Transgenic maize plant exhibiting increased yield and drought tolerance

Abstract

The present invention is directed to a transgenic maize plant or a part thereof comprising as transgene a nucleic acid capable of expressing a cell wall invertase or a functional part thereof, preferably a Chenopodium rubrum cell wall invertase or a functional part thereof, wherein as a result of the expression of the cell wall invertase or a functional part thereof the transgenic maize plant exhibits an enhanced tolerance to abiotic stress and/or an increased yield, to a method of producing such transgenic maize plant, to method of enhancing the tolerance to abiotic stress of a maize plant and/or of increasing yield potential of a maize plant, to a nucleic acid capable of expressing a cell wall invertase or a functional part thereof, preferably a Chenopodium rubrum cell wall invertase or a functional part thereof, to a vector comprising such nucleic acid, the use of the nucleic acid or vector for enhancing the tolerance to abiotic stress of a maize plant, for increasing yield potential of a maize plant and/or for protecting a maize plant against abiotic stress, and to a method for production of ethanol or methane from transgenic maize plant or a part thereof of the invention.

Claims

1. A transgenic maize plant comprising as transgene stably integrated into the genome of the maize plant: i) an expression cassette comprising a nucleic acid capable of expressing a Chenopodium rubrum cell wall invertase according to SEQ ID NO: 3, wherein the expression of the nucleic acid is controlled by an ubiquitin promoter comprising an intron and a 35S terminator sequence inducing constitutive overexpression of more than 10 fold relative to endogenous control gene ZmEF1, and wherein as a result of the expression of the cell wall invertase the transgenic maize plant exhibits an enhanced tolerance to drought and an increased biomass yield, optionally as compared to a reference.

2. A plant cell, a tissue, a harvestable part or a seed of the transgenic maize plant of claim 1, wherein the plant cell, the tissue, the part or the seed comprises the transgene.

3. A method of producing the transgenic maize plant of claim 1, comprising the following steps: introducing into at least a cell of a maize plant the expression cassette according to claim 1, or a vector comprising the expression cassette, and regenerating the transgenic maize plant from the at least a cell.

4. A method of enhancing the drought tolerance of a maize plant and of increasing biomass yield potential of a maize plant, comprising the following steps: introducing into at least a cell of a maize plant expression cassette according to claim 1, or a vector comprising the expression cassette, and causing constitutive overexpression of more than 10 fold relative to endogenous control gene ZmEF1.

5. A vector comprising the expression cassette according to claim 1.

6. A method for production of ethanol or methane comprising the following steps: cutting the transgenic maize plant according to claim 1, optionally treating the cut maize plant with an ensilage agent, optionally storing the cut maize plant optionally treated with an ensilage agent, and producing ethanol or methane from the cut maize plant by anaerobic digestion.

7. A method for production of ethanol or methane comprising the following steps: cutting the harvestable part according to claim 2, optionally treating the cut harvestable part with an ensilage agent, optionally storing the cut harvestable part optionally treated with an ensilage agent, and producing ethanol or methane from the cut harvestable part by anaerobic digestion.

Description

FIGURES

(1) FIG. 1A-C: Vectors used for Cloning CrCIN: Three vectors were used for cloning CrCIN. A: The first vector was received from GeneArt (ThermoScientific) containing a synthesised codon-optimized CrCIN gene (SEQ ID NO: 3). B: This gene was excised using the restriction enzymes BamHI and HindIII and cloned into the shuttle vector pABM containing the cloning cassette (ubi promoter and NosT terminator). C: This entire gene cassette was excised using the marked enzyme SfiI and cloned into the binary vector pZFNmcherb for transformation into Agrobacterium and finally maize.

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

(3) FIG. 3: T1 CrCIN plants winter 2015 (A) and T2 CrCIN plants winter 2016 (B): Photographs of transgenic CrCIN plants, selected events of Event 1, Event 5, Event 8 and Event 9 (E1, E5, E8 and E9) lined up with 2 controls, A188 WT (wildtype) and TC (transformation control) during 2 growth periods at Week 9. Event 1 is absent from experiment in 2016. All events shown here displayed significant increases in yield (biomass).

(4) FIG. 4: T1 CrCIN plant physiology measurements at week 8: Yield comparison of transgenic T1 CrCIN events E9, E5 and E8 plants compared to A188 and transformation control plants (n=5) using the Iowa State University Vegetative Stage leaf counting method at 8 weeks after sowing. Plants that were significantly different (student t-test) compared to A188 were marked with an asterisk while plants significantly different to the transformation control were marked with a hash.

(5) FIG. 5: T1 CrCIN plant physiology measurements at week 8: Plant height comparison of transgenic T1 CrCIN events E9, E5 and E8 plants compared to A188 and transformation control plants (n=5) at 8 weeks after sowing. Plants that were significantly different (student t-test) compared to A188 were marked with an asterisk while plants significantly different to the transformation control were marked with a hash.

(6) FIG. 6: T2 plant physiology measurements at week 8: Yield comparison of transgenic T2 CrCIN events E9, E5 and E8 plants (n=20) compared to A188 and transformation control plants (n=40) using the Iowa State University Vegetative Stage leaf counting method at 8 weeks after sowing. Plants that were significantly different (student t-test) compared to A188 were marked with an asterisk while plants significantly different to the transformation control were marked with a hash.

(7) FIG. 7: Experiment 1: CrCIN maize seedlings under simulated drought stress: Graph displaying the percentage of leaves of 25% PEG6000 treated versus untreated plants (n=10) that showed leaf rolling symptoms of leaves. All plants were grown for 1 week in ¼ strength Hoagland Solution and then treated for 1 day in added 25% PEG6000. Both control events showed high levels of leaf rolling. Event 5 showed reduction in leaf rolling symptoms. Event 8 and Event 9 showed a significant reduction in leaf rolling symptoms.

(8) FIG. 8: Experiment 1: CrCIN maize seedlings under simulated drought stress: Photo of Event 8 CrCIN seedlings in ¼ strength Hoagland for 1 week after germination followed by 2 days treatment with 25% PEG6000. Here it can be seen that the leaves of Event 8 show less leaf rolling symptoms than the WT.

(9) FIG. 9: Experiment 1: CrCIN maize seedlings under simulated drought stress: Photo of representative CrCIN plants after 2 days treatment with 25% PEG6000 versus control grow in ¼ strength Hoagland solution. All the plants were grown first for 1 week in ¼ strength Hoagland after germination before being transferred to the 25% PEG6000.

(10) FIG. 10: Experiment 2: CrCIN maize seedlings under simulated drought stress: Graph displaying the percentage of leaves of 25% PEG6000 treated versus untreated plants (n=10) that showed leaf rolling symptoms of leaves. All plants were grown for 1 week in ¼ strength Hoagland Solution after germination and then treated for 1 day in added 25% PEG6000. Both control events showed high levels of leaf rolling, Event 5 and Event 9 showed reduced levels of leaf rolling and Event 8 showed a significant reduction in leaf rolling symptoms.

(11) FIG. 11: Experiment 2: CrCIN maize seedlings under simulated drought stress: Photo of Event 8 CrCIN plants after 2 days treatment with 25% PEG6000. Here it can be seen that the leaves of Event 8 show less leaf rolling symptoms than the WT. All the plants were grown first for 1 week in ¼ strength Hoagland after germination before being transferred to the 25% PEG6000.

(12) FIG. 12: Experiment 2: CrCIN maize seedlings under simulated drought stress: Photo of three representative CrCIN plants after 2 days treatment with 25% PEG6000. The biggest difference is the development of the 3rd leaf in Event 8 and Event 9 plants versus the controls. All the plants were grown first for 1 week in ¼ strength Hoagland after germination before being transferred to the 25% PEG6000.

(13) FIG. 13: Data from wheat CrCIN transgenic plants: A: Plasmid map of wheat pABM-ubi-CrCIN (Apr_Ampicillin resistance) and B: Plasmid map of wheat pLHAB-ubi-CrCIN (aadA: Spectinomycin resistance, ColE1 ori: origin of replication for E. coli, pVS1 REP: origin of replication for Agrobacterium).

(14) FIG. 14: Wheat: CrCIN T1 screening, CrCIN expression: Mean±SE. Five biological replicates were used. CrCIN expression was analyzed from leaves of four week old plants grown in the greenhouse. Expression of TaEF was used as internal control. All plants were fully randomized in the greenhouse.

(15) FIG. 15: CrCIN overexpression does not increase yield or yield-related paramenters in wheat in the greenhouse. (A) Ear length, (B) Grain number per ear, (C) Grain weight per ear and (D) Grain weight was measured on the 4 first matured tillers of greenhouse-grown plants. Shown are Means±Standard error, N≥10 biological replicates. Statistical analysis was done by Two-way Anova. Other growth parameters (e.g. plant height) also did not show any significant difference.

(16) FIG. 16. CrCIN overexpression does not increase yield in the field. Numbers present yield in percentage of control plants (non-transgenic TAIFUN) at different locations. ANOVA analysis of single and multiple locations did not reveal any significant difference between transgenic CrCIN lines and control.

(17) FIG. 17. CrCIN overexpression in wheat (TAIFUN) does not lead to a detectable drought tolerance phenotype neither with respect to the leaf dry mass (top) nor to the root dry mass (bottom). black column: control without drought stress; white column: with drought stress simulated by application of 10% PEG; 1, 2, 3: transgenic CrCIN lines with CrCIN overexpression; 5 and 6: lines without CrCIN overexpression (control).

EXAMPLES

Results with Transgenic Maize Plants

(18) We first synthesized the Chenopodium rubrum cell wall invertase (CrCIN) gene and then transformed it into a shuttle vector cassette containing an ubiquitin promoter (containing an intron) from maize and a 35S terminator sequence to induce constitutive overexpression of the gene in a corn plant (FIGS. 1A and B). This cassette was then transformed into a binary vector containing for instance a herbicide gene (e.g.: BASTA resistance, glyphosate resistance or ALS inhibitor resistance) and spectinomycin resistance gene for subsequent transformation into Agrobacterium tumefaciens for Agrobacterium mediated plant transformation into maize (Zea mays) genotype A188 (FIG. 1C).

(19) These subsequently transformed maize embryos were then selected by herbicide treatment and regenerated into plants for seed production in the greenhouse. From this seed batch T1 homozygous plants were grown. The expression levels of the regenerated homozygous T1 CrCIN plants have been determined by means of RT-qPCR displaying relative expression of selected CrCIN events to endogenous control gene ZmEF1 (FIG. 2). Both non-transformed A188 and the transformation control (A188 transformed with empty vector) showed no expression, while those A188-lines containing CrCIN as transgene showed CrCIN expression at different levels.

(20) In addition, T1 homozygous plants were analyzed in the greenhouse for general physiological changes using primarily the leaf stage protocol comprising the counting of all leaves including the dead ones starting from the base of the plant to the first exposed leaf as per the Iowa State University protocol—also known as Leaf Collar Method (Abendroth et al., 2011, Corn Growth and Development, Iowa State University, Available Inventory: 9182).

(21) The Leaf Collar Method determines leaf stage in corn by counting the number of leaves on a plant with visible leaf collars, beginning with the lowermost, short, rounded-tip true leaf and ending with the uppermost leaf with a visible leaf collar. The leaf collar is the light-colored collar-like “band” located at the base of an exposed leaf blade, near the spot where the leaf blade comes in contact with the stem of the plant. Leaves within the whorl, not yet fully expanded and with no visible leaf collar are not included in this leaf staging method. The exception to this statement may be that leaves with barely visible leaf collars can be counted when you are staging plants early in the day, recognizing that the leaf collar may become completely visible by the end of the day. Leaf stages are usually described as “V” stages, e.g., V2=two leaves with visible leaf collars. The leaf collar method is generally the most widely used method by university and industry agronomists in the US. Mass accumulation in the CrCIN plants was observed to increase from the V8 stage of growth until reproductive stage compared to the control plants in all events that showed expression (FIG. 3A). This was measured by counting the V stages of the plants where the transgenic plants had significantly more leaves than the control A188 plants (FIG. 4).

(22) In one experiment, the plant height was also measured by bunching the leaves together and then pulling up and measuring plant height from the soil/plant stem base to the top of the tallest leaf (FIG. 5).

(23) T2 homozygous seeds collected from these plants were then grown a second time and the biomass phenotype was reconfirmed by determining V stages at 8 weeks growth under greenhouse and field conditions (FIG. 6). Plant height was not measured again in the T2 plants as this could be clearly seen by eye (cf. FIG. 3B).

(24) T2 seedlings were tested in a hydroponics experiment with 25% PEG6000 in 0.25× strength Hoagland solution to simulate drought stress (osmotic stress). Under such drought stress corn seedlings usually develop severe leaf dehydration and leaf rolling symptoms. Thus, leaf rolling in grasses like maize may be used as an estimate of obvious effects of water deficit (O'Toole, John C., and Rolando T. Cruz. “Response of leaf water potential, stomatal resistance, and leaf rolling to water stress.” Plant physiology 65.3 (1980): 428-432.). Investigating the levels of leaf rolling the seedlings with CrCIN Events E5, E8 and E9 showed enhanced tolerance to PEG6000 application compared to control A188 plants and transformation control in replicated experiments (experiment 1: FIGS. 7-9; experiment 2: FIGS. 10-12). of T2 seedlings (FIGS. 7-12). In these experiments there seems to be a dosage effect with the highest expressing events showing the strongest phenotype. As can be seen from the experiments, all maize plants into which the CrCIN nucleic acid has been introduced and which express CrCIN produce an increased yield under normal and drought conditions and have the drought tolerant phenotype.

Negative Results with Transgenic Wheat Plants

(25) CrCIN was overexpressed in wheat using an ubiquitin promotor (pABM-ubi-CrCIN and pLHAB-ubi-CrCIN; FIGS. 13A and B). Homozygous T1 plants were screened in the greenhouse. CrCIN expression was analyzed from leaves of four week old plants grown in the greenhouse. Expression of TaEF was used as internal control. All plants were fully randomized in the greenhouse. The non-transgenic control (TAIFUN transformed with empty vector) showed no expression, while those TAIFUN-lines containing CrCIN as transgene showed CrCIN expression at different levels. However, in contrast to the results observed in maize CrCIN overexpression in wheat surprisingly does not increase yield or yield-related parameters in the greenhouse. Even though different types of yield measurements have been executed, e.g., measuring plant height (data not shown), ear lengths (FIG. 15A), counting grain number per ear (FIG. 15B), measuring grain weight per ear (FIG. 15C) and grain weight measured on the 4 first matured tillers of greenhouse-grown plants, no significant difference have been determined. Measurements have been repeated with T2 and T3 lines in greenhouse and field. Field trials were done at 5 different locations with randomized complete block design (RCBD) in 4 replicates. even these trials revealed no significant difference in yield when compared to non-transgenic background TAIFUN (FIG. 16).

(26) Furthermore, the CrCIN overexpression in wheat does not show a significant effect on potential drought tolerance in wheat. There is no detectable difference in leaf dry mass or root dry mass between CrCIN overexpression lines and control lines without CrCIN overexpression in response to drought stress by PEG application (FIG. 17).