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  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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  • C12N9/14—Hydrolases (3)

Definitions

• the present invention relates to an improved method of transforming small grains such as wheat, barley, rice, oats, triticale, rye, sorghum and millet, using the dehal gene that encodes a dehalogenase that detoxifies the herbicide dalapon (2,2-dichloropropionate), in conjunction with dalapon, for selecting transformants of said small grains in an improved stepwise method with increased efficiency of transformant regeneration.
• the invention further relates to a redesigned dehal gene and to the use thereof to obtain transformants of small grains having field levels of resistance that allow controlling grass weeds with the herbicide dalapon.
• Glufosinate is an expensive organophosphorus herbicide whose synthesis is dangerous (via phosgene) making it inappropriate for small grains where production cost inputs must be low, and the herbicide synthesis be performed in the developing countries where it is used. Wheat has been transformed with resistance to the inexpensive herbicide ' glyphosate, but annual ryegrass has already evolved resistance to glyphosate in Australia (Gressel, 1996), making it unwise to use this herbicide alone in wheat crops.
• a herbicide resistance system in wheat should have the following traits: (1) the resistance should be to an inexpensive herbicide that controls a weed existing in the wheat land, e.g. Phalaris in India and annual ryegrass in Australia: (2) the herbicide should not be overly prone to have resistance to it evolved, as occurred with isoproturon in India; and (3) the herbicide should have acceptable toxicological and ecological characteristics.
• the dehal gene coding for resistance to the inexpensive herbicide dalapon pretty well fits these criteria. This herbicide presently kills most grassy species i.e. both small grains and the grass weeds that infest the small grains.
• the gene coding for a dehalogenase degrading dalapon has been isolated from Pseudomonas .and used to transform tobacco, where it provided a small differential of resistance in tobacco (Buchanan- Wollaston et al., 1992). This same gene was used a few years later in white clover (Derek White and Ian Gordon, Massey University, Palmerston North, New Zealand, pers.comm.1997). In neither case was expression sufficient enough for its being considered for use as a selective herbicide. Thus, the groups testing this gene were stymied, and stopped their projects due to lack of commercial significance, despite the large initial investment.
• the present invention thus relates, in one aspect, to an isolated DNA molecule encoding a dehalogenase that degrades the herbicide dalapon, said DNA molecule having codon usage suitable for small grain species.
• a dalapon-degrading dehalogenase gene can be obtained from a microorganism such as Pseudomonas by conventional methods. Once isolated, the gene is characterized using standard DNA manipulations, the open reading frame (coding sequence) is sequenced, and the gene is modified to allow integration and expression in plants of small grain species. The modification may be carried out by replacing codons of the bacterial gene by preferred codon usage of the small grain species coding for the same bacterial dehalogenase, or coding for a dehalogenase that has substantially the same amino acid sequence and is capable of rendering the small grain species resistant to dalapon.
• the small grain species may be wheat, barley, rice, oats, triticale, rye, sorghum and millet .
• the small grain species is wheat or rice.
• the DNA molecule comprises the nucleotide sequence depicted in Fig. 1 , but also genetic variants thereof having codon usage suitable for small grain species are encompassed by the invention.
• the DNA of Fig. 1 is a synthetic gene obtained by replacing codons of the Pseudomonas-de ⁇ ved dehal gene depicted in Fig. 4 by preferred small grain codon usage, which significantly enhances the level of resistance.
• the Pseudomonas-de ⁇ ved dehal gene contains several codons such as GGT (glycine), GTA (valine), AGT (serine), ATA (isoleucine), AGC (threonine),
• GGT glycine
• GTA valine
• AGT serine
• ATA isoleucine
• AGC threonine
• CTT and TTA leucine
• CGA and CTT arginine
• CCT and CCC proline
• the invention further relates to vectors and to expression cassettes comprising a DNA molecule of the invention, which are suitable for the transformation of plant cells and plants of small grain species in order to confer to the regenerated plants resistance to dalapon at field use levels of more than 1 kg dalapon/hectare, preferably 2-4 kg/Tiectare.
• the expression cassette contains the DNA molecule under the control of a plant promoter region and of regulatory elements allowing for the expression of said DNA molecule in plant cells.
• Any small grain- effective plant promoter such as, but not being limited to, CaMV 35 S promoter, rice actin promoter and ubiquitin promoter, may be added upstream of the coding sequence of the DNA molecule, possibly with other regulatory sequences such as transcriptional enhancer sequences. These 5' regions may be native to the small grain species or may be derived from other plants or chemically synthesized.
• the small grain-effective plant promoter is the CaMV 35S promoter with exon 1 from rice actin 1 promoter gene and intron 1 from the maize shrunken 1 gene.
• the expression cassette further contains a transcriptional termination sequence and may include polyadenylation signal sequences added downstream the DNA molecule.
• This 3' region may be derived from the same gene as the 5' sequences or from a different gene, or is chemically synthesized, and is preferably the octopine synthase terminator (osc. ter.).
• a vector for introducing the cassette containing the DNA molecule into plant cells will depend on the choice of the transformation method which, in turn, depends on the host plant and the choice of plant tissue source. Any kind of transformation protocol suitable for small grain species such as, but not being limited to, the use of electroporation, microprojectiles, micro injection, viral systems, and Agrobacterium- mediated transformation, can be used according to the invention.
• the plasmid containing the DNA molecule was transferred to competent E. coli cells, the cells were cultured, the plasmid DNA was then extracted from the precipitated cells and purified, and the transformation of wheat cells was carried out by ballistic bombardment protocol using a helium-driven delivery gun.
• the expression cassette used in this protocol contained the pCJVW14 plasmid as depicted in Fig. 3 wherein D ⁇ HAL is a DNA sequence having small grain codon usage encoding a dalapon-degrading dehalogenase inserted into the Bluescript(KS+) cloning vector.
• the expression cassette contained the pDM804 or pDM805 plasmid as depicted in Figs.
• the invention relates to transformed plant cells of a small grain species, particularly wheat, comprising a foreign DNA molecule having codon usage suitable for small grain species encoding a dehalogenase that degrades dalapon, which plants regenerated from said plant cells are substantially resistant to dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare.
• the foreign DNA encodes preferably a dehalogenase having the amino acid sequence depicted in Fig. 1 or a variant thereof that confers to small grain species resistance to dalapon at the desired field use levels.
• Said DNA molecule preferably comprises the coding nucleotide sequence depicted in Fig. 1 or a genetic variant thereof that encodes a dehalogenase that confers to small grain species resistance to dalapon at the desired field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare.
• the invention relates to seeds of small grain species, particularly wheat or rice, which possess, stably integrated in their genome, a foreign DNA molecule having codon usage suitable for small grain species expressing a dehalogenase capable of degrading the herbicide dalapon at a level sufficient to render the species resistant to dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare.
• the invention relates to plants of small grain species, particularly wheat, resistant to dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare, which possess, stably integrated in their genome, a foreign DNA molecule having codon usage suitable for small grain species encoding a dehalogenase capable of degrading dalapon.
• the invention provides a method for rendering a plant of a small grain species, particularly wheat or rice, resistant to the herbicide dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare, which comprises:
• step (c) regenerating from said plant cells of step (b) small grain plants resistant to dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare.
• Suitable plant tissue sources for transformation include, but are not limited to, callus, suspension culture cells, protoplasts, embryos and the like, such tissues being those that possess the ability to regenerate whole, fertile small grain plants following transformation.
• the transformation is carried out under conditions i.e. buffer, media, and periods of time, that are adapted to the tissue of choice.
• the plant cells or tissue may be cultivated for varying periods of time prior to selection, or may be immediately treated with dalapon as the selectable marker.
• Cells or callus growing in the presence of normally inhibitory concentrations of dalapon are presumed to be transformed and are subjected to known plant regeneration protocols, optionally after having been subcultured several additional times on the same medium to remove non-resistant sections.
• the regeneration is carried out in several steps in a suitable medium in the presence of varying concentrations of dalapon and plant growth hormones such as indoleacetic acid (IAA) and zeatin.
• IAA indoleacetic acid
• zeatin zeatin.
• a putatively dehal gene with small grain codon usage is used to transform embryogenic scutellar cultures of the small grain e.g.
• a method of increasing the regeneration efficiency of the transformed plants of small grain species comprises transferring the primary regenerants through a stepped series of plant hormone and dalapon concentrations, wherein the regenerants are floating on a liquid medium on a membrane raft.
• the invention relates to the use of dalapon as herbicide in fields containing small grain species, particularly wheat, resistant to dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare, and to a method for protecting a small grain plant species while destroying weeds in a field with the herbicide dalapon, comprising applying dalapon to a field of small grain species containing a foreign DNA having codon usage suitable for small grain species molecule, stably integrated in their genome, which expresses a dalapon-degrading dehalogenase at levels sufficient to render said small grain species resistant to dalapon at field use levels of more than 1 kg/Tiectare, preferably 2-4 kg/hectare.
• An additional aspect of the invention relates to the use of dalapon as a selectable marker in the genetic transformation of plants with a foreign gene imparting to the plant a desired trait, in the place of the usually used antibiotics, and to a method for introducing a desired trait in a plant, which comprises:
• step (c) regenerating a dalapon-resistant plant from said plant cells of step (b), whereby said plant cells also contain the DNA expressing the desired trait.
• a natural dalapon-degrading microbial dehalogenase gene may be used such as the Pseudomonas-derived dehal gene of the sequence depicted in Fig. 1, and it may be continuously or discontinuously present at most stages of the regeneration process of the primary transformed plant cells.
• Fig. 1 depicts the nucleotide sequence of the synthetic dehal gene with wheat codon usage encoding the dalapon-degrading dehalogenase and the amino acid sequence of the encoded dehalogenase.
• Fig. 2 depicts the cassetes pCJ14 (left) and pVW264 (right) used for the construction of the synthetic dalapon-degrading dehalogenase genes.
• Fig. 3 depicts the dalapon-resistance cassete pCJVW14 containing the synthetic dalapon-degrading dehalogenase (DEHAL) gene depicted in Fig. 1.
• DEHAL dalapon-degrading dehalogenase
• Fig. 4 depicts the nucleotide sequence of the Pseudomonas dehal gene encoding a dalapon-degrading dehalogenase and the amino acid sequence of the encoded dehalogenase. Rare codons in wheat are underlined and in bold.
• Fig. 5 depicts the dalapon-resistance cassete pDM804 containing the synthetic dalapon-degrading dehalogenase wss) gene depicted in Fig. 1.
• Fig. 6 depicts the dalapon-resistance cassete pDM805 containing the synthetic dalapon-degrading dehalogenase (wss) gene depicted in Fig. 1 as well as a gene coding for superoxide dismutase (Sod).
• Tungsten particles (60 mg, 1.0 ⁇ m diameter) were sterilized in 1 ml 100% ethanol overnight, then particles were washed 3 times with sterile water, and kept in 1 ml 50%) sterile glycerol. Tungsten particles (60 mg, 1.0 ⁇ m diameter) were kept in 1 ml 100% ethanol overnight, then they were washed 3 times with sterile water, and kept in 1 ml 50% sterile glycerol.
• the mixture used for bombardment contained: 1) 50 ⁇ l particle suspension; 2) lO ⁇ l plasmid DNA (l ⁇ g/ ⁇ l); 3) 25 ⁇ l 2.5 M CaCl2; 4) 20 ⁇ l 0.1 M spermidine.
• the suspension was incubated at room temperature for 10 minutes. After centrifugation for 5 seconds, the supernatant was discarded and the mixture washed with 140 ⁇ l 70% ethanol. The supernatant was removed after short centrifugation, then 50 ⁇ l 100%) ethanol were added to make the final suspension.
• a 6 ⁇ l drop of the suspension was placed in the center of the plastic macroprojectile. Forty to fifty embryos were placed in a 1 cm diameter center of each plate, 9 cm below the stop net, 1 100 p.s.i. (pounds per square inch) pressure was used and embryos were bombarded three times and left on the high osmotic medium for 24hours in the dark.
• the calli were transferred to rafts (Osmotek Lt, Rehovot 76120, Israel) with liquid MS medium containing 10 ⁇ g/ml zeatin and 20 ⁇ g/ml dalapon (0.12mM, the minimum concentration of dalapon that can block the untransformed calli growing in liquid medium) kept under 16-h light and slow shaking (50 rpm) conditions.
• the concentration of zeatin was decreased to 6 ⁇ g/ml in same selection medium, then used liquid MS medium containing also 1 ⁇ g/ml zeatin, 1 ⁇ g/ml IAA (indoleacetic acid) and 20 ⁇ g/ml dalapon to culture for 14 days, then the concentration of IAA was decreased to 0.2 ⁇ g /ml, and dalapon to 10 ⁇ g /ml.
• the shoots and roots were strong enough (about 5 cm each in length), the plantlets were transferred to solid medium with 0.5 MS containing 20 ⁇ g /ml dalapon.
• Example 1 As the transgenic plants in Example 1 could not withstand 2 kg/ha dalapon (as was with tobacco and white clover in the prior art as described previously herein), we analyzed whether the gene was being sufficiently transcribed, by northern blotting, and found it was. We then analyzed the codon sequence of the Pseudomonas-de ⁇ ved dehal gene (as provided by Dr. Buchanan- Wollaston) and compared it to the average codon usage of wheat for 45 genes, calculated from the data in Gen Bank 63. Many codon usages were distinctly not preferred or were exceedingly rare in wheat. The >30 rare codons for wheat interspersed along the gene are indicated in bold in Fig. 4.
• the new synthetic dehal gene with codon usage suitable for small grains was then ligated as performed in Example 1 to form the new constructs pDM804 and pDM805 shown in Figs. 5 and 6, respectively. These constructs are then transformed into wheat or other small grains by standard methods such as those described in Example 1 or by Agrobacterium-mediated transformation, in order to confer sufficient resistance to withstand field use rates of dalapon (2-4 kg/ha).
• the plasmid pDM804 has the following characteristics: plasmid size - 9.28 kb; the genes are labelled inside the plasmid and are indicated as follows: actin - actin promoter from rice; gus - ⁇ -glucoronidase; intron 1 - intron frm maize shrunken gene; nos3' - nopaline synthase terminator; rbcs3' - rubisco small subunit terminator; Ubil - ubiquitin promoter of maize; wss - synthetic dehalogenase gene.
• the plasmid restriction sites are shown on the outside.
• the plasmid pDM805 has the following characteristics: plasmid size - 8.15 kb; the genes are labelled inside the plasmid and are indicated as follows: actin - actin promoter from rice; gus - ⁇ -glucoronidase; intron 1 - intron frm maize shrunken gene; nos3' - nopaline synthase terminator; rbcs3' - rubisco small subunit terminator; Sod - Cu,Zn superoxide dismutase; Ubil - ubiquitin promoter of maize; wss - synthetic dehalogenase gene.
• the plasmid restriction sites are shown on the outside.
• the plasmid pDM804 (Fig. 5) containing the synthetic dehal gene (wss) of Fig. 1, was used to transform wheat as in Example 1, but the initial regeneration medium contained 0.6 mM dalapon, a rate that was toxic to and prevented shoot formation on transformants containing the native Pseudomonas dehal gene (Fig. 4). Copious shoot initials appeared on the transformed calli at this rate.

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• 1997
  • 1997-11-20 IL IL12227097A patent/IL122270A0/xx unknown
• 1998
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