AU1257099A - Dna molecules conferring dalapon-resistance to plants and plants transformed thereby - Google Patents

Description

WO 99/27116 PCT/IL98/00567 DNA MOLECULES CONFERRING DALAPON-RESISTANCE TO PLANTS AND PLANTS TRANSFORMED THEREBY 5 FIELD OF THE INVENTION 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), 10 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. 15 BACKGROUND OF THE INVENTION Weeds have been the maj or limiting factor in agriculture since the domestication of crops about 5000 years ago. Farmers spend much of their time preventing weeds from sharing the natural and human inputs of light, water and minerals with crops. Much of the 20 first world weeding is with selective herbicides, replacing much of the plowing, mechanical cultivation, hoeing and hand pulling still prevalent in much of the developing world. The advent of the green revolution wheat and rice varieties began the change in Asia. The basis of the green revolution is the use of dwarf varieties to increase the "harvest 25 index", namely the ratio of grain to straw. Biomass production of tall and dwarf varieties is similar, but the dwarfs produce grain at the expense of long straw. The green revolution doubled yield renders fertilizer use economical, as the fertilizer goes to producing even more grain than straw in dwarf varieties. Green revolution varieties were adopted as quickly as they could be propagated. 30 However, modem high yielding semi-dwarf and dwarf small grains, especially wheat, barley, as well as millet and sorghum and rice, are not as competitive with weeds as the lower yielding taller varieties, and cannot be cultivated commercially in most places without selective herbicides. The chemical industry has been very successful in 1 WO 99/27116 PCT/IL98/00567 developing herbicides that kill broad leaf weeds, however fewer herbicides have been found that control the grass weeds (graminocides) that are closely related to the small grains. The relatedness between grass weeds and small grains has facilitated the evolution of resistance to the best graminocides (Gressel, 1988a, b; Moss, 1992; Powles and 5 Matthews, 1992). The reason for the cross-resistance of grass weeds to herbicides used for grass weed control in wheat is probably due to evolution of cytochrome P450 mixed function oxidase systems similar to those utilized by wheat to degrade herbicides (Gressel 1988a, b). The situation is so bad that over a third of Australian wheat lands are presently infested with biotypes of annual (rigid) ryegrass (Lolium rigidum) that cannot be controlled 10 with selective post-emergence herbicides. Resistance has evolved to a graminocide in India's wheat lands, and the resistant weeds infest already millions of hectares, and there is a modicum of resistance to other graminocides (Gressel et al., 1994; Malik and Singh, 1995). Wild red rice and Echinochloa are major weeds that infest rice fields. Weed control is in a state of flux, with many new problems. Herbicides represent 15 more than 60% of the total pesticides used in developed countries. Due to ecological issues and political pressure, many herbicides are being removed from the market and fewer new ones are being released. One solution to overcome the weed control issue is to generate herbicide-resistant crops that may expand the use range of the compounds remaining in the market by conferring new selectivities. 20 It has been proposed that by genetically engineering novel resistances into small grain crops, preferably using non-plant genes, the evolutionary trend jeopardizing the world food supply could be staunched (Gressel 1988a, b). This, however, could not be easily done due to a lack of effective transformation/regeneration systems for wheat, and to the poor selectable markers available, which were often antibiotics (e.g. Li et al., 1993; 25 Fujimoto et al., 1993; Peng et al., 1992; Bercelo et al., 1994) despite a trend to limit the dissemination of antibiotic-resistance containing transgenic material in the environment. Five herbicide-resistant small grains have been reported, namely wheat, rice, barley, sorghum and oats containing the bar gene conferring resistance to glufosinate (Cao et al., 1992; Vasil et al., 1992; 1993; Becker et al., 1994; Nehra et al., 1994; Weeks et al., 30 1993; Rathore et al., 1993; Somers, 1992; Wan and Lemaux, 1994; Rathus et al., 1997). 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. 2 WO 99/27116 PCT/IL98/00567 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. In order to be appropriate, a herbicide resistance system in wheat should have the 5 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. 10 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., 15 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. 20 Nardi-Dei et al., 1997, disclose a dehalogenase gene from Pseudomonas, which has been mutated in order to study the results in enzyme activities. No reference to mutations related to codon usages suitable for small grain species is made. US Patent No. 5,780,708 discloses the use of the non-mutated Pseudomona dehal gene to transform Zea mays plants which are said to exhibit tolerance or resistance to normally toxic levels of 25 2,2-dichloropropionic acid. None of the above-mentioned references discloses or suggests isolated DNA molecules having codon usage suitable for small grain species encoding a dehalogenase that degrades dalapon, which DNA molecule is suitable to transform wheat, rice and other small grain species. 30 A major problem in achieving herbicide-resistant wheat has been the inability to transform wheat. The transformation of monocots, wheat in particular, has not been trivial (Potrykus,1990) and the few reports on such transformations describe low efficiencies because of regeneration problems (Vasil et al., 1992; Weeks et al., 1993). 3 WO 99/27116 PCT/IL98/00567 SUMMARY OF THE INVENTION It has now been found, according to the present invention, that the problems of poor selectable markers, low efficiency regeneration systems in small grains, and need for new 5 selective herbicides can be solved by using the dehal gene, in its natural or synthetic form, as a selectable marker in a novel staged small grains regeneration system, and in its synthetic form in order to confer resistance upon small grains so that dalapon can be used to selectively control grass weeds in small grains. The present invention thus relates, in one aspect, to an isolated DNA molecule 10 encoding a dehalogenase that degrades the herbicide dalapon, said DNA molecule having codon usage suitable for small grain species. In order to obtain a DNA molecule of the invention, 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 15 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 20 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 .In preferred embodiments, the small grain species is wheat or rice. In one preferred embodiment, the DNA molecule comprises the nucleotide sequence depicted in Fig. 1, but also genetic variants thereof having codon usage suitable 25 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-derived dehal gene depicted in Fig. 4 by preferred small grain codon usage, which significantly enhances the level of resistance. As shown in Fig. 4, the Pseudomonas-derived dehal gene contains several codons 30 such as GGT (glycine), GTA (valine), AGT (serine), ATA (isoleucine), AGC (threonine), CTT and TTA (leucine), CGA and CTT (arginine), and CCT and CCC prolinee), which usage frequency was found by the inventors to be rare (0.11 or less) in wheat, based on an analysis of 45 wheat genes found in the GenBank data base. 4 WO 99/27116 PCT/IL98/00567 The GenBank data base lists many bacterial dehal genes, but no dehalogenase genes for plants, so there are no known plant dehalogenase genes to which one can compare the synthetic gene depicted in Fig. 1. The invention further relates to vectors and to expression cassettes comprising a 5 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/hectare. 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 10 molecule in plant cells. Any small grain-effective plant promoter such as, but not being limited to, CaMV 35S 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. In 15 one preferred embodiment, 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 20 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.). The choice of 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 25 protocol suitable for small grain species such as, but not being limited to, the use of electroporation, microprojectiles, microinjection, viral systems, and Agrobacterium mediated transformation, can be used according to the invention. In one preferred embodiment, the plasmid containing the DNA molecule was transferred to competent E. coli cells, the cells were cultured, the plasmid DNA was then 30 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 DEHAL is a DNA sequence having small grain codon usage encoding a 5 WO 99/27116 PCT/IL98/00567 dalapon-degrading dehalogenase inserted into the Bluescript(KS+) cloning vector. In other protocols, the expression cassette contained the pDM804 or pDM805 plasmid as depicted in Figs. 5 and 6, respectively, wherein wss is a DNA sequence having small grain codon usage encoding a dalapon-degrading dehalogenase. 5 In another aspect, 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 lkg/hectare, preferably 2-4 kg/hectare. The foreign DNA encodes 10 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 lkg/hectare, 15 preferably 2-4 kg/hectare. In another aspect, 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 20 dalapon at field use levels of more than 1 kg/hectare, preferably 2-4 kg/hectare. In a further aspect, 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 25 degrading dalapon. In still a another aspect, 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: (a) transforming a plant cell of a small grain species with a DNA molecule having 30 small grain codon usage, encoding a dalapon-degrading dehalogenase; (b) selecting transformed cells whose growth is resistant to dalapon; and (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. 6 WO 99/27116 PCT/IL98/00567 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. 5 The transformation is carried out under conditions i.e. buffer, media, and periods of time, that are adapted to the tissue of choice. Following treatment with the plasmid DNA, 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 10 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. 15 According to one preferred embodiment of this aspect of the invention, a putatively dehal gene with small grain codon usage is used to transform embryogenic scutellar cultures of the small grain e.g. wheat, placed on media containing increasing concentrations of dalapon from 0.1 to 0.4 mM, as discerned with each species, as the tissue grows, and defined by whether a liquid or solid medium is used. 20 The transfers from one medium to another, with different herbicide, hormone and growth regulator concentrations, is facilitated by the use of floating rafts on liquid media with gentle agitation. Thus, according to another preferred embodiment of this aspect of the invention, a method of increasing the regeneration efficiency of the transformed plants of small grain species comprises transferring the primary regenerants through a stepped 25 series of plant hormone and dalapon concentrations, wherein the regenerants are floating on a liquid medium on a membrane raft. In still an additional aspect, 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 30 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 7 WO 99/27116 PCT/IL98/00567 said small grain species resistant to dalapon at field use levels of more than 1 kg/hectare, 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 5 desired trait, in the place of the usually used antibiotics, and to a method for introducing a desired trait in a plant, which comprises: (a) transforming plant cells with a DNA molecule encoding a protein conferring to the plant said desired trait together with a DNA molecule encoding a dehalogenase capable of degrading the herbicide dalapon; 10 (b) selecting transformed cells whose growth is resistant to dalapon; and (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. For this purpose 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 15 it may be continuously or discontinuously present at most stages of the regeneration process of the primary transformed plant cells. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts the nucleotide sequence of the synthetic dehal gene with wheat codon 20 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 25 dalapon-degrading dehalogenase (DEHAL) gene depicted in Fig. 1. 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 30 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). 8 WO 99/27116 PCT/IL98/00567 DETAILED DESCRIPTION OF THE INVENTION The invention will now be illustrated by the following non-limiting Examples. Example 1. Transgenic wheat containing the natural dehal gene 5 (i) Isolation of immature embryos. Immature embryos of wheat (Triticum aestivum cv. Deganit) were isolated from immature grains which were 11 days after anthesis. The whole spikes were surface sterilized continuously by stirring in a 3% sodium-hypochlorite solution for 24 minutes, then washed three times with sterile water. Embryos were isolated 10 from the grains under a binocular microscope and plated scutellum-side up on solidified (containing 0.6% agar pH 5.8) MS medium (Murashige and Skoog, 1962) supplemented with 2mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) in petri plates. (ii) Pre-culture before bombardment. Immature embryos were incubated in dark at 25 0 C 15 for 5-7 days. Embryos in which scutellar tissue started to proliferate were transferred to osmotic treatment medium on MS with 2 mg/L 2,4-D containing 0.25M mannitol for 4 hours before bombardment. (iii) Construction of a dalapon-resistance cassette - pCJVW4. A modular monomer 20 expression promoter was isolated from plasmid pCJ14 (Fig. 2) by cleavage with Dra III and BamHI. This 1.6 Kb DNA fragment contained the 35S CaMV promoter, exon 1 from rice actin 1 promoter gene, and intron 1 from the maize shrunken gene. Maas et al. (1993) reported that this promoter induced the expression of the marker gene CAT 100-fold more than the usual 35S promoter alone in protoplasts of barley. The DNA fragment from the 25 natural dehaI gene isolated from Pseudomonas (Fig. 4) coding for a dehalogenase capable of degrading dalapon, was isolated from plasmid pVW264 by cleavage with BaglI and SphI (described by Buchanan-Wollaston et al., 1992, and kindly provided by Dr. V. Buchanan-Wollaston, University of London). An octopine synthase terminator was used. The DNA fragment was then inserted to the cloning vector pBluescript (KS+). A fragment 30 cut from this new vector with Sal I and Pst I was inserted to plasmid pCJI4 which was cut with Sal I and Pst I. The produced plasmid pCJVW14 was transferred to competent E. coli cells (Fig. 3), e.g. line DH5a. The transformed cells were cultured in LB medium (Difco Labs, U.S.A.) by standard procedures. 9 WO 99/27116 PCT/IL98/00567 (iv) Preparation of DNA from plasmid- Overnight culture of 100 ml transformed competent cells, of ca 1.0 OD, were cooled on ice, and the cells were precipitated by low-speed centrifugations. The plasmid DNA was extracted and purified by Qiagen 5 Plasmid Purification kits, according to the manufacturer's instructions. (v) Transformation. A Dupont PDS 1000 Helium Biolistic Delivery bombardment system was used. Tungsten particles (60 mg, 1.0 tm diameter) were sterilized in 1 ml 100% ethanol overnight, then particles were washed 3 times with sterile water, and kept in 1 ml 10 50% sterile glycerol. Tungsten particles (60 mg, 1.0 jim 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 pd particle suspension; 2) 10pl plasmid DNA (1ptg/pl); 3) 25pl 2.5 M CaCl2; 4) 20pl 0.1 M spermidine. The suspension 15 was incubated at room temperature for 10 minutes. After centrifugation for 5 seconds, the supernatant was discarded and the mixture washed with 140 pl 70% ethanol. The supernatant was removed after short centrifugation, then 50pl 100% ethanol were added to make the final suspension. A 6 pl 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 20 each plate, 9 cm below the stop net, 1100 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. (vi) Regeneration. 24 hours after bombardment, the embryos were transferred to fresh MS 25 solid medium containing 2 mg/L 2,4-D and 60 mg/L dalapon (0.36 mM, the minimum concentration of dalapon that can block the growth untransformed calli growing in solid medium at this stage) in the dark at 250C. The embryos were maintained on this selection medium for 2-4 weeks and the medium was renewed every two weeks. At this stage, the cultured embryos produced callus. During the selection, the surviving calli were separated 30 and divided. The loose and light colored calli were then transferred to solid MS medium containing 1 0ptg/ml zeatin, and 60 jig/ml dalapon under 16-hour light and 250C. After the green shoots reached 0.5 cm long, the calli were transferred to rafts 10 WO 99/27116 PCT/IL98/00567 (Osmotek Lt., Rehovot 76120, Israel) with liquid MS medium containing 10 pig/ml zeatin and 20 ptg/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. After 14 days, the concentration of zeatin was decreased to 6 pig/ml in 5 same selection medium, then used liquid MS medium containing also 1p~g/ml zeatin, 1 ptg/ml IAA (indoleacetic acid) and 20 ptg/ml dalapon to culture for 14 days, then the concentration of IAA was decreased to 0.2 pg /ml, and dalapon to 10 ig /ml. When 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 20ptg /ml dalapon. After the plants 10 reached about 10 cm height (the plants touch the tops of the containers), they were transferred to Jiffy pots incubated in small boxes with little water for 2 days at room temperature and weak light with the box covered with a plastic bag. Finally the plants were planted in flower pots, covered with a plastic box in the greenhouse until the plants were sturdy. 15 (vii) Use of transgenic pollen to pollinate non-transgenic wheat. Pollen from putatively transformed wheat plants was collected 2 days after anthesis. The non-transformed wheat cv. Deganit plants were emasculated before anthesis. The pollen of the putatively transformed plants was used for pollinating with the non-transformed wheat plants. The 20 spikes were covered with bags after the cross. The putative transformants were then also bagged for self-pollination. (viii) Biological analysis of transgenic progeny. The RI transgenic grains from selfed Ro transformed plants were sown in pots. Three grains were sown in each pot. Grains of 25 non-transformed wheat was sown at the same time as the grains of control plants. Several concentrations were used for spraying the RI putative transgenic progeny plants 3 weeks after the first leaf appeared. Concentrations ranging between 0.12 mM and 1.2mM dalapon (with 0.2% Tween 20) were used to spray the plants. The non-transgenic plants sprayed with 0.24 mM dalapon or higher concentrations dried out and died 2 weeks after treatment. 30 Most putative transgenic plants sprayed with 0.24 mM up to 1.2 mM dalapon were green and survived for 2 weeks after dalapon treatment. After an additional 3-4 weeks, almost all the plants died except the plants treated with 0.24 mM dalapon. Therefore, we used this treatment with the pollinated progeny. The RI self-pollinated progeny had a near 3:1 11 WO 99/27116 PCT/IL98/00567 segregation of resistant to susceptible plants (Table 1). The backcross progeny were sprayed with 0.24 mM dalapon, as a discriminatory concentration. A near 1:1 segregation was observed after spraying the cross-pollinated progeny with 0.24 mM dalapon (Table 2). 5 Table 1. 3:1 Segregation of dalapon resistance in R1 self-pollinated progeny 0mM 0.12mM 0.24mM 0.40mM 0.75mM 1.2mM PNa Sb PNa Sb PNa Sb PNa Sb PNa gb Pna Sb 411 8 8 9 8 9 6 8 1 9 0 7 0 10 412 9 9 8 7 9 5 9 0 8 0 9 0 413 9 9 - - - - - - 9 0 9 0 414 9 9 8 7 9 7 9 2 9 1 8 0 4161 10 9 - - - - - - 10 0 8 0 4162 8 8 - - - - - - 9 0 9 0 15 42 12 11 10 8 11 7 10 1 12 0 10 0 Total 65 64 35 30 38 25 36 4 66 1 60 0 CK 5 5 6 2 6 0 6 0 6 0 6 0 Notes: PNa - numbers of plants that were planted in pots; Sb - surviving plants; CK 20 non-transformed plants. 12 WO 99/27116 PCT/IL98/00567 Table 2. Indication of gene integration by backcrossing RO with untransformed plants sprayed with 0.24 mM dalapon. Pollination number of number of number of 5 plants dead plants surviving plants 412XDeganit 8 5 3 413XDeganit 7 3 4 4161XDeganit 7 4 3 10 4162XDeganit 10 6 4 42XDeganit 11 5 6 Total treated 43 23 20 DeganitXDeganit(CK) 9 8 1 15 Example 2. 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. 20 We then analyzed the codon sequence of the Pseudomonas-derived 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. 25 We then redesigned the gene, based on the preferred codon usage of wheat, as well as adding desired restriction cutting sites needed to prepare constructs, as well as other changes, and had the sequence of the synthetic dehalogenase gene depicted in Fig. 1 custom synthesized (by Operon Technologies Inc., Alameda, CA., U.S.A.) The new synthetic dehal gene with codon usage suitable for small grains was then 30 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 13 WO 99/27116 PCT/IL98/00567 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: acting - acting promoter from rice; gus - p-glucoronidase; intron 1 - intron frm maize shrunken gene; nos3' 5 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 10 from rice; gus - p-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. 15 Example 3. 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 20 initials appeared on the transformed calli at this rate. 25 30 14 WO 99/27116 PCT/IL98/00567 References 1. Becker, D., Brettschneider, R. and Lorz, H. 1994. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J. 5: 299-307. 2. Bercelo, P., Hagel, C., Becker, D., Martin, A. and Lorz, H. 1994. Transgenic 5 cereal (tritodeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue. Plant J. 5: 583-592. 3. Buchanan-Wollaston, V., Snape, A. and Cannon, F. 1992. A plant selectable marker gene based on detoxification of the herbicide dalapon. Plant Cell Rep. 11: 627-631. 10 4. 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Claims (30)

1. An isolated DNA molecule encoding a dehalogenase that degrades the herbicide dalapon, said DNA molecule having codon usage suitable for small grain species. 5

2. An isolated DNA molecule according to Claim 1, wherein said dalapon-degrading dehalogenase has an amino acid sequence substantially as depicted in Fig. 1, and synthetic variants thereof having codon usage suitable for expression in small grain species. 10

3. The isolated DNA molecule according to Claim 2, consisting of the nucleotide sequence depicted in Fig. 1.

4. The isolated DNA molecule according to any one of Claim 1 to 3, wherein the small grain species is wheat, barley, rice, triticale, oats, rye, sorghum or millet. 15

5. The isolated DNA molecule according to Claim 4, having wheat codon usage.

6. A vector which contains a DNA molecule according to any one of Claims 1-5. 20

7. An expression cassette comprising a DNA molecule according to any one of Claims 1-5 or a vector according to claim 6, which is suitable for the transformation of plant cells and plants of small grain species and to confer dalapon-resistance to the regenerated plants. 25

8. The expression cassette according to Claim 7, wherein said DNA molecule is under the control of a plant promoter region and of regulatory elements allowing for the expression of said DNA molecule in plant cells of small grain species.

9. The expression cassette according to Claim 8, wherein a small grain-effective plant 30 promoter is added upstream of the coding sequence of the DNA molecule depicted in Fig. 1, and a terminator fragment is added downstream thereof.

10. The expression cassette according to Claim 9, wherein said small grain-effective 18 WO 99/27116 PCT/IL98/00567 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.

11. The expression cassette according to Claim 10 as depicted in Fig. 3, 5 or 6. 5

12. A transformed plant cell of a small grain species comprising a foreign DNA molecule having codon usage suitable for expression in small grain species encoding a dehalogenase that degrades dalapon, which plant cell is substantially resistant to dalapon at field use levels. 10

13. The transformed plant cells of Claim 11, wherein said foreign DNA encodes the dehalogenase having the amino acid sequence depicted in Fig. I or a variant thereof having small grain codon usage that confers to small grain species resistance to dalapon at field use levels. 15

14. The transformed plant cells of Claim 13, wherein said DNA molecule 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 field use levels. 20

15. The transformed plant cells according to any one of Claims 12-14, wherein said small grain species is wheat.

16. Seeds of small grain species which possess, stably integrated in their genome, a foreign DNA molecule having small grain codon usage and expressing a dehalogenase 25 capable of degrading the herbicide dalapon at a level sufficient to render the small grain species resistant to dalapon at field use levels.

17. The seeds of Claim 16, wherein said DNA molecule comprises the coding nucleotide sequence depicted in Fig. 1 or a genetic variant thereof having small grain 30 codon usage and that encodes a dehalogenase that confers to small grain species resistance to dalapon at field use levels.

18. The seeds according to Claim 16 or 17, wherein said small grain species is wheat. 19 WO 99/27116 PCT/IL98/00567

19. Plants of small grain species resistant to dalapon at field use levels which possess, stably integrated in their genome, a foreign DNA molecule having small grain codon usage and encoding a dehalogenase capable of degrading dalapon. 5

20. The small grain plants of Claim 19, wherein said DNA molecule comprises the coding nucleotide sequence depicted in Fig. 1 or a genetic variant thereof having small grain codon usage and that encodes a dehalogenase that confers to small grain species resistance to dalapon at field use levels. 10

21. The small grain plants according to Claim 19 or 20, wherein said small grain species is wheat.

22. A method for rendering a plant of a small grain species resistant to the herbicide dalapon at field use levels, which comprises: 15 (a) transforming plant cells of a small grain species with a DNA molecule according to any one of Claims 1-5 or an expression cassette according to any one of Claims 7-11; (b) selecting transformed cells whose growth is resistant to dalapon; and (c) regenerating from said transformed plant cells of step (b) small grain plants 20 resistant to dalapon at field use levels.

23. The method according to claim 21, wherein in the regeneration step c, the primary regenerants are transferred through a stepped series of plant hormone and dalapon concentrations, wherein the regenerants are floating on a liquid medium on a membrane 25 raft.

24. The method according to claim 23, wherein the dalapon concentration increases from 0.1 to 0.4mM. 30

25. A method according to any one of Claims 21 to 24, wherein said small grain species is wheat.

26. Use of dalapon as a selective herbicide in fields containing plants of small grain 20 WO 99/27116 PCT/IL98/00567 species resistant to dalapon at field use levels.

27. The use according to Claim 26, wherein said small grain species plants resistant to dalapon at field use levels are the transformed claimed in any one of Claims 19-21. 5

28. Use of dalapon as a selective herbicide in fields containing dalapon-resistant wheat.

29. A method for protecting plants of a small grain species and destroying weeds in a field with the herbicide dalapon, comprising applying dalapon to a field of small grain 10 species containing a foreign DNA molecule having codon usage suitable for expression in small grain species stably integrated in their genome, which foreign DNA expresses a dalapon-degrading dehalogenase at levels sufficient to render said small grain species resistant to dalapon at field use levels. 15

30. The method according to Claim 29, wherein said small grain species resistant to dalapon at field use levels have been transformed with a DNA molecule comprising 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 field use levels. 20 32. The method according to Claim 30 or 31, wherein said small grain species is wheat. 21