EA042149B1 - TRANSGENIC CORN PLANT SHOWING INCREASED YIELD AND DROUGHT RESISTANCE - Google Patents

Description

During their life span, plants are subjected to a number of abiotic stress conditions such as heat stress, frost stress, chill stress, salt stress, drought stress, etc. Such stressful conditions are important limiting factors for plant growth and productivity. Thus, exposure of plants to, for example, heat and/or drought conditions typically results in reduced yields of plant material such as leaves, seeds, fruits, and other edible or usable products. Such a decline in yields is an important economic factor for economically important crops such as corn, rice or wheat, and the result of such a decline in yields, especially in underdeveloped countries, can lead to food shortages that jeopardize the food supply of the population.

Corn is the most widely produced crop in the world. This cereal is grown in at least 164 countries around the world with a total production of over 1 billion metric tons. Maize is grown in latitudes spanning from the equator to just over 50 degrees north and south latitude, at altitudes from sea level to over 3,000 meters, in both cool and hot climates, with growing cycles ranging from 3 to 13 months. Therefore, in order to feed the world's population, it is important that the supply of corn plants remains high. However, especially in regions with extreme weather conditions such as extreme heat, extreme cold, extreme humidity, severe drought, etc., there is a danger that an adequate food supply will not be secured, and this threat, given the obvious changes in the weather in recent years has become increasingly critical. In addition, the importance of corn as a renewable resource has increased in recent years, as the burning of resources such as oil, coal and natural gas contributes to a warmer climate on the planet, and resources are required that, due to their regrowth, do not adversely affect the carbon dioxide balance. .

With this in mind, there is a scientific need to supply maize and other crop plants that withstand all forms of climate stress and other abiotic factors, and are able to produce consistently high yields despite heat, cold, drought, salinity, humidity, etc.

Cell wall invertases, also called extracellular invertases, are important enzymes for proper plant metabolism, growth and differentiation. They act by hydrolyzing sucrose to glucose and fructose outside of cells, which are subsequently imported into target cells by monosaccharide transporters. Monosaccharides not only serve as a source of carbon and energy for plants, but are also key signaling molecules that potentially regulate cell division, as well as growth, differentiation, metabolism, and resource allocation in plants. Cell wall invertases are considered critical for the supply of carbohydrate-accepting tissues through the apoplastic pathway.

Cell wall invertases are known in the art as a factor in the potential increase in grain yield and biomass for certain plants. For example, Li et al. (Li B. et al. Plant Biotechnology Journal, 2013, 11, 1080-1091) describe the constitutive overexpression of three cell wall invertase genes (AtCWIN1, OsGIF1 and ZmMn1) in transgenic maize plants, leading to an increase in grain yield. Schweinichen and Buttner (Schweinichen C. and Buttner M., Plant Biol. (Stuttg), 2005, 7, 469-475) describe root-specific expression of Chenopodium rubrum cell wall invertase in Arabidopsis resulting in early flowering and increased whole plant biomass , probably due to increased root growth. Albacete A. et al. Journal of Experimental Botany, 2015, 66, 863-878 describes fetal-specific expression of Chenopodium rubrum cell wall invertase in transgenic tomato, which may lead to improved drought tolerance, but the researchers did not observe increased shoot weight or leaf area, i.e. biomass.

Despite these successes in increasing plant yields, there is still a need to develop economically viable plants that produce high grain yields even under unfavorable abiotic conditions.

To solve this problem, the present inventors have been able to develop corn plants that overcome the shortcomings of previous corn plants in that these corn plants exhibit both increased drought tolerance and increased biomass production. Thus, the present inventors introduced Chenopodium rubrum species cell wall invertase (CrCIN) into corn plants and found that such corn plants have increased yield and increased drought tolerance.

The present invention is described below with reference to the claims.

Hereinafter, the present invention is described in detail. Features of the present invention are described in separate paragraphs. This, however, does not mean that the feature described in any paragraph exists in isolation from the feature or features described in other paragraphs. Conversely, a feature described in a paragraph may be combined with a feature or features described in other paragraphs.

The term contain/containing in the context of this document means

- 1 042149 include or cover disclosed features and additional features that have not been specifically mentioned. The term contain/comprising also implies the meaning of consisting of/consisting of the specified features, while not including additional features other than those specified. Thus, this product and method of the present invention may be characterized by additional features in addition to these features.

The first object of the invention relates to a transgenic corn plant containing as a transgene i) a nucleic acid capable of expressing Chenopodium rubrum cell wall invertase (CrCIN) or a functional part thereof, ii) a nucleic acid capable of expressing Chenopodium rubrum cell wall invertase or a functional part thereof from pos. . i) which is modified by degeneration of the genetic code, iii) a nucleic acid capable of expressing cell wall invertase or a functional portion thereof, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98 % or 99% amino acid identity or at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid homology to Chenopodium rubrum cell wall invertase or its functional part from pos. i), or iv) a nucleic acid capable of hybridizing under stringent conditions with a complementary nucleic acid sequence of any of the positions. i) to iii), whereby the nucleic acid of pos. iv) is capable of expressing a cell wall invertase, and by expressing Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, the transgenic corn plant exhibits improved abiotic stress tolerance and/or increased yield, possibly compared to the reference sample.

In an embodiment of said invention, such a nucleic acid is derived from a nucleic acid of any one of pos. i) to iv) by codon optimization.

In an embodiment of the above invention, the nucleic acid POS. i) contains the nucleic acid sequence of SEQ ID NO: 3 or encodes the amino acid sequence of SEQ ID NO: 4.

In an embodiment of the above invention, the transgenic corn plant contains, as a transgene, an expression cassette containing a nucleic acid.

In an embodiment of the above invention, the nucleic acid or expression cassette is tightly integrated into the genome of the corn plant or transiently expressed in such a corn plant, for example, present in the corn plant as part of a vector.

In an embodiment of the above invention, expression of the nucleic acid is controlled by a promoter, preferably a constitutive promoter.

In an embodiment of the above invention, the abiotic stress is selected from drought, salinity, heat or cold, and/or the yield is biomass yield or grain yield.

The present inventors have surprisingly demonstrated that Chenopodium rubrum cell wall invertase is effective in increasing the tolerance of a corn plant to drought stress and/or increasing the yield of a corn plant. This is also surprising because the present inventors have also demonstrated that the same gene introduced into wheat plants was not effective in increasing biomass or grain yield. This shows that the effect of cell wall invertase in general and Chenopodium rubrum cell wall invertase in particular is not predictable under heterologous conditions.

Thus, by introducing a gene encoding Chenopodium rubrum cell wall invertase, the present inventors have been able to increase the tolerance of a corn plant to abiotic stress and/or increase (by biomass) the yield of a corn plant under normal and/or stress conditions. In particular, the present inventors have shown that a gene encoding Chenopodium rubrum cell wall invertase introduced into a maize plant was expressed, and expression of the gene resulted in increased leaf production, development of taller plants, and maize plants with higher drought tolerance compared to reference sample.

The transgenic corn plant of the present invention expresses Chenopodium rubrum cell wall invertase (CrCIN). The gene encoding cell wall invertase CIN1 and derived from the species Chenopodium rubrum is known in the art and, for example, is characterized by an accession number available in the NCBI database (National Center for Biotechnology Information; National Library of Medicine - 38A, Bethesda, Maryland, 20894, USA ; www.ncbi.nih.gov): accession number X81792.1 (SEQ ID NO: 1), encoding protein accession number CAA57389.1 (SEQ ID NO: 2). Chenopodium rubrum cell wall invertase and the gene encoding cell wall invertase are not limited to SEQ ID NOs: 1 and 2, but include any Chenopodium rubrum cell wall invertase naturally expressed by the Chenopodium rubrum species, as well as a gene encoding cell wall invertase Chenopodium rubrum. In addition, the transgenic corn plant of the present invention contains a nucleic acid that expresses a Chenopodium rubrum cell wall invertase homologue. A homologue, as defined herein, is a cell wall invertase that has an amino acid identity with invert

- 2 042149 cell wall pelvis of Chenopodium rubrum, for example identified by SEQ ID NO: 2, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or which has an amino acid homology with Chenopodium rubrum cell wall invertase, for example identified by SEQ ID NO: 2, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% . Accordingly, the term amino acid homology refers to identical or homologous amino acids. Homologous amino acid residues have similar chemical and physical properties, for example, amino acids belonging to the same group: aromatic (Phe, Trp, Tyr), acidic (GIu, Asp), polar (GIn, Asn), basic (Lys, Arg , His), aliphatic (Ala, Leu, lie, Val), with a hydroxyl group (Ser, Thr) or with short side chains (Gly, Ala, Ser, Thr, Met). It is assumed that substitutions between such homologous amino acids do not change the phenotype of the protein (conservative substitutions). Alternatively, the homologue is a cell wall invertase that is encoded by a nucleic acid capable of hybridizing under stringent conditions to a complementary nucleic acid sequence encoding a Chenopodium rubrum cell wall invertase, such as identified by SEQ ID NO: 2, or to a complementary nucleic acid sequence, encoding a cell wall invertase that has amino acid identity or amino acid homology to a Chenopodium rubrum cell wall invertase, such as identified above.

A Chenopodium rubrum cell wall invertase, such as identified by SEQ ID NO: 2, or a homologue thereof, confers increased resistance to abiotic stress in a corn plant, and/or a corn plant carrying a nucleic acid encoding a Chenopodium rubrum cell wall invertase, such as identified by SEQ ID NO: 2, or its homologue, has an increased yield, as an option, in comparison with the reference sample. Preferably, a Chenopodium rubrum cell wall invertase, such as identified by SEQ ID NO: 2, or a homologue thereof, is not capable of conferring resistance to abiotic stresses and/or increased yield of a wheat plant in the wheat plant in which it has been transformed, more specifically, cell wall invertase. walls of Chenopodium rubrum or its homologue may not be able to provide an increase in wheat plant height or grain yield.

As used herein, the term functional portion of a Chenopodium rubrum cell wall invertase or homologue thereof refers to any portion of a protein that has the same activity as a full length Chenopodium rubrum cell wall invertase, such as that identified by SEQ ID NO: 2, namely a functional part of the hydrolysis of sucrose into glucose and fructose. In addition, the functional part gives the corn plant increased resistance to abiotic stress and/or the corn plant bearing such a functional part has an increased yield, alternatively, in comparison with the reference sample. Preferably, the functional portion may not be able to confer abiotic stress tolerance and/or increased yield on the wheat plant in the wheat plant in which it has been transformed, more specifically, the functional portion may be unable to provide increased wheat plant height or grain yield.

In the context of this document, the term corn plant means any plant of the species Zea mays.

In the context of this document, the term nucleic acid can mean any DNA or RNA, or a hybrid of DNA and RNA. Preferably, the DNA is double stranded. It may be genomic DNA containing intron sequences and possibly regulatory sequences in the 5' and/or 3' region, or cDNA without intron sequences. The term nucleic acid as used herein includes nucleic acids that encode Chenopodium rubrum cell wall invertases, or functional portions thereof, or homologues thereof, within the meaning of the term given above. In addition, the term nucleic acid includes a nucleic acid that is modified by degeneration of the nucleic acid genetic code encoding a naturally occurring Chenopodium rubrum cell wall invertase.

As used herein, the term nucleic acid also includes a nucleic acid portion encoding a Chenopodium rubrum cell wall invertase or a homologue thereof, whereby such nucleic acid portion encodes a functional portion of a Chenopodium rubrum cell wall invertase or a homologue thereof, within the meaning of the term given above.

The term degeneration of the genetic code refers to the degeneration of codons (a term known from the prior art) and means the redundancy of the genetic code, manifested as a plurality of combinations of tribasic pairs of codons that define a given amino acid. Therefore, such a codon encoding an amino acid can be specifically changed without changing the amino acid. This results in multiple nucleic acids encoding the same Chenopodium rubrum cell wall invertase.

Percent sequence identity or sequence homology, as used herein, refers to the percentage of amino acid residues that are, respectively, identical or homologous at their respective positions in two optimally expressed

- 3 042149 random sequences. This percentage is determined by comparing two optimally aligned sequences in the comparison window, where the amino acid sequence fragment in the comparison window may contain additions or deletions (e.g., dropouts or overhangs) compared to a reference sequence (which contains no addition or deletion), for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions at which an identical or homologous amino acid residue occurs in both sequences to obtain the number of matching positions, dividing that number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100, which gives the percentage sequence identity. Optimal alignment of sequences for comparison can be performed using the local homology algorithm developed by Smith T.F. and Waterman M.S., Add APL Math, 1981, 2, 482-489, as well as using the homology region alignment algorithm developed by Needleman S.B. and Wunsch C. D., J. Mol. Biol., 1970, 48, 443-453, Pearson W.R. and Lipman D. J., PNAS, 1988, 85, 24442448, Karlin S. and Altschul S. F. development algorithm, PNAS, 1990, 87, 2264-2268, modified by Karlin S. and Altschul S. F., PNAS, 1993, 90, 5873-5877, or using computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA as part of the Wisconsin Genetics software package from Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by survey. To determine the optimal alignment, it is preferable to use the GAP and BESTFIT software modules. In general, the default values of 5.00 for the deletion penalty and 0.30 for the continuation deletion penalty can be used.

As used herein, the term hybridize/hybridize refers to the formation of a hybrid between two nucleic acid molecules by base pairing of complementary nucleotides. The term hybridize under stringent conditions means hybridization under specific conditions. An example of such conditions includes conditions under which a substantially complementary strand, such as a strand consisting of a nucleotide sequence having at least 80% complementarity, hybridizes to the strand, while a less complementary strand does not hybridize. Alternatively, such conditions refer to specific hybridization conditions for sodium salt concentration, temperature, and washing conditions. As an example, extremely stringent conditions include incubation at 42°C, 50% formamide, 5 x SSC (150 mmol NaCl, 15 mmol trisodium citrate), 50 mmol sodium phosphate, 5 x Denhardt's solution, 10 x dextran sulfate, 20 mg / ml salmon seed nucleic acid with shear breaks and washing with 0.2 x SSC at about 65° C. (SSC corresponds to buffer with 0.15 mol sodium chloride and 0.015 mol trisodium citrate). Alternatively, such extremely stringent conditions may mean hybridization at 68° C. in 0.25 mol sodium phosphate, pH 7.2; 7% SDS, 1 mmol EDTA and 1% BSA for 16 hours and a double wash with 2 x SSC and 0.1% SDS at 68°C. In a further alternative, extremely stringent hybridization conditions are, for example: Hybridization in 4 x SSC at 65°C followed by multiple washes in 0.1 x SSC at 65°C for a total duration of about 1 hour, or hybridization at 68 °C in 0.25 mol sodium phosphate, pH 7.2; 7% SDS, 1 mmol EDTA and 1% BSA for 16 hours followed by a double wash with 2 x SSC and 0.1% SDS at 68°C.

The present inventors have shown that a nucleic acid encoding a Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, can be expressed in a corn plant. Expressed cell wall invertase confers increased resistance to abiotic stress in the corn plant and/or such corn plant exhibits increased yield. Tolerance to abiotic stress means that the introduction and expression of Chenopodium rubrum cell wall invertase, or a functional part thereof, or a homologue thereof, in a corn plant renders such corn less susceptible to adverse abiotic conditions, resulting in the typical symptoms of stress due to adverse abiotic factors not occur, or occur, but to a lesser extent than in the reference sample. In a further or alternative embodiment, the introduction and expression of Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, in the corn plant increases the yield of the corn plant, alternatively, relative to a reference sample. Increased yield in the context of this document means that the transgenic corn plant exhibits an increased growth rate under normal conditions that do not cause stress to the plant, or under abiotic stress conditions, alternatively, compared to a reference sample. The concept of increased growth rate includes an increase in the mass production of the whole plant or part thereof, such as an increase in the mass production of the above-ground part of the plant, for example, stem, leaves, inflorescences, cobs and/or grains, etc., and/or an increase in the mass production of the underground plant parts. The increased mass production may include any part of the transgenic corn plant and refers in particular to the stalk, leaves, cobs and/or grains. Increased yield also includes longer growth and survival, which also results in increased mass production.

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As used herein, the term reference may refer to a corn plant of the same genotype as the transgenic corn plant of the present invention, where such reference does not contain a transgene encoding a Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof. Reference sample experiments including (a) a reference corn plant(s) may be conducted in parallel with experiments to test the properties of the transgenic corn plant of the present invention. However, experiments with a reference sample may also be carried out at a different point in time under comparable conditions, and their results may be compared after all experiments have been completed. Alternatively, the reference sample may be a specific (pre-)targeted indicator of yield or some symptom, such as the percentage of leaves that exhibited the drought curl symptom, which characterizes the corn plant as having resistance to an abiotic stress factor or as a plant not resistant to abiotic stress factor. For example, the reference value may be a predetermined value or a generally accepted value that provides the skilled person with a threshold value and helps him/her decide that the transgenic corn plant is or is not resistant to an abiotic stress factor, or that it has an increased yield, depending on whether the value of the transgenic corn plant is below or above such a reference value. Based on such a benchmark (e.g., number of curled leaves), the skilled artisan can then identify a corn plant as stress-tolerant if the number of curled leaves in that corn plant is less than the benchmark, or as having increased yield if so. the corn plant shows a yield that exceeds the benchmark. Thus, the corn plant(s) used to set the benchmark need not be, but may be, a corn plant of the same genotype as the transgenic corn plant. For example, a reference corn plant(s) can be (a) a corn plant(s) that has an abiotic stress tolerance that reflects the average tolerance of a population of corn plants adapted to a particular environment. One skilled in the art who wishes to develop a corn plant that is better adapted to a particular environment can use this score as a benchmark and design a transgenic corn plant that performs better and therefore is better adapted to a particular environment. Or, the reference corn plant(s) may be (a) a corn plant(s) with a certain degree of resistance to an abiotic stress factor, and the goal is to create a transgenic corn plant that has a higher degree of resistance to an abiotic factor. Likewise, one of skill in the art may wish to develop a transgenic corn plant with high yields under certain conditions and may use benchmark comparison to determine if such a transgenic corn plant is up to the task.

To determine whether a transgenic corn plant exhibits abiotic stress tolerance or increased yield, CrCIN transcript and/or protein expression and/or the expression level of the transgene can be determined according to methods known in the art. Thus, determining whether a transgenic maize plant harboring Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, has abiotic stress tolerance or increased yield does not necessarily require comparison with a reference sample. The authors of the present invention found that the introduction and expression of Chenopodium rubrum cell wall invertase leads to increased yield and increased resistance to abiotic stress factors, as shown in the illustrative part of the present description.

The terms abiotic stress or abiotic stress conditions refer to stress conditions for a corn plant due to abiotic, ie non-living, factors. Such abiotic factors include drought, salinity (salt concentration), heat or cold. While not wishing to be limited to the following, it can be hypothesized that the effect of Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, in the maize plant is due to an increase in the carbohydrate pool that results from increased activity of Chenopodium rubrum cell wall invertase, or a functional portion thereof, or its homologue in the corn plant, where such enzyme is overexpressed. Sugars that are generally known to have a protective effect against osmotic stress can lead to the formation of a molecular cellular phenotype in the corn plant that protects the corn plant from stressful conditions such as drought, salinity and/or thermal conditions in which osmotic events play some role. role. In addition, sugars that are commonly known to have a protective effect against exposure to cold or freeze temperatures can lead to the formation of a molecular cellular phenotype that protects the maize plant from stressful conditions such as cold snap.

In a preferred embodiment of the present invention, the nucleic acid contained in the corn plant of the present invention is codon-optimized. By

- 5 042149 Once a cell wall invertase has been chosen to transform the maize plant, the codons can be modified and adapted to the specific requirements of the host to maximize expression. Those skilled in the art are aware of codon optimization of a nucleic acid for expression in heterologous host cells. There are many commercial vendors who have developed algorithms that take into account appropriate transcription and translation optimization parameters and deliver a nucleic acid sequence tailored to the requirements of the nucleic acid and the host. For example, codon optimization can be performed using GeneOptimizer™ software, GeneArt, ThermoFisher Scientific. A preferred nucleic acid that is within the scope of the present invention is the codon-optimized sequence of SEQ ID NO: 3 derived from SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 4. These codons are specifically adapted to codon usage in the corn plant.

The term expression or expression means (1) transcription of a nucleic acid that is within the scope of the present invention into RNA or mRNA and/or (2) translation of the RNA or mRNA into a Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof.

In the context of this document, the term expression cassette means a nucleic acid molecule that consists of one or more open reading frames or genetic sequences that are expressed in (a) the protein(s) of the corn plant into which the expression cassette has been introduced, and the regulatory element(s) ) at position 5' and, alternatively, 3', which controls their expression. Thus, an expression cassette may contain a promoter regulatory sequence as well as a designated promoter operably linked to an open reading frame or other genetic sequence, as well as a 3' terminator regulatory region which may contain a polyadenylation site. This promoter directs the cell's machinery to make RNA and/or protein. The regulatory element(s) may be derived from a cell wall invertase nucleic acid that is introduced into the corn plant, or may be derived from different genes, so long as such regulatory element(s) is capable of functioning in the corn plant. In addition, the regulatory element(s) at position 5' may be derived from the same gene as the regulatory element(s) at position 3', or may be derived from different genes. In the context of this document, the term operably linked means that the expression of linked nucleic acid sequences occurs in the corn plant. The expression cassette may be part of a vector used to clone and introduce the nucleic acid into a cell.

To introduce a nucleic acid molecule capable of expressing Chenopodium rubrum cell wall invertase, or a homolog or functional portion thereof, into a cell, the nucleic acid molecule or expression cassette carrying the nucleic acid may be inserted into a vector. Vectors carrying a nucleic acid molecule are known to those skilled in the art. In addition to the nucleic acid molecule, the vector may contain regulatory element(s) at position 5' and optionally 3' that are capable of functioning in the corn plant. The regulatory element(s) is preferably heterologous to Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof. Thus, the vector may contain a promoter regulatory sequence operably linked to the nucleic acid molecule and optionally a terminator regulatory sequence. Preferably, such a vector is a shuttle vector for transformation in Agrobacterium tumefaciens bacteria and subsequent transfer of a nucleic acid molecule encoding Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, into maize plant cells by infecting the maize plant with the transformed Agrobacterium tumefaciens bacteria. More preferably, the vector is a binary vector which is a standard means of transforming higher plants with an Agrobacterium tumefaciens intermediate. It consists of T-nucleic acid boundaries, multiple cloning sites, replication functions for Escherichia coli and Agrobacterium tumefaciens, selectable marker genes, reporter genes, and other ancillary elements that can increase efficiency and/or add additional capabilities to the system. Another more preferred vector is a superbinary vector that carries additional virulence genes from the Ti plasmid and exhibits a very high transformation rate, which is valuable for evolutionarily stable plants such as cereals. A number of useful vectors are known in the art. Particularly preferred is a binary vector that contains a maize ubiquitin promoter (e.g., US 5,510,474 A, US 6,020,190 A, US 6,054,574 A, US 6,878,818 B1, US 6,977,325 B2) and an Agrobacterium tumefaciens nopaline synthase terminator sequence or a cauliflower mosaic virus 35S terminator sequence. as transcriptional control sequences, and preferably a herbicide-extended resistance gene (eg pat gene to confer resistance to Basta (eg US 7,112,665 B1)) and/or spectinomycin resistance gene as selectable marker genes.

According to this invention, the term promoter regulatory sequence or pro

- 6 042149 motor is intended to refer to any gene promoter that can be expressed in a corn plant. Such a promoter may be a promoter that is naturally expressed in the corn plant or is of fungal, bacterial or viral origin. This promoter may include a constitutive promoter, a tissue-specific promoter, or an inducible promoter, whereby constitutive expression is preferred. A number of suitable promoters are known in the art. For example, the constitutive promoter used in the present invention is the ubiquitin promoter from maize. Another promoter is the Act-1 promoter from rice (eg, US 5,641,876 A). The NCR promoter from Soybean Chlorotic Mottle Virus (SoyCMV) (Hasegawa, A., et al. Complete Soybean Chlorotic Mottle Virus DNA Sequence and New Promoter Identification Nucleic acids research 17.23 (1989): 9993-10013.) has also shown that it can be useful for monocot plants. Other useful promoters are the 35S CaMV promoter (Franck A. et al., 1980, Cell 21:285-294) and the 19S CaMV promoter from cauliflower mosaic virus (US 5,352,605; WO 84/02913) or plant promoters such as those were derived from the small subunit of Rubisco (US 4,962,028).

The promoter used in the method of this invention may be an inducible promoter. An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of a nucleic acid sequence in response to an inducer. In the absence of an inducer, the nucleic acid sequence will not be transcribed. Inducible expression may be desirable. The stimuli for inducible promoters are varied and include environmental conditions such as light, temperature and/or abiotic stress conditions such as water stress, salinity stress, cold stress or heat stress. Other types of stimuli for inducible promoters are hormones (eg, gibberellin, abscisic acid, jasmonic acid, salicylic acid, ethylene, auxin) or chemicals (tetracycline, dexamethasone, estradiol, copper, ethanol, and benzothiadiazole). Thus, expression of Chenopodium rubrum cell wall invertase, or a functional fragment thereof, or a homologue thereof, under specific inductive conditions, preferably abiotic stress conditions, results in protection of the maize plant by preventing stress symptoms and allowing mass formation. Inducible promoters are, for example, benzylsulfonamide-inducible promoters (EP 0 388 186), tetracycline-inducible Gatz C. et al., Plant J. 2, 1992: 397-404), abscisic acid-inducible (EP 0 335 528) or ethanol or cyclohexenol (WO 93/21334).

In addition to the promoter sequence, the expression cassette or vector may also contain a terminator behind the structural gene to ensure efficient termination. In the present invention, the term terminator or terminator regulatory sequence is intended to mean any such sequence that is functional to terminate nucleic acid expression in a corn plant, and optionally containing polyadenylation sequences. The terminator may be derived from the same gene as the promoter sequence, or it may be derived from a different gene. Thus, it may be of viral origin, such as the CaMV 35S terminator, which is preferred, bacterial origin, such as the octopine synthase terminator or nopaline synthase terminator from the bacteria Agrobacterium tumefaciens, or plant origin, such as the histone terminator.

Polyadenylation sequences include, but are not limited to Agrobacterium signal octopine synthase.

The term introduction or introduction in the context of this document means the insertion of a nucleic acid into a corn plant by any method known in the art, such as transformation using non-viral methods of introduction or transduction using a virus intermediate to transfer the gene. Numerous methods are known in the art for introducing a nucleic acid molecule into a corn plant (see, for example, Miki et al., Procedures for Introducing Foreign nucleic acid into Plants in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds (CRC Press, Inc., Boca Raton, 1993), pages 67-88). In addition, expression vectors and in vitro culture methods are available for plant cell or tissue transformation and plant regeneration (see, for example, Gruber et al., Vectors for Plant Transformation in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds (CRC Press, Inc., Boca Raton, 1993), pages 89-119).

The preferred method used in the present invention is to transform the nucleic acid molecule, expression cassette or vector carrying the nucleic acid molecule by using bacteria of the genus Agrobacterium, preferably by infecting cells or tissues of a maize plant with the bacteria A. tumefaciens (Knopf U.C, 1979, Subcell. Biochem 6: 143173 Shaw C H et al 1983 Annu Rev Genet 16: 357-384 Tepfer M and Casse-Delbart F 1987 Microbiol Sci 4(1): 24-28 ). For example, the transformation of cells or tissues of maize plants using the bacterium Agrobacterium tumefaciens is carried out in accordance with the protocol described by Hiei Y. et al. (1994, Plant J. 6(2): 271-282).

Another method for introducing a nucleic acid into a corn plant is the bioleaf method.

- 7 042149 chemical transformation, characterized in that cells or tissues are bombarded with particles on which a nucleic acid, expression cassette or vector is adsorbed, which are included in the scope of the present invention (Brace WB et al., 1989, Proc. Natl. Acad. Sci. USA 86(24): 9692-9696; KleinTM et al., 1992, Biotechnology 10(3): 286-291; US Patent No. 4,945,050). Another method is the widely used protoplast transformation. Consequently, plant cells are separated by pectinases and, subsequently, the cell wall is cleaved to form protoplasts. For transformation, polyethylene glycol can be added or electroporation applied. In other methods, plant cells or tissues are contacted with polyethylene glycol (PEG) and a nucleic acid, expression cassette, or vector of the invention (Chang S. and Cohen SN, 1979, Mol. Gen. Genet. 168(1): 111115; Mercenier A. and Chassy BM, 1988, Biochimie 70(4): 503-517). Electroporation is another method which involves subjecting the cells or tissues to be transformed and the nucleic acid, expression cassette or vector that is within the scope of the present invention to an electric field (Andreason GL and Evans GA, 1988, Biotechniques 6(7): 650-660; Shigekawa K. and Dower WJ, 1989, Aust. J. Biotechnol. 3(1): 56-62). Another method is to directly introduce the nucleic acid, expression cassette or vector that is within the scope of the present invention into cells or tissues by microinjection (Gordon and Ruddle, 1985, Gene 33(2): 121-136). Another method for physically delivering a nucleic acid to plants is sonicating target cells (Zhang et al., Bio/Technology 9: 996 (1991)). Alternatively, liposome or spheroplast fusion can be used to introduce into plants a nucleic acid, expression cassette or vector that is within the scope of the present invention (Deshayes et al., EMBO J., 4: 2731 (1985), Christou et al. ., Proc Natl Acad Sci USA 84: 3962 (1987)). Direct capture of nucleic acid in protoplasts using precipitation of CaCl ₂ , polyvinyl alcohol or poly-L-ornithine has also been reported (Hain et al., Mol. Gen. Genet. 199: 161 (1985); Draper et al., Plant Cell Physiol 23:451 (1982)).

The selection step for identifying the transgenic corn plant containing the nucleic acid can be carried out using a selectable gene present in the vector as described above. The selectable gene may contain an operably linked promoter regulatory sequence and possibly a terminator regulatory sequence that are functional in maize cells. Among the selectable markers that can be used in the present invention are antibiotic resistance genes such as the spectinomycin resistance gene, the hygromycin phosphotransferase gene, the neomycin phosphotransferase II gene that induces kanamycin resistance, or the aminoglycoside 3'-adenyltransferase gene, the bar gene (White J. et al., Nucl. Acids Res., 1990, 18: 1062) for bialaphos resistance, EPSPS gene (US 5,188,642) for glyphosate resistance or HPPD gene (WO 96/38567) for isoxazole resistance, genes encoding identifiable enzymes such as the GUS enzyme, GFP protein, or genes encoding pigments or enzymes that regulate pigment production in transformed cells. Such selectable marker genes are specifically described in patent applications WO 91/02071, WO 95/06128, WO 96/38567 and WO 97/04103. In a preferred embodiment, in the present invention, the spectinomycin resistance gene and the pat gene are used as binary vector selectable genes.

Transformation without marker genes is another alternative for transferring a nucleic acid, expression cassette or vector, as mentioned above, into a maize plant.

In one embodiment, such a nucleic acid or expression cassette is stably integrated into the genome of the transgenic maize plant, preferably into a plant chromosome, such as a nuclear, plastid and/or mitochondrial chromosome. However, integration can also occur into an extrachromosomal element. Through stable integration into the plant genome, nucleic acid sequences can be passed on to subsequent generations of the transgenic plant. Stable integration and transmission to the next generation of corn plants in the present invention is preferred. Using the plant transformation method with the bacterium Agrobacterium tumefaciens intermediate as the preferred transformation method, the Chenopodium rubrum cell wall invertase nucleic acid is stably integrated into the maize plant genome. Alternatively, the nucleic acid or expression cassette or the vector carrying the nucleic acid or expression cassette can be converted into an autonomous replicon. Alternatively, the nucleic acid molecule or expression cassette is present in the plant cell on the vector used to introduce the nucleic acid molecule and is not stably integrated into the plant genome, or the nucleic acid is transiently expressed, for example, as a transformed mRNA. Therefore, nucleic acid sequences cannot be passed on to subsequent generations of the corn plant.

The term heterologous in the context of this document refers to conditions in which molecules are present in environments in which they are not present in nature. For example, a nucleic acid molecule that is expressed in a host cell in which it is not naturally expressed is a heterologous nucleic acid. Therefore, the host cell is a heterologous host cell. Heterologous regulatory elements are those

- 8 042149 which are associated with nucleic acid molecules with which they do not naturally bind.

The term transgenic corn plant as used herein refers to a corn plant that contains a nucleic acid capable of expressing Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, integrated into its nuclear genome or organelle genome, or present on an autonomous replicon or on the vector used to introduce the nucleic acid of the present invention, or present as a simple coding sequence without other elements. The term also includes the following generations of offspring, such as T1, T2 or subsequent generations, as well as their crossing with non-transgenic or other transgenic plants. The transgenic corn plant preferably contains at least one copy of the nucleic acid that is within the scope of the present invention.

Expression of Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, in a maize plant improves resistance to abiotic stress conditions. Preferably, the abiotic stress against which the transgenic corn plant of the present invention exhibits increased resistance includes drought, salinity (salt concentration), heat and/or cold.

Drought or drought conditions means conditions of water scarcity resulting from a long period of insufficient or no water supply (water stress conditions), especially conditions that adversely affect the conditions for the growth and/or life of a corn plant. Under drought conditions, the plant will show symptoms of injury such as wilting, browning and/or curling of leaves, growth will be difficult, and eventually the plant will die. Drought conditions can be created by growing a corn plant at stages V2, V3, V4, V5, V6, V7, or V8 (according to the leaf knot method described below) for one week in 1/4 strength Hoagland's solution, followed by soaking for days in a 25% PEG6000 solution. The term drought tolerance as used herein may mean that a transgenic corn plant exhibits significantly reduced leaf curl symptoms under drought conditions induced by treating the plants with Hoagland's solution and a 25% PEG6000 solution. The term significantly reduced means that the percentage of leaves showing the curl symptom is reduced compared to the reference sample by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. Alternatively, a corn plant is drought tolerant if at most 60, 50, 40, 30, 20, 10, or 5% or less of the corn plant's leaves are in stages V2, V3, V4, V5, V6, V7, V8, and/or VT (fully mature plant with inflorescence) exhibit curling symptoms when exposed to drought conditions.

The terms salinity or saline conditions mean conditions of high salt concentration, such as 100 mmol NaCl solution for irrigation, especially in the air and/or in the soil, especially conditions that adversely affect the growth and/or life of the corn plant. The ability of plants to tolerate salt is determined by several biochemical reactions that promote water retention and/or uptake, protect chloroplast function, and maintain ionic homeostasis. Major reactions include those that result in the synthesis of osmotically active metabolites, specific proteins, or certain free radical scavenging enzymes that control the flow of ions and water and support the removal of oxygen radicals or chaperones. The reason why cell wall invertases protect maize plants from the adverse effects of salinity may be their ability to synthesize osmotically active compounds. Under saline conditions, the yield of a corn plant will be lower than under non-salinized conditions. Under conditions of prolonged and/or very high salinity, the corn plant will eventually die. Salt tolerance may mean that a transgenic plant at stages V2, V3, V4, V5, V6, V7, or V8 survives and/or grows under saline conditions compared to a reference plant that stops growing or grows to a lesser extent, while in conditions of very high salinity and/or over a long period of salinity, the corn plant will eventually die. By survival is meant that such a transgenic corn plant survives for a longer period of time, such that at least 10, 11, 12, 13 or more days exceeds the survival period of the reference sample. By growth is meant that the increase in yield of the whole corn plant or parts thereof, such as stalk, leaves, ears or kernels, is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the yield reference sample.

Heat or heat conditions means high temperature conditions of about 3340° C. at cob level during the 15 day period prior to flowering, and especially conditions that adversely affect the conditions for growth and/or life of a corn plant. The rate of growth and development of plants depends on the ambient temperature. Extreme heat events that occur during the growing season are likely to have the most significant impact on plant productivity, with the result that extreme heat can lead to lower grain yields. In general, extreme temperatures during the reproductive stage can affect

- 9 042149 pollen viability, fertilization and grain formation. Prolonged exposure to extreme temperatures at the stage of pollination of the initial set of grain reduces the potential yield of the grain. Severe short-term exposure to extreme events can be most harmful during the reproductive stage of development (Hatfield J.L. and Praeger J.H., 2015, Weather and Climate Extremes, 10: 410). With rising temperatures and more frequent temperature extremes expected due to a warming global climate, developing maize plants with increased tolerance to heat stress conditions is an urgent need. Tolerance to heat in the context of this document may mean that a transgenic plant at stages V2, V3, V4, V5, V6, V7 or V8 or at the stage of pollination survives and/or grows in heat conditions compared to a reference sample, the growth of which is stopped or occurs to a lesser extent. By survival is meant that such a transgenic corn plant survives for a longer period of time, such that at least 10, 11, 12, 13 or more days exceeds the survival period of the reference sample. By growth is meant that the increase in yield of the whole corn plant or parts thereof, such as stalk, leaves, ears or kernels, is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the yield reference sample.

Cold snap or cold snap conditions means conditions at a low temperature of less than 10°C but above the freezing point, and especially conditions that adversely affect the conditions for the growth and/or life of a corn plant. A cold snap can cause damage (chlorosis) and interrupt nutrient and water flow paths. Under colder conditions, the plant will show a reduced yield. Cold tolerance may mean that a transgenic plant at stages V2, V3, V4, V5, V6, V7, V8 survives and/or grows under cold conditions compared to a reference sample that stops or grows to a lesser extent. By survival is meant that such a transgenic corn plant survives for a longer period of time than the reference sample. By growth is meant that the increase in yield of the whole corn plant or parts thereof, such as stalk, leaves, ears or kernels, is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the yield reference sample.

It will be appreciated by those skilled in the art that, due to the large number of different varieties of corn grown under a wide range of climatic and other abiotic conditions, it is difficult to specify specific values that characterize drought, salinity, heat or cold conditions, such as duration of drought, degree salinity or temperature value, which would indicate to the person skilled in the art under what conditions the resistance to the abiotic factor should be tested. For example, in order to assess whether the corresponding transgenic corn is more resistant or will have a higher yield, corn plants with high drought tolerance will need more drought conditions than corn plants with lower drought tolerance. Therefore, test conditions will depend on the maize plant used to introduce the transgene and/or the purpose for which such transgenic maize plant will be used.

Since the introduction and expression of Chenopodium rubrum cell wall invertase results in an increase in the yield of the transgenic corn plant under normal and drought conditions, as well as the formation of a drought-tolerant phenotype, it is not necessary to compare a transgenic corn plant that expresses Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, with a reference sample as specified herein, to determine whether such a transgenic corn plant is resistant to abiotic stress such as drought, salinity, heat and/or cold. It may be sufficient to determine the expression of Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, for example by quantifying the transcript or protein, to find that there is resistance to abiotic stress factors such as drought, salinity, heat and/or cooling.

Resistance to an abiotic stress factor can be determined by exposing a transgenic corn plant to an abiotic stress factor and determining the degree of manifestation of stress factor symptoms and/or yield. The obtained indicators can be compared with the reference sample. Resistance can also be detected by determining the expression or expression level of a transcript expressed from a transgene that is within the scope of the present invention.

A second aspect of the present invention relates to a plant cell, tissue, harvestable part or seed of a transgenic corn plant of the present invention, characterized in that the plant cell, tissue, part or seed contains a transgene that is within the scope of the present invention.

In principle, any part, tissue, or organ of a maize plant that is within the scope of the present invention contains, as a transgene, a nucleic acid encoding a Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof. Thus, vegetative organs/shoot structures, such as leaves, stems, roots,

- 10 042149 flowers or floral organs/structures, such as bracts, sepals, petals, stamens, carpels, anthers or ovules; seed, including germ, endosperm or seed coat; grain or mature ovary; plant tissue, such as vascular tissue or integumentary tissue; or cells, such as protective cells, eggs or trichomes; or offspring of the above. The term cell refers to a cell or aggregation of cells in a plant, as well as an isolated cell or an isolated aggregation of cells. The cell may have a cell wall or may be a protoplast. The present invention also relates to a seed that contains a nucleic acid, expression cassette or vector, which are included in the scope of the present invention. Preferably, the nucleic acid, expression cassette, or vector that is within the scope of the present invention contains the seeds of the transgenic corn plant, so that new plants derived from the seed continue to contain such nucleic acid, expression cassette, or vector.

A harvestable part is any part of a plant that can be harvested and used by humans. Preferably, the harvestable portion may be the entire aerial part of the corn plant, which may be crushed, possibly fermented, and used in livestock production as animal feed, or may be used in biogas plants as a source for the production of substances providing energy production such as biofuels such as ethanol or methane. Preferably, the harvestable portion may be the cob, especially grains that are used for human and animal nutrition.

The third object of the present invention relates to a method for producing a transgenic corn plant, comprising the steps of introducing, at least into a cell of a corn plant, a nucleic acid, or an expression cassette, or a vector included in the scope of the present invention, as well as regenerating the transgenic corn plant from at least one cells.

In the context of this document, the terms regeneration or regeneration means the process of growing an entire corn plant from a single cell, group of cells, part of a corn plant, or tissue of a corn plant. The person skilled in the art knows methods of introducing a nucleic acid into at least a cell of a corn plant and then growing a corn plant from it. The expression in at least a cell means one cell, a group of cells, a part of a corn plant, or tissue of a corn plant.

The fourth object of the present invention relates to a method for increasing the resistance of a corn plant to abiotic stress and/or increasing the potential yield of a corn plant, comprising the steps of introducing into at least a cell of a corn plant a nucleic acid, or an expression cassette, or a vector, which are within the scope of the present invention , and causing expression of the nucleic acid, expression cassette or vector.

In the context of this document, the term induction of expression means that under the conditions under which such a plant is stored and/or cultivated, transcription of the nucleic acid introduced into the corn plant occurs. For example, if the promoter is a constitutive promoter, expression occurs sequentially, while if the promoter is an inducible promoter, the activity of such a promoter may be induced by the presence or absence of specific biotic or abiotic factors.

In the context of this document, the term yield potential refers to the ability of a transgenic corn plant to increase yield. By expressing Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, the ability of the maize plant to increase its yield can be provided.

A fifth aspect of the present invention relates to the use of a nucleic acid or expression cassette or vector that is within the scope of the present invention to increase the resistance of a corn plant to abiotic stress, to increase the yield potential of a corn plant, and/or to protect the corn plant from abiotic stress.

In the context of this document, the expression protecting a corn plant from abiotic stress means making the corn plant resistant to abiotic stress. The resistant maize plant is not damaged by abiotic stress factors or is damaged, but to a lesser extent compared to the reference sample. Resistance can be defined as resistance to abiotic stress. This includes that resistance can be determined by assessing the expression of the transcript and/or protein, or the level of expression from the transgene.

In an embodiment of the invention, in the method of the fourth aspect or for use in the fifth aspect, the abiotic stress is selected from drought, salinity, heat or cold, and/or the potential yield is a potential biomass yield or a potential grain yield.

The terms "potential biomass yield" or "potential grain yield" have the meanings given above with respect to potential yield and mean biomass yield and grain yield, respectively.

The term biomass generally refers to the organic material obtained from a plant. The term biomass can be used to refer to an energy source and should not be applied to

- 11 042149 food or feed. Thus, in the context of this document, the term biomass refers to the parts of the corn plant, usually the aboveground parts such as the entire aboveground corn plant, that can be used as an energy source after it has been converted to various biofuels such as ethanol or methane.

A sixth aspect of the present invention relates to a nucleic acid which is derived from a nucleic acid encoding Chenopodium rubrum cell wall invertase, or a functional portion thereof, or a homologue thereof, which are within the scope of the present invention, by codon optimization, and preferably characterized in that such nucleic acid contains the nucleic acid sequence of SEQ ID NO: 3 or encodes the amino acid sequence of SEQ ID NO: 4. The invention also relates to an expression cassette containing said nucleic acid or a vector containing said nucleic acid or expression cassette.

A seventh aspect of the present invention relates to a vector containing a nucleic acid or an expression cassette as defined in the present invention.

The eighth object of the present invention relates to a method for the production of ethanol or methane, comprising the following steps: crushing a transgenic corn plant or part suitable for harvesting, according to the present invention, alternatively, processing the crushed corn plant or part thereof with an ensiling agent, alternatively, storage a crushed corn plant or a crushed harvestable part thereof, optionally treated with an ensiling agent; and producing ethanol or methane from the crushed corn plant or a crushed harvestable part thereof by anaerobic digestion.

The eighth object serves to provide a method in which a transgenic corn plant is used as an energy source for the production of biofuels such as ethanol or methane, which are used in motor fuels, for heating, for generating electricity, etc. Processes for obtaining energy from corn plants are known in the biogas art in which ground corn or other plant material is stored and fermented by anaerobic bacteria in a process called ensiling.

The treatment of the chopped biomass with an ensiling agent serves to improve ensiling results. By adding strong lactic acid bacteria or other bacteria useful for anaerobic digestion of biomass and/or chemicals, unwanted bacteria such as butyric acid generating bacteria are suppressed. Chemicals to reduce unwanted bacteria such as butyric acid generating bacteria can be sodium nitrite or hexamine, or sodium benzoate or potassium sorbate can be used to prevent yeast and mold growth. In this way, interference with the anaerobic digestion process and/or the post-heating process can be eliminated, and the anaerobic digestion process can be controlled.

Storing the shredded corn plant, or the shredded harvestable portion of it, means placing it in a container, storage, or well, and then compressing it in such a way as to leave as little oxygen as possible, or to maintain storage under anaerobic conditions that inhibit the growth of aerobic bacteria. In order to ensure anaerobic digestion, storage should preferably be carried out under appropriate conditions regarding suitable temperature, humidity, low or no oxygen, etc. The person skilled in the art will be aware of the conditions and devices to be used for storage and anaerobic digestion.

The present invention discloses a method for making a corn plant resistant to abiotic stress, comprising the following steps: introducing into at least a cell of a corn plant a nucleic acid capable of expressing a cell wall invertase, or a functional part thereof, or a homologue thereof, of an expression cassette containing the nucleic acid , or a vector containing the nucleic acid or expression cassette, and initiating expression of the nucleic acid, expression cassette, or vector.

The present invention discloses the use of a nucleic acid capable of expressing a cell wall invertase, or a functional portion thereof, or a homologue thereof, an expression cassette containing the nucleic acid, or a vector containing the nucleic acid or an expression cassette, in order to render a maize plant resistant to abiotic stress or to protect corn plants from abiotic stress.

The above method or application may contain drought, salinity, heat and/or cold snap as abiotic stress.

The cell wall invertase as indicated in the above method and use may be any cell wall invertase, not limited to Chenopodium rubrum cell wall invertase, or a homolog or functional portion thereof, with the function of hydrolyzing sucrose to glucose and fructose outside the cell, which are then are transported into cells and confer resistance to abiotic stress in maize plants. These functions also apply to part and homologue, which are otherwise defined as above. Definitions of other features contained in the method or application, such as resistance, abiotic stress, expression cassette, age

- 12 042149 tor, etc., are contained in the present description.

The present invention is further explained in the following illustrations and examples, which are included herein for illustrative purposes and are not intended to limit the present invention.

Figures.

Fig. 1A-C: Vectors used to clone CrCIN: Three vectors were used to clone CrCIN. A: The first vector was obtained from GeneArt (ThermoScientific) containing the synthesized codon-optimized CrCIN gene (SEQ ID NO: 3). B: This gene was separated using the restriction enzymes BamHI and HindIII and cloned into the pABM shuttle vector containing the cloning cassette (ubi promoter and NosT terminator). C: This whole gene cassette was separated using the labeled SfiI enzyme and cloned into the pZFNmcherb binary vector for transformation into Agrobacterium and finally maize.

Fig. 2: Expression levels of homozygous T1 CrCIN plants: RT-qPCR displays the relative expression of selected CrCIN events to the endogenous ZmEF1 control gene. Both the non-transformed A188 and the transformation control (A188 transformed with an empty vector) showed no expression, while A188 lines containing CrCIN as a transgene showed expression of CrCIN.

Fig. Figure 3: T1 CrCIN plants winter 2015 (A) and T2 CrCIN plants winter 2016 (B): Photographs of transgenic CrCIN plants, selected events: Event 1, Event 5, Event 8 and Event 9 (E1, E5, E8 and E9) aligned with 2 controls, A188 WT (wild type) and TC (transformation control) during 2 growth periods at Week 9. Event 1 is missing from the 2016 experiment. All of the events shown here have shown a significant increase in yield (by biomass).

Fig. 4: Physiological measurements of T1 CrCIN plant at week 8: Yield comparison of transgenic T1 CrCIN event E9, E5 and E8 plants compared to A188 and transformation control plants (n=5) using the Iowa State University vegetative stage leaf count method at 8 weeks after sowing. Plants that were significantly different (Student's t-test) compared to A188 were marked with an asterisk, while plants that were significantly different from the transformation control were marked with a bar.

Fig. 5: Physiological measurements of the T1 CrCIN plant at week 8: Comparison of the height of transgenic T1 CrCIN event E9, E5 and E8 plants compared to A188 and transformation control plants (n=5) 8 weeks after seeding. Plants that were significantly different (Student's t-test) compared to A188 were marked with an asterisk, while plants that were significantly different from the transformation control were marked with a bar.

Fig. 6: Physiological measurements of a T2 plant at week 8: Yield comparison of transgenic T2 CrCIN event E9, E5 and E8 plants (n=20) compared to A188 and transformation control plants (n=40) using the State University vegetative stage leaf count method Iowa 8 weeks after sowing. Plants that were significantly different (Student's t-test) compared to A188 were marked with an asterisk, while plants that were significantly different from the transformation control were marked with a bar.

Fig. 7: Experiment 1: CrCIN corn seedling under simulated drought stress: A graph showing the percentage of leaves treated with 25% PEG6000 versus untreated plants (n=10) that showed symptoms of leaf curl. All plants were grown for 1 week in 1/4 strength Hoagland's solution and then treated for 1 day in added 25% PEG6000 solution. Both control events showed a high level of leaf curl. Event 5 showed a reduction in the leaf curl symptom. Events 8 and 9 showed a significant reduction in leaf curl symptoms.

Fig. Figure 8: Experiment 1: CrCIN corn seedlings under simulated drought stress: Photo of Event 8 seedlings of CrCIN in 1/4 strength Hoagland's solution 1 week after germination followed by 2 days treatment in 25% PEG6000 solution. Here it can be seen that the leaves of Event 8 show fewer symptoms of leaf curl than in the WT sample.

Fig. Figure 9: Experiment 1: CrCIN corn seedling under simulated drought stress: Photograph of representative CrCIN plants after 2 days treatment with 25% PEG6000 compared to a control plant grown in 1/4 strength Hoagland's solution. Initially, after germination, all plants were grown for 1 week in 1/4 strength Hoagland's solution and then transferred to a 25% PEG6000 solution.

Fig. 10: Experiment 2: CrCIN corn seedling under simulated drought stress: A graph showing the percentage of leaves treated with 25% PEG6000 versus untreated plants (n=10) that showed symptoms of leaf curl. All plants after germination were grown for 1 week in 1/4 strength Hoagland's solution and then treated for 1 day in added 25% PEG6000 solution. Both control events showed a high level of leaf curl. Events 5 and 9 showed a reduction in leaf curl, and Event 8 showed a significant reduction in leaf curl symptoms.

- 13 042149

Fig. Figure 11: Experiment 2: CrCIN corn seedling under simulated drought stress: Event photo of 8 CrCIN plants after 2 days treatment with 25% PEG6000 solution. Here it can be seen that the leaves of Event 8 show fewer symptoms of leaf curl than in the WT sample. Initially, after germination, all plants were grown for 1 week in 1/4 strength Hoagland's solution and then transferred to a 25% PEG6000 solution.

Fig. 12: Experiment 2: CrCIN corn seedling under simulated drought stress: Photograph of three representative CrCIN plants after 2 days treatment with 25% PEG6000 solution. The biggest difference is the development of the 3rd leaf in Event 8 and Event 9 plants compared to controls. Initially, after germination, all plants were grown for 1 week in 1/4 strength Hoagland's solution and then transferred to a 25% PEG6000 solution.

Fig. 13: Transgenic wheat CrCIN data: A: Wheat plasmid map pABMubi-CrCIN (Apr_ Ampicillin resistance) and B: Wheat plasmid map pLHAB-ubi-CrCIN (aadA: Spectinomycin resistance, Co1E1 ori: origin of replication for E. coli , pVS1 REP: origin of replication for Agrobacterium).

Fig. 14: Wheat: CrCIN T1 screening, CrCIN expression: Mean ± SE. Five biological replicas were used. CrCIN expression was analyzed from leaves of 4 week old plants grown in the greenhouse. TaEF expression was used as an internal control. All plants in the greenhouse were completely randomized.

Fig. 15: Overexpression of CrCIN does not increase yield or yield-related parameters of greenhouse-grown wheat. (A) Head length, (B) Number of grains per head, (C) Weight of grains per head, and (D) Weight of grains was measured on the first 4 mature shoots of greenhouse plants. Mean values are shown ± SD, N>10 biological replicas. Statistical analysis was done by two-way analysis of variance. Other growth parameters (such as plant height) also showed no significant differences.

Fig. 16. Overexpression of CrCIN does not increase yield in the field. The numbers represent the yield as a percentage of control plants (non-transgenic TAIFUN) in different locations. Single and multiple site analysis of variance showed no significant differences between transgenic CrCIN lines and control plants.

Fig. 17. Overexpression of CrCIN in wheat (TAIFUN) does not result in a detectable drought tolerance phenotype either by leaf dry weight (top) or root dry weight (bottom), black bar: control plant without drought stress; white bar: with drought stress simulated with 10% PEG; 1, 2, 3: CrCIN transgenic lines overexpressing CrCIN; 5 and 6: lines without CrCIN overexpression (control).

Examples.

Results with transgenic corn plants.

We first synthesized the Chenopodium rubrum cell wall invertase (CrCIN) gene and then transformed it into a shuttle vector cassette containing the ubiquitin promoter (containing the intron) from maize and the 35S terminator sequence to induce constitutive overexpression of the gene in the maize plant (Figure IA and B) . This cassette was then transformed into a binary vector containing, for example, a herbicide gene (for example: BASTA resistance, glyphosate resistance, or ALS inhibitor resistance) and a spectinomycin resistance gene for subsequent transformation into the bacterium Agrobacterium tumefaciens, through which transformation into maize is performed (Zea mays) genotype A188 (Fig. 1C).

These successively transformed corn germs were then selected by herbicide treatment and regenerated into plants for seed production under greenhouse conditions. Homozygous plants were grown from a batch of these T1 seeds. Expression levels of regenerated homozygous T1 CrCIN plants were determined by RT-qPCR displaying the relative expression of selected CrCIN events to the endogenous ZmEF1 control gene (FIG. 2). Both the untransformed A188 and the transformation control (A188 transformed with an empty vector) showed no expression, while A188 lines containing CrCIN as a transgene showed different levels of CrCIN expression.

In addition, homozygous T1 plants were analyzed in the greenhouse for general physiological changes using primarily a leaf fixation protocol containing a count of all leaves, including those that died, from the base of the plant to the first open leaf according to the Iowa State University protocol, which also known as the leaf knot method (Abendroth et al., 2011, Corn Growth and Development, Iowa State University, Available Inventory: 9182).

The leaf node method determines the stage of corn leaf formation by counting the number of leaves per plant with visible leaf nodes, starting with the lowest, short, true leaf with a rounded tip, and ending with the highest leaf with a visible leaf node. The leaf node is a ring-like band, tinted in a lighter color and located at

- 14 042149 the base of the open leaf plate, near the place where the leaf plate comes into contact with the stem of the plant. Curled leaves, as well as leaves that have not fully expanded and do not have a visible leaf node, are not taken into account in this method of capturing changes in leaves. An exception to this rule would be leaves with barely visible leaf nodes, which can be included in the count when changes in plants are recorded early in the day, and it can be assumed that the leaf node may become fully visible by the end of the day. Leaf stages are usually described as V stages, eg V2=two leaves with visible leaf nodes. The leaf knot method is, in general, the most widely used by university and industrial agronomists in the United States. Compared to control plants, mass accumulation in CrCIN plants was observed to increase from the V8 growth stage to the reproductive stage in all events that showed expression (FIG. 3A). This was measured by counting stage V plants, with transgenic plants having significantly more leaves than control A188 plants (FIG. 4).

In one experiment, the height of the plant was also measured by gathering the leaves in a tuft and pulling it up, after which the height of the plant was measured from the soil/base of the plant stem to the tip of the tallest leaf (Fig. 5).

Homozygous T2 seeds harvested from these plants were then grown a second time and this biomass phenotype was confirmed by V-stage determination after 8 weeks of greenhouse and field growth (FIG. 6). Plant height T2 was not re-measured as everything was clear on visual evaluation (see Fig. 3B).

T2 seedlings were tested in a hydroponic experiment with 25% PEG6000 in 0.25x Hoagland's solution to simulate drought stress (osmotic stress). Under drought stress conditions, maize seedlings typically develop severe leaf dehydration and exhibit leaf curl symptoms. Thus, leaf curl in plants such as corn can be used as an assessment of the apparent effects of water stress. physiology 65.3 (1980): 428-432.). Examining seedling leaf curl levels with CrCIN events E5, E8 and E9 showed increased resistance to PEG6000 compared to A188 control and transformation controls in replicate experiments (experiment 1: Fig. 7-9; experiment 2: Fig. 10- 12) T2 seedlings (Fig. 7-12). In these experiments, there appears to be a dosage effect, with the most pronounced events exhibiting the strongest phenotype. As can be seen from the experiments, all corn plants injected with CrCIN nucleic acid and expressing CrCIN show increased yield under normal and drought conditions, and also have a drought tolerant phenotype.

Negative results with transgenic wheat plants.

CrCIN was overexpressed in wheat using the ubiquitin promoter (pABM-ubiCrCIN and pLHAB-ubi-CrCIN; Fig. 13A and B). Homozygous T1 plants were screened in the greenhouse. CrCIN expression was analyzed from leaves of 4 week old plants grown in the greenhouse. TaEF expression was used as an internal control. All plants in the greenhouse were completely randomized. Non-transgenic control plants (TAIFUN transformed with an empty vector) showed no expression, while TAIFUN lines containing CrCIN as a transgene showed different levels of CrCIN expression. However, in contrast to the results observed in corn, in the greenhouse, overexpression of CrCIN in wheat does not unexpectedly increase yield or related parameters. Although various types of yield measurements have been made, e.g. plant height (data not shown), spike length (Fig. 15A), grain per ear count (Fig. 15B), grain weight per ear (Fig. 15C) and grain weight measured on the first 4 mature shoots of greenhouse plants showed no significant differences. The measurements were repeated with the T2 and T3 lines in the greenhouse and in the field. Field trials were performed at 5 different locations in a randomized whole block design (RCBD) in 4 replications, however, even these trials did not reveal significant differences in yield compared to the non-transgenic progenitor TAIFUN (FIG. 16).

In addition, overexpression of CrCIN in wheat does not show a significant effect on the potential drought tolerance of wheat. There is no noticeable difference in dry leaf weight or dry root weight between CrCIN overexpressing lines and non-CrCIN overexpressing control lines in response to PEG-induced drought stress (FIG. 17).

Claims (26)

CLAIM 1. Transgenic corn plant containing as a transgene i) a nucleic acid capable of expressing Chenopodium rubrum cell wall invertase according to SEQ ID NO: 2, ii) a nucleic acid capable of expressing Chenopodium rubrum cell wall invertase according to SEQ ID NO: 2 of pos. i) which is modified as a result of the degeneracy of the genetic code, iii) a nucleic acid capable of expressing a cell wall invertase having at least 90% amino acid identity of a Chenopodium rubrum cell wall invertase according to SEQ ID NO: 2 or iv) a nucleic acid capable of hybridizing into strict conditions with a complementary nucleic acid sequence of any of the positions. i) to iii), wherein such a nucleic acid is capable of expressing cell wall invertase, wherein, as a result of cell wall invertase expression, the transgenic corn plant exhibits improved resistance to abiotic stress and/or increased yield compared to a corn plant of the same genotype as the transgenic corn plant. a maize plant that does not contain the transgene. 2. Transgenic corn plant according to claim 1, characterized in that the nucleic acid is obtained from the nucleic acid of any of the pos. i) to iv) by codon optimization. 3. Transgenic corn plant according to claims 1, 2, characterized in that such nucleic acid contains the nucleic acid sequence of SEQ ID NO: 3 or encodes the amino acid sequence of SEQ ID NO: 4. 4. A transgenic corn plant according to any one of claims 1 to 3, containing as a transgene an expression cassette containing a nucleic acid. 5. Transgenic corn plant according to any one of claims 1 to 4, characterized in that the nucleic acid or expression cassette is firmly integrated into the genome of the corn plant or transiently expressed in such a corn plant, for example, is present in the corn plant as part of a vector. 6. A transgenic corn plant according to any one of claims 1 to 5, characterized in that the expression of the nucleic acid is controlled by a promoter, preferably a constitutive promoter. 7. A transgenic corn plant according to any one of claims 1 to 6, characterized in that the abiotic stress is selected from drought, salinity, heat or cold, and/or the yield is biomass yield or grain yield. 8. A plant cell of a transgenic corn plant according to any one of claims 1 to 7, characterized in that the plant cell contains a transgene as defined in any one of claims 1 to 6. 9. A tissue of a transgenic corn plant according to any one of claims 1-7, wherein the tissue contains a transgene as defined in any one of claims 1-6. 10. A harvestable portion of a transgenic corn plant according to any one of claims 1-7, wherein the portion contains a transgene as defined in any one of claims 1-6. 11. Seeds of a transgenic corn plant according to any one of claims 1-7, wherein the seeds contain a transgene as defined in any one of claims 1-6. 12. A method for producing a transgenic corn plant according to any one of claims 1 to 7, comprising the steps of: introducing into at least a cell of a corn plant a nucleic acid as defined in any one of claims 1 to 3 or 6, or expression cassettes, as defined in claim 4 or 6, or a vector containing a nucleic acid as defined in any one of claims 1 to 3 or 6, or an expression cassette as defined in claim 4 or 6, and regenerating the transgenic corn plant from at least at least one cell. 13. A method for increasing the resistance of a corn plant to abiotic stress and/or increasing the potential yield of a corn plant, comprising the steps of introducing into at least a cell of the corn plant a nucleic acid as defined in any one of claims 1-3 or 6, or cassettes expression, as defined in claim 4 or 6, or a vector containing a nucleic acid, as defined in any one of claims 1 to 3 or 6, or an expression cassette, as defined in claim 4 or 6, and causing expression of the nucleic acid, expression or vector cassettes. 14. The method of claim 13, wherein the abiotic stress is selected from drought, salinity, heat or cold, and/or the potential yield is a potential biomass yield or a potential grain yield. 15. Use of a nucleic acid as defined in any one of claims 1 to 3 or 6 or an expression cassette as defined in claims 4 or 6 or a vector containing a nucleic acid as defined in any of claims 1 to 3 or 6, or an expression cassette as defined in claim 4 or 6, to increase the resistance of a corn plant to abiotic stress. 16. The use according to claim 15, characterized in that the abiotic stress is selected from the series: drought, - 16 042149 cold, heat or cold. 17. Use of a nucleic acid as defined in any one of claims 1 to 3 or 6 or an expression cassette as defined in claims 4 or 6 or a vector containing a nucleic acid as defined in any of claims 1 to 3 or 6, or an expression cassette as defined in claim 4 or 6 to increase the potential yield of a corn plant. 18. Use according to claim 17, characterized in that the potential yield is a potential biomass yield or a potential grain yield. 19. Nucleic acid containing the nucleic acid sequence of SEQ ID NO: 3 or encoding the amino acid sequence of SEQ ID NO: 4. 20. An expression cassette containing a nucleic acid according to claim 19. 21. A vector containing a nucleic acid according to claim 19 or an expression cassette according to claim 20. 22. A vector containing a nucleic acid as defined in any one of claims 1-3 or 6 or an expression cassette as defined in claim 4 or 6. 23. Method for the production of ethanol, comprising the following steps: grinding a transgenic corn plant according to any one of claims 1 to 7, or a harvestable part thereof, according to claim 10, and obtaining ethanol from a crushed corn plant or a crushed harvestable part thereof , by anaerobic digestion. 24. The method according to claim 23, comprising one of the additional steps: storing the crushed corn plant or crushed part suitable for collection before obtaining ethanol from the crushed corn plant or crushed part suitable for collection by anaerobic digestion, or treating the crushed plant corn, or harvestable portion thereof, with an ensiling agent prior to obtaining ethanol from the ground corn plant, or harvestable portion thereof, by anaerobic digestion, or treating the ground corn plant, or harvestable portion thereof, with an ensiling agent and storing the ground corn plant or the ground harvestable portion thereof prior to producing ethanol from the ground corn plant or the ground harvestable portion thereof by anaerobic digestion. 25. A method for producing methane, comprising the following steps: grinding a transgenic corn plant according to any one of claims 1 to 7, or a harvestable part thereof, according to claim 10, and obtaining methane from a crushed corn plant or a crushed harvestable part thereof , by anaerobic digestion. 26. The method according to claim 25, comprising one of the additional steps: storing the crushed corn plant or crushed part suitable for collection before obtaining methane from the crushed corn plant or crushed part suitable for collection by anaerobic digestion, or treating the crushed plant corn or harvestable part thereof with an ensiling agent prior to obtaining methane from the crushed corn plant or harvestable part thereof by anaerobic digestion, or processing the crushed corn plant or harvestable part thereof with an ensiling agent and storing the comminuted corn plant or the comminuted harvestable portion thereof prior to producing methane from the comminuted corn plant or the comminuted harvestable portion thereof by anaerobic digestion.