant, the transformation is improved. The present invention provides methods and compositions for increasing the frequency of stable transformation in plants using a direct involvement of a plant histone gene in the integration of T-DNA. Several mutants labeled with Arabidopsis T-DNA that are reluctant to Agrobacterium root transformation were identified. These are called rat mutants (resistant to the transformation of Agrobacterium). In most of these mutants the transformation of Agrro acteri is blocked at an early stage, either during bacterial placement in the plant cell or prior to nuclear importation of T-DNA. However, in some of the mutants, the integration stage of T-DNA is most likely blocked. Because the factors of plants involved in the illegitimate recombination of T-DNA in the plant genome are not previously identified, the characterization of a mutant of Arabidopsis labeled T-DNA, rat5, which is deficient in the integration of T- DNA, is an aspect of the present invention. Characterization of the rat5 mutant. Rat5, a tagged mutant of Arabidopsis T-DNA was previously identified as resistant to Agrobacterium root transformation. Rat5 mutants are also expected in other species, for example, maize. An in vitro root inoculation assay was performed using wild type Agrobacterium strain A208 (AtlO). After 1 month, the percentage of root bundles that formed tumors was calculated. More than 90% of the root bunches of wild-type plants (ecotype Ws) formed large green teratomas. In contrast, slightly more than 10% of the root bundles of rat5 plants respond to infection, forming small yellow calli (FIG. A homozygous rat5 (pollen donor) plant is crossed with a wild-type plant (egg donor) and the resulting progeny Fl is tested for susceptibility to Agrobacterium transformation. This analysis indicates that rat5 is a dominant mutation (FIG.1A). Additional analyzes of the F2 progeny indicate that the 3: 1 kanamycin resistant segregate indicates that a single location has been disrupted by a mutagenized T-DNA. The kanamycin-resistant co-segregated rat5 phenotype indicates that the gene that is involved in the transformation of Agrobacterium has been more possibly mutated by the insertion of T-DNA. Recovery of the T-DNA plant binding of rat5. The integration pattern of T-DNA in the rat5 mutant is determined by DNA Blot analysis. The results indicate that only two copies of the mutagenized T-DNA have been integrated into the rat5 mutant genome. Additional analyzes indicate that these two copies of T-DNA are presented as a tandem repeat as shown in FIG.1C. The left end (LB) of the T-DNA plant junction is recovered from rat5 using a plasmid rescue techniques (see materials and methods) and a restriction endonuclease map of this T-DNA plant junction is constructed. An EcoRI fragment of approximately 1.7 kbp containing DNA from both plants and LB is subcloned into pBluescript and subsequently processed sequentially at the center of sequence processing at Purdue University. The sequence of this fragment is shown in FIG.1B. The DNA sequence analysis of this binding region indicates that the T-DNA has been inserted into the 3 'untranslated region (UTR) of a histone H2A gene (FIG.1B). The histone H2A genes of Arabidopsis are further characterized by sequentially isolating and processing numerous cDNAs and genomic clones. Six different histone H2A gene variants have been identified, which indicates that the histone H2A genes of Arabidopsis comprise a small family of multiple genes. In a collection of lambda genomic DNA, a clone is identified that contains the wild type histone H2A gene corresponding to RAT5. The DNA sequence analysis of this genomic clone indicates that in the rat5 the T-DNA has been inserted in an upward direction of the consensus polyadenylation signal (AATAA). Blot DNA analysis of the Ws and rat5 DNAs indicate that the insertion of T-DNA into rat5 does not cause any major reconfiguration in the plant DNA immediately around the insertion site. The disruption of the 3 'UTR of the histone H2A gene of RAT5 is possibly the only cause for the rat phenotype in the rat5 mutant. Figures 1A-1C show the characterization of the rat5 mutant. (fig.lA) stable transformation of the wild type Arabidopsis Ws ecotype, the mutant rat5 and the progeny Fl. Sterile root segments infected with A were identified. Tumefaciens A208. Two days after the cocultivation, the roots were transferred to an MS medium that lacks phytohormones and that contains timentin as an antibiotic. Tumors were recorded after 4 weeks, (Fig. IB) Sequence of the T-DNA / rat5 binding region. (fig.lC) Pattern of the integration of T-DNA into rat5. LB, left end of the T-DNA, RB, right end T-DNA, the sequences pBR322, pBR322 contain the β-lactamase gene and the origin of replication ColEl; Tn903, kanamycin resistant gene for the selection of E. Coli; Tn5, kanamycin resistant gene for plant selection. 5 μg of genomic DNA from rat5 mutant was digested with either EcoRI or SalI and placed on a nylon membrane. An EcoRI-SalI fragment of pBR322 was used as the hybridization probe. The sizes of the restriction fragment shown above the T-DNA were detected by EcoRI digestion and the sizes shown below the T-DNA were detected by SalI digestion. Complementation of the rat5 mutant with the wild-type histone H2A gene (RAT5). Two different constructions are made to perform a complementation analysis of the rat5 mutant. First, a nopalina cintasa terminator (3 'NOS) is fused to the 3' region of the 1.7 bp binding fragment (the sequence of this 1.7 kbp fragment is shown in FIG. This construct contains the H2A gene of histone RAT5 with its own promoter and a 3 'NOS. This fragment (RAT5 plus NOS) was cloned into a binary vector pGTV-HPT from a beaker containing a hygromycin resistance gene between the left and right T-DNA ends, resulting in the binary vector pKM4 (FIG. 2A). For the second construct, a 90 kbp Sacl genomic fragment of wild-type Ws DNA containing the histone H2A (RAT5) gene plus at least 2.0 kbp of sequences upstream and downstream of RAT5 was cloned into the binary vector pGTV -HPT, which results in the binary vector pKM5 (FIG 2B). pKM4 and pKM5 were transferred separately in the non-tumorigenic Agrobacterium strain GV3101, resulting in strains of A. tumefaciens Atl012 and Atl062, respectively. Both strains Atl012 and Atl062 are used separately to transform rat5 plants using a germ line transformation method (Bent et al., 1998) and transgenic rat5 plants are selected for hygromycin resistance. (20 μg / ml). Several transgenic plants (IT) are obtained. These transgenic plants were allowed to self fertilize and collect IT seeds. Six transgenic lines obtained by transformation with Atl012 (histone H2A wild type with 3 'NOS) are randomly selected and their seeds are germinated in the presence of hygromycin. Tumorigenesis assays were performed as described in Na et al. (1999) using A. Tumefaciens AtlO and a sterile root inoculation protocol, in at least 5 different plants of each of the transgenic lines. The results indicate that in five of the six transgenic rat5 lines tested, it was recovered in the tumorigenesis susceptibility phenotype (FIG 2C, Table 1). The teratomas incited in the roots of these plants appear similar to the tumors generated in the wild-type plant. One of the transgenic plants tested did not recover the phenotype of susceptibility to tumorigenesis, probably due to an inactive transgene. Transgenic IT plants of rat5 obtained by transformation with Atl062 (containing a RAT5 genomic encoded wild-type plant) were also tested for restoration of the tumorigenesis susceptibility phenotype. Some of these plants are also capable of recovering the phenotype of susceptibility to tumorigenesis, indicating the completion of the rat5 mutation. The hygromycin-resistant transgenic plants generated by transforming the rat5 mutant with pGPTV-HPT alone do not form tumors during infection with A. Tumefaciens A208. To confirm the genetic basis of the experiment by complementation, a co-segregation analysis was performed on one of the rat5 transgenic lines (rat5 Atl012-6) obtained by transforming the rat5 mutant with A. Tumefaciens Atl012. To examine the co-segregation of complement T-DNA containing the wild-type RAT5 gene with the tumorigenesis susceptibility phenotype, seeds of a T2 plant homozygous for the rat5 mutation but heterozygous for hygromycin resistance are germinated and they are grown in a B5 medium without selection. The roots of these plants are subsequently tested for hygromycin resistance and susceptibility to crown gall tumorigenesis. All plants that are sensitive to hygromycin are also resistant to tumor formation in a manner similar to that of the rat5 mutant. Of the 25 plants resistant to hygromycin, at least 8 were susceptible to tumorigenesis. However, 17 remaining hygromycin-resistant plants are recalcitrant to Agrobac terium-mediated transformation, possibly due to the fact that these plants are heterozygous with respect to the complement RAT5 gene and do not express this gene at a sufficiently high level to restore Susceptibility to tumorigenesis. This possibly corresponds to the finding that the rat5 mutation is dominant, and that therefore an active copy of RAT5 is not sufficient to allow the transformation mediated by Agrobacterium. Taken together, the molecular and genetic data strongly indicate that in the disruption of the rat5 mutant of a histone H2A gene is responsible for the deficient phenotype (rat) to tumorigenesis. The overexpression of a histone H2A (RAT5) gene in wild-type plants improves the efficiency of Agrobacterium transformation. To further determine where the RAT5 gene plays a direct role in the Agrobacterium-mediated transformation, A. Tumefaciens Atl012 is used to generate several transgenic Arabidopsis plants (ecotype Ws) containing additional copies of the histone RAT5 H2A gene. These transgenic plants were allowed to self-pollinate, TI seeds are collected, and T2 plants are germinated in the presence of hygromycin. Tumorigenesis assays are performed as described herein to at least five plants out of four different transgenic lines. Because the Ws ecotype is normally highly susceptible to transformation by Agrobacterium, the tumorigenesis assay is altered to detect any of the subtle differences between the wild-type plant susceptible to transformation and the transgenic wild-type plants that over-express RAT5 . These alterations include the inoculation of root segments with concentrations 100 times lower (2x107 cfu / ml) of bacteria than those that are normally used (2x109 cfu / ml), and individual root segments scattered instead of bundles of root segments in MS means to observe tumor production. The results, shown in Table 1 and FIG.2D, indicate that the transgenics that over-express RAT5 are about twice as susceptible to root transformation as are wild-type Ws plants. These data indicate that the H2A gene of histone RAT5 plays a direct role in the transformation of T-DNA and that the overexpression of RAT5 can increase the susceptibility to transformation. The transient expression of H2A histone is sufficient to allow the transformation of rat5 and to increase the transformation efficiency of wild type Ws plants. The histone H2A gene expression of RAT5 from the input T-DNA complements the rat5 mutant. However, the transformation of this mutant with the Agrobacterium strain carrying pGPTV-HYG (which lacks a histone H2A gene) results only in some slow-growing calli in the hygromycin selection medium, Agrobacterium strains carrying pKM4 or pKM5 that stimulate the rapid growth of hygromycin-resistant calli in 60 + 21% and 54 ± 22% of the root segment bundles rat5, respectively. In addition, when wild-type plants (or a low bacterial density) are infected with a tumorigenic Agrobacterium strain (A208) that carries pKM4,78 ± 8% of the root segments develop tumors, compared to 36 ± 9% of the root segments infected with a tumorigenic bacterial strain carrying pGPTV-HYG. These transformation experiments indicate that the Agrobacterium strains contain the binary vectors pKM4, or pKM5 that are capable of transforming rat5 mutant plants or a relatively high efficiency, and in wild type plants are twice as tumorigenic, and are better able to incite hygromycin-resistant calli, which are Agrobacterium strains that contain the "empty" binary vector pGPTV-HYG. Histone H2A transiently produced improves the efficiency of stable transformation of plants by Agrobacterium strains.

The rat5 mutant is deficient in the integration of the T-DNA. The Agrobacterium-mediated transformation of the mutant Agrobacterium rat5 results in a high efficiency of transient transformation but a low efficiency of stable transformation, as determined by the expression of the gusA gene encoded by the T-DNA. This result suggests that rat5 is more possibly deficient in the integration of T-DNA. To test this hypothesis, segments of root Ws and rat5 plants are directly inoculated with A. Tumefaciens GV3101 that carries the binary vector of T-DNA pBISNI. pBISNl contains a gusA-intron gene under the control of a "super promoter" (Ni et al., 1995, Narasimhulu et al., 1996). Two days after co-cultivation, the root segments are transferred to a medium that induces the callus containing timentin (100 μg / ml) to kill the bacteria. Three days after infection, some segments were stained for GUS activity using the chromogenic pigment X-gluc. The mutant both wild type and rat5 show high levels of GUS expression (approximately 90% of the root segments stained blue, Figure 3A). The remaining root segments are allowed to form calluses in medium that induces callus containing timentin to eliminate Agrobacterium, but lack any antibiotic for the selection of plant transformation. After four weeks, numerous calli derived from at least five different plants Ws and rat5 are stained with X-gluc. Of the Ws callus shown, 92 + 12% shows large blue dyeing areas, while only 26 ± 10% of the rat5 callus show GUS activity, and most of these regions stained blue are small (Figure 3A). These data indicate that although mutant rat5 can transiently express the gusA gene at high levels, it fails to stabilize gusA expression. The suspension cell lines were generated from these callus Ws and rat5 and after an additional month the amount of T-DNA is evaluated (using as a hybridization probe the gene gusA-intron located within the T-DNA of pBISNl) integrated in the DNA of the high molecular weight plant of callus Ws and rat5 (Nam et al., 1997; Mysore et al., 1998). Figure 3B shows that although T-DNA integrated into the genome of wild-type Ws plants is easily detected, the T-DNA integrated into the rat5 genome does not. These data directly demonstrate that rat5 is deficient in the integration of T-DNA. To demonstrate equal loading of the plant DNA in each of the lanes, the gusA probe is purified from the immunoprecipitate and re-hybridizes the immunoprecipitate with an ammonia phenylalanine-lyase gene probe from Arabidopsis (PAL). Figures 2A-2D show the complement of the rat5 mutant and the RAT5 over-expression in wild-type Arabidopsis plants. The maps of the binary vectors pKM4 (fig.2A) and pKM5 (fig.2B). RB, right end of the T-DNA; LB, left end of the T-DNA; pAnps, polyadenylation signal sequence of nopaline tape; Histone H2A, sequence encoding histone H2A gene RAT5; pH2A, histone RAT5 H2A gene promoter sequence; Pnos, promoter of nopalina tape; hpt, in resistant to hydromycin; pAg7, polyadenylation signal sequence of agropin tape; uidA, gen gusA less promoter. The arrows above histone H2A, undA, and hpt genes indicate the direction of transcription. (fig.2C) complementation of the rat5 mutant. Rat5 mutant plants are transformed with an Agrobacterium strain that contains the binary vector pKM4 (Atl012). The hygromycin-resistant transgenic plants are obtained and self-pollinated to obtain T2 plants. The sterile root segments of T2 plants express RAT5, wild type Ws plants, and rat5 mutant plants are infected with tumorigenic strain A. tumefaciens A208. Two days after co-cultivation, the roots are removed to the MS medium that lacks phytohormones containing timentin. Tumors are recorded after four weeks. (fig.2D) Tumorigenesis assay of Ws transgenic plants that over express histone H2A gene RAT5. Plants Ws A are transformed. Tumefaciens Atl012 that contains the binary vector pKM4. The hygromycin-resistant transgenic plants are obtained and self-pollinated to obtain T2 plants. Sterile root segments of T2 plants over-expressing the Ws RAT5 and wild-type plants were infected at a low bacterial density with A. tumefaciens A208. After two days of co-cultivation, the roots are removed to the MS medium that lacks phytohormones and contains timentin. Tumors are recorded after four weeks. Teratomas incite the roots of these plants that look similar to the tumors generated in the wild-type plant. One of the transgenic plants tested does not recover the phenotype of susceptibility to tumorigenesis, probably due to an inactive transgene. Transgenic IT plants of rat5 obtained by transformation with Atl062 (which contains a RAT5 genomic coding of the wild-type plant) are also tested for restoration of the tumorigenesis susceptibility phenotype. Some of these plants are also capable of recovering the phenotype of susceptibility to tumorigenesis, indicating the complement of the rat5 mutation. The hygromycin-resistant transgenic plants generated by transforming the rat5 mutant with pGPTV-HPT alone do not form tumors during infection with A. tumefaciens A208. Figure 3 shows the T-DNA integration assays of plants rat5 and Ws; (A) Transient and stable GUS expression in Ws and rat5; sterile root segments of plants Ws and rat5 are identified with the non-tumorigenic Agrobacterium strain GV3101 which contains the binary vector pBISNl. Two days after co-cultivation, the roots are transferred to the callus-inducing medium (MIC) containing timentin. Three days after infection, half of the segments stained with X-gluc to determine the efficiency of transient GUS expression. The other segment group is allowed to form calluses in CIM. After four weeks these calluses are stained with X-gluc to determine the efficiency of stable GUS expression. (B) T-DNA integration in rat5 and Ws plants. Cell suspension cells derived from the root segments Ws and rat5 infected with the non-tumorigenic Agrobacterium strain GV3101 containing the binary vector pBISNl are derived. The suspension cell lines that grow for three weeks (without selection for transformation) in the presence of timentin or cefotaxime to eliminate Agrobacterium. The genomic T-DNA is isolated from these cells, subjected to electrophoresis through a 0.6% agarose gel, stained on a nylon membrane, and hybridized with a gusA gene probe. After the autoradiography, the membrane is purified and re-hybridized with a phenylalanine ammonia-lyase gene (PAL) probe to determine the equal charge of DNA in each lane.

Table 1. Complementation of rat5 mutant and on RAT5 expression in wild type Arabidopsis (Ws) plants On expression of RAT5 in Ws (plants T2) ab at least 5 plants were tested for each mutant and 40-50 root bunches were tested for each plant b Agrobacterium was diluted to a concentration 100 times lower than the one normally used, and single root segments were separated EXAMPLES Example 1: Results that indicate the value of the use of the gene H2A-1 of histone Arabidopsis to improve the transformation of the plant. Evidence from two independent experimental lines shows that histone H2A-1 from Arabidopsis is useful to improve the efficiency of plant transformation mediated by Agrobacterium in dicotyledons and monocots. 1. Many Arabidopsis ecotypes and mutants are not easily transformed by a root transformation method (although they can still be transformed by the flower soaking method). The flower soaking method is used to introduce a histone H2A cDNA, under the control of the CaMV 35S promoter, in a large number of recalcitrant and mutant ecotypes. A number of these transgenic lines are analyzed and the evidence emerges that all ecotypes / mutants tested to date can be made competent for root transformation when they over express the H2A1 gene. These mutants include in the Agrobacterium binding process (ratl and rat3), integration of T-DNA (ratl7, ratld, rat20, and rat22), a chromatin mutant (HAT6), and several other mutants with as yet uncharacterized lesions ( rat21 and ratJ7). Additionally, several recalcitrant ecotypes can become more susceptible to transformation when the H2A-1 cDNA is over expressed. These include the Ag-0 and Dijon-G ecotypes. Other appropriate mutants include (rat4, ratl, ratl 5, ratJl, al, T9 and T16) and ecotypes (Cal-0, UE-1, Ang-0, Petergof, and BI-1) when they express the H2A-1 gene They may also be more susceptible to Ag obacter-mediated transformation. Two different Agrobacterium strains were tested. One contains a binary vector of T-DNA with a gene resistant to the herbicide in the T-DNA (this is the control construct). The other strain contains a similar T-DNA binary vector, but in addition to the herbicide-resistant gene, the T-DNA contains histone Arabidopsis h2A-1 cDNA under the control of the maize and intron adhl promoter. These strains were used in four rounds of corn transformation experiments. Usually, the transformation and regeneration of corn requires an anti-oxidant (such as L-cysteine) to prevent tissue numbness and necrosis in response to bacteria. Several hundred transformations (which use the control vector without the histone gene) virtually do not produce transformants. In these experiments, there are no transformants (using the control strain) without L-cysteine. With L-cysteine, about 2-3% of infected immature embryos give transformants. Using the histone and L-cysteine gene, there is 2-3% transformation. However, with the histone gene and without L-cysteine, 2 transformants (0.2%) are obtained. Preliminary results suggest that the histone gene can sensitize maize embryos for transformation so that some transformants can be obtained in the absence of an antioxidant. Example 2: Improved Agrobacterium-based transformation of monocotyledonous plants with a H2A gene. Embryos from wild type maize plants were transformed with Agrobacterium containing a histone H2A gene and an antibiotic resistant gene in the presence of L-cysteine in the co-cultivation medium. The transgenic IT plants (founding lines) are obtained their seeds (T2) are collected. The T2 plants are selected in a growth medium resistant to the antibiotic and are based on histone H2A RNA expression data. The selected T2 plant embryos (T2 embryos) were retransformed with Agrobacterium um containing a standard binary vector and a gene of interest in the presence of L-cysteine in the co-cultivation medium. An increase in the number of embryos that respond indicating an increase in transformation efficiency over the transformation using histones only. Table 2 shows the overall efficiency of the transformation [total assumptions per event / total that respond to the event].

The Agrobacterium-mediated transformation results in a lower transgene copy number and a higher gene expression compared to the biolistic maize gun transformation (Figure 9A-9B). The expression of the transgene is more stable also in transformants derived from Agrobacterium than in those derived from the bombardment (Figures 9C-9D). Total RNA was extracted from the leaves of transgenic plants twice independently, and charges are run in duplicate per extract. These were four data points for each plant sampled. Plant number 9 was incorrectly identified as transgenic; It has no expression of H2A-1 construction therefore it can be used as a backup control.

In general, the H2A-1 gene was expressed more highly in AlO lines (containing H2A) than in Al7 (which lacks H2A) (Figure 5). The expression levels between the individual plants differ considerably within the AlO and Al7 groups. The plant 2 line was more intensely hybridized, followed by the group of lines 5, 8, 6 and 7 (average), which were similar to each other (Figure 5). The remaining lines were less intensely hybridized. Example 3: Repetition of transformation-retransformation of the genotype Hi II of corn. The H2A gene of Arabidopsis under a constitutive promoter (promoter of corn ubiquitin) is introduced into the genotype Hi II of corn as described (Frame et al., 2002). They are obtained on 20 independent transgenic events. These are denoted as A10S1, followed by the sequential numbers.

The control construct (the DNA vector without the H2A gene) also includes the experiments. These are not denoted as A11S1, followed by sequential numbers. These transgenic maize lines are brought to maturity and crossed with the connected pollen from the B73 inbred line. For the retransformation experiments, the immature embryos of seven A10SlxB73 transgenic lines (expressing H2A) and 12 AllSlxB73 transgenic lines (control vector) are reinfected with a DNA construct (pTOK233) carrying the hyg gene of the selectable marker ( that encodes the resistance to hygromycin) and the gus gene of separable marker by exclusion. The surviving calli of the medium containing hygro icines are recorded for the presence of the gus gene. A total of 5 experiments are conducted. The transformation efficiency is calculated as a percentage of positive callus lines gus on 100 infected embryos. The average retransformation efficiency was 15% for Hi II transgenic lines expressing H2A and 9% for the control vector Hi II transgenic lines, indicating an increase in histone levels by the constitutive expression of the H2A gene in corn that increases the efficiency of transformation (table 3). Example 4: Repetition of transformation - retransformation using an inbred corn line B104. Inbred corn lines are useful in genetic analysis and to explore genotypic qualities for crosses. These inbred lines also provide a fundamental source for mutant analysis and for establishing genetic relationships. A collection of approximately 260 inbred lines of corn and their structures and genetic diversity is described in Liu et al. , (2003). Some of the inbred lines of aíz include A679, A680, B73, B84, B104, B109, NC328, NC372, R229, B57, C103, C123. , DE2, L317, L1546, NC258, and NC262. The transformation / re-transformation experiments are conducted in an inbred corn line B104. B104 is a public elite plasma gene that is recalcitrant for genetic transformation. The efficiency was generally as low as 0.5%. Histone levels increase in the B104 inbred line. Two lines A10S1 (expressing H2A) B104 or three lines A11S1 (vector control) B104 were retransformed with pTOK233 as described herein. The comparison for its efficiency is summarized in Table 4. The average efficiency of re-transformation was 2.3% for the transgenic B104 lines expressing H2A and 0.5% for the B104 transgenic lines of the vector control. This intensification is important for a better addition of agronomically important corn crossing lines. In a similar way, the transformation efficiency of other corn crossing lines can also be increased by first transforming with H2A and then transforming again with the gene of interest. Example 5: Use of a global regulator of the transcription of histone gene expression to intensify the transformation. The overexpression of histone H2A cDNAs enhances the transformation efficiency of Arabidopsis and corn. The transformation of tobacco BY-2 cells induces the expression of all four classes of neutral histone genes (H24, H2B, H3, and H4). Therefore, an increased expression of all histone gene classes can intensify the transformation. One method to induce many genes simultaneously is to express a global transcriptional regulator of these genes. In wheat, the transcription factor of bZIP HBP-la is linked to the hexamer portion (hex) ACGTCA that is within a larger type I element (CCACGTCANCGATCCGCG) in the promoters of histone genes. Although this hex portion appears in the promoters of other genes, HBP-la binds more strongly to this hex portion in the histone promoters. The cDNA for the HBP-la protein can be cloned behind a corn ubiquitin promoter plus the ubiquitin intron, and this construct can be introduced into binary vectors in Agrobacterium strains deficient in integration or efficient in integration. The Agrobacterium strains can then be used to infect suitable plant tissues and the resulting transgenic plants that over-express HBP-la are tested for increased transformation efficiency. Other transcription factors that promote expression of the histone gene can also be used to activate multiple histone genes. Example 6: Increase in transformation efficiency when activating the endogenous expression of the histone genes. An activation labeling construct can also be introduced to selectively activate one or more genes and histone endogenously. Activation labeling has been successfully applied using T-DNA inserts and the Ac-Ds transposon system. These inserts are designed to carry strong activation sequences that can act on the genes adjacent to the insertion sites and modify their expression pattern. The well characterized cauliflower mosaic virus (CaMV) in the 35S promoter or enhancer sequences has been used as transcriptional activators in this type of insertion system. Their primary role has been in expressing the labeled genes to reveal dominant phenotypes that gain function. In several studies, the CaMV 35S enhancer used in the T-DNA inserts acts by quantitatively increasing the original expression pattern of a gene rather than by an ectopic or constitutive gene expression. The En-I (Spm-dSpm) system of maize (Zea mays) is also an efficient tool for the heterologous labeling of the transposon in Arabidopsis or other plant species. Labeled activation lines of a desired plant species can be created using standard activation labeling vectors that employ CaMV35S or other equivalent activators. A tagged line of activation of a suitable plant species is separated by exclusion for enhanced expression of histone genes. A transgenic plant line expressing a higher level of histones can be subjected to transformation experiments. The increased expression of histone genes (increase in histone levels) promotes an increased transformation efficiency. Example 7: Increase in histone protein levels increase the transformation efficiency. The increase in histone protein levels either through the overexpression of a histone gene or through the biochemical addition of histone protein or by any other means can also increase the transformation efficiency of the plants. When a histone gene such as H2A is over expressed, the levels of endogenous histone are increased. An increase in endogenous histone levels increases the transformation efficiency of the host plants. Therefore, the exogenous application of histones through a non-genetic method can also increase the transformation efficiency in host plants. MATERIALS AND METHODS Nucleic acid manipulation. Total plant genomic DNA was isolated according to the method of Dellaporta et al. (1983). Restriction endonuclease digestions, agarose gel electrophoresis, plasmid isolation and DNA staining analysis were performed as described (Sambrook et al., 1982). Plasmid Rescue Genomic DNA (5 μg) of rat5 was digested to termination with Sali. The DNA digested with phenol / chloroform was extracted and precipitated with ethanol. The DNA was self-ligated in a final volume of 500 μl in 1 x buffering solution (Promega) with 3 units of T4 DNA ligase at 16 ° C for 16 hr. The ligation mixture was precipitated with ethanol, transformed into electrocompetent cells of E. coli DH10B (mcrBBBC-; Life Technologies, Inc., Gaithersburg, MD) by electroporation (25 μF, 200 O, and 2.5 kV) and plated on an LB medium containing ampicillin (100 μg / ml). The ampicillin-resistant colonies were raised on a nylon membrane, the bacteria were used and the DNA was denatured in situ (Sambrook et al., 1982). A labeled radio labeled left border (LB) sequence (3.0 kbp EcoRI fragment of pE1461) was used as a hybridization probe to identify a plasmid containing the LB. The positive colonies were collected and isolated in plasmid DNA. By analysis of a restriction fragment, a plasmid containing both LB and the plant binding DNA was identified. The plant binding fragment was confirmed by hybridizing the DNA-binding fragment of wild-type plant. A restriction map of this plasmid containing the binding DNA of the LB plant was made. A fragment of A 1.7 kbp EcoRI containing the plant DNA plus 75 base pairs of the LB sequence was subcloned into pBluescript resulting in pE1509. This fragment subsequently formed sequences at the sequence formation center of Purdue University. The growth of Agrobacterium and the in vitro root inoculation of Arabidopsis thaliana. These were performed as previously described by Nam et al. (1997). Growth conditions in plants. The seeds of various Arabidopsis thaliana ecotypes were obtained from S. Leiser and E. Ashworth (originally from the inventory center of Arabidopsis Nottingham, UK, and from the Arabidopsis Biological Resources Center, Ohio State University, Columbus, respectively). The seeds were sterilized on the surface with a solution composed of 50% commercial bleach and 0.1% SDS for 10 minutes and then rinsed five times with sterile distilled water. The seeds germinated in Petri dishes contained a Gamborg B5 medium (GIBCO) solidified with 0.75% bactoagar (Difco) The plates were initially incubated at 4 ° C for 2 days and for 7 days under a light period of 16-hrs-light / 8-hr-darkness at 25 ° C. The seedlings were individually transferred into jars of food to drink containing solidified B5 medium and grown for 7 to 10 days for root culture. Alternatively, the seedlings were transferred to the soil for inoculation of the shoots. Growth of Agrobacterium Tumefaciens. All Agrobacterium strains were grown in a YEP medium (Lichtenstein and Draper, 1986) supplemented with the appropriate antibiotic (rifampin, 10 μg / mL, kanamycin, 100 μg / mL) at 30 ° C. Bacterial cultures overnight were washed with 0.9% NaCl and resuspended in 0.9% NaCl at 2 x 109 colony forming units per L for in vitro root inoculation or at 2 x 1011 colony forming units per mL for inoculation of the shoots. The in vitro transformation and inoculation tests of roots. Roots were grown on the surface of the agar and excised and cut into small segments (-0.5 cm) in a small amount of sterile water and stained on sterile filter paper to remove excess water. For some experiments, the excised roots were pre-incubated in a callus inducing medium (MIC, 4.32 g / L Murashige and Skoog [MS] minimum salts [GIBCO], 0.5 g / L Month, pH 5.7 lmL / L reserve solution of vitamin [0.5 mg / mL nicotinic acid 0.5 mg / mL pyridoxine, and 0.5 mg / mL thiamin-HCl], 100 mg / L myoinositol, 20 g / L glucose, 0.5 mg / L 2,4 Dichlorphenoxyacetic acid, 0.3 mg / L kinetin, 5mg / L indoleacetic acid, and 0.75% bactoagar) for 1 day before cutting them into segments. The dry sets of root segments were transferred to a basal medium MS (4.32 g / L minimum salts MS, 0.5 g / L Month, pH 5.7, 1 mL / L solution of vitamin reserve 100 mg / L myoinositol, lOg / L of sucrose and 0.75% of bactoagar), and 2 or 3 drops of bacterial suspension were placed in them. After 10 minutes, most of the bacterial solution was removed and bacteria and root segments were grown together at 25 ° C for 2 days. For transient transformation assays the root assemblages were infected with the Agrobacterium strain GV3101 was used (Koncz and Schell, 1986) containing the binary vector pBISNl (Narasimhulu et al., 1996). After various periods of time, the roots were rinsed with water, stained on filter paper and stained with X-gluc staining solution (50 mM NaH2HP04, 10 mM Na2 EDTA, 300 M mannitol, and 2 mM X-gluc, pH 7.0) for one day at 37 ° C. For quantitative measurements of β-glucuronidase (GUS) activity, roots were ground in a microcentrifuge tube containing GUS extraction buffer (50 mM Na2HP04, 5 mM DTT, 1 M Na2 EDTA, 0.1% sarcosil, and 0.1% Triton X-100, pH 7.0), and GUS specific activity was measured according to Jefferson et al. (1987). To quantify tumorigenesis, arrays of roots were infected with wild-type Agrobacterium strains. After 2 days, the root assemblies were rubbed on the surface of the agar to remove excess bacteria and then washed with sterile water containing timentin (100 μg / mL). The individual root segments (initial test) or small root set (5 to 10 root segments, modified assay) were transferred onto an MS basal medium that lacks hormones but contains (100 μg / mL) and was incubated by 4 weeks . For the transformation of root segments to resistance to kanamycin, the root assemblages were inoculated with the Agrobacterium strain GV3101 containing pBISNl. After 2 days, small root sets (or individual root segments) were transferred onto the MIC containing timentin (100 μg / mL) and kanamycin (50 μg / mL). The kanamycin-resistant calli were marked after 4 weeks of incubation at 25 ° C. To determine the stable expression of GUS, the roots were inoculated as given above and the root segments were transferred after 2 days to the timentin containing MIC (100 μg / mL) without any selection. After 4 weeks, GUS activity was tested either by staining with X-gluc or by measuring the specific activity of GUS when using the fluorometric assay of 4-methylumbelliferil β-D galactoside (MUG) as described above. To determine the kinetics of GUS expression, the root assemblages were infected, the root segments were transferred after 2 days to timentin containing MIC (100 μg / mL), and the calli were grown in MIC without selection. Root assemblies were tested at various times using a MUG fluorometric assay as described above to measure specific GUS activity. System Construction Simple Binaries.

The based constructs are optimized by monocotyledons (for example they use promoters of monocotyledons and enhancing introns) and contain proven markers of selection and separation by exclusion (for example bar, gusA, gfp). The gusA gene in all constructs contains an intron to avoid expression in Agrobacterium. A closure optimized by corn codon changed into synthetic red sgfp (S65T) is (Chiu et al., 1996). The CaMV 35S promoter (double promoter region) is used to boost markers that are separated by exclusion. The bar gene is a marker by selection (DeBlock et al., 1987). An intron promoter of corn ubiquitin (Ubi-1) is used to boost the expression of the bar gene (Christensen and Quail, 1996). The vectors are designed to reduce / eliminate the presence of repeated sequence within the constructs. If another promoter is needed, the "super promoter" is adequate (Ni et al., 1995). This promoter works well in corn has no homology with the CaMV 35S or ubiquitin promoters of corn, and is freely available to license from the Biotechnology Research and Development Corporation. T-DNA border sequences and multiple cloning sites are included in the base constructs.

Construction of the Ordinary Vectors pKM4 and pKM5. Plasmid pE1509 containing the 1.7 kbp binding fragment cloned into pBluescript was digested with EcoRI to release the binding fragment. The ends of the 5 'overlay are filled by using the Klenow fragment of DNA polymerase I and the deoxynucleotide triphosphates. The binary vector of T-DNA (pElOll) pGTV-HPT (Becker et al., 1992) was digested with the Sacl and Smal enzymes, releasing the non-promoter gene gusA from pGTV-HPT. The 3 '-sequence sequence of the major fragment containing the origin of replication and the hygromycin resistance gene (hpt) were removed using the 3'-5' exonuclease activity of the Klenow DNA polymerase, and the terminal fragment closed in its blind parts 1.7 kbp resultant is bound to the ends in its parts taps of the binary vector. A vector plasmid of the binary vector containing the 1.7 kbp fragment in the correct orientation (pAns downstream of the histone H2A gene) was selected and named pKM4 (strain E1547). A fragment of approximately 9.0 kbp genomic wild-type Sacl that contains the histone H2A gene (RAT5) from a lambda genomic clone was cloned into the Sacl site of the pBluescript plasmid. This 9.0 kbp Sacl fragment was subsequently released from pBluescript by digestion with Sacl and was cloned into the Sacl site of the binary vector pGTV-HPT, resulting in plasmid pKM5 (strain E1596). Both pKM4 and pKM5 were separately transferred by a triparental coupling (Ditta et al., 1980) within the non-tumorigenic strain of Agrobacterium GV3101, resulting in strains of A. tumefaciens Atl012 and Atl062, respectively. Transformation of the germinal line of Arabidopsis. Transformations of the germ line were carried out as described in (Bent and Clough, 1998). Transgenic plants were selected in n medium B5 containing hygromycin (20 μg / ml). Vector and strain of Agrobacterium tumefaciens. The A. tumefaciens strain of EH101 (Hodd et al., 1986) containing the standard binary vector pTF102 (12.1 kb) was used in all experiments. The T-DNA region of 5. 6 of this construct is shown in figure 1. The vector is a derivative of the binary vector pPZP (Haj dukiewicz et al., 1994) which contains the border fragments of right and left T-DNA from a nopaline strain of A. tumefaciens, a broad host origin of replication ( pVSl a spectinomycin resistant marker gene (aadA) for bacterial selection The CaMV 35S promoter (P35S) was used to boost both the selection marker gene in bar and the reporter gene gus The translational enhancer of the tobacco etch virus (Carrington and Freed, 1990) was included in the 5 'termination of the bar gene. The vegetative storage protein terminator of soybean (Glycine max L. Merrill) (Mason et al., 1993) was cloned into the 3 'end of the bar gene. The gus gene contained a portable intron in its codon region (Vancanneyt et al., 1990) to prevent GUS activity in A. tumefaciens cells. This vector system, pTF102 in EHA101, was maintained in a medium of pectone yeast extract (YEP) (An et al., 1998) containing 100 mg / L of spectinomycin (for pTF102) and 50 mg / L of kanamycin ( for EHA101). Bacterial cultures for the weekly experiments were initiated from reserve plates that were stored for up to 1 month at 4 ° C before being renewed from long-term glycerol stocks at 80 ° C. In all experiments, densities of bacteria cells were adjusted to an optical density (OD55o) between 0.35 to 0.45 using a spectrophotometer immediately before embryo infection. Plant Material Immature zygotic embryos F2 (1.5-2.0 mm) of maize (Zea mays) Hl II hybrid genotype (Armstrong et al., 1991) were dissected aseptically from ears grown in greenhouse harvested 10 to 13 d after pollination The ears were stored up to 3 d at 4 ° C before dissection. Means The means of infection, co-culture, resting and selection were after Zhaso et al. (1999) except that the co-culture medium was modified to contain Cys. All these media contained salts and vitamins N6 (Chu et al., 1975), 1.5 mg / L of 2,4-dichlorophenoxyacetic acid and 0.7 g Ll L-Pro in addition to the following ingredients: infection medium containing 68.4 g / L Suc and 36g / L Glc (pH 5.2) and supplemented with 100 μM AS (Sigma, St. Louis) before use; co-culture medium containing 30 g / L Suc, 0.85 mg / L silver nitrate, 100 μM AS, and 3 g / L gelrite (pH 5.8); resting media containing 30 g / L Suc, 0.5 g / L MES, 0.85 mg / L silver nitrate, 250 mg / L cefotaxin and 8 g / L purified agar (pH 5.8). The selection medium was identical to the resting medium with the addition of 1.5 to 3 mg / L bilafos (Shinyo Sanyo, Tokyo) The infection medium was sterilized by filter while all other media were treated by autoclaves. AS (100 mM) stock solutions were prepared by dissolving AS in 100% (v / v) of dimethyl sulfoxide (DMSO) to make a 200 mM stock which was then diluted (1: 1 [v / v]) with sterile water and stored in small aliquots at 20 ° C. Cys was added to the coculture medium after autoclaving from freshly prepared sterile filter stocks (100 mg / mL) and the co-culture medium was used within 2 to 5 d of preparation. The regeneration medium I contained the Murashige and Skoog salts and vitamins (Murashige and Skoog, 1962), 60 g / L Sic, 100 mg / L myo-inositol, without hormones and 3 g / L gelrite (pH 5.8) after Armstrong and Green (1985). Cefotaxime (250 mg / L and bialaphos (3mg / L) were added to this medium after autoclaving.Regeneration medium II differed from medium I containing 30 g / L suc and none of bilaf. Inoculation and cocultivation The cultures of A. tumefaciens were grown for 3 days at 19 ° C in a YEP medium amended with 100 mg / L of spectinomycin and 50 mg / L of kanamycin. A complete circuit (3 mm) of bacterial culture was scraped off from plates 3 days old and suspended in 5 mL of liquid infection medium (Inf) supplemented with 100 μM AS (Inf + AS) in a 50 falcon tube. The tube was fixed horizontally to a bench stirrer with a Vortex Genie platform head and was shaken at low speed (approximately 75 rpm) for 4 to 5 hours at room temperature This preinduction step was carried out for all For the infection, immature zygotic embryos were dissected (1.5-2.0 mm) to Inf + AS bacteria free medium (18 mL) in 2 mL eppendorf tubes (20-100 embryos per tube) and washed twice with this medium. The final wash was removed and 1 to 1.5 mL of a suspension A. Tumefaciens was added to the embryos. Embryo infection was achieved by gently inverting the tube 20 times before resting it vertically for 5 minutes with the embryos submerged. The embryos did not form vortices at any time during this procedure. After infection, the embryos were transferred to the surface of the co-culture medium and pipetted to remove the suspension in excess of A. tumef ci ens from the surface of the medium. The co-culture medium contained 4000 mg / L Cys unless stated otherwise. In the experiments in which the treatments with coculture medium were compared, the embryos were washed and infected in the same tube before being distributed between the media treatments. The embryos were oriented with the side of the embryo axis in contact with the medium (side of the scutellum upwards). The plates were wrapped with vending tape (Vallen Safety Supply, Irving, TX) and incubated in the dark at 20 ° C or 23 ° C for 3 days, after which the embryos were transferred to 28 ° C in a medium of rest.

The response of the embryos (%) was measured as the number of immature zygotic embryos cultured together that had initiated the formation of type II embryogenic callus at its base of scutellum after 4 to 7 days on a resting medium compared to the total number formed on plates. All embryos, with or without response, were transferred to the selection medium. Selection and Regeneration After 4 to 7 days in the resting medium (28 ° C, darkness), the embryos were transferred to the selection medium (30 per plate) containing 1.5 mg / L bialaphos. The selection was increased to 3 mg Ll bialaphos 2 weeks later. The supposedly transformed events were identified as early as 5 weeks after infection. The regeneration of the transgenic plants RO from the embryogenic callus of type II was achieved by a maturation stage of 2 to 3 weeks in a regeneration medium 1 followed by germination in the light of the regeneration medium II as described by Frame et al. (2000). The stable transformation efficiency (%) was calculated as the number of callus events resistant to bialaphos recovered per 100 infected embryos. Acclimatization and greenhouse care of transgenic plants Transplanting and acclimation of R0 regenerated plants was achieved as previously described (Frame et al., 2000). The transgenic plants were grown to maturity in the greenhouse. Statistical Analysis Data from 8 independent experiments were used to compare the stable transformation efficiency from pairs of similarly treated plates in addition to Cys exposure during cocultivation. A sign test was used to determine if the benefit in the rate of recovery of the transgenic event observed for the 400 mg / L Cys treatment was significantly greater than that by the treatment with 0 mg / L Cys. A Chi square test was used to determine if the segregation relationships observed for the progeny plants expressing the gus and bar gene are adjusted to the ratio, expected 1: 1. Histochemical analysis of transient and stable gus expression The GUS histochemical assays (Jefferson, 1987) were used to evaluate the transient expression of the gus gene in immature zygotic embryos 1 or 2 days after the 3-day joint culture (4 or 5 days after the infection) . The level of transient gus expression was evaluated on an embryo basis by estimating the number of blue foci visible on the scutellum side of each embryo. The embryos that display the blue foci only on the side of the embryo axis of the explant were recorded as non-expressing. The embryo was then categorized as follows: without expressing (without blue foci), low expression (one-25), moderate expression (26-100) or high expression (>101). The number of embryos in each of these four groups was compared with the total number of embryos evaluated to determine the percentage of total embryos in each of the expression categories. GUS histochemical assays were also used to evaluate the stable expression of the GUS gene in callus samples resistant to bialaphos and leaf tissue of transgenic plants in the Rl and R2 generations. Leaf segments (0.5 cm) were submerged in the substrate, infiltrated under vacuum (20 inches of mercury) for 10 min; and incubated at 37 ° C overnight. Cells stained in blue were visualized by rinsing leaf tissue in 75% followed by 95% (v / v) ethanol to remove chlorophyll and leaf pieces marked as positive or negative for GUS expression. Southern Blot Analysis The genomic DNA of the leaflet was prepared from 2 to 3 g of fresh leaf tissue from putative transgenic maize plants using cetyltrimethylammonium bromide (C ) as a method as described by Murray and Thompson. (1980). 10 micrograms of genomic DNA per sample were digested by the restriction enzyme HindIII at 37 ° C overnight and separated on an agarose gel of 0.8% (w / v). Gel-Blot analysis of DNA (Sambrook et al., 1989) was carried out on DNA samples using the bar or gus fragments labeled with 32P as shown in figure 1. Progeny segregation analysis for the expression of the bar bar A glufosinate spray test (Brettschnei der et al., 1997) was used to establish the segregation relationships for the expression of the bar gene in the progeny. The herbicide Liberty (Avent i s, Strasbourg, France) was dissolved in water (1.25 mL / L) together with 0. 1% (v / v) Tween 20 for a final glufosinate concentration of 250 mg / L. Beginning 9 days after planting, the seedlings were sprayed 3 times at 1 to 2 day intervals with a freshly prepared glufosinate solution and then labeled for herbicide (live) or herbicide (dead) sensitivity. Retransformation experiments A. T2 immature zygotic embryos The final results for experiments on T2 embryos from plants derived from IT seeds of an AlO event and an All event were presented in the last quarter. This method to evaluate the relative transformation of the AlO vs All events was generally unsuccessful due to the poor seeds affixed to the cobs gh T2.

B. Calluses A10S1 and A11S1 All the supposed events collected from this callus bombing experiment have been tested with gus and no blue was observed in any of the events. It is noted, however, that this construct of the Nobel Foundation, which carries the gus gene based on the map, also produced no transients (although the positive control pBGF throughout bb did so). These supposed events were maintained in hygromycin 50 or 75 mg / L per week without dying but without being convincingly real (the clones are collected for section C below they are as transparent as a corduroy). BF took a subset of the best view for regeneration to see if they hardened. At present, it is likely that they will recover from a few to no clones of this massive amount of work. Although no conclusions can be drawn about the benefit for the biolistic transformation of H2A protein expression in these lines, it can be concluded that the selection of hygromycin for fast growing callus needs to be applied early and strongly to minimize excessive growth during the treatment. later selection. C. AlO and All IT Embryos The assumed clones of the IT embryos of AlO plants (experimental and All (control) TO returns to transform with pTOK233 have been collected, tested by gus and some named this quarter.

The number of ears per event AlO and All, the numbers of embryos per infected ear and the number taken for the selection of hygromycin after resting the embryos in 2 mg / L bialaphos to exterminate the embryos without segregation are summarized in Table 1. We have named 33 AlO assumptions and 23 assumptions All of which 15/17 and 10/22 of those tested to date are positive for gus respectively. When using the number of embryos that respond after resting in the medium containing 2 mg / L bialaphos (to kill the IT embryos that do not secrete AlO or All), the overall efficiency for the retransformation of A10S1 using A18S6 was 33 events / 401 infected, embryos without dying (8.2%), and for A11S1 it was 23 events / 753 embryos (3%). (Table 2). This suggests that on the 16 ears of A10S1 and the 17 ears of A11S1, the average transformation efficiency was higher for the AlO than for the All in this selection. Table 2: Summary of the retransformation experiment to A11S1-27 NONE 261 136 1.5 X B73 A11S1-30 NONE 275 184 3.8 X B73 Table 3: Comparison of the efficiency of retransformation in a genotype of Hi II corn with or without the H2 gene Table 4: Comparison of the efficiency of retransformation in an inbred line (maize B104) with or without the H2A gene DOCUMENTS CITED The following documents are incorporated by reference to the extent permitted by the present invention: Armstrong CL, Green CE (1985), stablishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline. Plant 164: 207-214 Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high type II culture formation response. Maize Genet Coop Newslett 65: 92-93 Bailas, N. & Citovsky, V. Nuclear localization signal binding protein from Arabi dopsi s mediates nuclear import of Agrobac terium VirD2 protein. Proc. Nati Acad. Sci. USA 94, 10723-10728 (1997). Bent, A. F. & Clough, S.J. in Plant Molecular Biology Manual, (eds Gelvin, S.B. &Verma, D. P.S.) vol. 3, p. B7 / 1-14 (Kluwer Academic Publishers, Netherlands, 1998). Brettsclmeider R, Becker D, Lorz H (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94: 737-748 Carrington JC, Freed DD (1990) Cap-independent enhancement of translation by a plant potyvirus 5 'nontranslated region. J Virol 64: 1590-1597 Chiu et al. (1996) Curr. Biol. 6: 325-330 Christensen and Quail (1996) Transgenic Res. 5: 213-218 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen source. Sci Sin 18: 659-668 DeBlock et al. (1987) The EMBO J 6: 2513-2518 Dellaporta, S.L., Wood, J., & Hicks, J. B. Plant Mol. Biol. Rep. 1.19-22 (1983). Ditta, G., Stanfield, S., Corbin, D., & Helinski, D. R. Proc. Nati Acad. Sci USA 77,7347-7351 (1980). Enriquez-Obregon GA, Prieto-Samsonov DL, of the Riva GA, Pérez M, Selman-Housein G, Vazquez-Padron Rl (1999) Agroiacterium-ediated Japan rice transformation: a procedure assisted by an anti-necrotic treatment. Plant Cell Tissue Organ Cult 59: 159-168 Frame B, Zhang H, Cocciolone S, Sidorenko L, Dietrich C, Pegg S, Zhen S, Schnable P, Wang K (2000) Production of transgenic maize from bombarded Type II callus. effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol Plant 36: 21-29 Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium and vectors for plant transformation. Plant Mol Biol 25: 989-994 Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervírulence of Agrobacterium tumefaciens A281 is encoded in the region of pTiBo542 outside of T-DNA.

J Bacteriol 168: 1291-1301 Jefferson RA (1987) Assaying chimeric genes in plants. The gus gene fusion system. Plant Mol Biol Rep 5: 287-405 Jefferson, R. A. m Kavanagh, T. A., Bevan, M. W. GUS fusions -. Beta-glucuronidase as to sensitive and versitile gene fusion marker in higher plants. EMBO J. 6, 391-3907 (1987). Koncz, C. and Schell, J. Mol. Gen. Genet. 204, 383-396 (1986). Lichtenstein, C. and Draper, J. Genetic engeneering of plants. In Glover, D. M. (ed.) DNA Cloning: A Practical Approach, vol. 2, pp. 67-119 (IRL Press, Oxford, 1986). Liu et al (2003) Genetic structure and diversity among maize inbred lines as inferred from DNA icrosatellites. Genetics 165 (4): 2117-28. Mason HS, DeWald D, Mullet JE (1993) Identification of a methyl j asmonate-responsive domain in the soybean vspB promoter. Plant Cell 5: 241-251 Meshi et al., (1998) Conserved Ser residues in the basic region of the bZIP-type transcription factor HBP-la (17): importance in DNA binding and possible targets for phosphorylation. Plant Mol Biol. 36 (1): 125-36. Mikami et al., (1995) Developmental and tissue-specific regulation of the gene for the wheat basic / leucine zipper protein HBP-la (17) in transgenic Arabi dopsi s plants. Mol Gen Genet. 248 (5): 573-82. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobáceo tissue cultures. Physiol Plant 15: 473-497 Murray MG, Thompson WF (1980) Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8: 4321-4325 Mysore, K.S., Yi, H.C. & Gelvin, S. B. Molecular cloning, characterization and mapping of histone H2A genes in Arabi dopsi s. In preparation. Mysore, K. S. et al. Role of the Agroba c terium tumefaciens VirD2 protein in T-DNA transfer and integration. Mol. Plant-Microbe Interact. 11, 668-683 (1998). Nam J. et al. Identification of T-DNA tagged Arabi dopsi s mutants that are resistant to transformation by Agrroj acteriuip. Mol. Gen. Genet. 261, 429-438 (1999). Nam, J., Matthysse, A. G. & Gelvin, S. B. Differences in susceptibility of Arabi dopsi s ecotypes to crown gall disease may result from a deficiency in T-DNA integration. Plant Cell 8, 873-886 (1997). Narasimhulu, S.B., Deng, X. -B. Sarria, R. & Gelvin, S. B. Early transcription of Agrobacterium um T-DNA genes in tobaceous and maize. Plant Cell 8,873-886 (1996). Ni, M. et al. Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J. 7,661-676 (1995). Sambrook, M.A., Fritsch, E. F. , & Maniatis, T. (1982) in Molecular cloning: A laboratory manual. 1st st. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) Sambrook J, Fritsch EF, eds (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Vancanneyt G, Schmidt R, O 'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrrojbacterium-mediated plant transformation. Mol Gen Genet 220: 245-250 Zhao ZY, Gu W, Cai T, Pierce DA, inventors. Nov. 9,1999. Methods for Agrrojbacterium-mediated transformation. United States Patent No. 5,981, 840 Zhao ZY, Gu W, Cai T, Tagliani LA, Hondred DA, Bond D, Krell S, Rudert ML, Bruce WB, Pierce DA (1998) Molecular analysis of TO plants transformed by Agrobacterium and comparison of AcjroJbacterium-mediated transformation with bombardment transformation in maize. Maize Genet Coop Newslett 72: 34-37 It is noted that with this date, the best method known to the applicant to carry out the practice of said invention, is that which is clear from the present description of the invention.

Claims (28)

Claims Having described the invention as above, the content of the following claims is claimed as property.

1. A method for increasing the transformation efficiency of Agrobac terium in a monocotyledonous host plant, characterized in that it comprises: (a) an increase in histone levels in the host plant compared to normal levels of histone in the host plant; (b) infecting the host plant with a DNA molecule of interest by infection with a strain of Agrobacterium. (c) providing at least one antioxidant in a co-culture medium and; (d) transform the host plant with Agrobacterium um.

2 . The method according to claim 1, characterized in that the histone is a histone H2A.

3. The method according to claim 2, characterized in that the histone H2A is encoded by Arabi dopsi s RAT5.

4. The method according to claim 1, characterized in that the host plant is corn.

5. The method according to claim 4, characterized in that the corn belongs to an inbred line.

6. The method according to claim 2, characterized in that the histone H2A is H2A-1.

7. A cell of a transgenic monocot plant characterized in that it comprises an increased level of histone sufficient to increase the transformation efficiency of the plant cell by Agrobac terium in comparison with a wild-type monocot plant cell.

8. The plant cell according to claim 7, characterized in that the histone belongs to a family of histone H2A.

9. The plant cell according to claim 8, characterized in that a histone H2A is encoded by Arabi dopsi s RAT5.

10. A method for increasing the transformation efficiency by Agrobac terium in a host monocot plant, characterized in that it comprises: (a) introducing at least one copy of a polynucleotide sequence encoding a plant histone protein to the host plant; (b) selecting a host plant that expresses the polynucleotide sequence encoding a histone protein of the plant; and (c) transforming the host plant expressing the polynucleotide sequence encoding a histone protein of the plant with a DNA molecule of interest using Agrobac terium by supplying at least one antioxidant in a co-culture medium.

11. The method according to claim 10, characterized in that the monocotyledonous plant is corn.

12. The method according to claim 11, characterized in that the corn belongs to an inbred line. The method according to claim 10, characterized in that the polynucleotide sequence encoding a histone protein of the plant is a member of a family of the Arabidopsis H2A gene. 14. The method according to claim 13, characterized in that the member of the family of the H2A gene of the Arabi dopsi is RAT5. 15. The method according to claim 10, characterized in that the antioxidant is L-cysteine. 16. A transgenic monocot plant comprising at least one exogenous copy of a polynucleotide sequence encoding a histone H2A protein of a plant characterized by being capable of increasing transformation efficiency. 17. A method for increasing the stable transformation efficiency of Agrobac terium in monocotyledonous host plants, characterized in that it comprises: (a) selecting a host plant that expresses a polynucleotide sequence encoding a plant histone H2A protein; (b) infecting the host plant with a DNA molecule of interest by infection with a strain of Agrobacterium; (c) providing at least one antioxidant in a co-culture medium; and (d) selecting the infected cells for the transformants expressing the DNA molecule of interest. 18. The method according to claim 17, characterized in that the monocotyledonous plant is corn. 19. The method according to claim 17, characterized in that the antioxidant is L-cysteine. 20. The method according to claim 19, characterized in that the L-cysteine is used at a concentration of about 100 mg / L up to 400 mg / L of the co-culture media. 21. The method according to claim 17, characterized in that the infection of the host plant in the co-culture medium is for about 3 days. 22. The method according to claim 17, characterized in that the host plant is in an embryo stage. 23. A method for increasing the transformation efficiency of Agrobac terium in a host plant, characterized in that it comprises: (a) transforming a host plant with a transcription activator of a histone gene; (b) selecting a transgenic host plant that overexpresses at least one histone gene; and (c) transforming the transgenic host plant overexpressing at least one histone gene with a DNA molecule of interest. 24. The method according to claim 23, characterized in that the host plant is a monocotyledonous plant. 25. The method according to claim 23, characterized in that the transcription factor is a transcription factor of the b-ZIP family. 26. The method according to claim 25, characterized in that the transcription factor of b-ZIP is HBP-la. 27. A method for increasing the transformation efficiency in a host plant, characterized in that it comprises: (a) transforming the host plant with an activator. (b) separating by exclusion a progeny of the transformed host plant to increase expression of the histone gene; and (c) transforming the progeny of the host plant that has increased expression of the histone gene with a 7DNA molecule of interest. 28. The method according to claim 27 'characterized in that the activator is a CaMV 35S enhancer element.