BR122017004832B1 - Transgenic Plant Production Process - Google Patents

Description

(54) Title: PROCESS OF PRODUCTION OF TRANSGENIC PLANTS (73) Holder: MONSANTO TECHNOLOGY LLC, Legal entity. Address: 800 NORTH LINDBERGH BOULEVARD, SAINT LOUIS, MO 63167, UNITED STATES OF AMERICA (US), North American (72) Inventor: ANISHA AKULA; DAVID R. DUNCAN; BRENDA LOWE; MICHAEL T. MANN; WILLIAM L. PETERSEN; JYOTI R. ROUT; DAVID D. SONGSTAD; JOEL B. WILKS; WANGGEN ZHANG

Validity Term: 10 (ten) years from 10/02/2018, subject to legal conditions

Issued on: 10/02/2018

Digitally signed by:

Liane Elizabeth Caldeira Lage

Director of Patents, Computer Programs and Topographies of Integrated Circuits

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Invention Patent Descriptive Report for

PROCESSES OF PRODUCTION OF TRANSGENIC PLANTS. Divided from PI0716373-8, deposited on 08/31/2007.

Background to the Invention [001] This patent application claims the priority of provisional US Patent Application No. ² Series 60 / 841,519 filed on August 31, 2006, the entire description of which is incorporated herein by reference.

Field of Invention [002] The micropropagation of plants has been done routinely in large batches and in an automated way. In micropropagation, an explant is usually collected and placed in a regeneration medium that has to be kept fresh for the duration of the regeneration process to produce a series of plants. This is in contrast to the transformation processes, which are designed to produce new transgenic events and require the integration of foreign DNA within a plant cell. The automation of the plant tissue culture process, particularly the transformation process, has been difficult. Plant tissues go through different stages that require different types of growth media and conditions. Transformation processes require several steps and means. For example, in Agrobacterium-mediated transformation, the process begins with the isolation of an explant that can be regenerated and transformed. Then the explant is inoculated with Agrobacterium in an inoculation medium. After inoculation, excess Agrobacterium is typically removed and the explant and

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Agrobacterium are co-cultivated together to allow DNA transfer. After co-cultivation, the presence of Agrobacterium is deleterious for plant tissue culture (for example, it causes unwanted contamination during the subsequent manipulation and tissue culture steps), so that explants are typically transferred to fresh medium containing antibiotics for inhibit the growth of Agrobacterium. This medium may or may not contain selection agents. If not, then it is called a means of delay or rest. The explants can be placed in a delay medium to allow them to grow for some time before being optionally placed in the selection medium. Other protocols place explants directly in the selection medium for the selection of transgenic events. Selection regimes vary widely depending on the selection agent and the explant system. Multiple selection steps are often used and varying amounts of selection agent may be required in different steps. After the selection of the transgenic events, the transgenic live events are then transferred to the regeneration medium in seedlings which can then be transferred to the soil. To date, transformation processes have been time-consuming and laborious and are not capable of being carried out on a large scale. Automating the transformation process would allow large numbers of transgenic plants to be produced with reduced labor, material and ergonomic overhead.

[003] The present invention has overcome the previous limitations in transformation by providing methods and apparatus for carrying out some or all of the transformation steps and optionally some of the regeneration steps, in a single container. Thus, the present methods have overcome the deficiencies of the current transformation protocols by eliminating steps

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3/65 long for subculture of plant tissue and change of medium. The methods and apparatus are particularly suitable for the automation of transformation, for the automation of regeneration and / or for the large-scale production of transformed cells, tissues and plants.

[004] The invention of genomics enabled the identification and isolation of a large number of genes and had the need for reliable and efficient high-throughput production systems to test the usefulness of these genes by transforming them into crop plants economically important such as maize. Current corn processing methods require at least four processing steps from the stage of selecting a transformed cell to the stage of transferring transgenic plants to the soil, thus requiring higher material costs, for example, plates and media. culture and labor costs. Several manual tissue transfers also assess the risk of ergonomic damage from repeated movement.

[005] Thus, there is a need in the corn transformation technique for a high-performance automated system for the transformation, selection and regeneration of plants that produces a large number of transgenic plants to test genes and create useful plants while costs materials and labor are reduced. There is still a need in the art for methods that can decrease the risk of ergonomic damage by making the workplace safer.

[006] Here, the inventors provide a method of maize transformation for the selection and regeneration of transformed maize plants suitable for the high-throughput automation system. The method employs a suitable support matrix in combination with selection and liquid regeneration medium. The use of this liquid culture method eliminates the need for several transPetition 870170015706, from 10/03/2017, p. 12/86

4/65 injuries that are normally needed when a solid medium is used for the selection and regeneration steps. In addition, this method makes it possible to increase regeneration, which has been a problem in the liquid culture medium until now. In addition, the step of selecting a transformed cell and regeneration can be accomplished in a single container such as a sundae cup until the plants are transferred to the soil.

Summary of the Invention [007] The present invention provides new methods for automating plant transformation processes by providing a stable transformation method and optionally selecting and partially regenerating a plant in a container. In some embodiments, a single container can be used to carry out the methods of the present invention. In other embodiments, various containers (for example, an interconnected vessel system) can be provided to facilitate the use and optimization of the present invention.

[008] In one aspect of the invention, a method for producing transgenic corn plant is provided. The method comprises obtaining a corn explant that can be transformed; the transformation of the corn explant that can be transformed; the selection of a transformed corn cell starting from the corn explant that can be transformed into a selection medium; and the regeneration of the cell transformed into a plant in a regeneration medium, in which the transformation, selection and regeneration are carried out in the same container. Optionally, the selection in the container can be omitted and the transgenic plant or the transgenic plant can be subjected to selection after being placed in the soil (for example, sprayed with a selection agent).

[009] The container can be a bioreactor, a petri dish,

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5/65 a multi-cavity plate, a flask, a jar, a bottle, a moringa, a PlantCon®, a temporary immersion system and a combination thereof and is provided with a way to supply and remove the medium. In some embodiments, the container is a Petri dish, a multi-well plate, a PlantCon or a temporary immersion system.

[0010] The explant can be selected from the group consisting of a callus, an embryo and a cell suspension.

[0011] The medium can be a liquid medium, a solid medium or a combination thereof. In one embodiment, the selection medium is a solid medium and the regeneration medium is a liquid medium that covers the solid selection medium.

[0012] The explant can be placed in contact with the medium temporarily. In one embodiment, the explant is placed in contact with the selection medium and the regeneration medium for approximately 1 to approximately 5 minutes approximately every 12 to 24 hours.

[0013] In yet another aspect of the present invention, a method is provided for obtaining an explant for the production of a transgenic corn plant. The method involves dividing a callus into smaller pieces of callus. In one embodiment, the callus is a type I callus.

[0014] In yet another aspect of the present invention, a method is provided for preparing a suspension of Agrobacterium cells for the inoculation of an explant. The method involves cultivating a frozen glycerol stock of Agrobacterium directly in an induction medium.

[0015] In yet another aspect of the present invention, a corn cell culture is provided that comprises cells that can be transformed and embryogenic.

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[0016] In yet another aspect of the present invention, a method is provided for producing a culture of corn cells that can be transformed and embryogenic. The method comprises obtaining a callus from corn embryos and cultivating the callus for approximately 5 days to 30 days in a liquid medium to produce the cell culture. In one embodiment, the callus is a type II callus.

Brief Description of the Drawing [0017] Figure 1 Example of plasmid map of pMON30113. Detailed Description [0018] The following definitions will assist in understanding the description of the invention.

[0019] Callus refers to a proliferating mass of differentiated cells or tissue.

[0020] Explant refers to a part of the plant that is capable of being transformed and subsequently regenerated into a transgenic plant. Typical explants include immature embryos, calluses, cotyledons, meristems, leaves or stems.

[0021] Tissue culture medium refers to liquid, semi-liquid or solid media used to support plant growth and development in an environment other than the soil. Suitable plant tissue culture media are known to one skilled in the art, as discussed in detail subsequently. The components of the medium can be obtained from suppliers other than those identified here and can be optimized for use by those skilled in the art according to their requirements.

[0022] Coding sequence, coding region or open reading frame refers to a region of continuous sequential nucleic acid triplets encoding a protein, polypeptide or peptide sequence.

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7/65 [0023] Endogenous refers to materials that originate from within the organism or cell.

[0024] Exogenous refers to materials that originate from the outside of the organism or cell. Refers to nucleic acid molecules used in the production of transformed or transgenic host cells and plants. As used here, exogenous is intended to refer to any nucleic acid that is introduced into a recipient cell, regardless of whether a similar nucleic acid may already be present in such a cell.

[0025] Genome refers to the chromosomal DNA of an organism. The genome is defined as a haploid set of chromosomes of a diploid species. For the purposes of this patent application, the genome also includes the genome of organelles.

[0026] Monocot or monocot refers to plants that have a single cotyledon. Examples include cereals such as corn, rice, wheat, oats and barley.

[0027] Nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0028] Phenotype refers to a characteristic exhibited by an organism that results from the interaction of the genotype and the environment.

[0029] Polyadenylation signal or polyA signal refers to a nucleic acid sequence located 3 'from a coding region that promotes the addition of adenylated nucleotides at the 3' end of the transcribed mRNA starting from the coding region.

[0030] Promoter or promoter region refers to a nucleic acid sequence, generally found 5 'from the coding sequence, which controls the expression of the coding sequence by controlling the production of messenger RNA (mRNA) by supplying the site of recognition for RNA polymerase or other factors necessary for transcription to begin at the correct site.

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8/65 [0031] Recombinant nucleic acid vector or vector refers to any agent such as a plasmid, a cosmid, a virus, an autonomously replicating sequence, a phage, a nucleotide segment of DNA or single-stranded RNA or linear or circular double strand, derived from any source, capable of integrating into the genome or replicating autonomously, comprising a nucleic acid molecule in which one or more nucleic acid sequences have been linked in a functionally operational manner . Such recombinant nucleic acid vectors or constructs are capable of introducing a 5 'regulatory sequence or promoter region and a DNA sequence for a selected gene product in a cell in such a way that the DNA sequence is transcribed into a functional mRNA, which is subsequently translated into a polypeptide or protein.

[0032] Regeneration refers to the process of growth of a plant starting from a plant cell.

[0033] Regeneration medium refers to a plant tissue culture medium necessary to contain a selection agent.

[0034] Callus that can be regenerated refers to the callus from which whole plants can be produced, but that the mode of regeneration (embryogenesis or organogenesis) has not been determined or is not relevant for discussion.

[0035] Marker that can be selected or marker that can be verified refers to a sequence of nucleic acid whose expression confers a phenotype that facilitates the identification of cells that contain the sequence of nucleic acid.

[0036] Selection refers to the contact of an inoculated explant with a selection medium to obtain a transformed cell, tissue or plant.

[0037] Selection medium refers to a tissue culture medium

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9/65 vegetable that contains a selection agent.

[0038] Transcription refers to the process of producing a copy of RNA from a DNA template.

[0039] Transformation refers to a process of introducing an exogenous nucleic acid sequence (vector or construct) into a cell or a protoplast, in which the exogenous nucleic acid is incorporated into nuclear DNA, plastoid DNA or is capable to replicate autonomously.

[0040] Transgenic refers to organisms in which an exogenous nucleic acid sequence has been integrated.

[0041] Explant that can be transformed refers to any part of a plant that is receptive to transformation.

[0042] The present invention provides a system in which the stable transformation can be performed in a single container. The transformation process begins with the inoculation of the explant that can be transformed with Agrobacterium and results in a stable transformed plant with suitable roots for transfer to the soil.

[0043] There are many containers that can be used for this purpose. Bioreactors, including the temporary immersion system, can be used. Many different containers have been used for liquid culture of plant tissues, including, but not limited to Petri dishes of various sizes, plates of various cavities, flasks, jars, bottles, jars and PlantCons. These containers are generally provided with means such as an inlet and an outlet for supplying the fresh medium and removing the spent medium. A large number of containers can be connected to obtain a high performance system.

[0044] These containers may include some support for the explant. Such support can be, but is not limited to, filter paper,

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10/65 felt, barquets, glass beads, zirconia / silica beads, foam or solid medium. The liquid medium is usually placed in the container and then changed when necessary. This exchange can be done manually or mechanically.

[0045] The containers can contain many explants at once or they can be small enough to contain only a single explant. In the case of multi-well plates, an arrangement of small wells each containing an explant is used to grow large numbers of explants. The advantages of multi-cavity plates include the isolation of any contaminated explants. Explants can be prepared manually or mechanically.

[0046] The purpose of the invention is to have a system that can be easily automated from the beginning to the end; however, any of these liquid culture systems and containers can be used in combination with other steps of transformation, selection and regeneration known to one skilled in the art.

[0047] A high-performance transformation system can be developed in which the containers can be manipulated by robotic arms on a freely configurable work table that can include incubators and shakers in addition to standard laboratory items. Various liquid handling tools equipped with one or more pipetting tips can be used to supply fresh media and remove spent media. The work table, robotic arms and liquid handling tools can be controlled by software via a computer. Alternatively, the liquid medium for the selection and regeneration of the transformed cell can be supplied to the container via one or more tubes connected to a media storage container and removed via one or more connected tubes

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11/65 to a disposal container. The supply and removal of the medium can be controlled manually or mechanically.

[0048] In order to start a transformation process according to the present invention, it is necessary to first select the genetic components that will be inserted in the cells or in the plant tissues. The genetic components can include any nucleic acid that is introduced into a cell or plant tissue using the method according to the invention. The genetic components can include non-plant DNA, plant DNA or synthetic DNA.

[0049] In a preferred embodiment, the genetic components are incorporated into a DNA composition such as a recombinant double-stranded plasmid or vector molecule comprising at least one or more of the following types of genetic components: (a) one promoter that works in plant cells to cause the production of an RNA sequence, (b) a structural DNA sequence that causes the production of an RNA sequence that encodes an agronomic utility product, and (c) an untranslated DNA sequence the 3 'that works on plant cells to cause the addition of polyadenylated nucleotides at the 3' end of the RNA sequence.

[0050] The vector can contain a number of genetic components to facilitate the transformation of the cell or plant tissue and regulate the expression of the desired gene (s). In a preferred embodiment, the genetic components are oriented to express an mRNA, which in one embodiment can be translated into a protein. The expression of a plant structural coding sequence (a gene, cDNA, synthetic DNA or other DNA) that exists in the form of a double strand involves the transcription of messenger RNA (mRNA) starting from a DNA strand by the enzyme RNA polymerase and O

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12/65 subsequent processing of the primary mRNA transcription product within the nucleus. This processing involves a 3 'untranslated region that adds polyadenylated nucleotides to the 3' ends of the mRNA.

[0051] Means for preparing plasmids or vectors containing the desired genetic components are well known in the art. Vectors typically consist of a number of genetic components, including, but not limited to, regulatory elements such as promoters, leaders, introns and terminator sequences. Regulatory elements are also referred to as cis or trans regulatory elements, depending on the proximity of the element to the sequences or gene (s) they control.

[0052] DNA transcription in mRNA is regulated by a region of DNA generally referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with DNA and initiate transcription in mRNA using one of the DNA strands as a template to produce a corresponding complementary RNA strand.

[0053] A number of promoters that are active in plant cells have been described in the literature. Such promoters would include, but are not limited to, the promoters of nopaline synthase (NOS) and octopine synthase (OCS) that are loaded on Agrobacterium tumefaciens tumor-inducing plasmids, kaolinimovirus promoters such as 19S and 35S promoters of the mosaic virus from cauliflower (CaMV) and the 35S promoter of the scrofular mosaic virus (FMV), the enhanced CaMV35S promoter (e35S), the promoter that can be induced by the light of the small subunit of the ribulose bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide ). All of these promoters were used to create various types of DNA constructs that were expressed in plants.

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13/65 [0054] Hybrid promoters may also be constructed to enhance transcriptional activity (U.S. Patent No. 5,106,739 ²⁾ or to combine desired transcriptional activity, inducibility and tissue specificity or specificity stage development. Promoters that work on plants include, but are not limited to, promoters that can be induced, viral, synthetic, constitutive as described and regulated in a temporal way, regulated in a spatial way and regulated in a spatiotemporal way. Other promoters that are tissue-enhanced, tissue-specific or regulated in relation to the stage of development are also known in the art and envisaged as having utility in the practice of this invention.

[0055] Promoters can be obtained from a variety of sources such as plants and plant DNA viruses and include, but are not limited to, CaMV35S and FMV35S promoters and promoters isolated from plant genes such as ssRUBISCO genes. As described below, it is preferred that the particular promoter selected is capable of causing sufficient expression to result in the production of an efficient amount of the gene product of interest.

[0056] The promoters used in the DNA constructs (for example, chimeric / recombinant plant genes) of the present invention can be modified, if desired, to affect their control characteristics. Promoters can be derived through linkage with operator regions, random or controlled mutagenesis, etc. In addition, promoters can be altered to contain several enhancer sequences to assist in increasing gene expression.

[0057] The mRNA produced by a DNA construct of the present invention can also contain a 5 'untranslated leader sequence.

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This sequence can be derived from the promoter selected to express the gene and can be specifically modified in order to increase mRNA translation. The 5 'untranslated regions can also be obtained from viral RNAs, suitable eukaryotic genes or a synthetic gene sequence. Such enhancer sequences may be desirable to increase or to alter the translation efficiency of the resulting mRNA. The present invention is not limited to constructs in which the untranslated region is derived from the 5 'untranslated sequence that accompanies the promoter sequence. Instead, the untranslated leader sequence can be derived from unrelated promoters or genes (see, e.g., US Patent 5,362,865 ^2). Other genetic components that serve to increase expression or to affect the transcription or translation of a gene are also envisaged as genetic components.

[0058] The 3 'untranslated region of the chimeric constructs must contain a transcription terminator or an element that has equivalent function and a polyadenylation signal that works in plants to cause the addition of polyadenylated nucleotides at the 3' end of the RNA. Examples of suitable 3 'regions are (1) the untranslated 3' transcribed regions that contain the polyadenylation signal from Agrobacterium (Ti) tumor-inducing plasmid genes, such as the nopaline synthase (NOS) gene and ( 2) plant genes such as soy storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase gene (ssRUBISCO). An example of a preferred 3 'region is that of the pea ssRUBISCO gene E9 (European Patent Application 0385 962). [0059] Typically, DNA sequences located a few hundred base pairs downstream from the polyadenylation site serve to terminate transcription. DNA sequences are referred to here as transcription termination regions. Regions are required. 870170015706, from 03/10/2017, p. 23/86

15/65 for efficient polyadenylation of the transcribed messenger RNA (mRNA) and are known as 3 'untranslated regions. RNA polymerase transcribes a DNA sequence encoding through a site where polyadenylation occurs.

[0060] In a preferred embodiment, the vector contains a marker gene that can be selected, verified and classified. These genetic components are also referred to here as functional genetic components, as they provide a product that has a role in identifying a transformed plant or a product of agronomic utility. The DNA that serves as a sorting device works in plant tissue that can be regenerated to produce a compound that would give plant tissue resistance to a compound that would otherwise be toxic. Genes of interest for use as a marker that can be selected, verified or classified would include, but are not limited to, GUS, green fluorescent protein (GFP), genes related to anthocyanin biosynthesis (C1, Bperu), luciferase (LUX) , antibiotics such as kanamycin (Dekeyser et al., 1989) and herbicides such as glyphosate (Della-Cioppa et al., 1987). Other selection devices can also be implemented including, but not limited to, tolerance to phosphinothricin, bialaphos, dicamba and positive selection mechanisms and would still fall within the scope of the present invention.

The present invention can be used with any suitable plasmid or plant transformation vector that contains a selectable or verifiable marker and associated regulatory elements as described, together with one or more nucleic acids expressed in a manner enough to confer a particular characteristic. Examples of suitable structural genes of agronomic interests provided for by the present invention would include, but are not limited to, genes for insect tolerance

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16/65 or pests, tolerance to herbicides, genes for quality improvements such as yield, nutritional improvements, environmental or stress tolerances or any desirable changes in plant physiology, growth, development, morphology or product (s) (s) of the plant.

[0062] Alternatively, DNA coding sequences can affect these phenotypes by encoding an untranslatable RNA molecule that causes targeted inhibition of the expression of an endogenous gene, for example, through antisense or co-mediated mechanisms - suppression (see, for example, Bird et al., 1991). The RNA could also be a catalytic RNA molecule (for example, a ribozyme) engineered to cleave a desired endogenous mRNA product (see, for example, Gibson and Shillitoe, 1997). More particularly, for a description of antisense regulation of gene expression in plant cells, see US Patent 5,107,065 and ² for a description of gene suppression in plants by transcription of a dsRNA see US Patent 6,506,559 ^2, US Patent Application Publication No. ² 2002/0168707 A1 and US Patent Applications Serial No. 09 / 423,143 (see WO 98/53083), 09 / 127,735 (see WO 99/53050) and 09 / 084,942 (see WO 99/61631), all of which are incorporated herein by reference. Thus, any gene that produces a protein or an mRNA that expresses a phenotype or a change in the morphology of interest is useful for the practice of the present invention.

[0063] Examples of nucleic acids that can be introduced using the methods covered by the present invention include, for example, DNA sequences or genes from another species or even genes or sequences that originate from or are present in the same species, but they are incorporated into the recipient cells through genetic engineering methods instead of reproduction techniques 870170015706, from 10/03/2017, p. 25/86

17/65 classic or crossover. However, it is also intended that the term exogenous refers to genes that are not normally present in the cell that will be transformed or perhaps simply that are not present in the form, structure, etc., that is found in the transforming DNA segment or the gene or genes that are normally present despite what is desired, for example, to be overexpressed. Thus, it is intended that the term exogenous gene or DNA refers to any gene or segment of DNA that is introduced into a recipient cell, regardless of the fact that a similar gene may already be present in such a cell. The type of DNA included in exogenous DNA can include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or DNA generated externally, such as a DNA sequence that contains an antisense message of a gene or a DNA sequence that encodes a synthetic or modified version of a gene.

[0064] In consideration of this description, several other marker genes that can be selected or that can be verified as possible, regulatory elements and other sequences of interest will be evident to those skilled in the art. Therefore, the previous discussion is intended to be an example rather than a complete one.

[0065] Technologies for introducing DNA into cells are well known to those skilled in the art and can be divided into categories that include, but are not limited to: (1) chemical methods; (2) physical methods such as microinjection, electroporation and bombardment of microprojectiles; (3) viral vectors; (4) receptor-mediated mechanisms; and 5) Agrobacterium-mediated plant transformation methods.

[0066] For Agrobacterium-mediated transformation, after the construction of the vector or plant transformation construct, the

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18/65 said nucleic acid molecule, prepared in the form of a DNA composition in vitro, is introduced into a suitable host such as E. coli and crossed with another suitable host such as Agrobacterium or transformed directly into competent Agrobacterium. These techniques are well known to those skilled in the art and have been described for a number of plant systems including soybeans, cotton and wheat (see, for example, US Patent Nos. 5,569,834 and 5,159,135 and WO 97/48814, incorporated here as a reference in its entirety).

[0067] The present invention encompasses the use of bacterial strains for the introduction of one or more genetic components in plants. Those skilled in the art would recognize the usefulness of Agrobacterium-mediated transformation methods. A number of wild and unarmed strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes that carry the Ti or Ri plasmids can be used for gene transfer to plants. Preferably, Agrobacterium hosts contain unarmed Ti and Ri plasmids that do not contain the oncogenes that cause tumorigenesis or rhizogenesis, respectively, which are used as the vectors and contain the genes of interest that are subsequently introduced into the plants. Preferred strains would include, but are not limited to, the C58 strain of Agrobacterium tumefaciens, a nopaline type strain that is used to mediate the transfer of DNA to a plant cell, octopine type strains such as LBA4404 or succinamopine type strains, for example , EHA101 or EHA105. Other bacteria such as Sinorhizobium, Rhizobium and Mesorhizobium that interact with plants naturally can be modified to mediate gene transfer to a number of different plants. These symbiotic bacteria associated with plants can be made competent for gene transfer by acquiring both a

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19/65 Ti plasmid disarmed as well as a suitable binary vector (Broothaerts et al., 2005).

[0068] The use of these strains for the transformation of plants has been reported and the methods are familiar to those skilled in the art.

[0069] The explants can be of a single genotype or a combination of genotypes. Any corn seed that can germinate is a viable starting material. In a preferred embodiment, superior explants of plant hybrids can be used as explants. For example, a fast-growing cell line with a fast culture response (increased frequency of embryonic callus formation, growth rate, frequency of plant regeneration, etc.) can be generated using hybrid embryos containing various genotypes. In a preferred embodiment, an F1 hybrid or the first generation offspring of individuals of different origins can be used as a donor plant and crossed with another genotype. Those skilled in the art are aware that heterosis, also referred to here as hybrid vigor, occurs when two crosses of individuals of the same origin are crossed. The present invention also covers the use of an explant resulting from a three-way crossing, in which at least one or more of the crossings of individuals of the same origin can be highly regenerated and transformed and the frequency of transformation and regeneration of the crossing explant in three ways it exceeds the frequencies of the crossings of individuals of the same origin individually. It is also envisaged that other fabrics have utility in the practice of the present invention. Explants can include mature embryos, immature embryos, meristems, callus tissue or any other tissue that can be transformed or regenerated.

[0070] Any suitable plant culture medium can be used during the transformation process. Examples of means

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Suitable 20/65 would include, but are not limited to, MS-based media (Murashige and Skoog, 1962) or N6-based media (Chu et al., 1975) supplemented with additional plant growth regulators including, but not limited to auxins such as picloram (4-amino-3,5,6-trichloropico-linic acid), 2,4-D (2,4-dichlorophenoxyacetic acid) and dicamba (3,6-dichloroanisic acid); cytokinins such as BAP (6-benzylaminopurine) and kinetin; TAB; and gibberellins. Other media additives may include, but are not limited to, amino acids, macroelements, iron, microelements, inositol, vitamins and organic compounds, carbohydrates, undefined media components such as casein hydrolysates, ethylene antagonists including silver nitrate with or without a appropriate gel forming agent such as an agar form, such as a low melting point agarose or Gelrite®, if desired. Those skilled in the art are familiar with the variety of tissue culture media, which when properly supplemented, support the growth and development of plant tissue and are suitable for plant transformation and regeneration. These tissue culture media can be obtained in the form of a commercial preparation or prepared and modified by the user. Examples of such media would include, but are not limited to, Murashige and Skoog (1962), N6 (Chu et al., 1975), Linsmaier and Skoog (1965), Uchimiya and Murashige (1962), Gamborg (Gamborg) media et al., 1968), medium D (Duncan et al., 1985), McCown's Woody plant media (McCown and Lloyd, 1981), Nitsch and Nitsch (1969) and Schenk and Hildebrandt (1972) or derivations of these media supplemented in a corresponding. Those skilled in the art are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation Petition 870170015706, of 10/03/2017, p. 29/86

21/65 tion, pH and incubation temperatures can be optimized for the particular variety of interest.

[0071] Once the plant tissue that can be transformed is isolated, the next step in the method is to introduce the genetic components into the plant tissue. This process is also referred to here as transformation. The plant cells are transformed and each plant cell transformed independently is selected. Independent transformants are referred to as transgenic events. A number of methods have been reported and can be used to insert genetic components into plant tissue that can be transformed. The bombardment of microprojectiles and the gene supply mediated by Agrobacterium are the two most commonly used plant transformation methods, but other methods are known.

[0072] Those skilled in the art are aware of the typical steps in the plant transformation process. Those skilled in the art are familiar with the procedures for growth and with the appropriate culture conditions for Agrobacterium as well as with subsequent inoculation procedures. The density of the Agrobacterium culture used for inoculation and the proportion of Agrobacterium cells in relation to the explant can vary from one system to the next and, therefore, the optimization of these parameters is expected for any transformation method. Agrobacterium can also be induced directly from frozen stocks or can be grown in a variety of ways known to one skilled in the art.

[0073] The next stage of the transformation process is inoculation. At this stage, the explants and suspensions of Agrobacterium cells are mixed together. This can be achieved by incubating the explants in the cell suspension of AgrobacPetição 870170015706, of 10/03/2017, p. 30/86

22/65 terium. In other modalities described here, the explants were inoculated while still attached to the donor tissue, while the explants were removed from the donor tissue, after plating the explants in the selection medium or through a combination. The duration and condition of the inoculation and the density of Agrobacterium cells will vary depending on the plant transformation system.

[0074] After inoculation, any excess Agrobacterium suspension can be removed and the Agrobacterium and the target plant material are co-cultivated. Co-cultivation refers to post-inoculation time and before transfer to a delay or selection medium. Any number of plant tissue culture media can be used for the coculture stage. Plant tissues after inoculation with Agrobacterium can be grown in a liquid medium or in a semi-solid medium. Coculture is typically carried out for approximately one to three days at a temperature of approximately 18Ό - 30Ό. Co-cultivation can be carried out in light or in conditions of limited light. The lighting conditions can be optimized for each plant system known to those skilled in the art.

[0075] After co-cultivation with Agrobacterium, explants can typically be placed directly in selective medium. Alternatively, after co-cultivation with Agrobacterium, the explants could be placed in a medium without the selective agent and subsequently placed in a selective medium. Those skilled in the art are aware of the various changes in the selection regimes, the media and the growing conditions that can be varied depending on the plant system and the selective agent. Typical selective agents include, but are not limited to, antibiotics such as geneticin (G418), kanamycin, paromomycin or other chemical compounds such as gliPetition 870170015706, from 10/03/2017, p. 31/86

23/65 phosphate, phosphinothricin, bialaphos and dicamba. Additional appropriate media components can be added to the selection or delay media to inhibit the growth of Agrobacterium. Such components of the medium may include, but are not limited to, antibiotics such as carbenicillin or cefotaxime.

[0076] In one embodiment, the stages of inoculation, co-cultivation and selection were combined in a single step by plating the inoculated explants directly onto a medium containing selective agents for suppressing the growth of Agrobacterium and killing cells from untransformed explants to increase the efficiency of the transformation production system.

[0077] The cultures are subsequently transferred to a suitable medium for the recovery of the transformed seedlings. Those skilled in the art are aware of the number of methods for recovering transformed plants. A variety of means and transfer requirements can be implemented and optimized for each plant system for the transformation of plants and for the recovery of transgenic plants. Consequently, such culture media and conditions disclosed in the present invention can be modified or replaced by nutritionally equivalent components or similar processes for the selection and recovery of transgenic events and still fall within the scope of the present invention.

[0078] The present invention includes all the steps described above; however, modifications are made when appropriate to facilitate the process in a single container. Liquid culture in various types of support is used to facilitate the exchange of the medium from step to step. A bioreactor with a temporary immersion system or other device that provides similar results can be used to replace the medium.

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24/65 [0079] In the case of the callus as the explant, the callus can be cut finely using various devices including, but not limited to, a garlic press, scissors, scalpel or other cutting devices. The callus can be cut very thinly to fit within a plate system of several small cavities, where the expectation is to obtain a single event in each cavity. These modifications provide ergonomic relief and can be used to recover many transgenic events from a single piece of callus.

[0080] The transformants produced are subsequently analyzed to determine the presence or absence of a particular nucleic acid of interest contained in the transformation vector. Molecular analyzes may include, but are not limited to, Southern blots (Southern, 1975) or PCR (polymerase chain reaction) analyzes, immunological diagnostic approaches and field assessments. These and other well-known methods can be performed to confirm the stability of the transformed plants produced using the disclosed methods. These methods are well known to those skilled in the art and have been reported (see, for example, Sambrook et al., 1989).

[0081] Those skilled in the art will consider the many advantages of the methods and compositions provided by the present invention. The following examples are included to demonstrate preferred embodiments of the invention. It should be considered by those skilled in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to work well in the practice of the invention and thus can be considered to constitute the preferred modes for their practice. However, those skilled in the art should, in the light of this description, consider that many changes can be made in the specific modalities that are disclosedPetition 870170015706, of 10/03/2017, p. 33/86

25/65 and still obtain an equal or similar result without departing from the spirit and scope of the invention. All references cited here are incorporated here by reference to the extent that they supplement, explain, provide a basis for or teach a methodology, techniques or compositions employed here.

Examples [0082] The following examples are provided for the purpose of illustrating various embodiments of the invention and are not to be construed as limiting the present invention in any way. One skilled in the art will easily consider that the present invention is well adapted to achieve the objectives and to obtain the purposes and advantages mentioned, as well as the objectives, purposes and inherent advantages contained herein. The present examples, together with the methods described here are now representative of the preferred embodiments, are examples and are not intended as limitations on the scope of the invention. The changes contained herein and other uses that fall within the spirit of the invention that is defined by the scope of the claims will occur to those skilled in the art.

Example 1

Source of Explants and Culture Conditions [0083] Eight-day-old calluses (eight days after subculture) obtained from immature corn embryos were grown in 211V gelled medium (mixture of N6 basal salts, 1 mg / L of 2 -4-D, 1 mg / L Thiamine HCI, 1 mg / L Nicotinic Acid, 0.91 g / L LAsparagine Monohydrate, 0.1 g / L myo-inositol, 0.5 g / L MES , 1.6 g / L of MgCl2 6H ₂ O, 0.1 g / L of casein hydrolyzate, 0.69 g / L of proline, 20 g / L of sucrose, 0.1 mM of silver nitrate, 6 g / L of Phyta agar).

[0084] Whenever the use of gelled medium is mentioned, Petri dishes of 25 X 100 mm containing 50 mL of

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26/65 medium with 12 explants per plate and all transfers were made manually.

[0085] The liquid medium consisted of 212V (basal salts of N6 with 2 mg / L of 2-4-D, 1 mg / L of Thiamine HCl, 1 mg / L of Nicotinic Acid, 0.91 g / L of L -Asparagine Monohydrate, 0.1 g / L of myo-inositol, 0.5 g / L of MES, 1.6 g / L of MgCl2 6H ₂ O, 0.1 g / L of casein hydrolyzate, 0.69 g / L of Proline, 20 g / L of sucrose, 0.01 mM of silver nitrate, pH adjusted to 5.8 and autoclaved at 121Ό and 105 kPa for 20 min. After autoclaving, Carbenicillin and Paromomycin sterilized by filtration at 250 mg / L or 500 mg / L and 100 mg / L or 200 mg / L concentration was added to the medium as a selective agent.

[0086] For liquid culture, a Temporary Immersion System (TIS) container, Petri dishes of 240 x 240 mm in size or Petri dishes of 25 x 100 mm or plates of several cavities were used.

[0087] All cultures were incubated at 27-28Ό in the dark in a growth chamber (Percival scientific Series 101 US). [0088] Infection and co-cultivation with Agrobacterium tumefaciens. Eight-day-old calluses of uniform size in active growth were selected and inoculated with a suspension of Agrobacterium (0.25 X 10 ⁹ cfu / mL) for 30 minutes. The calluses were washed with 212V liquid medium containing 0.1 mM AgNO ₃ and transferred to a TIS container or a 25 X 100 mm Petri dish containing filter paper or a large shallow Petri dish (240 X 240 mm) containing two layers of (220 X 220 mm) polyester felt. Co-cultivation treatments were carried out in the dark at 23Ό.

[0089] A. tumefaciens strains and plasmids. The C58 ABI strain of Agrobacterium was used to mediate the transfer of DNA into plant cells. Plasmid pMON30113 (figure 1) contained the neomycin phosphotransferase II (NPTII) gene as the marker

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27/65 that can be selected and a green fluorescence protein (GFP) gene as the marker that can be verified controlled by the 35S promoter.

Example 2

Temporary Immersion System (TIS) [0090] A modified TIS was built as follows. Two chambers of a 250 ml autoclavable filter unit (Nalge Nunc Intl., Rochester, NY) were connected by inserting a glass tube into the base of the support plate normally used to support the membrane filter. Air hoses were connected in the two chambers. The air flow was sterilized by passing through hydrophobic PTFE membrane filters with a diameter of 50 mm and a pore size of 0.2 pm (Millipore, Billerica, MA). The liquid medium was placed in the lower chamber and the explant materials were placed on a support disc in the upper chamber. Air was pumped into the lower chamber to move the liquid medium through the glass tube to the upper chamber, covering the callus with liquid. The air pumped into the upper chamber forced the medium to return to the lower chamber (only a thin film of liquid medium remains on the tissues). An electronic timer was used to operate the solenoids that control the air flow to the upper or lower chamber. The timer was programmed in relation to the air flow into the lower chamber, the air flow to the upper chamber or idle time (without air flow). The air flow emission times were adjusted from 1 to 5 min each while the idle time could vary. The program was then adjusted to run in a continuous cycle through different stages of the transformation process. In this way, the tissues were completely immersed for approximately 1-5 min at the adjusted time intervals. This is called the immersion cycle frequency.

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28/65 [0091] Optimization of the frequencies of the immersion cycle. The tissue was cultured in an immersion lasting 1 min and four different immersion cycle frequencies (once every 6 h, 8 h, 12 h and 24 h) each, over a period of 8 weeks were tested. As a control, half of the calluses derived from the same ear were grown in 211V gelled medium with selection.

[0092] It has been observed that the immersion time interval has a significant impact on the callus proliferation rate in the TIS. The maximum rate of increase of callus proliferation and biomass occurred using an immersion frequency of 12 h or 24 h. With these two immersion cycle frequencies, 70-80% of the calluses produced positive sectors for GFP. Increasing the frequency of immersion for one cycle every 6 or 8 hours, there was no sign of callus proliferation and within 10 to 14 days, the callus turned brown and eventually died.

[0093] Effect of Carbenicillin on the growth of A. tumefaciens. The calluses were inoculated with pMON30113 as previously described. After inoculation, the infected calluses were transferred to the callus proliferation medium (212V) supplemented with 0, 100, 250, 500 or 1000 mg / L carbenicillin. The growth of Agrobacterium was evaluated visually by observing the turbidity of the liquid medium in the lower chamber of the TIS apparatus. A separate TIS unit with uninoculated control treatment was included. An additional control of uninoculated callus was also grown on gelatin medium supplemented with carbenicillin (211V) to study the effect of carbenicillin on the callus culture.

[0094] No significant difference in callus quality was observed when uninoculated callus grown in 211V (gelled) and 212V (liquid) medium with or without carbenicillin supplementation, indicating that there is no negative effect of carbenicillin on growth

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29/65 and the development of uninoculated callus. However, differences in the growth of A. tumefaciens and positive sectors for GFP were observed as the carbenicillin concentration was increased from 0.0 to 1000 mg / L. Carbenicillin at a concentration of 100 mg / L could not suppress the growth of Agrobacterium in liquid or gelled medium. It was observed that the optimal carbenicillin concentration was 500 mg / L for gelled and liquid media.

[0095] Effect of Paromomycin on the Proliferation of Calluses and Shoots. To inhibit the proliferation of non-transgenic calluses, the culture media, both gelled (control) and liquid, were supplemented with the selective agent paromomycin (25, 50, 75, 100 or 200 mg / L). The control treatments were (1) callus not inoculated in selective medium, (2) callus not inoculated in medium without selection and (3) callus inoculated in medium without selection. The basic culture medium consisted of 211V or 212V. For this experiment, deep Petri dishes of 25 X 100 mm were used. With the liquid culture, two layers of felt of 60 X 60 mm were used to support the explants. The selective agent for the culture inoculated with Agrobacterium contained carbenicillin and paromomycin.

[0096] Paromomycin in low concentrations more efficiently inhibited the growth of embryogenic calluses and the regeneration of the plant in liquid than in gelled medium. No plants were obtained from uninoculated callus in a liquid medium containing as little as 50 mg / L of paromomycin, while 50% of the calluses produced plantlets in a gelled medium in the same concentration of paromomycin. Paromomycin at a concentration of 100 mg / L inhibited callus growth in both liquid and gelled media.

[0097] Optimization of the amount of liquid medium used in TIS. The developing calluses were grown in one of four

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30/65 different amounts of liquid medium (20 ml, 30 ml, 50 ml and 100 ml / container).

[0098] Selection and Regeneration at TIS. After 3 days of co-cultivation in the TIS, 20 mL of 212VRT selection medium (basal salts of N6 with 2 mg / L of 2,4-D, 1 mg / L of Thiamine HCI, 1 mg / L of Nicotinic Acid, 0, 91 g / L of L-Asparagine Monohydrate, 0.1 g / L of myo-inositol, 0.5 g / L of MES, 1.6 g / L of MgCl ₂ 6H ₂ O, 0.1 g / L of hydrolyzate casein, 0.69 g / L proline, 20 g / L sucrose, 0.01 mM silver nitrate, 500 mg / L Carbenicillin and 100 mg / L Paromomycin) were injected into the lower chamber of the TIS. The calluses were immersed for one minute once every 12 hours. After two weeks of incubation, 212VRT medium was aspirated from the lower chamber using a vacuum pump and replaced with 20 ml of 212RT medium (without silver nitrate). The calluses were immersed for one minute once every 24 hours for another two weeks. After completing 4 weeks of the selection phase, the selection medium was replaced with pulse medium of BAP 217A (basal salts of N6 with 1 mg / L of Thiamine HCI, 1 mg / L of Nicotinic Acid, 3.52 mg / L BAP, 0.91 g / L of L-Asparagine Monohydrate, 0.1 g / L of myo-inositol, 0.5 g / L of MES, 1.6 g / L of MgCl ₂ 6H ₂ O, 0, 1 g / L of casein hydrolyzate, 0.69 g / L of proline, 20 g / L of sucrose, 250 mg / L of Carbenicillin). The calluses were treated with 217A medium for 7 days with a minute of immersion once every 12 hours. [0099] For plant regeneration, medium 217A was replaced by medium 632AG (mixture of MS salts, MS vitamins, 50 mg / L of myo-inositol, sucrose at 60 g / L, paromomycin at 50 mg / L and carbenicillin at 250 mg / L). The medium was injected into the upper chamber of the TIS and two quick one-minute immersion rounds were performed to dilute any residual 217A medium. The lights in Percival were turned on in a cycle of 16 hours on / 8 hours off. The TIS was kept idle (without any immersion) for 72 hours and, after that, the calluses were 870170015706, of 10/03/2017, p. 39/86

31/65 were immersed once every 24 hours until shoots develop to a size of 5-20 mm.

[00100] After 10-12 weeks of selection and regeneration in a container, transgenic plants were obtained. A transformation frequency of 5.9% was achieved in TIS. This compares to 17.7% in the control.

Example 3

Use of Petri dishes [00101] The calluses obtained as in Example 1 were washed with 212V liquid medium containing 0.1 mM AgNO ₃ and transferred to a 25 x 100 mm Petri dish containing filter paper or a Large shallow petri (240 x 240 mm) containing two layers of acrylic felt (220 X 220 mm).

[00102] The explants were then cultured as described in the TIS in Example 2 with the differences being that exchanges of liquid media were done manually. The liquid medium was compared with the gelled medium. In the smaller Petri dish, the liquid medium provided a transformation frequency of 4.2% compared to 6.6% with gelled medium. In the larger Petri dish, the liquid medium provided a transformation frequency of 7.8% compared to 11.5% with gelled medium. This demonstrates that the liquid medium is a viable alternative to standardized solid medium protocols. The liquid medium will allow for greater automation.

Example 4

Using Multi-Cavity Plates as a Container [00103] Multi-cavity plates can also be used as a single container system. Experiments were performed as previously described except for the use of multi-well plates instead of Petri dishes. Costar multi-well plates (Corning Life Sciences, Acton, MA) were used with or

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32/65 without its Transwell® inserts. The size of the membrane pores used (0.1 μ) allows the medium to penetrate through the membrane into the corn tissue when necessary, without submerging the tissue. The pipette tips access doors allow the medium to be quickly removed and added to the wells for changing the medium. During medium transfers, the medium inside the cavities is changed by vacuuming the old medium with a vacuum hose and pipetting the new medium there.

[00104] The multi-well plates used in this invention come in a size of 24 separate wells (with inserts) per plate. Alternatively, plates of several cavities with an opposite medium reservoir for separation can be used. The plates are typically wrapped with Nescofilm® or Parafilm® or left unwrapped and stored in incubators (27 ° C) in the same way as with solid selection. The fact that the cavities are separated from each other is particularly beneficial because bacterial contamination does not spread to other explants, as could be the case in common medium reservoirs, liquid felt selection or solid medium selection.

[00105] The vector pMON 68410 was used for the transformation and contained the npt \\ gene as a marker that can be selected and gfp as a marker that can be classified. Callus tissue from LH244 was used as an explant. The transformation steps were the same as those disclosed in Example 8. The experiments were also carried out on plates of several Costar cavities with and without different matrices to provide support for the explant in liquid medium. Glass beads, silicon beads, felt, foam and the solid medium with additional liquid medium were used. The control treatments were: culture in solid medium in Petri dishes (trt # 1; Table 1) and only liquid medium in plates of several caviPetition 870170015706, of 03/10/2017, p. 41/86

33/65 (trt # 2; Table 1). Different matrices were used in four experiments by four different people. All treatments provided transgenic plants. However, the use of silicon sphere matrix and small felts were in accordance with the culture control treatment in solid medium in Petri dishes.

Table 1. Use of plates from several cavities with and without matrices for the production of transgenic corn plants from callus explants.

Trt N ⁹ Matrix / Medium M ⁹ requests for: hello for each experiment TF% Ex. 1 Ex. 2 Ex. 3 Ex. 4 Total 1 Petri dish gel / solid 48 39.6 50.0 50.0 25.0 41.1 2 None / Net 24 4.2 25.0 16.7 8.3 13.5 3 Glass / liquid spheres 24 25.0 12.5 33.3 16.7 21.9 4 Silicon / liquid spheres 24 33.3 25.0 29.2 50.0 34.4 5 Small Felt / liquid 24 41.7 25.0 54.2 29.2 37.5 6 Foam 24 0.0 16.7 0.0 4.2 5.2 7 Solid medium with liquid medium additional 24 16.7 16.7 12.5 4.2 12.5

[00106] Table 2 shows that the multi-cavity system with (trt n ° 3 to 5) and without (trt n ° 6) matrices can also be used for the production of transgenic corn plants using immature embryos like explants and glyphosate as a selective agent. The pMON 92690 vector for this study contained the cp4 and gus genes. The transfer of medium in several wells was done on a weekly basis.

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Table 2. Use of plates from several cavities with and without matrices for the production of transgenic corn plants from embryo explants.

Trt N ° Container Middle Matrix No. of embryos TF% 1 Petri dish Solid Gel 40 60.0% 2 Petri dish Liquid Felts 40 45.0% 3 Several cavities Liquid Silicon Spheres 40 25.0% 4 Several cavities Liquid Glass Spheres 40 15.0% 5 Several cavities Liquid Small Felt 40 37.5% 6 Several cavities Liquid None 48 2.1%

Example 5

Use of a Single Petri Dish with Solid Medium Followed by Medium

Liquid [00107] Another example of a single vessel system is the use of a single Petri dish with solid medium and subsequently the addition of liquid medium to facilitate selection and regeneration on the single plate without replacing the old medium. Corn callus was obtained and inoculated as described in Example 7.

[00108] The callus was then co-cultured in a 25 X 100 deep Petri dish containing Whatman filter paper and 100 pL of 1/2 MSPL medium at 23Ό. After 3 days, the callus was transferred to the solid 850QRT medium (4.33 g / L of MS salts, 0.5 mg / L of thiamine HCI, 0.5 mg / L of pyridoxine HCI, 0.5 mg / L nicotinic acid, 100 mg / L myoinositol, 0.5 g / L casein hydrolyzate, 1.38 g / L proline, 30 g / L sucrose, 0.5 mg / L 2.4 -D, 10 pg / L BAP, 20 pM AgNO3, 500 mg / L carbenicillin, 100 mg / L paromomycin, pH 5.8, 6 g / L agar).

[00109] After 14 days, 7-10 mL of fresh 850QRTT medium (850QRT

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35/65 without the agar and with 200 mg / L of paromomycin) were added and the plates were incubated in low light. After an additional 14 days, 7-10 mL of fresh 850RT medium (4.33 g / L of MS salts, 0.5 mg / L of thiamine HCI, 0.5 mg / L of pyridoxine HCI, 0.5 mg / L of nicotinic acid, 100 mg / L of myo-inositol, 0.5 g / L of casein hydrolyzate, 1.38 g / L of proline, 30 g / L of sucrose, 0.5 mg / L, 10 pg / L BAP, 500 mg / L carbenicillin, 100 mg / L paromomycin, pH 5.8) were added and the plates were further incubated in low light.

[00110] After another 14 days, the regenerated shoots were transferred to the 632AT medium (4.33 g / L of MS salts, 0.5 mg / L of thiamine HCI, 0.5 mg / L of pyridoxine HCI, 0 , 5 mg / L nicotinic acid, 50 mg / L myo-inositol, 60 g / L sucrose, 250 mg / L carbenicillin, 100 mg / L paromomycin, pH 5.8, 6 g / L agar ). The regenerated plants were then transferred to phytatrays containing the same medium after 14 days and then transferred to the soil in two weeks. [00111] The plants selected and regenerated in solid medium produced approximately 46% of transformed plants that could be used compared to 47% of transformed plants that could be used starting from the control method using liquid medium on a felt support.

Example 6

Maize Transformation mediated by Agrobacterium Using a New Suspension Culture Method [00112] It is known in the art that producing suspension culture of cells that can be transformed is time consuming and often results in the production of non-embryonic cells. This example describes a highly reproducible method (referred to here as short suspension culture or SSC) for producing a fast suspension culture that is highly embryogenic and very competent for Agrobacterium-mediated gene transformation. THE

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SSC as an explant combines the desirable characteristics of callus (embryonic and easy to use) and suspension cultures (uniform, which can be highly regenerated and which can be perfected for high yield production). In this non-limiting example, neomycin phosphotransferase II (npt \\) was used as a marker that can be selected and a standardized binary vector system was used for the efficient selection and regeneration of stable transgenic events mediated by Agrobacterium in corn.

[00113] Agrobacterium strain, plasmid and culture. Agrobacterium tumefaciens EHA 101 disarmed carrying the binary vector pMON25457 was used in this experiment. PMON25457 contained selectable genes (npt \\) and reporters (uidA), each controlled by an enhanced 35S promoter (e35S) and followed by an untranslated hsp70 intron. The uidA gene has an additional intron within its coding sequence to minimize bacterial expression. The plasmids were introduced into the Agrobacterium strain by electroporation with a Bio-Rad Gene Pulser operated at 2.5 kV and 400 Ohms. The transformed colonies were selected in solid Luria-Bertani (LB) medium (Sambrook and Russell, 2001) containing 100 mg / L of each kanamycin and gentamycin.

[00114] Induction and cultivation of Agrobacterium. The Agrobacterium cells used for the transformation were pre-induced with acetosyringone (200 μΜ) and glucose (2%) in an AB-based induction medium (0.1 M MES, 0.5 mM NaH ₂ PO ₄ , 2% glucose, 1 g / L NH ₄ CI, 300 mg / L MgSO ₄ * 7H ₂ O, 150 mg / L KCI, 10 mg / L anhydrous CaCI ₂ , 2.5 mg / L FeSO ₄ '7H ₂ O, pH adjusted to 5.4 with NaOH). [00115] A general procedure for inducing Agrobacterium cells is as follows. A loop full of bacterial colonies was collected from a fresh plate and cultured at 280 in 50 mL of LB medium containing appropriate antibiotics. The optical density at 660

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37/65 nm of the bacterial culture after approximately 15 to 24 hours of culture was approximately 1.4. A 10 ml aliquot of the culture was transferred to 50 ml of fresh LB medium containing appropriate antibiotics and cultured for an additional 6 to 8 hours to an optical density at 660 nm of approximately 1.2. Agrobacterium cells were centrifuged at 4Ό for 10 min at 3250 x g. The resulting pellet was resuspended in the induction medium to a final optical density at 660 nm of approximately 0.2 and incubated at 28Ό for approximately 12 to 15 hours. Before use for transformation, Agrobacterium cells were centrifuged at 4Ό for 10 min at 3250 x g. After decanting the supernatant, the pellet was resuspended in 1/2 MSVI medium (2.2 g / L of MS salt (Gibco), 1 mL / L of 1000x DM vitamin stock, 115 mg / L of proline, 10 g / L of glucose, 20 g / L of sucrose, pH 5.4, sterilized by filtration) and supplemented with 200 μΜ of acetosyringone. At least 100 mL of 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone was used for each 1 L of Agrobacterium suspension. The suspended cells were aliquoted in smaller volumes, centrifuged at 4 degrees C for 10 min at 3250 x g, the supernatant was discarded and the pellets were stored on ice until use (up to 4 hours). The pellets were resuspended to a desired optical density with 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone, with a suspension of approximately 109 cells / mL providing an optical density at 660 nm of approximately 0.2.

[00116] Growth of stock plants and callus formation. The Hi-ll and FBLL maize genotypes were grown in the greenhouse over a 16 h day period. Crosses involving these genotypes were made and the immature embryos were cut on a modified N6 medium (Chu et al. 1976) supplemented with 1.0 mg / L of 2.4D, 1 mg / L of thiamine HCI, 0.5 mg / L of nicotinic acid, 0.91 g / L of LPetition 870170015706, of 10/03/2017, p. 46/86

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Asparagine Monohydrate, 100 mg / L of myo-inositol, 0.5 g / L of MES, 1.6 g / L of MgCl ₂ '6H ₂ 0, 100 mg / L of casein hydrolyzate, 0.69 g / L proline and 20 g / L of sucrose; the modified N6 medium was solidified with 2 g / L of Gelgro (catalog number 150180, ICN Biomedicals) and the pH of the medium was adjusted to pH 5.8 with KOH (pre-autoclave). The same growth conditions were used for the elite genotype RBDQ2. The strains of the hybrid FBLL x Hi-ll and callus RBDQ2 were subcultured at two-week intervals and kept at 28Ό in the dark for up to four months in the same medium. This subculture protocol also worked with Hi-ll and FBLL-MAB strains.

[00117] Formation of culture in short suspension (SSC). A general protocol for producing a callus SSC is as follows. To start SSC formation, approximately 2 g of calli, two weeks after subculture, were transferred to a 250 mL flask containing 80 mL of MS Fromm medium supplemented with 2.0 mg / L of 2.4-D , 20 g / L of sucrose, 150 mg / L of LAsparagine, 100 mg / L of myo-inositol, 1 mL of vitamin stock 1000x of MS Fromm containing 650 mg / L of nicotinic acid, 125 mg / L of pyridoxine HCI , 125 mg / L of thiamine HCI and 125 mg / L of calcium pantothenate (pH of the medium adjusted to 5.8 with KOH). The SSC were generated on a rotary shaker adjusted to 170 rpm and 28 ° C. During each subsequent transfer, the tissues were allowed to settle for a short period of time in the bottom of the flasks to remove unwanted cell types (for example, cells that were elongated, thick-walled, low in cytoplasmic content or that were not in division), before the suspension is transferred with a wide mouth sterile Falcon® disposable pipette. On day 1 after initiation, tissue from the flask was transferred to a new flask containing 80 ml of modified MS medium and grown for a further

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39/65 two days; this transfer step was repeated on day 3 after the start. On day 5 after the start, the tissue was divided equally between two flasks, providing a volume of approximately 2.5 ml of compacted cells (PCV) per flask. After that, the medium was replaced every 3 days. On day 14 after initiation, the volume of resulting compacted cells in each flask was approximately 6 ml (approximately 4 g per flask or approximately a four-fold increase in total PCV over two weeks). These cells (SSC) were transferred to fresh medium on day 14 after onset and transformation was started on day 15 after onset. The relatively frequent change of medium throughout this SSC procedure allowed for rapid tissue proliferation. An additional advantage of the SSC procedure was that no visual selection of tissues was required at each transfer step, making this system both practical and reproducible.

[00118] Inoculation and coculture. Three flasks containing a combined total of approximately 18 mL of PCV were used in a study of transformation of explants into SSC of FBLL x Hi-ll hybrids. Pilot experiments were carried out to improve the parameters involved in the various stages of transformation. A modified coculture technique was used as described by Cheng et al. (2003), which is incorporated here as a reference. A desiccation step was employed after infection with Agrobacterium, which was observed to greatly increase the transfer of TDNA as well as increasing the recovery from transgenic events. [00119] The SSC tissue of the FBLL x Hi-ll hybrid from each vial together with part of the liquid medium was equally divided and transferred to two wells of a six well tissue culture plate (Corning CoStar®, untreated, piece number 9088) or, alternatively, to a 20 X 60 mm Petri dish. The liquid medium was

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40/65 removed from each cavity or plate. Five mL of the Agrobacterium suspension (DO660nm ~ 0.5) in 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone was added to each well or plate, followed by an inoculation period of one hour. At the end of the inoculation period, most of the Agrobacterium suspension was carefully removed and the cells in inoculated SSC were washed with 5 ml of 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone. The cells in each well or plate were transferred to a Petri dish containing 3 layers of sterile filter paper to absorb excess liquid from the cells and then divided equally and transferred to two 60 X 20 mm Petri dishes, each containing a piece of sterile filter paper 5.5 cm in diameter (Baxter). A total of 12 coculture plates containing filter paper were obtained in this way (4 from each original bottle). The transferred cells were arranged in 6 to 8 groups on the filter paper. A one-day coculture under drying was performed using a modified protocol by Cheng et al. (2003), which is incorporated here as a reference in its entirety. One hundred pL of 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone was added to each filter paper and the plates were wrapped with Parafilm® and incubated in the dark at 23 degrees C. The transformation frequency was calculated as the number of events regenerated by mL of PCV.

[00120] Transformation experiments were carried out similarly with SSB explants of RBDQ2, using three volumes of compacted cells in replicates (4.5 mL each). The transformation procedure was generally the same as that used with the SSC explants of FBLL x Hi-ll hybrids, except for the fact that, for one of the two cavities per flask, the 1/2 MS VI medium supplemented with 200 μΜ of acetosyringone and containing 20 μM AgNO ₃ was used 870170015706, dated 10/03/2017, p. 49/86

41/65 during the inoculation and washing steps.

[00121] Selection and regeneration of transgenic plants. The protocol for the transformation of SSC with nptll using selection with paromomycin involved a simple filter paper holder for the transfer of explants during selection, which decreased the work, guaranteed the rapid elimination of Agrobacterium, increased the rate of tissue growth and resulted in faster selection.

[00122] This procedure was performed in SSC of FBLL x Hi-ll hybrids as follows. On day 0 of the transformation (which is at the end of the coculture step), a washing step was carried out with 5 ml of MS Fromm medium supplemented with 1000 mg / L of carbenicillin, 100 mg / L of ticarcillin, 100 mg / L of vancomycin and 40 mM AgNO ₃ which were added directly to each of the coculture plates and the groups of cells were gently agitated using a sterile spatula to ensure submersion. The washing medium was removed from the plates and the filter papers carrying the cells were transferred to Petri dishes containing fresh solid Duncan's D medium containing 3.0 mg / L of 2,4-D supplemented with 1000 mg / L of carbenicillin , 100 mg / L of ticarcillin and 40 micromolar AgNO ₃ as a delay medium. Duncan's medium D contains basal salts and vitamins of medium D as described by Duncan et al. (1985), which is incorporated here by reference; usually 500 mL of a 2X stock of this medium was prepared and added to 500 mL of autoclaved sterile distilled water containing 6 g of Phytagar. The cells were kept in the delay medium for 6 days. On day 6 of transformation, a first selection step was performed with the filter papers loading the cells transferred to Petri dishes containing Duncan's D medium with 1.5 mg / L of 2.4-D supplemented with 750 mg / L of carbenicillin, 100 mg / L of ticarcillin, 40 micromolar AgNO3 and 50 mg / L of paromomycin as a first

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42/65 selection. Two weeks after the end of the coculture step, a second selection step was performed with the filter papers loading the cells transferred to the Petri dishes containing Duncan's D medium with 1.5 mg / L of 2.4-D supplemented with 750 mg / L carbenicillin, 100 mg / L ticarcillin, 40 micromolar AgNO3 and 100 mg / L paromomycin as a second selection medium. Three weeks after the end of the coculture step, a third selection step was carried out with pea-sized groups of tissue removed from each filter paper and plated on a solid Duncan D medium with 1.5 mg / L of 2, 4-D supplemented with 750 mg / L carbenicillin, 100 mg / L ticarcillin and 100 mg / L paromomycin as a third selection medium. There were approximately 4 to 6 groups resulting from cells plated on each filter paper and each group of cells was treated individually thereafter in the experiments. A group plated with filter paper was analyzed histochemically in relation to the size of the transgenic sector, which was observed to be approximately 1 to 2 mm in diameter. Four weeks after the end of the coculture stage, a fourth selection stage was performed with each group transferred to Petri dishes containing Duncan's D medium supplemented with 1.5 mg / L of

2,4-D supplemented with 750 mg / L carbenicillin, 100 mg / L ticarcillin and 100 mg / L paromomycin. At the end of this fourth selection cycle, a total of 232 hybrid lines FBLL x Hi-ll resistant to paromomycin were obtained. Seven weeks after the end of the coculture stage, a selection and pre-regeneration stage was performed with the resistant calluses transferred to deep Petri dishes (20 X 100 mm) containing 3.52 mg / L of 6-BAP, 500 mg / L carbenicillin and 100 mg / L paromomycin for one week. Finally, the regenerating tissues were transferred to an MS regeneration medium for two weeks in Petri dishes, followed by the transfer

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43/65 for a Phytatray for an additional three weeks. The MS regeneration medium consisted of modified MS medium supplemented with 10 g / L glucose, 20 g / L sucrose, 100 mg / L myo-inositol, 150 mg / L L-Asparagine and 1 mL / L stock 1000x vitamins from MS Fromm containing 650 mg / L nicotinic acid, 125 mg / L pyridoxine HCI, 125 mg / L thiamine HCI and 125 mg / L calcium pantothenate and 6 g / L Phytagar; the pH of the medium was adjusted to 5.8 with pre-autoclave KOH and 250 mg / L of carbenicillin and 100 mg / L of paromomycin were added post-autoclave. Thirteen weeks after the end of the coculture stage, the resulting seedlings were transferred to the soil.

[00123] Analysis of the progeny of transgenic plants. The activity of uidA was analyzed in several stages of selection and transformation, following the histochemical procedure described by Jefferson (1987), which is incorporated here as a reference, in various stages of transformation and plant growth. Detection and analysis of the number of nptll copies was performed with INVADER® assays (Third Wave Technologies, Madison, Wl). At least under the experimental conditions described above, it was observed that a delay step followed by a step increase in selection pressure was necessary to recover transgenic sectors of explants in SSC. In general, it was observed that the competence for Agrobacterium-mediated transformation of maize cells was increased by subjecting the callus to at least a short liquid phase before the start of the transformation. For example, it was observed that explants in SSC of FBLL x Hi-ll hybrids were highly competent at transferring TDNA between approximately 5 days to approximately 14 days after the initiation of SSC with 80% of the tissues expressing gus 3 days after transformation. It was also observed that older SSC cultures (2 months) were competent for transformation, with approximately 20% of the explants showing histochemical staining in relation to Petition 870170015706, from 03/10/2017, p. 52/86

44/65 uidA activity 3 days after transformation. In contrast, it was observed that the calluses of FBLL x Hi-ll hybrids maintained in 216 modified N6 medium were poorly competent, with less than 20% of explants exhibiting histochemical staining in relation to UidA activity 3 days after transformation.

[00124] In the case of the FBLL x Hi-ll hybrid genotype, a total of 232 strains resistant to paromomycin were obtained from the three original flasks. Of the 76 strains of resistant calluses tested, it was observed that 56 (approximately 74%) expressed uidA. A subset of 35 strains resistant to paromomycin has been regenerated; of these, it was observed that 24 out of 25 events that were regenerated in plants contained uidA. Southern blots and histochemical tests of plants from various SSC experiments confirmed the integration of the transgene in the R1 generation and the inheritance of the transgenes by the RO and R1 generations. In the case of the RBDQ2 genotype, 3 replicate vials (each containing approximately 4 mL of PCV) provided a total of 37 purported positive events in relation to paromomycin-resistant uidA. The transformation frequency was underestimated because some tissues were sacrificed for histochemical assays for UidA. In this genotype, the use of AgNO ₃ during inoculation or washing did not increase the frequency of transformation, suggesting that the suppression of Agrobacterium growth by drying was sufficient to minimize the toxicity of Agrobacterium. A total of 29 supposed calluses resistant to paromomycin were generated, of which 18 supposed events were transferred to the soil. It was observed that seventeen of the 18 events were positive in relation to uidA through histochemical tests of leaf tissue. Thus, the frequency of transformation into SSC of RBDQ2 (based on the supposed events) was estimated to be approximately 1.4 event / 1 mL of PCV (17 events / 12 mL of PCV).

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45/65 [00125] This SSC procedure was thus able to quickly establish type II suspension cultures that could be regenerated, suitable for stable transformation mediated by Agrobacterium, starting from both the hybrid FBLL x Hi-ll and the RBDQ2 genotypes of elite property. SSC explants can be especially useful in the development of a high performance gene evaluation system.

Example 7

Preparation of Agrobacterium for Transformation Through Direct Inoculation of a Stock of Frozen Agrobacterium Glycerol in an Induction Medium [00126] This is a non-limiting example of a method of preparing Agrobacterium for transformation. More specifically, this example describes the direct inoculation of a frozen glycerol stock of Agrobacterium in an induction broth to come.

[00127] The transfer of T-DNA to plant cells using the Ti plasmid requires activation of the vir genes encoding the VirA and VirG proteins. Signs for activation of VirA include, for example, acidic pH, phenolic compounds (eg, acetosyringone) and certain sugars that act synergistically with phenolic compounds. Conventionally, pre-induction of vir genes involves cultivating Agrobacterium cells (usually from a frozen stock) in a culture medium for a variable period of time, measuring Agrobacterium density, adjusting to a desired density, growing in minimal AB induction medium or other suitable medium for induction, centrifuging and washing the cells and finally adjusting the Agrobacterium culture to a desired density before inoculation. The various steps involved require considerable time and effort and provide opportunities for undesirable variability in the experiments.

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46/65 [00128] A procedure for direct induction of Agrobacterium in a single fast step that reduces time, effort and variability in transformation experiments is disclosed here. The reduction in the number of steps required for induction is also advantageous for the purposes of quality control and for the reduction of ergonomic overload.

[00129] In non - limiting embodiments disclosed herein, we used the vector pMON 70801 which carries a gene (CP4) for resistance to glyphosate (see US Patent 5,633,435 ^2, which is incorporated herein by reference in its entirety); however, many other Agrobacterium vectors could be used, as is known in the art. Optical densities (OD) were measured at 660 nanometers. Additional suitable procedures, including descriptions of media and reagents, for the transformation of plants using glyphosate selection, have been disclosed, for example, in US Patent Application Publication No. ² 2004/0244075 to Cai et al., Which is incorporated herein as reference in its entirety.

[00130] Direct induction with modified MS induction medium. In a non-limiting modality, the procedure may involve pre-induction of a stock of frozen Agrobacterium glycerol in an MS induction medium containing necessary induction components. After pre-induction, Agrobacterium cells are used directly in the transformation experiments.

[00131] Agrobacterium was grown from a frozen stock with MS medium (% MS salts, 100 μΜ MES, 2% glucose with 25% (v / v) glycerol). The frozen stock made with MS salts was resuspended in a modified MS induction broth containing acetosyringone (% MS salts, 100 μΜ MES, 2% glucose) supplemented with appropriate antibiotics (Spec / Strep 50 pg / milliliter, Kanamycin 50 pg / milliliter and Chloramphenicol 25 micrograms / milliliter and

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200 μM acetosyringone). A total of 5 hours was followed up with an initial OD of 0.4. For induction, a flask (250 mL flask flask) with 50 milliliters of induction medium was used. At the end of the induction, the Agrobacterium suspension was used directly for inoculation and was supplemented with 20 μM AgNO ₃ just before inoculation.

[00132] The modified direct induction protocol in MS described above was compared with a conventional protocol involving the following steps: Agrobacterium was grown from a frozen stock for 8 hours in liquid LB with appropriate antibiotics (Spec / Strep 100 micrograms / milliliter, kanamycin 100 micrograms / milliliter and chloramphenicol 25 micrograms / milliliter). The Agrobacterium was centrifuged and resuspended in a minimum AB induction broth with appropriate antibiotics (spec / Strep 50 micrograms / milliliter, kanamycin 50 micrograms / milliliter and chloramphenicol 25 micrograms / milliliter) and 200 micromolares of acetosyrrone. A total of 13 hours of induction was accompanied with an initial OD of 0.2. For induction, a flask (flask with 1 liter fins) containing 300 milliliters of induction medium was used. At the end of the induction, Agrobacterium was centrifuged and resuspended with% MSVI supplemented with 200 micromolar acetosyringone to an OD of 0.4. AgNO ₃ was added to a final concentration of 20 micromolar just before inoculation.

[00133] The explants for this study were calluses derived from an almost elite maize strain subcultured in fresh medium 8 days before the start of transformation in 201 medium based on N6. Inoculation was performed in deep 25 x 100 mm plates. Approximately 6 grams of calluses were transferred to a deep plate containing 50 milliliters of% MS VI (one plate per treatment). The explants were washed and all the liquid was removed. Twenty milliliters

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48/65 suspension of Agrobacterium (OD 0.4) supplemented with acetosyringone (final concentration of 200 micromolar) and AgNO ₃ (final concentration of 20 micromolar) and an inoculation of 1 hour were used. The Agrobacterium suspension was removed after inoculation and the explants were washed with 15 milliliters of% MSVI supplemented to the concentration of 20 micromolar AgNO ₃ and 200 micromolar acetosyringone. The calluses from each inoculation plate were transferred to a deep plate containing 10 filter papers (7.5 mm; Baxter catalog number 28313-046) to remove excess liquid. Approximately 0.5 gram (wet weight) or approximately 2.5 gram (dry weight) of callus was transferred to a single coculture plate (25 x 60 mm) containing a single filter paper (5.5 mm; catalog number of Baxter 28297-868). The explants were arranged in four groups facing the edge of the coculture plate. Coculture was carried out with or without desiccation (1 milliliter of% MSVI was added) at 230 ° C for 18 hours. For each treatment, which is conventional or modified MS induction, seven replicates were used (4 without desiccation, 3 with desiccation). No statistically significant difference was observed between the two induction protocols. It was shown that the direct induction protocol with modified MS produced approximately the same number of supposed events as the longest conventional protocol.

[00134] Direct induction with AB induction medium. In another non-limiting modality, the procedure involved the pre-induction of a stock of frozen Agrobacterium glycerol in an AB induction medium containing the necessary induction components. After pre-induction, the Agrobacterium cells were adjusted to the desired density and used directly in the transformation experiments. Agrobacterium was grown from a stock

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49/65 frozen in regular LB with an optical density (OD) of Agrobacterium of 3.0 measured at 660 nanometers (final concentration).

[00135] An Agrobacterium culture was prepared and divided into two parts. A portion was cultured for an additional 4.5 hours to an OD ~ 0.6 in 36 milliliters of 2XYT medium, centrifuged, resuspended in DO ~ 3.0 in minimal AB medium containing 20% glycerol and frozen for use in a protocol of direct induction. For induction, 5 milliliters of the frozen stock were thawed, added to 70 milliliters of minimal AB medium containing acetosyringone and induced overnight.

[00136] The second part of the Agrobacterium culture was cooled on plates and incubated for 3 days; a first sowing culture prepared from these plates was grown overnight. This was followed by a second sowing culture prepared starting from the first sowing culture. Grown for one day and induced overnight at OD ~ 0.2 in minimal AB medium containing acetosyringone. [00137] The two induced Agrobacterium preparations were used to inoculate corn embryos in parallel experiments. Corn embryos from half of each ear were cut into each of the Agrobacterium preparations (DO ~ 1.0) for 15 minutes and then allowed to settle for 5 minutes. The embryos were removed and placed on coculture plates. Using the conventional protocol, a total of 883 embryos were inoculated, resulting in a total of 174 (20%) plants in the soil and an average transformation frequency of 17% (standard deviation = 11.4). Using the direct induction protocol, a total of 814 embryos were inoculated, resulting in a total of 153 (19%) plants in the soil and an average transformation frequency of 19% (standard deviation = 11.6). It was observed that the number of copies of the cp4 transgene was similar between treatments.

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Example 8

Methods for the Preparation of Corn Callus for Subculture Using Mechanical Means [00138] One of the most time-consuming and ergonomic processes in a corn processing production system is the establishment and maintenance of the embryonic callus for Agrobacterium-mediated transformation which requires manual callus rupture during the subculture stage. In addition, during the transformation process, many cells are transformed into a single immature embryo or a single piece of callus. However, current selection and regeneration practices treat each population of cells transformed into a single immature embryo or piece of callus as a single event, generally regenerating one plant per piece of tissue.

[00139] To alleviate the ergonomic overhead created by manual compression using callus forceps, especially type I callus, various mechanical means such as coffee grinders, baby food grinders, peppercorn grinders, herb grinders, Garlic cutters and knives for decorative cutouts have been tested as a means of breaking or cutting the subculture callus. Among these means, it was observed that the herb grinder and the knife for decoration cutouts are the most suitable for breaking or cutting the callus and for producing pieces of callus suitable for subculture and transformation.

[00140] The use of mechanical means for the preparation of corn callus for the subculture can also be done to separate many transformed cells within a certain tissue unit by cutting or breaking the tissue into smaller units of individually transformed cells starting of which a transformed plant can be regenerated, thus obtaining more transgenic plants from the same tissue unit.

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51/65 [00141] The callus for this example was derived from the cultivation of immature corn embryos in 1074 callus medium (Table 3), which were isolated from the developing grains approximately 10 days after pollination. A callus from five to nine weeks of age was treated with an herb grinder and placed in contact with Agrobacterium containing a vector carrying the nptll gene for selection for up to 60 minutes. The inoculated callus was blotted dry and co-cultured / dried for 2-3 days at 23 ° C in the dark. The callus tissue was then transferred to pieces of felt in 1086 liquid selection medium (Table 3) containing approximately 50 mg / L of paromomycin and cultured for up to 30 days at 27-28 ° C in the dark to select the transformed tissue containing the marker gene that can be selected npt \\. The transformed tissue was then regenerated in plants by cultivating the tissue that survived the selection in budding medium 1087 (Table 3) containing BAP to induce the formation of sprouts for 5-7 days at 27-28 ° C in 16 h of light . The growing tissue was incubated in 1067 medium (Table 3) for an additional 2-3 weeks to induce roots. Healthy shoots with or without roots were transferred to Phytatrays containing 1067 medium and subsequently to the soil for growth.

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Table 3: Compositions of the media used.

Media Components / L (Supplier) 1074 1086 1087 1067 Basal MS Salts (Phytotech) 4.33 g 4.33 g 4.33 g 4.33 MS Vitamins (100X) (Phytotech) 10 mL 10 mL 0 0 Vitamins from MS Fromm (1000X) * 0 0 1 mL 1 mL BAP (Sigma) 0 0 3.5 mg 0 Thiamine HCI (Sigma) 0.5 mg 0.5 mg 0 0 2,4-D (Phytotech) 0.5 mg 0.5 mg 0 0 Sucrose (Phytotech) 30 g 30 g 30 g 0 Glucose (Phytotech) 0 0 0 10g Maltose (Phytotech) 0 0 0 20 g Proline (Sigma) 1.38 g 1.38 g 1.38 g 0 Casamino Acids (Difco) 0.5 g 0.5 g 0.5 g 0 Asparagine Monohydrate (Sigma) 0 0 0 0.15 g Myo-inositol (Sigma) 0 0 0 0.1 g Phytagel (Sigma) 3g 0 3g 0 Phytagar (Gibco) 0 0 0 6g Carbenicillin (Phytotech) 0 500 mg 250 mg 250 mg Picloram (Sigma) 2.2 mg 2.2 mg 0 0 Paromomycin (Phytotech) 0 100 mg 100 mg 100 mg Silver Nitrate (Sigma) 3.4 mg 3.4 mg 0 0 pH 5.8 5.8 5.8 5.8 comprising 1250 mg / L nicotinic acid (Sigma), 250 mg / L pyridoxine HCI (Sigma), 250 mg / L of thiamine HCI (Sigma) and 250 mg / L of calcium pantothenate (Sigma).

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53/65 [00143] The results indicate that the herb grinder can be used successfully to break the callus into pieces suitable for subculture and processing (Table 4).

Table 4. Use of herb grinder to break up pieces of callus for subculture and for processing.

Expt Treatment & Description Grams of callus used Number of pieces of No. of plants produced TF (%) callus placed selection in 1. Cut manually, subcultured manually 2.5 192 53 27.6 1 2. Cut manually, mechanically subcultured 2.5 185 39 21.1 1. Cut manually, subcultured manually 1.5 144 9 6.3 2 2. Cut manually, mechanically subcultured 1.5 116 17 14.7

[00144] A decorating knife (Pickle Slicer, No. 4428 from International Culinary, Mystic, CT) was also used as a means of cutting callus to reduce the ergonomic burden of manual subculture methods. The decorating knife consisted of 8 parallel blades attached to the end of a plastic handle. The knife was sterilized by immersion in a mild bleach, followed by immersion in ethanol and then allowed to dry for approximately 30 minutes. While the knife was drying, the 4-week-old calluses (manually subcultured in 2 weeks) were placed in a 150 x 15 mm Petri dish and divided into 3 to 4 smaller piles inside each plate.

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54/65 containing approximately 40 - 60 pieces of callus. Working with one pile at a time, the knife was used to cut along the callus. With a sterile utensil, the callus was pushed out of the knife that had collected it between the blades to the plate, where it was cut a second time. Other piles of calluses were treated in the same way. The cut callus was used for subculture and processing.

[00145] The data from various experiments with the knife for decoration cutouts showed a transformation frequency of 13.16%. In additional tests, the transformation frequency of the manually subcultured callus of 21.5% was comparable to that of the callus prepared through the knife for decorative cutouts (18.9%). In yet another 74 experiments using a decorating knife as a means to cut the callus by 5 different users, the average transformation frequencies among all users were 19.4%, indicating the usefulness of this method.

Example 9

Methods for Inoculation of Explants with Agrobacterium for

Transformation [00146] The present methods for Agrobacterium-mediated transformation, for example, of immature corn embryos use many steps that involve cutting an embryo at a time of the grains, incubating it in suspension of Agrobacterium cells, removal of the Agrobacterium suspension, the transfer of the embryos to a coculture plate for infection to occur, the orientation of the embryos and the placement of the embryos with the scutellum side facing up and finally the transfer of the embryos to a selection medium or delay for additional manipulations such as selection and regeneration. Many of these steps are ergonomically adverse and also increase the possibility of

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55/65 damage the cut embryos and the higher incidence of cell death related to Agrobacterium thus resulting in a reduced transformation frequency. Therefore, there is a need in the corn processing technique to simplify the inoculation step, such that it is ergonomically favorable and results in a higher frequency of transformation.

[00147] The inventors have now simplified the inoculation step by reducing the total number of steps by inoculating the explants that can be transformed, for example, immature embryo, by 1) immersing a spatula in suspension of Agrobacterium before cutting the embryo with the spatula; 2) by soaking the ear of corn with the Agrobacterium suspension before cutting the embryo; or 3) by combining the two steps, for example, by soaking the corn cob with the Agrobacterium suspension followed by cutting the embryos with a spatula immersed in the Agrobacterium suspension.

[00148] In general, the following transformation method was used. Immature corn embryos from a recipient corn strain were desiccated from developing grains approximately 10 days after pollination and inoculated by incubation, immersed spatula (18.0%) or soaking the ear with Agrobacterium containing the construct. The inoculated embryos were co-cultured in 1514 co-culture medium (Table 5) for approximately 24 h at 230 in the dark. The embryos were then transferred for selection in 1278 selection medium (Table 5) containing 0.1 mM glyphosate to select the transformed tissue containing the marker gene that can be selected cp4 and 500 mg / L of Carbenicillin to inhibit the growth of Agrobacterium through incubation for 2-3 weeks at 270 ° in the dark. The transformed corn tissue was then regenerated into plants by transferring

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56/65 of the callus pieces transformed into the first 1073 regeneration medium (Table 5) and cultivated for 5-7 days with a photoperiod of 16 hours of light / 8 hours of darkness and a temperature of 27Ό. The tissue was then transferred to the second 1071 regeneration medium (Table 5) for approximately two weeks. The regenerated shoots were transferred to the 1084 rooting medium (Table 5) in Phytatrays. The developing seedlings were then transferred to the soil, allowed to stiffen in a growth chamber at 27Ό, 80% humidity and low light intensity for approximately one week and then transferred to a greenhouse and grown under standard greenhouse conditions.

[00149] Agrobacterium carrying 92 689 pMON plasmid containing a modified CP4 aroA gene for glyphosate selection (U.S. Patent 5,627,061 ²⁾ for inoculation was grown starting from a frozen stock made with MS induction medium as described in Example 7 The grains on the cob were sterilized with 80% ethanol and cut in the crown and then washed with 100 ml of% MSVI and 20 ml of Agrobacterium suspension were applied. A spatula was immersed in the Agrobacterium suspension and the immature embryos were cut and plated in the 1514 coculture medium for 24 h to 23Ό in the dark. These treatments were compared with a standardized treatment in which the embryos were cut in a moment and incubated with the Agrobacterium suspension for up to 5 min. The size of the embryos was 1.5-1.8 mm. However, it was found that the method worked well with other embryo sizes as well, for example, embryo sizes ranging from 1.4 to 2.2 mm. Transgenic events could be obtained by directly inoculating the explants with Agrobacterium while the explants were still adhered to maternal tissue and by inoculating the explants while they were being removed from the

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57/65 source. The following transformation frequencies were obtained for each treatment: inoculation by incubation (20.7%), inoculation by immersed spatula (18.0%) and inoculation by soaking the ear (17.0%).

[00150] Other means of placing the explants in contact with Agrobacterium and the duration of the contact can be predicted by those skilled in the art. For example, a knife immersed in the Agrobacterium suspension can be used for inoculation by cutting the leaf tissue or callus into smaller pieces, or a submerged needle can be used to inoculate the plant tissue by inserting the needle into the plant tissue. .

[00151] It was observed that the method increases the frequency of transformation allowing less tissue manipulation and less cell death related to Agrobacterium. With 6 different constructs, the immersed spatula method produced an average TF of 14.3% above the standardized incubation method which produced a TF of 9.2%.

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Table 5: Compositions of the media used.

Media Components / L (Suppliers) 1233 1278 1514 1524 1073 1071 1084 Basal MS Salts (Phytotech) 2.165 g 4.33 g 4.33 g 4.33 g 4.33 g 4.33 g 2.165 g MS Vitamins (100X) (Phytotech) 10 mL 10 mL 10 mL 10 mL 0 0 0 Vitamins from MS Fromm (1000X) * 0 0 0 0 1 mL 1 mL 0 BAP (Sigma) 0 0.01 mg 0 0 3.5 mg 0 0 Thiamine HCL (Sigma) 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0 0 0 2,4-D (Phytotech) 3 mg 0.5 mg 0.5 mg 0.5 mg 0 0 0 NAA (Sigma) 0 0 0 0 0 0 0.5 mg IBA (Sigma) 0 0 0 0 0 0 0.75 mg Sucrose (Phytotech) 20 g 30 g 30 g 30 g 30 g 0 20 g Glucose (Phytotech) 10g 0 0 0 0 10g 0 Maltose (Phytotech) 0 0 0 0 0 20 g 0 Proline (Sigma) 115 mg 1.38 g 1.38 g 1.38 g 1.38 g 0 0 Casamino Acids (Difco) 0 0.5 g 0.5 g 0.5 g 0.05 g 0.5 0 Asparagine Monohydrate (Sigma) 0 0 0 0 0 0.15 0 Myo-inositol (Sigma) 0 0 0 0 0 0.1 g 0 Low EEO Agarose (Sigma) 5j5 9 0 0 0 0 0 0 Phytagel (Sigma) 0 3g 3g 3g 3g 3g 3g Acetosyringone (Aldrich) 200 uM 0 0 0 0 0 0 Carbenicillin (Phytotech) 500 mg 500 mg 500 mg 500 mg 250 mg 250 mg 0 Glyphosate (Gateway Chemical) 0 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM Silver nitrate (Sigma) 3.4 mg 3.4 mg 3.4 mg 3.4 mg 0 0 0 PH 5.2 5.8 5.8 5.8 5.8 5.8 5.8 'Comprising 1250 mg / L nicotinic acid (Sigma), 250 mg / L pyridoxine HCI (Sigma), 250 mg / L thiamine HCI (Sigma) and 250 mg / L calcium pantothenate (Sigma). For liquid media, Phytagel was excluded from the composition.

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Example 10

Transformation Mediated by Maize Agrobacterium Performing Inoculation, Co-Culture and Selection in a Single Stage [00152] Normally, the methods of transformation mediated by Agrobacterium involve 3 distinct stages: inoculation of an explant with Agrobacterium, co-cultivating the explant inoculated in a medium without an antibiotic to allow the survival of Agrobacterium and to increase the infection of the explant and selection of the transformed cell in a medium containing a selective agent such as kanamycin and glyphosate. There is always a need to reduce the number of steps required in a transformation method to increase production efficiencies. The inventors have now provided a method for Agrobacterium-mediated transformation of maize in which the stages of inoculation, co-culture and selection can be combined in a single step by plating the explants inoculated directly onto a medium containing selective agents for growth suppression. Agrobacterium and to kill untransformed explant cells thus allowing inoculation, co-culture and selection in a single step, thereby increasing the efficiencies of the transformation production system.

[00153] In general, we used the transformation method disclosed in Example 9. The Agrobacterium containing pMON65375 comprising a CP4 aroA gene modified for glyphosate selection (U.S. Patent 5,627,061 ²⁾ for inoculation was prepared starting from a frozen stock done in minimal AB induction medium as disclosed in Example 7.

[00154] The spatula was immersed in the Agrobacterium suspension before cutting each embryo and the cut embryos were plated in the breeding medium 1233 and then in the selection medium 1278 or plated directly in the selection medium 1278. The results show 870170015706, of 10 / 03/2017, p. 68/86

60/65 show that the transformation can be done by combining inoculation, co-culture and selection in a single step with a variety of Agrobacterium concentrations and embryo sizes.

[00155] In another embodiment, the transformation was also achieved by first cultivating the embryos in the selection medium and then applying the Agrobacterium suspension immediately to the embryos instead of first applying Agrobacterium and then cultivating the embryos in the selection medium. Transgenic plants could also be obtained through the application of additional Agrobacterium during breeding and selection.

Example 11

Selection of Sorbarod-Transformed Cells and Development of High Yield System [00156] Suitable explants such as calluses and immature embryos can be obtained by mechanical and manual cutting means known in the art and can be inoculated with Agrobacterium containing a plasmid comprising any gene of interest known in the art. In one embodiment, the selection of a transformed cell is made on a piece of felt placed in the liquid medium and regeneration is done on a block of Sorbarod® (Baumgartner Papiers SA, Crissier-Lausanne, Switzerland) placed in the liquid medium. In another mode, both the selection and the regeneration of the transformed cell are carried out on a block of Sorbarod® placed in the liquid medium.

[00157] The usefulness of these modalities is demonstrated through several examples of data provided in Table 6. The compositions of the media used are shown in Table 5 except that for liquid media Phytagel was excluded. Immature corn embryos of the recipient strain were desiccated from developing grains and

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61/65 inoculated with Agrobacterium containing the binary vector pMON42073 comprising a CP4 gene for selection with glyphosate. The inoculated embryos were co-cultured in 1514 co-culture medium (see Table 5 for the composition) for approximately 24 h at 23Ό in the dark. The inoculated embryos were then transferred to a 5 X 5 cm square piece of felt (Consumer Products Enterprises (CPE) Inc., Union, South Carolina) or a Sorbarod block (ILACON Limited, Kent, UK) placed in the selection medium liquid 1278 (see Table 5 for composition) containing 0.1 mM glyphosate to select the transformed tissue. After this step, the used liquid selection medium 1278 was removed and replaced with the first liquid regeneration medium 1073 (see Table 5 for the composition). The tissue was grown for 5-7 days with 16 hours of light at 27Ό. Typically, this step requires transferring the selected tissue to the first solid regeneration medium for further cultivation. This method eliminates this step. The partially regenerated tissue in the first liquid regeneration medium was then transferred to a Sorbarod block placed inside a second liquid regeneration medium (see Table 5 for the composition) in a Sundae cup for complete regeneration. This transfer step was eliminated if the inoculated embryos had been grown directly on the Sorbarod block to start. In such a case, the first liquid regeneration medium was simply removed and replaced by the second liquid regeneration medium. The tissue was grown for approximately two weeks. Typically, this step requires transferring the regenerating tissue from the first solid regeneration medium to the second solid regeneration medium for further cultivation. This method eliminates this step. Smaller corn seedlings completely regenerated in the Sorbarod block were transferred directly to the soil. Usually, this step requires the transfer of

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62/65 a smaller seedling completely regenerated from the second solid regeneration medium to the 1084 solid rooting medium (see Table 5 for composition) for further cultivation. This method eliminates this step. The developing seedlings were allowed to stiffen in a growth chamber at 27Ό, 80% humidity and low light intensity for approximately one week and then transferred to a greenhouse and grown under standardized greenhouse conditions and tested for the presence of the CP4 gene. Table 6 shows the production of transgenic corn using liquid culture in combination with felt and Sorbarod block and eliminating several transfer steps.

[00158] A high-throughput processing system for corn is developed in which the containers are handled by robotic arms on a freely configurable work table that includes incubators and shakers that can be used in addition to standard laboratory utensils . Various liquid handling tools equipped with one or more pipette tips are used to supply fresh media and to remove spent media. The work table, robotic arms and liquid handling tools are controlled by software via a computer. Alternatively, the liquid medium for the selection and regeneration of the transformed cell can be supplied to the container via one or more tubes connected to a medium storage container and removed via one or more tubes connected to a disposal container. The supply and removal of the liquid medium is controlled manually or mechanically.

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Table 6. Frequency of transformation (TF) of corn using liquid medium in combination with different matrices. The frequency of transformation is calculated as the percentage of cultivated embryos that produced transgenic plants.

Middle Matrix Culture guidance during selection N ⁵ Explants N ⁵ Regenerated plants % TF Es- stud 1 Solid None 14-21 d, 27 ° C darkness 20 2 10 Solid None 10-d, 30 ° C darkness; 7-d, 28 ° C light 20 3 15 Liquid plug 10-d, 30 ° C darkness; 7-d, 28 ° C light 72 5 6.9 Liquid felt / block 10-d, 30 ° C darkness; 7-d, 28 ° C light 63 5 7.9 Es- stud 2 Solid None 14-21 d, 27 ° C darkness 20 2 10 Solid None 10-d, 30 ° C darkness; 7-d, 28 ° C light 20 4 20 Liquid block 10-d, 30 ° C darkness; 7-d, 28 ° C light 72 7 9.7 Liquid felt / block 10-d, 30 ° C darkness; 7-d, 28 ° C light 63 8 12.7 Es- stud 3 Solid None 14-21 d, 27 ° C darkness 30 6 20 Solid None 10-d, 30 ° C darkness; 7-d, 28 ° C light 30 9 30 Liquid block 10-d, 30 ° C darkness; 7-d, 28 ° C light 70 7 10 Liquid felt / block 10-d, 30 ° C darkness; 7-d, 28 ° C light 63 8 12.7

REFERENCES

The following references, to the extent that provide examples of procedures or other details supplementary to those presented here, are incorporated here specifically for reference.

US Patent No. ² 5,159,135

US Patent 5,362,865 ²

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US Patent No. ² 5,569,834

US Patent 5,106,739 ²

US Patent 5,107,065 ²

US Patent 5,627,061 ²

US Patent 5,633,435 ²

US Patent 6,506,559 ²

US Patent Application Publication No. ² 2002/0168707 US Patent Application Publication No. ² 2004/0244075 US Patent Application Series 09 / 423,143

Bird et al., Biotech Gen. Engin. Rev., 9: 207-227, 1991. Broothaerts et al., Nature, 433: 630, 2005.

Cheng et al., Dev. Biol.-Plant, 39: 595-604, 2003.

Chu et al., Scientia. Sinica., 18: 659, 1975.

Dekeyser et al., Plant Physiol., 90: 217-223, 1989. Della-Cioppa et al., Bio / Technology, 5 579-584, 1987. Duncan et al., Planta, 165: 322-332, 1985.

Duncan et al., Planta, 165: 322-332, 1985.

European Patent Application 0385 962 Gamborg et al., Exp. Cell Res., 50: 151, 1968.

Gibson and Shillitoe, Mol. Biotech.,. 7: 125-137, 1997. Jefferson, Plant Mol. Biol. Rep., 5: 287-405, 1987. Linsmaiere Skoog, Physiol. Plant., 18: 100, 1965. McCown and Lloyd, HortScience, 16: 453, 1981.

Murashige and Skoog, Physiol. Plant, 15: 473-497, 1962. Nitsch and Nitsch, Science, 163: 85-87, 1969.

PCT Patent Application WO 09/084942 PCT Patent Application WO 09/127735 PCT Patent Application WO 97/48814 PCT Patent Application WO 98/53083 PCT Patent Application WO 99/53050

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PCT Patent Application WO 99/61631

Sambrook and others In: Molecular Cloning, A Laboratory Manual, 1989.

Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, cold Spring Harbor Laboratory Press, NY, 2001.

Schenke Hildebrandt, Can. J. Bot., 50: 199-204, 1972.

Southern, Mol. Biol. 98: 503-517, 1975.

Uchimiya and Murashige, Plant Physiol., 15: 473, 1962.

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Claims (24)

1. Production process of a transgenic plant, characterized by the fact that it comprises: inoculating a plant embryo by contact of the embryo with recombinant Agrobacteria comprising a plant transformation vector, before excision, during excision, or before and during embryo excision to produce an inoculated embryo explant. 2. Process according to claim 1, characterized by the fact that the inoculated embryo explant comprises an immature embryo. 3. Process according to claim 1, characterized by the fact that the plant embryo is a corn embryo. 4. Process according to claim 1, characterized by the fact that the inoculation comprises contacting the embryo with a spatula, knife or needle having the recombinant Agrobacteria. 5. Process according to claim 4, characterized in that the inoculation of the regenerable plant explant with recombinant Agrobacteria comprises contacting the explant with a spatula having the recombinant Agrobacteria. 6. Process according to claim 1, characterized by the fact that the spatula, knife or needle is used to excise the embryo. Process according to claim 4, characterized by the fact that it comprises: immersing the spatula in a suspension comprising the recombinant Agrobacteria before contacting the embryo. 8. Process according to claim 1, characterized by the fact that it also includes the culture of the embryo with the recombinant Agrobacteria. Process according to claim 8, characterized Petition 870180055055, of 06/26/2018, p. 7/15 2/4 due to the fact that said coculture is carried out in a container selected from the group consisting of a bioreactor, a Petri dish, a plate of several cavities, a flask, a jug, a bottle, a moringa, a PlantCon, a temporary immersion system and a glass of sundae. 10. Process according to claim 8, characterized by the fact that it further comprises placing the inoculated explant in a delay medium. Process according to claim 10, characterized in that the retarding means comprises an agent that suppresses the growth of Agrobacteria. 12. Process according to claim 1, characterized by the fact that it further comprises placing the plant embryo in a selection medium comprising a selection agent that is selective for transgenic cells of the embryo explant. 13. Process according to claim 12, characterized by the fact that the plant transformation vector comprises a selectable marker gene conferring tolerance to the selective agent. 14. Process according to claim 9, characterized by the fact that it further comprises regenerating a transgenic plant from the embryo explant. 15. Process according to claim 14, characterized by the fact that said coculture and said regeneration are carried out in the same container. 16. Process according to claim 15, characterized by the fact that the same container is a bioreactor, a Petri dish, a multi-cavity dish, a flask, a jug, a bottle, a moringa, a PlantCon, a system of immersion or a glass of sundae. Petition 870180055055, of 06/26/2018, p. 8/15 3/4 17. Process according to claim 10, characterized in that said delay means is a liquid medium. 18. Process according to claim 10, characterized in that said delay means is a solid medium. 19. Process according to claim 5, characterized in that contacting the explant comprises soaking a plant part comprising the embryo in a suspension of the recombinant Agrobacteria. 20. Process according to claim 19, characterized by the fact that it also comprises contacting the explant with a spatula, knife or needle having the recombinant Agrobacteria. 21. Production process of a transgenic plant, characterized by the fact that it comprises: a) inoculating a plant tissue by cutting the plant tissue with a device having recombinant Agrobacteria comprising a plant transformation vector, to produce an inoculated plant tissue explant; b) co-cultivation of the plant tissue explant tissue with the recombinant Agrobacteria; and c) regeneration of the transgenic plant from the explanted embryo explant. 22. Process according to claim 21, characterized by the fact that the cutting device is selected from the group consisting of: coffee grinders, baby food grinders, herb grinders, garlic cutters, a garlic press, scissors , a knife, a trim knife, a needle and a spatula. 23. Process according to claim 21, characterized in that the plant tissue is leaf tissue. Petition 870180055055, of 06/26/2018, p. 9/15 4/4 24. Process according to claim 21, characterized in that the plant tissue is callus tissue. Petition 870180055055, of 06/26/2018, p. 10/15