A METHOD OF SELECTIVELY PRODUCING MAT F OR FEMALE STFRITE PLANTS
Heterosis in crop plants can have a marked effect on yield improvement In general, hybrids exhibit increased yields in comparison with non-hybrid varieties Hybrids usually give a greater return unit for growth factors such as water and fertilizer Hybrids often offer superior stress tolerance, uniformity in product and maturity and also afford a simple breeding opportunity to combine charactensties or traits that may be difficult to combine in other ways Hybnd vigour in plants is generally of sufficient magnitude to warrant commercial exploitation Commercial hybrids are used extensively in many crops including corn, sorghum, sugar beet, sunflower and canola However, owing mainly to the lack of economical hybnd seed production methods, wheat, barley and rie are still grown mainly as inbreds
Traditionally, hybnd seed production involves planting out separate blocks of female and male parent lines with only the seed from the female parents being harvested To ensure that this seed is hybnd, self pollination of the female parent line must be minimised by rendenng the line male-stenle Methods for making the female parent line male stenle include mechanical, chemical and genetic methods In monoecious plants, such as maize, male stenlity can be readily achieved mechanically by detasselling of the male infloresence However most crops are diecious and having male and female organs within the same flower makes such physical emasculation impractical Genetic approaches have therefore sometimes been used
Genetic male stenlity traits which occur are normally controlled by nuclear genes in which the alleles associated with the stenle phenotype are generally expressed recessively with respect to the corresponding alleles associated with fertility Where genetic male stenlity occurs it is normally associated with a single recessive gene that must be homozygous in order for male stenlity to be expressed In order to make practical use of such genetic male stenlity traits, breeders usually develop a phenotypically uniform female line that segregates into male-stenle and male-fertile plants The male fertile plants, once identified, need to be rogued out which is labour intensive There is always a problem with maintaining the parental line since male fertile plants cannot be eliminated from the population because they are essential for maintenance of the population Rather than rely on the existence of natural male stenlity alleles it is also possible to use molecular biological methods Plants may be engineered which express, for example, anti-sense or nbozyme
genes that decrease or eliminate expression of key genes necessary for the formation of viable pollen Such transgenic lines of plants are male-stenle and are used for the production of hybnd seed by crossing using pollen from male-fertile plants The main problem with such lines is that they can only be maintained in a heterozygous state in subsequent generations, via crosses with the isogenic fertile lines This can be a problem in hybnd seed production where yield is cntical Although, for example by linking herbicide resistance to male stenlity, it may be possible to selectively rogue out the male-fertile plants this still necessitates that the plants are planted initially at extra high densities
The use of cytoplasmic male stenlity for commercial hybnd production requires a stable male-stenle cytoplasm and a source of pollen The cytoplasmic-genetic system of male stenlity requires the existence of three types of line for hybnd production, the A line (cytoplasmic male-stenle), B line (male-fertile maINtainer) and R line (male fertile with restorer genes) Three-way crosses produced with this system involve maintenance and production of four lines, an A and a B line of one inbred and male-fertile inbreds of the other two Reliance on a single source of male-stenle cytoplasm can minimise breeding flexibility and lead to progeny with wholesale susceptibility to particular diseases
Hybnd seed can also be produced through the use of chemicals that inhibit viable pollen formation These chemicals, called gametocides, are used to impart transitory male-stenlity However the expense, registerability and reliability of gametocides has limited their use
A shortcoming of traditional hybnd seed production systems is the need to plant separate rows or blocks of the male and female parent lines Here low efficiency pollination is an especially acute problem in crop species, such as wheat, that release small amounts of pollen which does not travel far on the wind In such crops as much as two/thirds of the hybnd-producing field needs to be dedicated to male pollen-donor plants and then hybnd seed production therefore becomes uneconomic
In order to achieve more economic seed production in wheat and other crops it is necessary to move male and female plants closer together for more efficient pollen transfer, most efficiently by mterplantmg males and females within centimetres of each other in the same rows In such a system it would be impractical to harvest only the seed from the (male-stenle) female parents The contamination with non-hybnd seed ongmatINg from the male parent can be minimised by using as low a percentage of such male parent plants in the planting mix as possible and/or by using male plants which are female sterile A method for
constructmg a dominant female sterile line has been described (EP 412,006 Al (1990), Goldman et al, (1994) EMBO J , 13, 2976-2984) but, as with the male sterile lines, the line has to be maintained as a heterozygote
Accordingly there remains a need for simple economic methods of hybrid seed production In particular, in order efficiently to produce hybrid seed there remains a need to provide both male-stenle female parental lines and female-sterile male parental lines which can be easily maintained as pure homozygous lines and which are useful for efficient hybrid seed production Methods which are described in the art for achieving this include methods wherein hybrid seed is produced from male and female parent lines at least one of which comprises a heterologous chimeric gene, preferentially expressed in floral tissue, which renders the line conditionally sterile dependent upon the exogenous application of a non-phytotoxic substance which can be specifically and locally converted to a phytotoxin by an enzyme which is encoded by the heterologous chimeric gene and which is preferentially expressed in either the male or female reproductive structures The non-phytotoxic substance may be a pro-herbicide The advantage of having such conditionally sterile parent lines is that it allows them to be maintained as homozygotes with respect to the sterility trait Fertility is only disrupted upon exogenous application of the non-phytotoxic substance In one such example of a conditional male sterility system a gene encoding a deacetylase enzyme is preferentially expressed in tapetal cells of male flower tissue where it converts the exogenously applied pro-herbicide N-acetyl L phosphinothncin to the phytotoxin L phosphmothricin and thus prevents viable pollen formation In further similar examples (I) tapetum preferential expression of a bacterial cytochrome P450 catalyses conversion of pro-herbicide R7402 to a sulphonylurea phytoxm which prevents the production of viable pollen, and (ii) tapetum preferential expression of a phosphonate monoester hydrolase catalyses conversion of glyceryl glyphosate pro-herbicide to the phytotoxin glyphosate which also prevents production of viable pollen WO 98/03838 describes examples of a conditional female sterility system wherein enzymes capable of converting the pro-herbicides to phytoxins are preferentially expressed in female reproductive structures
Despite the existence of these methods for making male and female parent lines that are conditionally sterile, hybrid seed production remains far from routine in crops such as wheat The current inventions concern, inter alia, improvements in the art with respect to the generation of female parent lines which are conditionally male sterile and male parent lines which are conditionally female stenle
The current invention relates to improvements in methods for the production of crop hybrid seed In particular the invention relates to a method of hybrid seed production from male and female parent lines at least one of which is conditionally female or male sterile dependent upon the exogenous application of a substance which is non-phytotoxic to the crop and which include pro-herbicides The invention further relates to a method in which the said non-phytotoxic substance is applied at a time and in sufficient amount that self fertilization is minimised or prevented in the conditionally sterile parent lme(s) The current invention also relates to a method of generating conditionally male or female-stenle plants by 1) transforming plant material with one or more chimeric genes which, singly or together, encode one or more enzymes capable of reacting with a non-phytotoxic substance, preferably in the form of a pro-herbicide, to produce a phytotoxic one Enzymes are expressed under operable control of one or more promoters which, in the case of conditionally male sterile plants, causes the enzyme(s) to be expressed preferentially in the male reproductive structures or which, in the case of conditionally female sterile plants, causes the said enzyme(s) to be expressed preferentially in the female reproductive structures The plant materia] is regenerated into morphologically normal fertile plants which are conditionally male or female sterile The invention also includes the use of conditionally male-sterile plants in combination with conditionally female-stenle plants to produce more efficiently hybnd seed, the use, as non-phytotoxic substances, of certain pro-herbicides and the use of chimenc genes to produce more efficiently hybnd seeds, chimenc genes and enzymes useful for the invention The invention also provides conditionally male-stenle, conditionally female-stenle plants, seeds of these plants and hybnd seeds produced by the method In preferred embodiments of the invention the crop plants to which the method for making hybnd seed is applied are maize, nee, sorghum, wheat, millet, oats, canola and barley
According to the present invention there is provided a method of producing male or female stenle plants compnsing the steps of transforming plant matenal with a polynucleotide which encodes at least one enzyme which reacts with a non-phytotoxic substance to produce a phytotoxic one, and regenerating the thus transformed matenal into a plant, wherein the said non-phytotoxic substance is applied to the plant up to the time of male or female gamete formation and/ or maturation, so that the non-phytotoxic substance provides for the production of a phytotoxic one which selectively prevents the formation of or otherwise renders the said gametes non-functional, wherein the enzyme is expressed preferentially in either male or female reproductive structures, charactensed in that (1) the
non-phytotoxic substance is selected from the group consisting of ester derivatives of non-phosphonate herbicides which herbicides are directly phytotoxic to non-green tissue, D-alpha amino acids, peptide derivatives of non-protein D-alpha amino acids, S-enantiomers of aryloxyphenoxypropionates and S-enantiomers of ester derivatives of aryloxyphenoxypropionates and (11) the enzyme is selected from the group consisting of carboxylesterases, D-amino acid oxidases, D-amino acid dehydrogenases, D-amino acid racemases, 2-arylpropionyl-CoA epimerases, alpha-methylacyl-CoA racemases, thioesterases and acyl-CoA synthetases
Since it is a desirable objective to maximise the yield of hybrid seed and therefore to minimise any crop damage, in preferred embodiments, the non phytotoxic substance is a pro-herbicide selected from amongst compounds which are relatively non-phytotoxic to the crop In order to be capable of an effect against floral tissues it is also desirable that pro-herbicides be progenitors of phyto-toxins that are effective in 'non-green' tissues Thus, in preferred embodiments of the invention, pro-herbicides are selected from those which are progenitors of phyto-toxms which are directly phytotoxic to non-green tissues rather than those which have a principle site of action in photosynthesis or in the generation of photosynthetic pigments It is also a desirable objective to minimise the costs of hybrid seed production Thus, in preferred embodiments, pro-herbicides are selected from amongst those chemical substances for which approval from appropriate regulatory authorities for use in crops is either already granted or is pending Nomenclature Definitions
'Gene' as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter and a 5' regulatory region, a coding sequence and an untranslated 3' region comprising a polyadenylation site
'Chimeric' when referring to a gene or DNA sequence is used to refer to the fact that in nature, the coding sequence is not associated with the promoter or with at least one other regulatory region of the DNA in the gene
'Chimeric gene' as used herein refers to a gene wherein, in nature, the coding sequence is not associated with the promoter or with at least one other regulatory region of the DNA in the gene
'Expression cassette' as used herein refers to a transferable region of DNA comprising a chimeric gene which is flanked by one or more restriction or other sites which facilitate precise excision from one DNA locus and insertion into another
'Non-phytotoxic substances' are, in the context of the current invention, substances which are relatively non-phytotoxic to plants, cells or tissues of any particular crop to which the method of the invention is applied Non-phytotoxic substances need not be non-phytoxic in all plant tissues of all plants Non-phytotoxic substances include pro-herbicides which are substances with no appreciable direct toxic effect on plant tissues but which are progenitors of active phyto-toxins In susceptible plant species such pro-herbicides act indirectly as herbicides through the action of endogenous enzymes which convert them in planta to a phyto-toxm
'Phyto-toxms' are, in the context of the current invention, substances which are toxic to plants, plant tissues and plant cells of the particular crop to which the method of the invention is applied Such phyto-toxms need not be phyto-toxic to all plant tissues from all plant species
'Female reproductive structure' means the female gametes and those portions of the plant that are specialised for the production, maturation and viability of female gametes Normally this comprises those portions of a plant that compnse the carpel or gynoecium ("pistill'') The carpel of a plant includes but is not limited to, a stigma, style, ovary and cells or tissues that are comprised by the stigma, style and ovary
'Male reproductive structure' means the male gametes and those portions of the plant that are specialised for the production, maturation and viabihty of male gametes This comprises those portions of a plant that compnse, for example, microspores, stamens, tapetum, anthers and the pollen
'Female-stenle plant' as used herein is a plant that is incapable of supporting viable seed formation when pollinated with functional or viable pollen Such female stenlity can be the result of breeding selection or the presence of a transgene A 'conditionally female-stenle plant' refers to a plant which under normal growing conditions is female fertile and which can become female-stenle under specific conditions In the context of the ctinent invention the said conditions compnse the exogenous application of a pro-herbicide or other non-phytotoxic substance In the context of the current invention such a 'female-stenle plant' or 'conditionally female-stenle plant' remains male fertile and able to produce viable pollen
'Male-stenle plant' as used herein is a plant that is incapable of supporting viable pollen formation Such male stenlity can be the result of breeding selection or the presence of a transgene A 'conditionally male-stenle plant' refers to a plant which under normal
growing conditions is male fertile and which can become male-stenle under specific conditions For example the conditions might comprise physical emasculation or application of a specific chemical gametocide In the context of the current invention the said conditions particularly comprise the exogenous application of a pro-herbicide or other non-phytotoxic substance In the context of the current invention such a 'male-stenle plant' or 'conditionally male-stenle plant' remains female fertile and able to produce viable seeds when pollinated with functional or viable pollen
'Promoter region' as used herein is a region of DNA compnsing at least a functional promoter and, optionally, some or all of its associated upstream regulatory sequences including enhancer sequences and/or associated downstream sequences including some or all of the 5' untranslated region of the gene endogenous to the promoter
'Inter-planting' as used herein refers to a method of planting seeds or plants in a field that ensures adequate cross-pollination of male stenle or conditionally male-stenle plants by the male-fertile plants This can be achieved either by random mixing of female and male parent seed in different blends (80/20, 90,10, etc) before planting or by planting in specific field patterns whereby different seeds are alternated When separate harvesting from different plants is required planting in alternating blocks or rows is preferred
'Carboxylesterase' as used herein only encompasses enzymes that are properly classified as EC 3 1 n
In the method according to the invention the said non-phytotoxic substance may be applied in mixture along with at least one further substance which may be selected from the group consisting of amino acids, safeners, gametocides, glutathione-S-transferase inducers, cytochrome P450 inducers, fertilizers, herbicides, nematocides, synergists, insecticides, fungicides, hormones, plant-growth regulators and cytochrome P450 inhibitors In particular embodiments the said non phytotoxic substance may be applied in mixture with piperonyl butoxide or malathion In particular embodiments the said non-phytotoxic substance may be applied in a mixture with the same phytotoxic substance that the non-phytotoxic substance is a progenitor of
The enzyme used in the method of the invention may be a carboxylesterase and the non-phytotoxic substance may be an ester of imazamethabenz or of flamprop In a particularly preferred form of the invention which relates specifically to wheat, the non-phyto-toxic substance is a pro-herbicide selected from the group consisting of imazamethabenz methyl, flamprop methyl or flamprop isopropyl
The said enzyme may be a D-amino acid oxidase, a D-amino acid dehydrogenase or a D-amino acid racemase and the non-phytotoxic substance may then be a D amino acid and, in particular, it may be the D enantiomer of phosphmothncin, the D enantiomer of bialaphos or selected from the group consisting of D-alamne, D serine, D isoleucine, D methionine, D leucine or D valine As used herein "D amino acid oxidase" means any enzyme capale of oxidising a D-amino acid to produce a 2 keto acid and includes enzymes with specificity for aspartate known as "D-aspartate oxidases"
Alternatively, the enzyme used in the present inventive method may be selected from
the group consisting of 2-arylpropionyl-CoA epimerases, alpha-methylacyl-CoA racemases,
thioesterases and acyl-CoA synthetases and the non-phytotoxic substance may then be an S
enantiomer of an aryloxyphenoxypropionate or an S enantiomer of an
aryloxyphenoxypropionate ester
Chimeric genes encoding enzymes capable, singly or in combination with others, of
reacting with a non-phytotoxic substance to produce a phytotoxic one may be selected from
amongst genes comprising DNA coding sequences which encode one or more of the
following enzymes
(1) Carboxylesterases capable of catalysing the hydrolysis reaction
imazamethabenz methyl → imazamethabenz + methanol
(2) Carboxylesterases capable of catalysing the hydrolysis reaction
flamprop methyl → fiamprop + methanol and/or flamprop isopropyl → flamprop + isopropanol
(3) D-amino acid oxidases capable of catalysing the oxidation
D-amino acid + O2 + H2O → NH3 + H2O2 + 2-oxo acid and in certain embodiments particularly the reaction D-phosphmothncin + O2 + H2O → NH3 + H2O2 + 2-oxo-4-methylphosphmobutyrate
(4) D-amino acid dehydrogenases capable of catalysing the oxidation
D-phosphmothncin + electron acceptor + H2O → NH3 + 2e- reduced electron acceptor + 2-oxo-4-methylphospninobutyrate The D-amino acid dehydrogenases may be membrane-associated enzymes which couple electrons via an electron acceptor to a membrane-bound electron transport chain from which the ultimate electron recipient may, for example, be NAD+ or O2
(5) Ammo acid racemases capable of catalysing the mterconversion
D-phosphmothncin oL-phosphinothncin
(6) 2-arylpropionyl-CoA epimerases or alpha-methylacyl-CoA racemases capable of
catalysing one or more of the following reactions
S-Fluazifop-CoA → R-Fluazifop-CoA and/or S-Quizalofop-CoA → R-Quizalofop-CoA and/or S-Propaquizafop-CoA → R-Propaquizafop-CoA and/or S-Haloxyfop-CoA → R-Haloxyfop-CoA and/or S-Fenoxaprop-CoA → R-Fenoxaprop-CoA and/or S-Diclofop-CoA → R-Diclofop-CoA and/or S-Cyhalofop-CoA → R-Cyhalofop-CoA and/or S-Clodinafop-CoA → R-Clodmafop-CoA
(7) Thioesterases capable of catalysing the hydrolysis reaction
R-Fluazifop-CoA → R-FIuazifop + CoA and/or R-Quizalofop-CoA → R-Quizalofop + CoA and/or R-Propaquizafop-CoA → R-Propaquizafop + CoA and/or R-Haloxyfop-CoA → R-Haloxyfop + CoA and/or R-Fenoxaprop-CoA → R-Fenoxaprop + CoA and/or R-Diclofop-CoA → R-Diclofop + CoA and/or R-Cyhalofop-CoA → R-Cyhalofop + CoA and/or R-Clodmafop-CoA → R-Clodinafop + CoA
(8) Acyl-CoA synthetases capable of catalysing the reaction
S-Fluazifop + CoA + ATP → S-Fluazifop-CoA + PPi + AMP and/or S-Quizalofop + CoA + ATP → S-Quizalofop-CoA + PPi + AMP and/or S-Propaquizafop + CoA + ATP → S-Propaquizafop-CoA + PPi + AMP and/or
S-Haloxyfop + CoA + ATP → S-Haloxyfop-CoA + PPi + AMP and/or S-Fenoxaprop + CoA + ATP → S-Fenoxaprop-CoA + PPi + AMP and/or S-Diclofop + CoA + ATP → S-Diclofop-CoA + PPi + AMP and/or S-Cyhalofop + CoA + ATP → S-Cyhalofop-CoA + PPi + AMP and/or S-Clodinafop + CoA + ATP → S-Clodmafop-CoA + PPi + AMP The carboxylesterase enzyme may be selected from carboxylesterase B (EC 3 111) type enzymes, especially those that are denved from from Arthrobacter sp, Bacillus sp, pig
liver, Saccharomyces sp or Synechocystis sp Preferred such enzymes may be selected from amongst proteins having the Swissprot accession numbers Q01470, P37967, Q29550 (the mature peptide sequence from 60-1703), P40363 or SEQ ID number 2 (this application), and the DNA sequence encoding the carboxylesterase enzyme may be selected from amongst DNA sequences composed within EMBL accessions M94965, BSO6089, SSCE , Z34288 and SEQ ID number 1 (this application) Further carboxylesterase enzymes and DNA coding sequences suitable for working the invention are selected from amongst plants and microorganisms which, in a minimal medium, are found to exhibit similar sensitivity to growth inhibition by imazamethabenz methyl as by imazamethabenz Candidate esterase genes from DNA libraries of such organisms are identified using suitable DNA probes and isolated by subcloning Alternatively, genes encoding suitable enzymes are identified and selected from expression libraries in suitable imazamethabenz methyl insensitive host organisms via screemng for transformation to the imazamethabenz methyl sensitive phenotype Equally, suitable and improved genes and enzymes are selected on the basis of expression in E cob and, either in vivo or in vitro, assay for the desired flamprop ester or imazamethabenz ester esterase activity via the usual methods including detection of imazapyr or flamprop by inhibition of target enzymes such as acetohydroxyacid synthase, by HPLC/ UV and/ or by denvitization and GC MS
The D-amino acid oxidase (DAMOX) enzyme may be selected from amongst those produced by Rhodospondium sp (Rhodotorula sp ), Tngonopsis sp, pig, Fusanum sp, Candida sp,, Schizosasaccharomyces sp and Verticillium sp, and may selected from proteins having sequences corresponding to Swissprot accession numbers P80324, Q99042, PO0371, P24552 or SPTREMBL numbers Q9HGY3 and Q9Y7N4 The DNA sequences which encode the D amino acid oxidase may be selected from sequences comprised within EMBL accessions A56901, RGU60066, Z50019, SSDA04, D00809, AB042032, RCDAAOX, A81420 and SPCC1450 D-amino acid oxidases are ubiquitous flavoenzymes
Where the non-phytotoxic substance is D phosphinothncin or D-bialaphos or D-aspartate or D-ghitamate then particularly preferred D-amino acid oxidases are obtained from Rhodotorula gracilis mutants or is a D-aspartate oxidase Such mutants, whatever the non-phytoxic substance, may comprise single and double amino acid substitutions at positions 213 and 238 when compared with the wild type sequence Preferably at position 213 the wild type methionine is replaced by Arg, Lys, Ser, Cys, Asn or Ala, and the wild type Tyr at position 238 is replaced by His, Ser, Gys, Asn or Ala.
However, the enzyme may comprise substitutions in addition to, or at other than, the positions mentioned in the preceding paragraph In particular, the Phe at position 58 in the wild type sequence may be replaced by a residue selected from the group consisting of His, Ser, Lys, Ala, Arg, and Asp, and preferably is either His, Ser or Ala In addition, or alternatively, the Met at position 213 in the wild type sequence may be replaced by a residue selected from the group consisting of His, Ser, Lys, Ala, Arg, and Asp, and preferably is either Ser or Ala In addition, or alternatively, the Tyr at position 223 in the wild type sequence may be replaced by a residue selected from the group consisting of His, Ser, Ala, Arg, and Asp In addition, or alternatively, the Tyr at position 238 in the wild type sequence may be replaced by a residue selected from the group consisting of His, Ser Lys, Ala, Arg, and Asp
A particularly preferred mutant form of the enzyme comprises at least two of the above mentioned mutations A first embodiment of such a double mutant has His at position 58 (rather than Phe in the wild type sequence), and Ser at position 213 (rather than Met) A second embodiment of such a double mutant has Ser at position 58 (rather than the wild-type Phe) and Arg at position 213 (rather than the wild-type Met)
Where the non-phytotoxic substance is a D-amino acid other than D phoshmothncin or D-bialaphos then the enzyme is a D-amino acid oxidase The D amino acid is preferably, not an endogenous plant metabolite and is selected to be one that is phloem mobile, metabohcally stable in the plant (preferably having a t ¥i in the plant of greater than ~ 1 week) and an efficient substrate of the said oxidase Oxidation of the D-amino acid by the enzyme is concomitant with reduction of oxygen to phytotoxic peroxide and/or superoxide anions
In a preferred embodiment the oxidase enzyme is targeted to a subcellular location other than the peroxisome This is achieved, for example, by modifying the gene so that three C-termmal amino acids (e g SKL in the case of the the Rhodotorula gracilis D amino acid oxidase) are deleted or modified and/or by addition of sequence to add a chloroplast or mitochondrial transit peptide to the N-termmus
Further suitable D amino acid oxidases may be obtained preferably from fungal sources, by the mutation and selective procedures known to the skilled man and augmented by the present disclosure
Further D amino acid oxidase (or equally, phosphinothncin racemase) enzymes and DNA coding sequences suitable for working the invention are selected from amongst those
organisms, optionally subjected to mutagenesis, where it is found that growth on a N-limited media, under conditions where D-amino acid oxidase (or phosphmothricin racemase) is induced (for example grown on D-alamne) is selectively inhibited in the presence of D-phosphinothncin D-amino acid oxidase genes suitable for the invention are then, for example, obtained by probing gene libranes of such organisms with suitable degenerate DNA probes (for example based upon established D-amino acid oxidase concensus sequences such as PROSITE, PSO0677) and subcloning Alternatively, genes encoding suitable enzymes are obtained by screening gene expression libranes in a suitable host cell such as E coh or a yeast (suitable host strains lack an endogenous oxidase or dehydrogenase activity versus D-phosphmothncin) for transformation to a phenotype with increased sensitivity to growth inhibition by D-phosphinothncin on a minimal medium This method relies upon the ability of transformed E coh clones to produce L-PPT from D-PPT via the combined action of their endogenous L transaminase activity and the heterologous oxidase Alternatively, suitable and improved genes are selected on the basis of in vitro assay of the expressed enzyme for the desired ability to oxidise D-phosphinothncin There are many methods for directly assaying the activities of D-amino acid oxidases such as based upon detection of peroxide (Enzyme Microb Technol, (2000), 27(8), 605-611), depletion of oxygen using an oxygen electrode or based on direct detection of aminonia
In an embodiment of the invention, a preferably fungally-denved DAMOX gene is cloned into a shuttle vector under operable control of a promoter (e g GAL promoter) capable of expression in the host organism in which the selection will be earned out (preferably yeast) This gene is then subjected to mutagenesis , for example by Mn2+-poisoned PCR, plasmid DNA replication in a strain which is defective in DNA repair/ editing processes such as E coh strain XL1 red, or by plasmid DNA replication in a host strain which is subjected to mutagenesis using, for example X-Rays, UV light, addition of a chemical mutagen and transformed into a host organism (preferably yeast) The desired DNA encoding a DAMOX having the desired property of an enhanced ability to oxidise D-PPT is selected for (following an optional, initial selection step for transformants based upon selectable markers present on the shuttle vector allowing, for example, selection via restoration of prototrophy or growth in presence of hygromycin etc) via, for example
a) Selection of transformed cells having the ability to utilise amino acids which are chemically similar to D-phosphinothricin as sole nitrogen source For example,
transformed yeast colonies are selected which are able to grow on analogues of D-PPT (and its esters) where the phosphmic acid moiety is replaced with a carboxylate (1 e D-glutamate), sulphonate, phosphonate, sulphone, or sulfoxide moiety (or esters of these) as sole N source E g
(Formula Removed)
b) Selection of transformed cells capable of utilizing D-PPT itself as sole N source For this selection to work, the host cell must also be transformed with a gene capable of negating the inhibitory effect of L-phosphinothncin on glutanune synthetase For example the shuttle vector may also comprise a gene which encodes an enzyme such as PAT which inactivates L-PPT
Cycles of mutation and selection may be iterated D-amino acid oxidases may further be cloned, expressed, part punfied and charactensed bnetically in order to identify genes and DAMOXs with the most suitable properties (e g enzyme stability, high kcat/ Km value for oxidaUon of D-PPT, minimal oxidation of any endogenous plant substrates, optimum pH optimum etc)
Where the non-phytoxic substance is D-phosphmothncin (PPT) it may be obtained from a mixture of D and L PPT For example, DL PPT may be added to a culture medium (preferably minimal) of E coli cells (optionally an arg E mutant to minimise the background level of N-acetyl PPT deacetylase activity) transformed and induced to express a PAT gene
(encoding an enzyme which transfers an acetyl group from acetyl CoA to L-PPT) at a high
level After allowing a suitable time for the L component to substantially all be N-
acetylated, (judged, for example, by monitoring the conversion using 31-P NMR) D-PPT is
recovered and purified from the cell-free medium using successive steps of, for example,
solvent extraction at high and low pH, anion and cation exchange chromatography, selective
crystallisation with chiral cations such as chmchocine or other procedures known in the art
such as liquid/ liquid extraction with two non-miscible aqueous phases as the phase system
(cf methods in USP 5,153,355) Typically a late step is cation exchange chromatography
from which D-PPT is recovered as the aminonium salt
Alternatively, D-PPT may be obtained by an enzymatic method wherein DL PPT + 2
ketoglutarate is converted to primarily a mixture of D PPT, 2-oxo PPT (and its
decarboxlation products) and GABA by the combined actions of (I) L-aminotransferase (e g
from E coli) and (II) glutamate decarboxylase The desired pure D PPT is resolved from the
reaction mixture using methods known in the art and as outlined above
D PPT may also be obtained using an enzymatic method wherein DL PPT + 2
ketoglutarate + NAD is converted to primarily a mixture of D PPT, 2-oxo PPT (and its
decarboxylation products) NADH, and aminonia by the combined actions of (I) L-
aminotransferase and (IT) glutamate dehydrogenase The desired D PPT is purified from the
reaction mixture
t
In a yet further method of making D-PPT, DL PPT is treated with a L amino acid oxidase so that the only remaining amino acid is the desired D form This D-PPT is then purified from the reaction mixture
A still further method involves (I) conversion of DL PPT to N-acetyl DL PPT (using acetic anhydride or other acetylatmg reagents and methods well known in the art) and (H) treatment of N-acetyl DL PPT with D-aminoacylase so that only N acetylD-PPT is deacetylated The resultant D-PPT is purified from the reaction mixture For example, D-PPT is resolved from N-acetyl-L-PPT by binding to Dowex anion exchange resm and elution with 40 raM formic acid Under suitable loading conditions this acid elutes the D-PPT whilst leaving the N-acetyl L-PPT bound to the column
A still further method involves treatment of DL PPT with L-aminoacylase and an acylatmg agent in a non-aqueous solvent so that only the desired D-PPT is left in a non -acetylated form
A yet further method of preparing pure D-PPT involves enantioselective crystallisation from DL PPT using a chiral base such as chinchocme and addition of a seed crystal of the chiral base with pure D-PPT
A yet further method of preparing pure D-PPT from DL-PPT by direct chiral chromatography using a chiral base column
A detailed method for the production of pure D-PPT is given in one of the Examples following
The 2-arylpropionyl-CoA epimerase or alpha-methylacyl-CoA racemase (EC 5 1 99 4) enzyme may be selected from amongst those produced by rat liver, Acremomium sp or Neurospora crassa The 2-arylpropionyl-CoA epimerase or alpha-methylacyl-CoA racemase (EC 5 1 99 4) enzyme may be selected from proteins having sequences corresponding to AAR49827 in the GENESEQP Derwent database, P70473 in Swissprot or SEQ ID number 4 (this application) and the 2-arylpropionyl-CoA epimerase or alpha-methylacyl-CoA racemase (EC 5 1 99 4) enzyme may be encoded by a DNA coding sequence selected from sequences comprised witKm GENESEQN Derwent database accession AAQ44447, EMBL accessions RN2ARYLCO and RNU89905 and SEQ ID number 3 (this application)
The acyl CoA synthetases for use in the invention may be 'long-chain' acyl CoA synthetases (EC 6 2 13) selected from those produced by Brassica napus, rat liver, Saccharomyces sp or Arabidopsis The said synthetases may be selected from proteins having sequences corresponding to SPTREMBL sequence Q96338, Swissprot P18163, Swissprot P39518, SPTREMBL Q9C5U7 or SPTREMBL Q9TOAO and the DNA sequences which encodes the acyl CoA synthetases may be selected from sequences comprised within EMBL accessions BNAMPBP2, J05439, X77783 and AB030317 The acyl CoA synthetases , 2-arylpropionyl-CoA epimerases and thioesterase enzymes and/or DNA sequences which encode them which are suitable for working the method of the invention may be selected on the basis of the capability of the source organism to convert S-aryloxyphenoxypropionates to R-aryloxyphenoxypropionates and/or to convert S-ibuprofen to R-ibuprofen Such organisms can, for example, be obtained from soil samples and methods for assaying and detecting such chiral inversions in cell cultures and microbial broths are well-known (cf Menzel-Soglowek et al (1990) J Chromatogr , 532, 295-303, Bewick (1986) Pestic Sci, 17, 349-356) Accordingly, the acyl CoA synthetase, 2-arylpropionyl-CoA epimerase and/or thioesterase enzyme and, optionally, the DNA
sequences which encode them may be sourced from, Arthrobacter simplex NCIB 8929, Arthrobacter roseoparaffmeus ATCC 15584, Bacillus subtilis ATCC 15841, Botrytis cmerea CM1 124882, Brevibactenum butamcum ATCC 15841, Brevibactenum healii ATCC 15527, Brevibactenum ketoglutamicum ATCC 21004, Brevibactenum paraffinolyticum ATCC 21195, Corynebactenum fascians, Corynebactenum fnikoense ATCC 21496, Methanomonas methanohca NRRL B-5758, Micrococcus roseus, Mycobactenum aurum NCTC 1043, Mvcobactenum petroteophilum ATCC 21497, Mycobactenum phlei NCTC 10266, Mycobactenum smegmatis ATCC 19420, Nocardia opaca NCIB 9409, Nocardiopsis asteroids ATCC 21943, Psuedomonas dimimuta NCIB 9393, Psuedomonas lemoignei NCIB 9947, Rhodococcus rhodocrous ATCC 13808, Rhodococcus rhodocrous ATCC 21197, Rhodococcus sp ATCC 21499, and Rhodococcus sp ATCC 31337 Using methods well known in the art, candidate and improved genes comprising DNA coding sequences are readily cloned and selected from suitable gene libranes of these organisms by the use of suitably degenerate probes based upon the known sequences of other acyl CoA synthetase , 2-arylpropionyl-CoA epimerase and thioesterase enzymes Alternatively and additionally suitable genes are selected on the basis of prepanng expression libranes in a suitable host and screening the library, using either in vitro or whole organism culture assays, for ability of clones to carry out the overall chiral conversion or, alternatively, for ability to catalyse each of the individual acyl CoA synthetase (using the microsomal fraction), 2-arylpropionyl-CoA epimerase or thioesterase partial reactions Suitable methods for in vitro assays of these activities are analogous to or the same as those descnbed in the literature for ibuprofen (e g Shieh and Chen (1993) JBC, 268, 3487-3493
The enzyme for use in the present inventive method may be a phosphmothncin racemase, the DNA coding sequence for which is produced by mutagenesis and/or recombmatonal shuffling of glutamate racemase genes followed by iterative rounds of selection and further evolution toward increasing levels of phosphmothncin racemase activity Glutamate racemases are ubiquitous amongst bactena and are of two types, those that are dependent on pyndoxal phosphate as a cofactor and those which are cofactor-mdependent and contain two active-site cysteine residues In one embodiment of the invention, sequences encoding glutamate racemases of Pediococcus pentosaceus, Lactococcus lactis, Lactobacillus brevis. Staphylococcus hemolyticus and Bacillus sphaencus are selected for mutation and/or recombmatonal family shuffling In a particular embodiment the genes selected for shuffling encode proteins having sequences
corresponding to (Swissprot) sequences 082826, P94556 and 031332 Genes suitable for working the current invention are selected by screening expression libranes in a suitable host eel] such as E coh or yeast those colonies which exhibit increased sensitivity to growth inhibition by D-phosphmothxicm in minimal medium L-PPT inhibits glutamine synthetase while D-PPT does not A glutamate racemase mutant clone which converted D- to L-PPT will not grow on minimal medium unless supplemented with glutamine In suitable host strains, endogenous D-armno acid oxidase or D-amino acid dehydrogenase activities are either not expressed or do not encompass D-phosphinothncin as a substrate Alternatively suitable genes may be selected on the basis of assay, in vitro or in vivo, of the ability of the encoded enzyme to mterconvert D and L phosphmothncin Suitable such assays may be based upon exchange of the alpha proton, the use of bioassays to detect L-phosphinothncin formation from D-phosphmothncin or, for example, detection of conversion of L-phosphmothncin to D-phosphinothncin using coupling to a suitable D-armno acid oxidase
DNA sequences encoding the enzymes used in the present invention may, optionally, be further mutated and selected in order to generate further useful enzymes having improved utility Many charactenstics of enzymes are thus improved including catalytic activity (kcat/ Km) versus the desired substrate, temperature stability and pH optimum Methods for generating, screening and selecting for such improved vanants are well known For example, suitable vanant DNA sequences are generated by a process of mutagenesis (e g by passaging DNA through bactenal or yeast strains with error-prone DNA replication such as E coh XL1 red, by UV, chemical or targeted oligonucleotide PCR mutagenesis) In particular such genes are produced by any of a number of alternative processes of DNA shuffling or 'sexual PCR' as, for example, summansed in WO 0061740 fom pages 28-41 all of which are included by reference herein Many methods are suitable for selecting such improved genes Genes may be suitably expressed in a suitable host cell such as E coh or yeast and selected for improvement using suitable such assays as, for example, desenbed herein
The chimenc genes encoding enzymes for use in the invention which are capable, singly or in combination with others, of converting a non-phytotoxic substance to a phytotoxic one, may each compnse a DNA sequence which encodes one of said enzymes operably linked to a 5' promoter region which preferentially directs expression to either the male or the female reproductive structures This specificity of expression ensures that the effect of the expressed enzyme(s) will be exerted only within the locality of the tissues and
cells necessary for formation of viable seed or viable pollen and will not be deletenous to the plant beyond its effect on fertility in the presence of a suitable non phytotoxic substance, perhaps a pro-herbicide In addition to promoter regions chimeric genes according to the current invention also comprise a 3' transcriptional terminator sequence This is responsible for the termination of transcription and correct mRNA polyadenylation Many such 3' transcriptional terminator sequences are known in the art and are suitable for use in the chimeric genes of the current invention In particular embodiments the 3' transcriptional terminator sequence is selected from the CMV 35S terminator, the tml terminator, the nopalme synthase (nos) terminator and the pea rbcS EO terminator
5' Promoter regions suitable for use in certain embodiments of the said chimeric genes include 5' regions of genes which are preferentially expressed in female floral tissues In certain embodiments the 5' promoter region is selected from the group consisting of the stig 1 promoter of tobacco (Goldman et al, (1994) EMBO J, 13, 2976-2984), a modified S13 promoter (Dzelkalns et al (1993) Plant Cell, 5, 8555), the AGL5 promoter (Savidge et al (1995) Plant Cell, 7, 721-733 and the promoter region 5' of the maize-carpel specific ZAG2 gene (Thiessen et al (1995) Gene, 156, 155-166) Optionally, further suitable promoter regions are obtained from regions upstream of the coding sequences of genomic DNA corresponding to cDNA sequences known in the art to be preferentially expressed in female reproductive structures In certain embodiments such probe cDNAs are selected from the group consisting of the Arabidopsis Fbp7 and Fbpll genes (Angenent et al, (1995) Plant Cell, 7,1569-1582) and the orchid-specific cDNAs 040,0108, 039,0126 and 0141 (Nadeau et al, (1996) Plant Cell, 8, 213-239) In particular embodiments 5' promoter regions comprising genomic DNA associated with preferential expression in female reproductive structures is selected from DNA regions comprised within the group consisting of the genomic DNA clone pSH64 having the accession number NRRL B-21920, genomic clone, pCIB 10302 hybridising to the cDNA P26-A4 having the accession number NRRL B-21655 and genomic DNA clone X2-1 hybridising to cDNA clone P19-QA having the accession number NRRL B-21919 In further particular embodiments these promoter regions comprise nucleotides 1 to 1390 of SEQ ID No 11, SEQ ID No 2 and nucleotides 1 to 1093 of SEQ ID No 4 in WO 98/39462 In further embodiments, further 5' promoter regions suitable for use in the chimeric genes of the invention are isolated and cloned by methods which are familiar to one skilled in the art For example, novel transcripts expressed in female reproductive structures are identified by isolating RNA from tissues such as maize
silks or wheat pistils followed by differential screening using techniques such as differential display, PCR select cDNA subtraction and subtractive cDNA library construction cDNA clones that are preferentially expressed in the female tissues and not in other parts of the plant such as the leaves, roots and tassels The tissue specificity of expression is, optionally, further confirmed by Northern blotting The cDNA clones are used as probes for genomic library screening 5' promoter regions and,optional]y, 3' untranslated DNA regions associated with tissue preferential expression are obtained from the genomic DNA clones and used in the construction of chimenc genes for preferential expression in female reproductive structures
5' Promoter regions suitable for use in certain embodiments of the said chimenc genes include 5' regions of genes which are preferentially expressed in male floral tissues These include promoter regions for expression in pollen, the tapetum or other structures in the anther In certain embodiments these 5' promoter regions are selected from the group consisting of the LAT52 promoter (Twell et al, (1989) Dev , 109,705-713), the tomato A127 promoter (Dotson et al, (1996) Plant J , 10, 383=392), the maize Zmg promoter (Hamilton et al, (1989) Sex Plant Reprod 2,208-212), the maize CDPK promoter (Guerro et al, (1990) Mol Gen Genet, 224,161-168) and the anther specific ant32 and ant43D promoters disclosed in USP 5477002 herein incorporated by reference in its entirety In certain further embodiments the 5' promoter region is selected from the group consisting of the tapetum-specific promoter CA55 from maize ("Pca55" descnbed in WO 92/13956), the tapetum-specific promoter El from nee (descnbed in USP 5639948), the tapetum-specific promoter T72 from nee (descnbed in USP 5639948), the RA8 anther-specific promoter from nee (EMBIVGenbank accession number AF042275, Jean Js et al,(1999) PMB, 39, 35-44) the anther-specific Tapl promoter (Spena et al (1992) Theor Appl Genet 84, 520-527) and the ZmC5 - pollen specific promoter from maize (EMBL/Genbank accession number Y13285 , Wakeley et al, (1998) PMB, 37,187-192) Optionally, further suitable promoter regions are obtained from regions upstream of the coding sequences of genomic DNA corresponding to cDNA sequences known in the art to be preferentially expressed in male reproductive structures In certain embodiments such probe cDNAs are selected from the group consisting of the orchid pollen-tube specific cytochrome P450 gene (Nadeau et al, (1996) Plant Cell, 8, 213-239), the Bcpl gene of Arabidopsis (Xu et al (1995) P N A S , 92, 2106-2110) and the male-flower specific MFS14 gene of maize (Wnght S Y et al, (1993) Plant J 3,41-49) In further embodiments, further 5' promoter regions suitable for use in the chimenc genes of the
mvention are isolated and cloned by methods which are familiar to one skilled in the art For example, novel transcripts expressed in male reproductive structures are identified by isolating RNA from tissues such as tassels, pollen tubes, anther or tapetum followed by differential screening by techniques such as differential display, PCR select cDNA subtraction and subtractive cDNA library construction cDNA clones that are preferentially expressed in the male tissues and not in other parts of the plant such as the leaves, roots and stigma are isolated The tissue specificity of expression is, optionally, confinned by Northern blotting The cDNA clones are used as probes for genomic library screening 5' promoter regions and 3' untranslated DNA regions associated with tissue preferential expression are obtained from the genomic DNA clones and used in the construction of chimeric genes for preferential expression in male reproductive structures
Further promoter regions useful in the chimeric genes of the invention include the regions upstream of the Osmads 13 gene of nee, the OSG gene of nee anther, and the YY2 gene of nee Generally, promoter regions yielding high, early, sustained and preferential expression in male or female reproductive structures are selected as most suitable Promoter regions may also further compnse chimenc combinations with each other and with further enhancer regions
Chimenc genes may optionally compnse a region, immediately preceding the DNA sequence encoding the enzyme involved in the conversion of non-phytotoxic substance to phytotoxm, which encodes a peptide sequence capable of targeting the said enzyme to subcellular organelles such as the chloroplast, peroxisome (other than when the phytotoxm is a peroxide or super oxide anion) or mitochondna and the said targeting protein may have the sequence of (I) a chloroplast transit peptide or (a) a chloroplast transit peptide-N-terminal portion of a chloroplast protein - chloroplast transit peptide This may be particularly advantageous where, for example, the said DNA sequence encodes a D-amino acid dehydrogenase enzyme which would be expected to function best in a compartment such as the mitochondrion or chloroplast compnsing a membrane electron transport chain or where, for example, the DNA sequence encodes an enzyme catalysing only a partial step in the overall desired transformation and where the full reaction requires combination with compartmentalised metabolites and endogenous activities In particular, for targeting to the mitochondnon, the said region of DNA which immediately precedes the enzyme-coding DNA sequence, encodes a mitochondnal transit peptide sequence In certain embodiments the transit peptide sequence may be selected from the group consisting of the endogenous
transit peptide sequences of the faeta-subunit of Nicotima plumbaginifolia mitochondnal ATP synthase, mitochondria-specific NADP-dependent isocitrate dehydrogenase, NADPH-binding subunit of respiratory chain complex I and yeast mitochondrial tryptophanyl-tRNA-synthetase (WO 6121513)
Polynucleotides for use in the present inventive method may comprise one or more chimenc genes which encode enzymes which catalyse reactions involved in the generation of phytotoxms from non-phytotoxic substances Optionally such polynucleotides compnse yet further genes and chimenc genes, such as a chimenc marker gene A chimenc marker gene as used herein compnses a marker DNA under expression control of a promoter which is active in plant cells The marker DNA encodes an RNA, protein or polypeptide which, when expressed in a plant, plant tissue or plant cell allows such plant matenal to be distinguished from plant matenal not expressing the marker DNA Examples of marker genes are genes that provide a specific colour to a cell such as the Al gene (Meyer et al (1987) Nature 330, 667) or genes that render plant cells resistant to otherwise lethal selection with antibiotics (e g the aac(6') gene encoding resistance to gentamycm, WO 94/01560 or hygromycm phosphotransferase genes providing resistance to hygromycin) or herbicides such as glyphosate (e g EPSPS genes such as in USP 5510471 or WO 00/66748), phenmedipham (e g pmph gene USP 5347047, USP 5543306), bromoxynyl (e g genes descnbed in USP 4810648) sulphonylureas (e g genes descnbed in EP 0360750), dalapon (genes descnbed in WO 99/ 48023), cyanarmde (genes descnbed in WO 98/48023, WO 98/56238) and genes encoding resistance to glutamme synthetase inhibitors such as L-phosphmothncin (such as, for example, N-acetyl-transferase genes descnbed in EP 0242246, EP 0242246 and EP 0257542) In a preferred embodiment of the polynucleotide of the current invention which compnses a herbicide resistance gene as a marker gene, the said herbicide is a herbicide which is useful for weed control in the crop and, additionally, the herbicide resistance gene is expressed sufficiently to provide robust tolerance to field rates of the said herbicide In a further prefened embodiment the herbicide is glyphosate and the herbicide resistance gene is an EPSP synthase However the marker gene may be a gene that provides for positive selection wherein the marker gene encodes an enzyme which provides, in the context of a particular medium, the transformed plant cells with a positive metabolic advantage USP 5767378 descnbes a number of suitable positive selection systems and genes
Where the polynucleotide of the current invention compnses a herbicide resistance gene the herbicide is exogenously applied to crop plants which are mterplanted at a sufficient
density to eliminate the production of non-hybrid seed originating from non-transgemc self-fertile parent plants In a preferred embodiment the herbicide is glyphosate or an agronomically useful salt thereof and the said herbicide resistance marker gene is selected from amongst those glyphosate resistance conferring genes described in WO 00/66748
Where a marker gene is present, means for the removal of said marker gene may also be provided This is desirable where, for example, it is decided to combine traits In addition it is also desirable to remove herbicide-resistance marker genes which could interfere with the operation of the pro-herbicide-dependent conditional fertility mechanism of the present invention For example, it might be desirable to remove a phosphmothncin N acetyl transferase (PAT) herbicide-resistance marker gene from a polynucleotide also comprising a chimeric gene, useful for providing conditional male or female sterility dependent on the exogenous application of D-phosphmothncin pro-herbicide The presence of the PAT gene could potentially interfere with successful conditional sterility by inactivating the L phosphmothncin phytotoxm Thus, polynucleotides which compnse marker genes may optionally compnse specific recogmtion sites for specific recombinases in positions which flank the marker gene and which allow the sequence to be 'kicked out' Crossing of a plant carrying the so-flanked marker gene with a plant carrying a gene which encodes the corresponding specific recombmase results in progeny plants from which the marker is specifically excised Examples of suitable such site-specific homologous recombination systems are the flp/ fit system (Lyznik et al, (1996), Nucleic Acids Res 24, 3784-3789) and the Cre/Lox system (Bayley, C C et al, (1992) PMB, 18, 353-361)
Polynucleotides used in the present inventive method may optionally compnse one or more translational enhancers located within the non translated regions 5' of the protein-encoding sequences The skilled man is aware of the identity of such suitable translational enhancers - such as the Omega and Omega pnme sequences denved from TMV and that denved from the tobacco etch virus, and how such translational enhancers can be introduced into the polynucleotide so as to provide for the desired result of increased protein expression Further examples include translational enhancers denved from maize chlorotic mottle virus and alfalfa mosaic virus (Galhe et al ,(1987) NucI Acids Res , 15, 8693-8711, Skuzeski et al , (1990) PMB , 15, 65-79) To further optimise expression of proteins from chimenc genes and chimenc marker genes the said polynucleotides may also further compnse elements such as enhancers, scaffold attachment regions (SARS or MARS) and nitrons Vanous mtron sequences such as the maize adhl mtron 1 have been shown to enhance
expression when mcluded into the 5' untranslated region of genes and, optionally, are used in the chimeric genes of the current invention
Plants which have been transformed according to the invention so as to exhibit the desired male/female sterility characteristics may also have been transformed with a polynucleotide which comprises regions encoding proteins capable of conferring upon plant material containing it at least one of the following agronomically desirable traits resistance to insects, fungi, viruses, bacteria, nematodes, stress, dessication, and herbicides
Herbicide resistance conferring genes may, for example, be selected from the group encoding the following proteins glyphosate oxidase (GOX), EPSP synthase, phosphmothncin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-CoA carboxylase (ACCase), Acetolactate synthase (ALS), protoporphynnogen oxidase (PPO), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil nitnlase, phytoene desaturase (PDS), the product of the tfdA gene obtainable from Alcahgenes eutrophus, and known mutagenised or otherwise modified variants of the said proteins The skilled man will recognise the need to close such genes, and the promoters which drive their expression, carefully, having regard to the nature of the enzyme he uses to convert the non-phytoxin substance In the case that the polynucleotide provides for multiple herbicide resistance such herbicides may be selected from the group consisting of a dimtroaniline herbicide, tnazolo-pynmidines, a uracil, a phenylurea, a tnketone, an isoxazole, an acetanilide, an oxadiazole, a tnazmone, a sulfonarulide, an amide, an anilide, an lsoxaflutole, a flurochlondone, a norflurazon, and a tnazohnone type herbicide and the post-emergence herbicide is selected from the group consisting of glyphosate and salts thereof, glufosmate, asulam, bentazon, bialaphos, bromacil, sethoxydim or another cyclohexanedione, dicamba, fosamme, flupoxam, phenoxy propionate, quizalofop or another aryloxy-phenoxypropanoate, picloram, fluormetron, butafenacil, atrazme or another tnazme, metnbuzin, chlonmuron, chlorsulfuron, flumetsulam, halosulfuron, sulfometron, lmazaqum, imazethapyr, isoxaben, imazamox, metosulam, pynthrobac, nmsulfuron, bensulfuron, nicosulfuron, fomesafen, fluroglycofen, K3H9201, ET751, carfentrazone, mesotnone, sulcotnone, paraquat, diquat, bromoxynil and fenoxaprop
In the case that the polynucleotide comprises sequences encoding msecticidal proteins, these proteins may be selected from the group consisting of crystal toxins derived from Bt, including secreted Bt toxins such as those known as "VIP", protease inhibitors, lectins and Xenhorabdus/Photorhabdus toxins The fungus resistance conferring genes may
be selected from the group consisting of those encoding known AFPs, defensins, chitmases, glucanases, and Avr-Cf9 Particularly preferred insecticidal proteins are crylAc, crylAb, cry3A, Vip 1A,Vip 1B, Vip3A, Vip3B, cysteine protease inhibitors, and snowdrop lectin In the case that the polynucleotide compnses bactenal resistance confemng genes these may be selected from the group consisting of those encoding cecropins and techyplesin and analogues thereof Virus resistance confering genes may be selected from the group consisting of those encoding virus coat proteins, movement proteins, viral replicases, and anti-sense and nbozyme sequences which are known to provide for virus resistance, whereas the stress, salt, and drought resistance confemng genes may be selected from those that encode Glutathione-S-transferase and peroxidase, the sequence which constitutes the known CBF1 regulatory sequence and genes which are known to provide for accumulation of trehalose
Polynucleotides used in accordance with the present invention may have been "modified" to enhance expression of the protein encoding sequences comprised by them, in that mPvNA instability motifs and/or fortuitous splice regions may have been removed, or -crop preferred codons may have been used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the protein encoding regions of the unmodified polynucleotide in the organism in which such regions of the unmodified polynucleotide are endogenous The degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein may be such as to prevent co-suppression between the modified and endogenous sequences In this case the degree of identity between the sequences should preferably be less than about 70% In addition the sequence around a translational start position may be modified such that it is "Kozack preferred" What is meant by this is well known to the skilled man
The invention still further includes morphologically normal conditionally fertile whole plants which result from the crossing of plants which have been regenerated from matenal which has been transformed with the nucleic acid in accordance with the present invention and which therefore provides fopr such a trait The invention also includes progeny of the resultant plants, their seeds and parts
Plants of the invention may be selected from the group consisting of field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat,
barley, nee, sorghum, mangel worzels, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing plants insofar as they are not already specifically mentioned, their progeny, seeds and parts
Particularly preferred such plants include wheat, barley, oats, nee, maize, millet and sorghum
The invention still further provides a preferred method of producing hybrid wheat seed which comprises the steps of
(I) transforming plant material with a polynucleotide or vector which comprises a gene conferring male sterility conditional upon exogenous application of a pro-herbicide or other non-phytotoxic substance,
(II) selecting the thus transformed material, and
(m) regenerating the thus selected material into morphologically normal
conditionally male-sterile whole plants (IV) breeding a homozygous conditionally male-sterile female parent line (v) transforming plant material with a polynucleotide or vector which composes a gene conferring female sterility conditional upon exogenous application of the same pro-herbicide or non-phytotoxic substance as in (1), (vi) selecting the thus transformed material, and (vn) regenerating the thus selected material into morphologically normal
conditionally female-sterile whole plants (vm) Breeding a homozygous conditionally female-sterile male parent line (IX) Interplanting said conditionally-stenle male and female parent lines at such a
ratio as to ensure efficient pollination (x) Applying said pro-herbicide or other non-phytotoxic substance to the mterplanted parent lines at such a dose and stage in development as to minimise self-fertilisation (xi) Harvesting hybrid wheat seed from the mterplanted parent plants The current invention also provides variants of the above method wherein the male parent is female sterile by any means, the female parent is male sterile by any means, male and female parent lines are conditionally sterile dependent upon the application of different pro-herbicides both of which are applied, and the crop is other than wheat
The present invention also includes a diagnostic kit comprising means for detecting the proteins, or DNA sequences encoding them, which are present in plants produced in accordance with the present inventive method and therefore suitable for identifying tssues or samples which contain these The DNA sequences can be detected by PCR amplification as is known to the skilled man - based on primers which he can easily derive from the enzyme encoding sequences which are disclosed or mentioned in this application The enzymes per se can be detected by, for example, the use of antibodies which have been raised against them for diagnostically distinguishing the antigenic regions which they contain
The present invention also provides a method of producing enantiomencally pure D-Phosphmothncin (D-PPT), comprising the steps of
(a) Providing cells which contain an enzyme capable of selectively N-
acylating PPT,
(b) Growing said cells in a medium which contains D-L PTT to produce conditioned medium,
(c) Separating the cells from the conditioned medium of (b),
(d) Optionally extracting the conditioned medium with a non-aqueous, non miscible solvent, at various pHs, so that the PPT containing fraction is separated from the fraction that contains molecules more water soluble than is PPT,
(e) Optionally admixing with the conditioned or PPT-containmg extracted media of step (d) a cation exchange resin in its protonated form, in an amount, and at pH, sufficient to absorb a substantial proportion of the cations - other than PTT, from the medium,
(f) Admixing with the conditioned medium, extracted medium or medium to result from step (e) a cation exchange resin in its protonated form, in an amount, and at a pH, sufficient to bind the bulk of the PPT in the medium,
(g) Harvesting the cation ion exchange resin from step (f) to which the PPT is bound and selectively elutmg PPT from it using an elutmg medium having a sufficient pH and ionic strength, with the proviso that the pH of the said elutmg medium is not so low as to cause racemisation of the thus eluted PPT
In respect of the transformation of plant material, those skilled in the art will recognise that although particular types of target material (e g embryogemc cell suspension culture or de-differentiating immature embryos) and particular methods of transformation (e g using Agrobactenum or particle bombardment) are specified in the examples below, the present invention is not limited to these particular embodiments and such target matenals and methods may be used interchangeably Furthermore the term "plant cells" as used throughout this description of the invention can refer to isolated cells, including suspension cultures as well as to cells in an intact or partly intact tissue such as embryo, scutella, microspore, microspore-denved embryo or somatic cells from plant organs Similarly, although the specific examples are limited to maize and wheat, the invention is equally applicable to a broad range of agncultural crops which can be transformed using suitable methods of plant cell transformation
The present invention will be further apparent from the following non-limiting examples taken in conjunction with the associated Sequence Listing and Drawings
SEQ ID NO 1 shows a DNA sequence, isolated from Synechocystis sp which encodes an enzyme (depicted as SEQ ID NO 2) having the activity of an esterase B
SEQ ID NO 3 shows a DNA sequence, isolated from Neurospora crassa which encodes an enzyme (depicted as SEQ ID NO 4) having the activity of an acyl-methylacyl-CoA racemase sequence
SEQ ID NO 5 and 6 depict the PCR pnmers used to obtain the TA29 promoter region
SEQ ID NO 7 depicts a DNA sequence, isolated from Rhodotorula gracilis which encodes an enzyme having the activity of a D-amino acid oxidase
SEQ ID NO 8 and 9 depict degenerate oligos used to provide vanant D-amino oxidase
SEQ ID NO 10 and 11 depict motifs where alternative amino acids may substituted in order to provide vanant D-amino acid oxidases
Figure 1 is a schematic representation of a construct for tobacco transformation having Rhodotorula D-amino acid oxidase under operable control of the stig 1 promoter region The components indicated are LB (left border sequence), AOPR1 (AoPRl promoter), PSTTG1 (EMBL accession no X77823), RGDAO (OPT) (SEQ ID NO 7), PC PROMOTER (EMBL accession no X16082), PAT (EMBL accession no A02774), NOS (nos terminator obtained from EMBL accession no ATU237588) and RB (right border sequence)
Figure 2 is a schematic representation of a construct for tobacco transformation where the Rhodotorula D-amino acid oxidase coding sequence is truncated by 3 codons at the 3' terminus and, at the 5' terminus (RGDAO (OPT)-SKL), is fused to a region encoding an optimised transit peptide (FR2673643)
Figure 3a is a map of the plasmid Ubi-CoA synthetase, wherein PUB 11-01-01 has EMBL accession number SM29159 and CoA synthetase has number J05439
Figure 3b is a map of the plasmid Ubi-Epimerase, wherein Epimerase has EMBL accession number Y0817Z
General molecular biology methods are earned out according to well established methods
For the most part the following examples each comprise multiple exemplifications of the current invention Where the term 'promoter region of a gene' is used this is taken to mean DNA sequences which comprise the promoter, sequences upstream of the promoter and also, optionally, all or part of the DNA sequence encoding the 5' untranslated leader region of the mRNA
Example 1 Tobacco plants which are conditionally female sterile dependent upon exogenous apphcation of D-phosphmothncin or D alanine or D leucine or D methionine or D asparagine or D-aspartate or D-glutamate
The DNA sequence encoding the D-amino acid oxidase protein sequence Q99042 (Swissprot) withm the EMBL sequence Z50019 is either obtained by RT-PCR from
Trigonopsis vanabilis mRNA or is obtained synthetically Alternatively the DNA sequence encoding the D-amino acid oxidase protein sequence P80324 (Swissprot) within the EMBL sequence A56901 is either obtained by RT-PCR from Rhodospondium tolruloides (Rhodotorula gracilis) mRNA or is obtained synthetically (which makes it easier to control which internal restriction enzyme sites are present and to create flanking sites to facilitate cloning) as, for example, SEQ ID # 7 which is designed to account for plant (in this case wheat) codon usage and to minimise DNA features potentially immicable to expression Alternatively, the DNA sequence (e g denved from EMBL Accession X95310) encodes a 'D-aspartate oxidase' such as P31228 (Swiss Prot) and, again, is synthesised to account for plant codon usage and to minimise features immicable to expression Alternatively D-amino acid oxidase encoding sequences obtained are the same as in example 2 Flanking PCR-pnmer and synthetic DNA sequences are designed to place useful unique restriction sites for cloning Preferably and in the case where the oxidase coding sequence does not contain confounding internal sites, an Ncol or Ndel site is placed at the 5' end to facilitate the cloning of in-frame fusions with sequences added 5' to the ORF such as chloroplast transit peptide encoding sequences In some variants of the example the D-amino acid oxidase (in some variants named 'D aspartate oxidase') gene is cloned in such a way that the terminal 3 amino acids are truncated and the encoded enzyme is therefore no longer peroxisomally targeted In an additional series of variants of the method the gene is engineered by PCR so as to encode the Rhodotorula gracilis D amino acid oxidase with alternative amino acids at positions 213 and 238 and, in particular, with an argraine, serine, cysteine, lysine, asparagine or alanine replacing the methionine at position 213 and/ or a histidme, serine, cysteine, asparagine or alanine replacing the tyrosine at position 238 The methionine at the '213' position is identified as the M in the native protein sequence motif RCTMDSS The tyrosine at position 238 is identified as the 'Y' within the native protein sequence motif GGTYGVG These variants of the D amino acid oxidase from Rhodotorula or the 'D-aspartate oxidase' are used when female sterility is to be made conditional upon the application of D-aspartate, D-glutamate or D-phosphmothncin
Restriction sites can be placed upstream of the ATG translational start site intervening sequences to conform to plant translational concensus sequences such as according to Kozak
The 'delta S13 promoter ' is a promoter region useful for obtaining preferential expression in female flower parts This comprises a region -339 to -79 from the SLG13
promoter region fused to the -46 to +8 of the CMV 35S core promoter (Dzelkalns et al (1993) Plant Cell, 5, 833-863) This S13 promoter region is cloned into bluescnpt sk which plasmid is then further restricted and ligated with restriction fragments comprising the nos 3' transcriptional terminator region and one or other of the amino acid oxidase coding sequences so as to create a 'delta S13-D-amino acid oxidase-Nos terminator' expression cassette within a bluescnpt sk plasmid This is then suitably restricted out as, for example, an EcoRl fragment and, as such hgated back into a suitable site in a vector such as pBIN19 (Bevan (1984) Nucleic Acids Res ) or pCIB200 or pCIB2001 (WO 98/39462) for use for transformation using Agrobactenum As described in WO 98/39462 pCTB200 contains the following unique polyhnker restriction sites EcoRl, Sstl, Kpnl, Bgin, Xbal and Sail PCEB2001 contains an insertion in the polyhnker which adds further unique restriction sites including Mlul, Bell, AvrE, Apal, Hpal and StuI PCEB200 and pCIB2001 also provides selectable marker genes for plant and bactenal selection on kanamycm, left and nght T-DNA borders, the RK2-denved trfA function for mobilization between E coli and other hosts and the onT and onV functions from RK2 Alternatively the binary vector pCIB 10 which incorporates sequences from the wide host range plasmid pRK252 is used (Rothstein et al (1987) Gene 53,153-161) or one of its denvatives which incorporates both kanamycm resistance genes and the hygromycm phosphotransferase gene such as pCIB715 is used (Gntz et al (1983) Gene 25, 179-188)
Alternatively the ~ 1 6 kb Stigl promoter region (denved from EMBL accession X77823) is used For example the coding region of the GUS gene in the stigl-GUS construct desenbed by Goldman et al (1994) in EMBO J , 13, 2976-2984, is replaced with the DNA sequence encoding either the P80324 or Q99042 coding sequences using suitable restnction enzymes and the resultant stigl-D-amino acid oxidase expression construct cloned into in a suitable vector such as pCIB200 at a position upstream of a 3' terminator sequence adjacent to a suitable marker gene and between T-DNA border sequences
In a further particular example the T-DNA insert within the binary vector is constructed according to Figure 1 A construct compnsing the synthetic DNA sequence (SEQ ID # 7) encoding Rhodotorula gracilis D-amino acid oxidase under operable control of the stigl promoter region and also the DNA sequence (A02774) encoding L-phosphmothncin N-acetyl transferase (PAT) under operable control of the pea plastocyanm promoter region is cloned into a site between the LB/ npt II gene and the RB of the T-DNA of the binary vector In bnef, sequence ID # 7 is cloned into plasmid pFse4-Stiglnos
(described in W09942598) behind the Sbgl promoter and in front of the nos terminator region (comprised within EMBL ATU237588) as an NcoI/PstI fragment The pea plastocyamn promoter region (derived from EMBL Accession number X16082) is obtained from pea genomic DNA by PCR and cloned in front of the PAT gene/nos terminator The resultant PC-PAT-nos cassette is cloned behind the Stigl-RGDAMOX-nos as a Not! fragment and this whole two gene construct is transferred to a binary vector (pVB6, a Bml9 derivative) as an Fsel fragment
In a further variant of the method the construct used is according to the schematic representation in Figure 2 The Rhodotorula D-amino acid oxidase coding sequence, SEQ ID# 7, optionally site-directed mutated to encode the M213R form, is truncated by 3 codons at the 3' terminus and, at the 5' terminus, is cloned to place it immediately downstream of a region encoding a chloroplast transit peptide so that a chloroplast transit peptide/ D-amino acid oxidase fusion protein is encoded The chloroplast transit peptide encoding sequence is derived from the Arabidopsis gene encoding the small subumt of EPSP synthase (Klee et al 1987 in Mol Gen Genet, 210,437) Optionally this is modified to include an Sphl site at the CTP processing site thereby replacing the Glu-Lys at this location with Cys-Met (SEQ in Fig 9 of WO 92044490) Correspondingly, an SPh 1 site may be engineered at the N-terminus of the D-amino acid oxidase coding sequence (converting the amino acid following the methiomne to a leu) Alternatively the chloroplast transit peptide encoding sequence is derived from the Petunia gene encoding EPSP synthase (Fig 11 of WO 92044490) Alternatively the chloroplast coding sequence is any one of a large number of possibilities including those derived from genes encoding the small subumt of Rubisco and including the so-called 'optimized' chimeric transit peptide sequence (FR 2673643) In all cases, rather than rely on subclomng, the whole desired DNA sequence encoding the chloroplast transit peptide/ Rhodotorula D-amino acid oxidase fusion polypeptide may simply be obtained synthetically This sequence is cloned into a site downstream of the sugl promoter region and upstream of an (e g nos) terminator sequence within a suitable vector (e g replacing the GUS coding sequence in the vector containing the stigl→ GUS construct described by Goldman et al (1994) in EMBO J , 13, 2976-2984) The whole gene expression construct is then cloned into a suitable site between the right and left borders of the T-DNA of a PVB6 vector
Tobacco leaf discs are transformed with the recombinant binary vectors using methods similar to those described in Horsch et al (1985) Science, 227,1229-1231 Many
vanations of the method may be used The binary vector can be transformed into, for example, Agrobactenum tumefaciens strain LBA 4404 using the freeze thaw method of transformation Tobacco transformation and whole plant regeneration is performed using Nicotiana tabacum var Samsun according to protocols described by Draper et al (Plant Genetic Transformation, Blackwell Sci Pub 1989) Transformation events are selected on MS-media containing kanamycm or other suitable antibiotic The presence of integrated transgenes is confirmed by PCR Plants are regenerated and allowed to reach maturity and selfed on to produce seed Northern and/or Western analysis is used to confirm tissue-specific expression of the D-amino acid oxidase genes The plants are self-fertile but have the condition of conditional female sterility Seeds of the Tl generation are planted out Once plantlets have grown to a sufficient size they are tested by PCR for the presence of transgene PCR positive plants are transferred to the greenhouse These plants are fully fertile in the absence of exogenously applied proherbicide A subset of these (putatively) conditionally sterile plants are treated with D-phosphmothncin or D-alanine or D leucine or D asparagme or D methionine or D-aspartate or D-glutamate in various amounts and at varying growth stages Such treatments are earned out on the Tl plants confirmed as PCR positive for the D-amino acid oxidase gene, or, equally, such treatments are earned out directly on plants of the To generation (which are vegetatively cloned so that untreated clones of each event may be set aside for seed production) The observed fertility is then used as a basis to select suitable plant lines exhibiting the clearest conditional sterility phenotype For example these amino acids are pure D enantiomers or, alternatively, are DL racemates For example, they are applied as a foliar spray, prior to or during the early stages of flower formation, at rates usually between 0 25 and 20 kg/ ha Amino acids which may crystallise out of solution on the leaves following foliar application may be redissolved and remobilised for leaf uptake by further applications of water as a spray mist Amino acids are, for example, also applied as a root drench and optionally, further applied as ~ 50 ul of a 10-200 mM solution flooded directly into the buds of emerging florets Pollen from the treated plants is collected and viability is tested Plants are obtained which produce relatively little or no seed after treatment with D phosphmothncin or D alanine or D leucine or D asparagme or D methionine or D aspartate or D glutamate but which, nevertheless, under the same treatment conditions do produce near normal levels of viable pollen Control plants are both transgenic and non-transgenic and are grown under identical conditions and under an
identical regime of physical treatments except that treatment solutions are either water or an equivalent concentration of pure L-amino acid
In one variant of the method, the amino acid applied is racemic DL phosphmothncin In this case, the DNA construct used for transformation comprises, in addition to the DNA sequence encoding a D-amino acid oxidase under operable expression control of a tissue specific female floral promoter region such as 'stig 1' , also a DNA sequence (EMBL A02774) a 'PAT' gene under operable control of a promoter region such as the region 5' of the translational start of the plastocyanm gene of the Pisum sativum plastocyamn gene (EMBL accession number X16082) For example, the construct is the same as depicted in Fig 1 except that the DNA sequence encoding D-amino acid oxidase is site-specifically mutated so that the M213R form of the enzyme is encoded The plastocyamn promoter region provides for preferential expression in the green tissues of the plant It is found, unexpectedly, that such a promoter which, unlike for example, the 35S promoter region, is substantially expressed only in certain tissues of the plant and most notably in green tissues, does, nevertheless, when used in combination with the PAT gene provide for substantially complete reproductive tolerance to the herbicide DL PPT even at rates in excess of 2 kg/ ha Furthermore, in the absence of any suitable heterologous D-amino acid oxidase or similar D to L converting activity being co-expressed in the floral tissues, the plastocyamn/ PAT gene combination provides essentially complete reproductive tolerance witii no significant loss of yield despite the PAT expression level being low or non-existent in many of the cntical floral tissues when expressed under control of this promoter region Thus, in this variant of the example, the non-phytotoxic substance D phosphmothncin is applied in its least costly and most readily available form as the commercial herbicide DL phosphmothncin racemate At appropnate spray timings and rates between 250 g/ ha and 5 kg/ ha of DL phosphmothncin the treated plants are not visibly damaged but are rendered conditionally female stenle whilst remaining of normal or near-normal male fertility
Example 2. Tobacco plants which are conditionally male sterile dependent upon exogenous application of D-phosphinothricm or D alanine or D leucine or D methionine or D asparagme or D-aspartate or D-glutamate
The DNA sequence encoding the D-amino acid oxidase protein sequence Q9HGY3 (Sptrembl) within the EMBL sequence AB042032 is either obtained by RT-PCR from
Candida boidini mRNA or is obtained synthetically Alternatively the DNA sequence encoding the D-ammo acid oxidase protein sequence P24552 (Swissprot) within the EMBL sequence D00809 is either obtained by RT-PCR from Fusanum solani mRNA or is obtained synthetically Hanking PCR-primer or synthetic DNA sequences are designed to place useful unique restnction sites for cloning Preferably and in the case where the oxidase coding sequence does not contain confounding internal sites, an Ncol or Ndel site is placed at the 5' end to facilitate the cloning of in-frame fusions with sequences added 5' to the ORF Alternatively, where restnction sites are placed upstream of the ATG translational start site intervening sequences are designed to conform to plant translational concensus sequences such as according to Kozak Alternatively D-ammo acid oxidase encoding sequences obtained are the same as in example 1 Again, as in the previous example, where stenlity is to be made dependent upon the application of D-aspartate, D glutamate or D phosphmothncin then, preferably, D-amino acid oxidases are vanants at amino acid positions 213 and/or 238
The TA29 promoter region (Knete et al (1996) Plant J , 9,*808-818) is cloned from tobacco genomic DNA by PCR using the pnmers 5'-
CAGACTAGTTTTAGCTAATTTCTTTAAGTAAAAAC-3, (SEQ ID # 6) Through a senes of restnction and subclonmg steps the PCR fragment so obtained is placed upstream of the D-ammo acid oxidase coding sequence and a nos transcnptional terminator is added 3' of the coding region The resultant TA29-D-ammo acid oxidase -nos terminator expression cassette is then cloned, obtained as as a suitable restriction fragment and cloned into a binary vector as in Example 1 As in example 1, where stenlity is to made conditional upon application of D-aspartate, D-glutamate or D phosphmothncm it is preferred that vanants of Rhodotorula gracilis D-ammo acid oxidase be used with mutations at positions 213 and/or 238
Alternatively, any of the above D-ammo acid oxidase coding sequence regions are cloned as a suitable restnction fragment (for example BamH1, Bgl/n where synthetic vanants of coding sequences are designed so as to remove internal restnction sites) and fused to the CaMv 35S promoter and the nopahne synthase terminator regions by insertion into (for example) the BamHl site of the binary vector pROK1 (Baulcombe et al (1986) Nature, 321, 446-449) in a sense configuration The EcoRl-BamH1 fragment carrying the 35S promoter region is then excised and replaced with an EcoRl-BamHl fragment from pAP30 (Knete et
al (1996) The Plant Journal 9, 809-818) carrying the TA29s promoter region fragment (-810 to + 54) The resultant vectors can be termed pGKTA29_ Q99042, pGKTA29_ P80324, pGKTA29_ Q9HGY3 and pGKTA29_ P24552 etc according to the protein sequence encoded
Tobacco plant material is transformed, via Agrobactenum, with vector and transgenic plants are regenerated in a similar manner to that described in the previous example The plants produced are self-fertile but are conditionally male stenle Seeds of the Tl generation are planted out into soil Once plantlets have grown to a sufficient size they are tested by PCR for the presence of transgene PCR positive plants are transferred to the greenhouse These plants are fully fertile in the absence of exogenously applied proherbicide A subset of these putatively conditionally stenle Tl plants, or, alternatively plantlets of TO 'events' (direct regenerants from transformation) are treated with D-phosphmothncin or D alanine or D leucine or D methionine or D asparagme or D-aspartate or D-glutamate in vanous amounts and at varying growth stages Where To plants are treated they are vegetatively cloned so that untreated siblings of the events are set aside for seed production The observed fertility is then used as a basis to select suitable plant lines exhibiting the clearest conditional stenlity phenotype For example these amino acids are pure D enantiomers or, alternatively, are DL racemates For example, they are applied as a foliar spray, pnor to or dunng the early stages of flower formation, at rates usually between 0 25 and 20 kg/ ha Ammo acids which may crystallise out of solution on the leaves following foliar application may be redissolved and remobihsed for leaf uptake by further applications of water as a spray mist Ammo acids are, for example, also applied as a root drench and optionally, further applied as a 10-200 mM solution directly into the buds of emerging florets
Pollen from the treated plants is collected and viability is tested Plants are obtained which shed no or relatively little pollen and/or pollen which is not viable Pollen collected from some of the treated plants is tested and found to be malformed and non-viable However, such male infertile plants remain female fertile and produce (hybnd) seed when pollinated with pollen collected from other, untreated non-transgemc or conditionally female-stenle tobacco plants Control plants are both transgenic and non-transgemc and are grown under identical conditions and under an identical regime of physical treatments except that treatment solutions are either water or an equivalent concentration of pure L-amino acid
Analagous to example 1, in one variant of the example, the amino acid applied is racemic DL phosphinothncin In this case the DNA construct used for transformation comprises, in addition to the DNA sequence encoding a D-amino acid oxidase under operable expression control of a tissue specific male floral promoter region such as 'TA 29', also a DNA sequence encoding a phosphinothncin N-acetyl transferase gene such as the 'PAT' gene under operable control of a promoter region such as that from the plastocyamn gene (m this case the region from the Pisum sativum plastocyamn gene) At appropriate spray timings and rates between 250 g/ ha and 5 kg/ ha of DL phosphinothncin the treated plants are not visibly damaged but are rendered conditionally male stenle whilst remaimng of normal or near-normal female fertility
Example 3 Chimeric genes preferentially expressed in male reproductive structures and encoding enzymes capable of hydrolysmg unazamethabenz methyl or flamprop M methyl or flamprop M isopropyl to their respective carboxyhc acids
The DNA sequence encoding the carboxylesterase protein sequence Q01470 (Swissprot) within the EMBL sequence M94965 is either obtained by PCR from genomic DNA of Arthrobacter oxydans or is obtained synthetically Alternatively the DNA sequence encoding the carboxylesterase protein sequence P37967 (Swissprot) withm the EMBL sequence BSO6089 is either obtained by PCR from Bacillus subtiks genomic DNA or is obtained synthetically Alternatively the DNA sequence encoding the carboxylesterase protein sequence P40363 (Swissprot) within the EMBL sequence Z34288 is either obtained by RT-PCR from Saccharomyces cervisiae mRNA or is obtained synthetically Flanking PCR-pnmer or synthetic DNA sequences are designed to place useful unique restnction sites for cloning Preferably and in the case where the carboxylesterase coding sequence does not contain confounding internal sites, an Ncol or Ndel site is placed at the 5' end to facilitate the cloning of m-frame fusions with sequences added 5' to the ORF Alternatively, where restnction sites are placed upstream of the ATG translational start site intervening sequences are designed to conform to plant translational concensus sequences such as according to Kozak
Plasmid pGK73 carnes the TA29s promoter region EcoRl-BamHlfragment from -810 to +54 (Knete et al (1996), 9, 809-818) This restnction fragment or a similar suitable PCR-generated fragment is cloned, preferably as an m-frame fusion, at a position upstream of the DNA sequence encoding either carboxylesterase Q01470 or P37967 into bluescnpt sk
Using a suitable series of restriction, ligation and subcloning steps a nos transcriptional terminator is added 3' of the coding region to generate, according to the coding sequence, alternative expression cassettes of the type TA29-carboxylesterase-nos in Bluescnpt sk plasmids, pBLTA_Q01470, pBLTA_P37967 and pBLTA_P40363
In a further example, the anther specific SGB6 promoter region seq ID number 1 of USP 5470359 is used For example, pSGBNEl containing a 3 kb genomic EcoRl-Nhel subcloned fragment from pSGB6gl (USP 5470359) is further subcloned to place a 1558 bp ApaH/Xbal fragment blunt cloned into bluescnpt ks at the Smal site As before, through further restriction and cloning steps this fragment is fused in frame upstream of either the P37967 , Q01470 or P40363 DNA coding sequences Again a nos terminator is added 3' of the coding region to create, alternative, Bluescnpt sk plasmids, pBLB6_Q01470, pBLB6_P37967 and pBLB6_P40363 compnsing the alternative SGB6-carboxyIesterase-nos expression cassettes
In a similar set of examples the RA8 anther-specific promoter region from nee (EMBIV genbank accession AF042275, Jean Js et al (1999) PMB, 39,35-44) is similarly also fused at a site m-frame and upstream of one or other of the DNA sequences encoding carboxylesterase and a nos 3' terminator to compnse alternative RA8-carboxylesterase-nos expression cassettes in a senes of bluescnpt sk vectors, pBLRA8_Q01470, pBLRA8_P37967 andpBLRA8_P40363
Example 4 Chimeric genes preferentially expressed in female reproductive structures and encoding enzymes capable of oxidising D phosphinothricin and/or D alanine and/or D leucine and/or D methionine and/or D asparagine and/or D-aspartate and/or D-glutamate
DNA sequences encoding D-amino acid oxidase protein sequences are obtained as desenbed in Examples 1 and 2
The genomic clone pSH64 was deposited under the terms of the Budapest treaty on 27/02/1998 with NRRL and assigned the number NRRL B-21920 It was detected as a genomic clone hybridising to the silk-specific cDNA clone B200i4-2 (WO 98/39462) Chimenc genes which are expressed preferentially in female reproductive structures are constructed as follows A bluescnpt ks-denved plasmid similar to pSH70 having an 'empty' expression cassette compnsing, from 5' to 3', the B200i 5' promoter region consisting of nucleotides 1-3790 of SEQ JD No 11 of WO 98/39462, a BamH1 site and the B2001 3'
untranslated terminator region compnsing nucleotides 4427-6397 of sequence ID No 11 of WO 98/39462 is constructed as described in WO 98/39462 Using a partial BamH1 digestion or, alternatively by further subcloning, PCR and ligation steps alternative D-amino acid oxidase coding sequences are Iigated into the position at or adjacent to the BamH1 site such that they are immediately 3' of the B200i promoter region and 5' of the B2001 terminator region Accordingly, a series of bluescnpt vectors pBLB200_ Q99042, pBLB200_ P80324, pBLB200_ Q9HGY3 and pBLB200_ P24552 encoding the alternative B200i-D-amino acid oxidase-B200i expression cassettes are created
Alternatively, as described in WO 98/39462, a Pst V Nco I fragment of the 5' promoter region of the P19 gene is excised from the genomic clone X2-1 which was deposited under the terms of the Budapest treaty on 27/02/1998 at NRRL and assigned accession number B-21919 The Nco I site at nucleotide 1088 of SEQ ID No 14 of WO 98/39462 corresponds with the ATG translational start of the P19 gene Using appropriate subcloning, restriction, ligation and PCR steps this fragment is Iigated to form a m-frame fusion with one or other of the DNA sequences encoding D-amino acid oxidase and a nos terminator sequence is added 3' of the coding sequence Accordingly, a series of bluescnpt vectors pBLP19_ Q99042, pBLP19_ P80324, pBLP19_ Q9HGY3 and pBLP19_ P24552 etc encoding the alternative P19-D-amino acid oxidase-nos expression cassettes are created Alternatively, using similar standard methods, similar plasmids are obtained having the 5' promoter region (composing some or all of nucleotides 1-3987 of SEQ ID No 2 of WO 98/39462) of the P26 gene in place of the P19 promoter region The genomic P26-A4 clone, pCIB 10302 deposited under the terms of the Budapest Treaty on Jan 21 1997 with the Agricultural Research Service patent culture collection, (NRRL) accession number NRRL B-21655 is subcloned as described in WO 98/39462 Accordingly, a series of bluescnpt vectors pBLP26_ Q99042, pBLP26_ P80324, pBLP26_ Q9HGY3 and pBLP26_ P24552 encoding the alternative P19-D-amino acid oxidase-nos expression cassettes are created
Example 5 Chimeric genes preferentially expressed in male reproductive structures and encoding enzymes capable of oxidising D phosphuiothncin and/or D alanine and/or D leucine and/or D methionine and/or D asparagine and/or D-aspartate and/or D-glutamate
DNA sequences encoding D-amino acid oxidase protein sequences are obtained as described in Examples 1 and 2
Plasmid pGK73 caraes the TA29s promoter region EcoRl-BamH1fragment from -810 to +54 (Knete et al (1996), 9, 809-818) This restriction fragment or a similar suitable PCR-generated fragment is cloned, preferably as an m-frame fusion, at a position upstream of the DNA sequence encoding the D amino acid oxidase into bluescnpt sk Using a suitable series of restriction, ligation and subclomng steps a nos transcriptional terminator is added 3' of the coding region to generate, according to the coding sequence, alternative expression cassettes of the type TA29-D-amino acid oxidase-nos in Bluescnpt sk plasmids
In a further example, the anther specific SGB6 promoter region seq ID number 1 of USP 5470359 is used For example, pSGBNEl containing a 3 kb genomic EcoRl-Nhel subcloned fragment from pSGB6gl (USP 5470359) is further subcloned to place a 1558 bp ApaJJ/Xbal fragment blunt cloned into bluescnpt ks at the Smal site As before, through further restnction and cloning steps this fragment is fused in frame upstream of the D amino acid oxidase coding sequence Again a nos terminator is added 3' of the coding region to create, alternative, Bluescnpt sk plasmids, compnsing the alternative SGB6-D-amino acid oxidase-nos expression cassettes
In a similar set of examples the RA8 anther-specific promoter region from nee (EMBL/ genbank accession AF042275, Jean Js et al (1999) PMB, 39, 35-44) is similarly also fused at a site in-frame and upstream of one or other of the DNA sequences encoding D-amino acid oxidase and a nos 3' terminator to compnse alternative RA8-D-amino acid oxidase-nos expression cassettes in a senes of bluescnpt sk vectors
Example 6 A pair of complementary constructs useful in a method to provide (a) a female inbred parental line which is conditionally male-sterile dependent upon the application of DL phosphinothricm and (b) a complementary male inbred parental line which is conditionally female sterile dependent upon the application of DL phosphinothricm
The first DNA construct suitable for providing a female inbred parental cereal or rice plant line which is conditionally male-stenle dependent upon the application of DL phosphinothncin compnses three genes A), B) and C) A) consists of a DNA sequence encoding a PAT enzyme capable of N-acetylatmg L-phosphmothncin under operable control of the - 1kb promoter region from the barley plastocyanin gene (EMBL Z28347) and a suitable terminator region such as that from the nos or 35S gene, B) consists of a PAT encoding sequence similar to the first but this time under operable control of a tissue specific
female floral promoter region (such as P19 or P26 as described in example 4) plus a suitable terminator and C) consists of a suitable DAMOX encoding sequence as described in examples 1,2, 12 and 13, encoding, for example, a mutant form of the Rhodotorula gracilis D amino acid oxidase having an arginine, serine, cysteine, lysine, asparagine or alanine replacing the methionine at position 213 and/ or a histidine, serine, cysteine, asparagine or alanine replacing the tyrosine at position 238 under operable control of a tissue specific male floral promoter region (such as SGB6 or RA8 as described in example 5) and a suitable terminator region This construct is assembled using methods which are standard in the art and informed by the previous examples
The second DNA construct suitable for providing a male inbred parental cereal or nee plant line which is conditionally female-sterile dependent upon the application of DL phosphinothricin comprises three genes A), D) and F) A) consists of a DNA sequence encoding a PAT enzyme capable of N-acetylatmg L-phosphmothncin under operable control of the promoter region from the barley plastocyanin gene and a suitable terminator region such as that from the nos or 35S gene, D) consists of a PAT sequence similar to the first but this time under operable control of the same tissue specific male floral promoter region (such as SGB6 or RA8 as used in example 5) as used in construct 1 plus a suitable terminator and F) consists of a suitable DAMOX gene as described in examples 1, 2, 12 and 13 and, for example, encoding a mutant form of the Rhodotorula gracilis D amino acid oxidase having an arginine, serine, cysteine, lysine, asparagine or alanine replacing the methionine at position 213 and/ or a histidine, serine, cysteine, asparagine or alanine replacing the tyrosine at position 238 under operable control of the same tissue specific female floral promoter region (such as P19 or P26 as described in example 4) as used in construct 1 and a suitable terminator region This construct is assembled using methods which are standard in the art and informed by the previous examples
A pair of DNA constructs of this example contain, for example, the following elements Construct 1
A = Barley plastocyanin promoter region → PAT encoding sequence, Nos terminator, B = P19 promoter region → PAT encoding sequence, 35S terminator, C = RA8 promoter region → Rhodotorula D-amino acid oxidase (M213R mutant) encoding sequence, Nos terminator Construct 2
A = Barley plastocyamn promoter region → PAT encoding sequence, Nos terminator, D = RA8 promoter region → PAT encoding sequence, 35S terminator , E = P19 promoter region → Rhodotorula D-armno acid oxidase (M213R mutant) encoding sequence, Nos terminator
Example 7 Polynucleotide vectors for transformation of wheat
Examples 3,4, 5 and 6 describe the construction of various chimeric genes in expression cassettes which are usually cloned into bluescnpt sk (for example, pBLRA8_Q01470, pBLRA8_P37967, pBLRA8_P40363, pBLB200_ Q99042, pBLB200_ P80324, pBLB200_ Q9HGY3 and pBLB200_ P24552 etc ) Optionally these vectors are prepared in bulk for direct DNA transformation for use with a co-bombarded selectable marker such as pSOG35 (DHFR/methotrexate) or pUb1-Hyg (hygromycin phosphotransferase/ hygromycin) as described in WO 98/39462 Preferably, after bulk preparation, the vectors are linearised using a suitable restriction enzyme to remove the ampicillm resistance gene of bluescnpt
Optionally, rather than use co-bombardment the said bluescnpt vectors are further engineered by standard methods so that they further compnse a plant selectable marker gene such as kanamycin resistance , hygromycin resistance, methotrexate resistance or glyphosate resistance gene and are used directly In some of the foregoing examples a PAT gene is integral to the design of the vector and, in these cases, DL phosphimthncin may optionally be used for selection at some stage after transformation
Alternatively, expression cassettes are excised within a suitable restriction fragment and cloned into pIGPD9 denved vectors (descnbed in Figure 12 of WO 00/66748) The use of this vector for transformation avoids transfer of antibiotic marker genes to the plant since its maintenance in bactena relies on complementation of an auxotrophic his B E coli mutant The vector compnses a gene expressing IGPD (the hisB product) and is further engineered to compnse a plant selectable marker gene such as an EPSPS gene cloned into the Xma I site as, for example, in pZEN161 and pZEN181 of WO 00/66748 Alternatively a marker gene which provides positive selection on mannose or xylose is used (USP 5767378)
In particular examples of using pIGPD9 vectors, plasmids for wheat transformation are constructed Illustrative examples are pZENl 8_ BLB200_ Q99042 and pZENl 8_ BLRA8_Q01470 These are pIGPD9-denved vectors compnsing the pZEN18 EPSPS gene
(WO 00/66748) and, in this case either the B200i-(Q99042)D-amino acid oxidase-B200i or the RA8-(Q01470)carboxylesterase-nos expression cassettes, respectively
Large-scale DNA preparations for use in plant transformation are obtained using the Max1-prep procedure (Qiagen) using protocols supplied by the manufacturer
Example 8. Transformation/ regeneration of wheat with polynucleotides comprising chimeric genes preferentially expressed in either male or female reproductive structures and which encode enzymes capable of oxidising D-phosphinothricm and/or D alanine and/or D leucine and/or D methionine and/or D asparagme and/or D-aspartate and/or D-glutamate
In one example, immature embryos (0 75 -1 0 mm in length) of genotype UC703 are plated on MS medium contaimng 3 mg/1 2,4-D and 3% sucrose After approximately 4h the embryos are plated onto MS medium contaimng 15% maltose, 3% sucrose and 3 mg/12,4-D overlaid with a filter paper supported slab of agarose contaimng the same components The embryos are allowed to plasmolyze for 2-3h before bombardment
DNA prepared as described in example 7 and in the foregoing examples is precipitated onto micrometer size gold particles using standard procedures Four target plates with 16 embryos per target are shot twice with a DuPont Biolistics helium device using a burst pressure of 1100 psi Plates are shot with an 80 mesh screen in place between the carner stage and the target After bombardment targets are placed in the dark at 25 C for 24h before the slabs with the embryos are laid onto plates of MS medium contaimng 3% sucrose and 3 mg/1 2,4-D The individual embryos are removed from the slabs and placed directly on fresh medium of the same composition after another 48h Approximately 6 weeks after gene delivery the tissue is placed on MS medium with 3 mg/1 2,4-D, 3% sucrose and 0 2 mg/1 of methotrexate for a 3 week period The tissue is then placed on regeneration medium comprised of MS medium contaimng 1 mg/1 zeatm nboside and 1 mg/1 methotrexate After 2 weeks regenerating plantlets are placed in sterile containers with half-strength MS medium containing 2% sucrose, 1 mg/1 napthylacetic acid and 4 mg/1 methotrexate
In particular variants of the example the vectors comprising chimeric genes preferentially expressed in male reproductive structures are co-bombarded with alternative selectable marker genes Thus, for example, DNA of plasmids such as pBLRA8_P24552, made analogously to Example 3 (but expressing a D-armno acid oxidase rather than a
carboxylesterase sequence) under operable control of the RA8 promoter region is prepared and coated onto gold particles along with pUbiHyg (a plasmid encoding hygromycin phosphotransferase under operable control of the maize polyubiquitm promoter) In this case transformation and regeneration is earned out as described above except that, following bombardment, the regeneration media contain increasing concentrations of hygromycm between 2 and 20 mg/1
In a further example wheat is transformed with pZEN18_ BLB200_ Q99042, selected using glyphosate and regenerated as described in example 15 of WO 00/66748
DNA is extracted from leaf tissues of plants derived from transformation and PCR is run for the presence of selectable marker gene and the gene encoding D amino acid oxidase PCR positive plants are propagated During flowering, pistils and anthers are collected and RNA is prepared DNA expression is confirmed by Northern analysis In addition, D-amino acid oxidase genes are expressed using pET vectors in E coll and part purified The protein bands of the expressed protein is cut out of an SDS gel and used to generate polyclonal antibodies These antibodies are used to detect expression in flower tissues and other tissues by Western analysis
Example 9 A method of efficiently producing hybrid cereal crops wherein DL phosphinothricin is applied both for weed control and at the same time as the chemical hybridising agent and wherein the Fl hybrid generation of plants resulting from the so-produced hybrid seed is both vegetatively and reproductrvely substantially tolerant to the application of DL phosphinothricin
Chemical hybndismg agents are expensive It would be desirable to use a relatively cheap substance such as a commercial herbicide as a chemical hybndismg agent This would also achieve further efficiency since weed control could be combined with chemical hybndisation However there are a number of problems to overcome in order that this proposition be realised Firstly, male and female parental lines would need to be established which are tolerant to the herbicide in question Furthermore, in order to achieve the desired 'conditional' fertility in response to application of the herbicide the two lines would need to be engineered in such a way that the tolerance to the herbicide did not extend to all tissues but was expressed in a tissue specific manner so that each one of the required floral tissues remained selectively susceptible Thus, in one line (the female parent line), the bulk of the plant plus the female tissue must be rendered tolerant whilst some critical part of the male
floral tissue must remain susceptible to the application whereas in the other (the male parent line), the converse is needed with only some cntical part of the female gamete forming tissue remaining susceptible Even given that this can be achieved there remains a further problem to overcome in respect of the hybnd seed and Fl generation Given that this generation of the crop would, necessarily, contain at least two genes capable of conferring resistance to the herbicide it would be desirable that this same herbicide could also be used for weed control in the crop However, it is very difficult to conceive of a combination of herbicides, tissue specific promoter regions and tolerance genes that would permit this use of the same herbicide in the Fl generation It would be likely that the hybnd crop would display vegetative tolerance but little or no gram yield after herbicide application to the Fl generation For example, for the herbicide glyphosate the usual mechanism of resistance is the expression of a resistant form of EPSP synthase It is difficult to identify a promoter region or combination of promoter regions that would permit sufficient expression of a R-EPSPS in all tissues and at all times other than, say, at a cntical stage in the development of stamens or stigmas The most straightforward way around this would be to use an antisense or similar approach wherein expression of the R-EPSPS is dnven by a tissue nonspecific/constitutive promoter and only locally and transiently suppressed in, for example, the stamens due to expression of an antisense EPSPS gene (see for example WO 9946396) However, in that case the suppression of expression in the stamen (or stigma) would be dnven by a dominant gene It is clear that, for any such mechanism, the application of the herbicide to the Fl generation would result in a stenle non-yielding crop due to the additive effects of the dominant male and female conditional stenlity genes
The current invention provides a method of overcoming the problem of enabling the use of a cheap commercial herbicide, DL phosphmothncin, as both weed control and hybndismg agent in the production of hybnd cereals and which method, furthermore, provides resultant hybnd cereals or nce in which DL phosphinothncin (or L phosphmothncin) can be safely used for weed control without substantial loss of yield As a yet further benefit, selfed seed from the Fl generation which may later anse as volunteers in subsequent crops will be easier to manage since they, themselves, will generally be stenle if sprayed with controlling amounts of DL phosphinothncin The same will hold for the progeny of pollen outcrossing from the Fl plants to weeds (e g red nce) or other cereals The current invention provides genes and enzymes that convert a non-phytotoxic component, D-phosphmothncin, of a commercial herbicide formulation DL phosphmothncin, into the
active L form The PAT gene which converts L -phosphmothncin to N-acetyl L-phosphinithncin is known already and is used commercially to provide tolerance to DL phosphmothncin in crops A further cntical observation germane to the current example is that, surpnsingly, wheat containing a PAT gene under operable expression control of the barley plastocyamn promoter region is found to be substantially reproductively tolerant to the application of DL phosphinothncin at rates up of at least 2kg/ ha Thus a cntical feature of the constructs descnbed in example 6 which are used to provide the plants of the current example is that the PAT gene which provides the resistance trait is expressed under operable control of a promoter region which provides for expression in substantially only the green tissues A charactenstic of such a useful promoter region is that it should express PAT in such a way that it protects adequately all the non-green floral tissues from foharly applied DL phosphmothncin whilst, at the same time, providing only a minimal level of PAT expression in the floral tissue itself and especially low in those parts targeted for conditional stenlity With PAT expressed under operable control of the barley plastocyamn promoter region this condition appears to be met since substantially all of the L-phosphinothncin which is sprayed enters via the leaves is intercepted and converted to non-phytotoxic N-acetyl-L-phosphmothncin before it translocated to developing floral tissues Thus, in the current invention, the L phosphmothncin which causes the tissue selective stenlity effects in the parental lines is only generated transiently and locally from phloem mobile non-phytotoxic D-phosphmothncin via D amino acid oxidase By exactly matching the floral control elements driving expression of PAT to those elements which dnve expression of D-amino acid oxidase in the complementary pair of constructs (example 6) it is ensured that, in the Fl hybnd, the transient burst of L-phosphmothncin in the target floral tissue is rapidly neutralised by a corresponding burst of PAT expression at the same time and in the same local tissue Thus application of the herbicide induces no stenlity effect in the hybnd However, in further generations, the florally corresponding PAT and D-amino acid oxidase of the hybnd will segregate apart and thus, once again, the resulting plants will be male or female stenle upon application of controlling amounts of DL phosphmothncin
Using the methods descnbed in examples 7 and 8, the constructs descnbed in example 6 are transformed into wheat or (using standard superbmary vector methods) into nee which is selected and regenerated into plantlets TO transformant events are selected (using clonal propagation of tillers to maintain untreated lines) and suitable events for breeding on as, alternatively, male inbred parental lines which are conditionally female
stenle dependent upon the application of DL phosphinothncin or female mbred lines which are conditionally male sterile dependent upon the application of DL phosphinothncin are selected using methods essentially as descnbed in examples 1 and 2 The best lines exhibit the best herbicide tolerance, minimum yield loss, cleanest conditional stenhty phenotype etc The alternative male parent and female parent lines are selected and, optionally, backcrossed into suitable elite lines for a number of generations The genetic inserts in these finally selected events are fully charactenzed as are the genetics of the inhentance of the conditional fertility and herbicide resistance traits and the charactenstics of expressed gene products
The, thus selected, female and male parental lines are then mterplanted together in suitable ratios in a field and sprayed with DL phosphinothncin at a suitable rate between 0 05 and 5 kg/ ha and timing up to the penod of early flowenng selected to optimise the production of hybnd seed The seed thus produced have the advantage that they will give nse to plants which not only benefit from hybnd vigour but which are also tolerant to the herbicide formulations containing DL phosphimthncin which may thus be used for weed control The hybnd seed also have the advantage that the herbicide tolerance trait that they express will be only incompletely passed onto future selfed generations or outcrossed into related weeds Thus, for example, the hybnd nee resulting from this invention can be grown using DL phosphinothncin as weed control agent without significant loss of yield However future generations of red nee plants which anse as the progeny of pollen from the hybnd nee outcrossing with red nee female parents will be vegetatively tolerant to treatment with DL phosphinothncin but have reduced self-fertility (owing to the expression of a D-amino acid oxidase in the floral tissue) and thus produce little gram Hence using hybnd nee of the current invention DLphosphmothncin may be used for weed control with much reduced future nsk of gram contamination with red nee as a result of the herbicide resistance trait having outcrossed into the closely related red nee Similarly, second generation volunteers of nee or wheat which anse from the hybnd crop will, for the most part, not produce gram after spraying with DL phosphinothncin
Example 10 Transformation/ Regeneration of maize with a polynucleotide comprising a chimeric gene preferentially expressed in male reproductive tissue and which encodes an enzyme capable of hydrolysing vmazamethabenz methyl or flamprop methyl or flamprop isopropyl to their respective acids
RA8-carboxylesterase-nos expression cassettes are cloned into a series of bluescnpt sk vectors, pBLRA8_Q01470, pBLRA8_P37967 and pBLRA8_P40363 as descnbed above Optionally, these are combombarded with DNA comprising selection markers such as pUbiHyg or pSOG35, selected and regenerated using hygromycm or methotrexate as descnbed, for example, in example 11 of WO 98/ 39462
Alternatively, pZEN18_ BLRA8_Q01470 is directly bombarded or transferred on silicon carbide whiskers into maize cells and maize plants are selected and regenerated on glyphosate as, for example, descnbed in examples 12 and 13 of WO 00/ 66748
Alternatively maize transformation is earned out usmg Agrobactenum tumefaciens containing a superbmary vector For example, the pZEN18 expression cassette and the BLRA8_Q01470 chimenc gene is excised from, pZEN18_ BLRA8_Q01470 and cloned into positions between the nght and left T-DNA borders of a pSBl-denved superbmary vector through a senes of subclonmg and homologous recombination in a senes of steps similar to those descnbed in WO 00/ 66748 Plant matenal denved from immature embryos is infected with Agrobactenum containing superbmary vector compnsing the glyphosate marker gene and the chimenc gene of the current invention Plants are selected and regenerated using glyphosate as descnbed in WO 00/ 66748
DNA is extracted from leaf tissues of plants denved from transformation and PCR is run for the presence of selectable marker gene and the gene encoding carboxylesterase PCR positive plants are propagated Dunng flowenng pistils and anthers are collected and RNA is prepared DNA expression is confirmed by Northern analysis In addition, carboxylesterase genes are expressed using pET vectors in E cob and part punfied The protein bands of the expressed protein is cut out of an SDS gel and used to generate polyclonal antibodies These antibodies are used to detect expression in flower tissues and other tissues by Western analysis
Example 11 Transformation of maize cells to a phenotype which exhibits enhanced sensitivity to growth inhibition by S-Fluazifop acid
DNA sequences encoding the 2-arylpropionyl-CoA epimerase protein sequence AAR49827 in the GENESEQP Derwent database or P70473 (Swissprot) compnsed within the DNA sequences of GENESEQN Derwent database accession AAQ44447 or EMBL accession RN2ARYLCO, respectively are obtained either by RT-PCR or synthetically to optimise expression in plant tissues Flanking PCR-pnmer or synthetic DNA sequences are
designed to place useful unique restnction sites for cloning Preferably and in the case where the epimerase coding sequence does not contain confounding internal sites, an Ncol or Ndel site is placed at the 5' end to facilitate the cloning of in-frame fusions with sequences added 5' to the ORF Alternatively, where restnction sites are placed upstream of the ATG translational start site intervening sequences are designed to conform to plant translational concensus sequences such as according to Kozak
DNA sequences encoding the 'long-chain' acyl CoA synthetases protem sequence P18163 or P39518 (Swissprot) comprised within the DNA sequences of EMBL accessions J05439 or X77783, respectively are obtained either by RT-PCR or synthetically Flanking PCR-pnmer or synthetic DNA sequences are designed to place useful unique restnction sites for cloning Preferably and in the case where the epimerase coding sequence does not contain confounding internal sites, an Ncol or Ndel site is placed at the 5' end to facilitate the cloning of in-frame fusions with sequences added 5' to the ORF Alternatively, where restriction sites are placed upstream of the ATG translational start site intervening sequences are designed to conform to plant translational concensus sequences such as according to Kozak
Similar to Examples 1 and 2, the above coding sequences are cloned initially into pUC19 or into bluescnpt sk The coding sequences are then excised with suitable restnction enzymes, preferably using an Nco 1 site at the 5' end of the coding sequence, into pMJBl to create alternative in-frame fusion expression cassettes, compnsing in a 5' to 3' direction, CaMV35S promoter, TMV translational enhancer, acyl CoA synthetase or epimerase coding sequence-nos terminator pMJBl is a pUC19-denved plasmid which contains a plant operable double-enhanced CaMV35S promoter, a TMV omega enhancer and a nos terminator sequence A schematic representation of pMJBl is depicted in Figure 2 of WO 98/20144
In this way a senes of pMJBl denvatives are created, pMJ35S_ AAR49827 etc and pMJ35S_ P18163 etc compnsing alternative epimerase and acyl CoA synthetase expression cassettes, respectively Using standard techniques these are, optionally, further cloned into vectors such as pUbiHyg which compnse plant selectable marker genes
Alternatively, two constructs, one for expression of 'long-chain' acyl CoA synthetases and the other for expression of 2-arylpropionyl-CoA epimerase are built according to the schematic designs of Fig 3 A and Fig3B In 3 A, the DNA construct compnses, in the 5' to 3' direction, a maize polyubiquitm promoter region (EMBL
ZM29159), the DNA sequence encoding acyl-CoA synthetase (EMBL J05439), a nos terminator region, a CMV 35S promoter region, a region encoding a 5' untranslated leader sequence comprising the maize ADH intron, a DNA sequence encoding phosphonothncin acetyl transferase and a nos terminator As usual, this entire DNA construct is cloned into a suitable site in a vector (e g a pUC derivative) comprising an E cob. origin of replication and an ampicillin resistance gene Construct 3B is the same except that the DNA sequence encoding acyl-CoA synthetase is replaced with a DNA sequence encoding 2-arylpropionyl-CoA epimerase (EMBL Y08172)
These vectors, singly and in combination are transformed into maize plant cell culture using whiskers For example, cell suspensions of BMS cells are transformed by contacting cells with silicon carbide whiskers coated with DNA using methods essentially as described by Frame et al (1994), Plant J, 6, 941-948 Transformed callus so generated is selected on the basis of differential growth in medium containing a range of concentrations of selecting agent which, depending on the DNA used for transformation might, for example, be glyphosate, hygromycm, L-phosphmothricin or kanamycin In the case of the constructs -depicted in Figures 3A and 3B, the selection is carned out on DL phosphinothncin or a derivative thereof Stably transformed lines are selected as callus which is propagated and continues to grow in selection agent
For example, following transformation using silicon carbide whiskers, the BMS cells are grown on MS media supplemented with 1mg/L Bialaphos After 2 weeks the cells are transferred to MS based media supplemented with 5mg/L Bialaphos, where they stay for the 6 -8 weeks Resistant calli are formed are transferred to MS media supplemented with 2mg/L Bialaphos Stably transformed calli are transferred into a liquid MS based media where they were allowed to grow for 2 weeks After this period the cells are pelleted and re-suspended into a 1 10 dilution of medium They are then distnbuted evenly into a 6 well assay plate and exposed to 2 5ppm and 10ppm of the R or S fluazifop After 4 days in the presence of either R or S fluazifop, 0 1ml of the settled volume of cells is removed from the wells, washed with fresh liquid MS media and plated onto solid MS based media The ability of the cells to actively grow and divide was scored after 7 days
The transformed lines are compared with untransformed lines in respect of sensitivity to S-fluazifop, S-fluazifop butyl or similar S-aryloxyphenoxypropionates and derivatives DNA coding sequences encoding enzymes preferable for use in the method of the invention are selected as those sequences which, when expressed in BMS cells, encode an enzyme or
combmation of enzymes transform the phenotype of the transformed maize cells from only being sensitive to growth inhibition by relatively high concentrations of S fluazifop or S-fluazifop butyl to being sensitive to much (at least 2-3 fold) lower concentrations
DNA coding sequences so selected are then used, as described in the other examples, to create wheat plant lines which are either male or female stenle dependent upon exogenous application of S-fluazifop or S-fluazifop esters
Example 12 Site-directed mutagenesis to generate genes encoding D-amino acid oxidases which oxidise D-phosphmothncin
This example concerns the production of genes which encode vanants of R gracilis D-amino oxidase having improved ability to oxidise D-phosphmothncin These genes are used in preferred embodiments of the invention, described in the other examples, where sterility is made conditional upon application of D-phosphinothncin In the current example these genes encode enzymes having a single amino acid change at position '213' and/ or at position '238' The methionine at the '213' position is identified as the M in the native protein sequence motif RCTMDSS The tyrosine at position 238 is identified as the 'Y' within the native protein sequence motif GGTYGVG There are many approaches known in the art to providing a senes of genes encoding a senes of D-amino acid oxidase vanants with amino acid changes at one or both of these positions The choice of DNA template for mutagenesis also depends upon the use Thus, for example, where the intended use of the mutant gene is for expression in plants then a synthetic DNA which encodes an R gracilis D amino acid oxidase such as SEQ ID#7 is a suitable starting point On the other hand, where the intended immediate use of the mutant gene is to use as a starting point for further rounds of mutagenesis and improvement in a yeast-based selection system (as in Example 13) then the native DNA sequence (optionally improved for expression in S cerevisiae) is more suitable
A preferred method for providing suitable vanants of R gracilis D amino acid oxidase is through the use of degenerate oligonucleotides using Strategenes Quickchange mutagenesis kit Methods used are according to the manufacturers instructions
For example (in the case that the native R gracilis DNA sequence encoding D-amino acid oxidase be the template DNA for mutagenesis) pairs of 'top' (RGMUTTOP) and 'bottom' (RGMUTBOT) degenerate oligonucleotides may suitably be of 50 -250 nucleotides in length and designed to compnse, within them, sequence regions as follows

(Sequence Removed)
In addition, these two oligonucleotides, RGMUTTOP and RGMUTBOT comprise at each end, sequences which, once the two oligonucleotides are annealed with eachother will constitute 5' and 3' ends which will exactly match the ends created when the template DNA is cut at a suitable pair of unique restriction sites (l e designed so that the annealed oligonucleotides can replace a unique restriction fragment cut out of the D-amino acid oxidase encoding template DNA)
0 5 to 1 0 ug of each oligonucleotide is transferred to a 0 5 ml Eppendorf centrifuge tube and heated at a suitable temperature (e g 94 C , depending on calculated melting points) for 5 minutes and annealed slowly by cooling to room temperature Template DNA (for example pYES6/CT yeast shuttle vector) is then cut with two restriction enzymes (according to the two unique restnction sites in the template DNA which span the region including the two codons to be replaced and that characterise the ends of the annealed DNA), gel purified, hgated with the annealed oligonucleotide, and transformed into yeast as, for example, described in example 13, so that the alternative D-amino acid oxidases created by mutagenesis are expressed in yeast Then, as described, yeast clones which yield the best growth on analogues of D phosphmothncin (such as D-homocysteic acid) or on D-phosphmothncin (when the PAT gene is co-expressed) as sole nitrogen source are selected as those containing the vanant D-amino acid oxidase encoding sequences with the desired properties Alternatively D-amino acid oxidase expression is earned out in some microorganism other than yeast and, for example, under expression control of the t7 promoter of a pET vector in an E coli lysogen In this case, following transformation, individual colonies may be picked, replica plated, grown, induced, lysed and screened for the desired substrate activity versus D phosphmothncin using methods known in the art (for example, a fluonmetnc screen for peroxide generation or a colonmetnc assay for aminonia generation) The yeast or other microbial clones thus selected are grown up, DNA is prepared and the full length D-amino acid oxidase DNA sequence cloned via proofreading PCR and cloning into pCRBlunt II using Invitrogens Zero Blunt TOPO kit The D-amino
acid oxidase encoding sequences characterising the selected clones are determined These D-amino acid oxidase coding sequences are further subcloned for expression in a pET vector (e g Novagen pET 24a) and transformed into E coli BL21 DE3 The cells are grown in a fermenter on LCM50 medium containing 100 ug/ ml kanamycm, induced with IPTG, harvested, broken and the extract part-punfied and assayed for D-amino acid oxidase activity (as detailed below) D-amino acid oxidase genes are selected which encode D-armno acid oxidase enzymes yielding acceptable stability and the highest activity (kcat/ Km) per mg of pure protein versus D-phosphmothncin at pH 7 0
Additionally a series of particular DNA sequences encoding particularly targeted D-amino acid oxidase enzymes are generated In particular, genes encoding Rhodotorula gracilis D amino acid oxidase with an arginine, serine, cysteine, lysine, asparagine or alanine replacing the methionine at position 213 and/ or a histidine, serine, cysteine, asparagine or alanine replacing the tyrosine at position 238 The methods used are the same as described above except that, rather than a mixture of oligonucleotides, individual oligonucleotide pairs are designed and used to effect each single or double amino acid change Each resulting mutant D-amino acid oxidase coding sequence is cloned for (untagged) expression behind the T7 promoter in Novagen pET 24A and transformed into E coli BL21 DE3 The cells are grown ina10 1 fermenter in LCM50 medium supplemented with 100 ug/ ml kanamycm, induced for expression with 1 mM IPTG and harvested by low-speed centnfugation
LCM50 Medium contains (in 1 litre)
KH2PO4 (3g), Na2HPO4 (6g), NaCl (0 5g), Casern hydrolysate (Oxoid) (2g), (NH4)2SO4 (10g), Yeast Extract (Difco) (lOg), Glycerol (35g) (these mgredienst are made up in solution and autoclaved) The following additional ingredients are filter sterilised as solutions and added to the media MgSO4 (2 5ml of 246 5mg/ml solution), Thiamine HC1 (1ml of 8mg/ml soln ) CaCl2 2H2O (0 2ml of 147g/l solution), *Fe SO4 7H20 / Citnc acid stock (2ml), * Trace element solution (5ml) and make up to 1 litre
*Fe SO4 7H2O / Citnc acid stock per 100ml consists of Fe SO4 7H2O (0 415mg), Citnc acid (0 202mg)
** The Trace element solution composition per lml is AlCl3 6H2O (20mg), CoCl2 6 H2O (8mg), KCo(SO4)2 12 H2O (2mg), CuCl2 H2O (2mg), H3BO3 (lmg), KI (20mg), MnSO4 H2O (0 8mg), Na2MoO4 2H2O (4mg), ZnSO4 7H2O (4rng)
Approximately 7g wet weight of cells is washed in water The cells are resuspended in an equal volume of 50 mM Mops/ KOH buffer at pH 7 0 containing 2 mM EDTA, 2 mM DTT and 0 01 mM FAD Cells are evenly suspended using a glass homogeniser and then disrupted using a one shot head in the Constant Systems (BudBrooke Rd, Warwick U K) Basic Z cell disrupter at 13500 psi The crude extract is kept cold (~ 4 °C) centnfuged at 30,000 gav for 1 h and the pellet discarded Some of the extract protein is run out on an SDS PAGE gel stained with Coomassie Blue and, through side by side comparison with similarly prepared extracts of cells containing only 'empty' pET vector it is estimated that 2-50% of the total soluble protein in the extract is D-amino acid oxidase Some of the extract protein is exchanged into 50 mM Mops/ KOH buffer at pH 7 0 containing 0 01 mM FAD This is diluted with the same buffer in a standard oxygen electrode cell (calibrated at 25°C between zero and a saturated concentration of oxygen) Optionally, the D-amino acid oxiadse is further purified using ion-exchange, phenyl sepharose, fractional aminonium sulphate precipitation and gel filtration Assays, at 25 °C, are started by addition of a 200 mM solution of the aminonium salt of DL phosphinothncin to the diluted enzyme The final reaction volume in the oxygen electrode cell is 2 ml Rates of oxygen consumption (after substraction of any drift in the bases line) are measured The M213R (arginine replacement for methionine) mutant form of R gracilis D amino acid oxidises DL phosphinothncin at a rate of - 14 nmol/ mm/ mg of protein of crude extract (the estimated purity of the D-amino acid oxidase in the extract being 35 +/-15% of the total protein) The M213S (serine replacement for methionine) mutant form of R gracilis D amino acid oxidises DL phosphinothncin at a rate of ~ 4 nmol/ mm/ mg of protein of crude extract (the estimated punty of the D-amino acid oxidase in each extract being 35 +/-15% of the total protein) In control expenments the pure L-form is not oxidised at all and, depending on concentration, the pure D form is oxidised at up to twice the rate that the DL is Under similar conditions, the native (unmutated) R gracilis D-amino acid oxidase exhibits no significant (< 0 4 nmol/mm /mg) ability to oxidise DL or D-phosphmothncin
Example 13 Mutation and selection to generate D-amino acid oxidase genes encoding enzymes with improved specificity (kcat/Km) for the oxidation of D-phosphmothncin
The native Rhodotorula gracillis D-amino acid oxidase coding sequence is cloned into Invitrogen's pYES6/CT shuttle vector as a HmdIII/Pmel fragment downstream of the GALl promoter Similarly the native Rhodotorula D-amino acid oxidase coding sequence is cloned into the pAUR123 protein expression shuttle vector (Panvera) as an Xbal fragment downstream of the ADHI constitutive promoter Construction of these vectors is performed in E coli followed by transformation into S288C Saccharomyces cerevisiae Where appropnate, the PAT gene is used to replace the blasticidin or aureobasidin antibiotic resistance genes on the pYES6/CT/pAUR123 vectors respectively and DL phosphinothncin rather than antibiotic used to maintain selection In addition, sequences encoding the M213R or M213S gene or M213S, Y238S mutant forms of Rhodotorula D-amino acid oxidase are cloned in place of the wild-type coding sequence
Further mutant variants of D-amino acid oxidase are created using various methods of mutagenesis For example, multiple variants of the the D-amino acid oxidase coding sequence are generated by Mn2+-poisoned PCR, the mixed population is cloned in front of the GALl or ADH1 promoters of the two shuttle vectors, transformed into yeast and selection made based upon the ability of the new sequence to confer upon yeast the ability to grow on D-homocysteic acid as sole nitrogen source Alternatively mutation and selection is earned out directly on the transformed yeast For example, yeast transformed with the above plasmids are grown up in a fermenter in the presence of a chemical mutagen such as EMS in a nitrogen-limited culture medium which contains 10-50 mM D-homocysteic acid or (m the case that the PAT gene is expressed) 20-100 mM DL phosphmothncin and induced for D-amino acid oxidase expression (e g grown on galactose as carbon source) After successive subcultunngs, subcultures growing on the D-homocysteic acid or phosphmothncin as sole N source are identified, plated out and the D-amino acid oxidase coding sequences subcloned, sequenced and expressed in E coli for further charactensation
In a further, preferred, method mutagenesis is carned out on the two shuttle vectors by using amplification and passage through E coli strain XLl-red This strain is deficient in three primary DNA repair pathways, mut S, mut D and mut T This results in ~ a 5000 fold increase in mutation rates dunng DNA replication The protocol used is according to Stratagene For example, 10 ng of shuttle vector is transformed into E coli strain XLl-red, cells are grown up and then plated out onto L-Broth agar containing ampicillin for 24h
From each plate > 200 transformant lots of colonies are pooled by scraping the colonies off the plate into L broth and then 1 in 100 and 1 in 1000 dilutions are grown and successively subcultured in L-broth/ ampicillin at 37°C for 1-2 weeks so that a large number of cell-divisions have ensued A similar procedure is earned out starting from a number of plates Minipreps of shuttle vector DNA are prepared from cells grown overnight and transformed back into yeast The transformed yeast are grown up and colonies containing improved D-armno acid oxidases selected as described above
Alternatively, D-amino acid oxidase expression and selection is carned out in some microorganism other than yeast and, for example, under expression control of the t7 promoter of a pET vector in an E coli lysogen In this case, the D-amino acid oxidase coding sequence (optionally mutagenised by Mn2+-poisoned PCR) is cloned into a pET vector, transformed into E coli XLlred and after passage for a number of generations, transformed back into an E coll lysogen such as E coli BL212 DE3 Individual colonies may then be picked, replica plated, grown, induced with IPTG, lysed and screened for the desired substrate activity versus D phosphmothncin using methods known in the art (for example, a fluonmetnc screen for peroxide generation or a colonmetnc assay for aminonia generation) Alternatively, mutagenesis and selection for improved D-amino acid oxidase coding sequences is earned out directly in Rhodotorula gracilis R gracilis are grown in minimal medium with D-alanme or D-glutamate as sole nitrogen source, subjected to successive rounds of mutagenesis with EMS and selection via subcultunng into media of increasing stnngency where, the sole nitrogen source is shifted from D-glutamate towards D-homocysteic acid In a vanant of this example the Rhodotorula gracilis is transformed with one of the yeast vectors descnbed above so that it expresses PAT (either when grown on galactose or constitutively) and the final stage of stringent selection is made on DL phosphinothncin or D phosphmothncin as sole nitrogen source
Optionally the media used for selection of yeast contain a low concentration of solvent (e g 0 1% DMSO)
Example 14 Production of D-phosphinothncin in an enantiomencally pure form
E coli BL21 DE3 codon plus RIL is transformed with Novagen pET 24A having the PAT coding sequence (A02774) cloned for (untagged) expression behind the T7 promoter These cells are grown to a density of ~ 40 OD600nm in a 10 litre fermentor of LCM50 medium containing kanamycin , induced with 0 2mM IPTG, harvested by low speed centnfugation
and quickly transferred into minimal media containing 9 91g of the aminonium salt of D/L phosphinothncin (PPT)
Minimal media (in 1 litre) is
Na2HPO4 (6g), KH2PO4 (3g), NaCl (lg), NH4Cl (lg) were dissolved in water and autoclaved and the following solutions were added after filter sterilisation CaCl2 (1ml of 14 7g/l),MgSO4 (lml of 246 5g/l), Thiamine HC1 (5ml of 1mg/ml) Glucose (30ml of 20% solution autoclaved separately), DMSO 0 5ml
Fermentation details are as follows A 10 litre fermenter of LCM 50 medium is inoculated with an LB broth-grown inoculum (200ml) of E coh BL21 DE3 codon plus RIL containing the PAT gene and is maintained at 30°C, 200 rpm stirring rate, pH6 5, oxygen concentration 50% air-saturated After ~ 12 h the culture grows to an OD 600nm of - 30 The culture is then induced for PAT expression by the addition of
0 2mM IPTG After 1 5 h , the culture typically grows further to an OD 600nm of ~ 40, before the cells are harvested by centnfugation and washed in 8 litres of minimal medium The cells are spun once again and resuspended to a final volume of 10 litres in the fermenter in minimal media containing 9 91g of the commercially available aminonium salt of D/L -phosphinothncin and a further 0 2mM IPTG The temperature is increased to 37°C and samples of the fermenter medium monitored by proton and phosphorous NMR in order to determine a) when the glucose levels have dropped substantially and need replenishing and b) the extent of conversion of phosphinothncin to n-acetyl phosphinothncin Over the course of ~ 12 h, ~ 500g of glucose are added to the fermenter The formation of n-acetyl phosphinothncin is observed to start after a few hours and by ~ 20 h reaches > 93 % conversion of the L-PPT (46 5%v of the D/L) to N-acetyl-L-PP The fermentation medium is harvested soon thereafter with the cells being remove by low-speed centnfugation
D-PPT is punfied from the fermentation medium using ion-exchange chromatography The fermentation medium (-9 51) is stored at 4°C It is mixed with 900ml of Dowex 50W-X8 200-400 mesh cation exchange resin (pre-prepared with HC1) in the H+ form such that the pH of the supernatant above the resin drops to - pH 3 0 The Dowex resin is allowed to settle out under gravity and the supernatant together with a 21 water nnse of the Dowex resin is
decanted off and then centnfuged to clanfy The washed Dowex is discarded (to eventually be recycled) The clanfied supernatant is then extracted via a separating funnel with ethyl acetate (1/4 of the volume of supernatant) and the aqueous fraction (-121) retained A further 2 3 I of Inform Dowex 50W-X8 resm is then added and stirred with the -121 The resm is then allowed to settle out The pH of the supernatant above the resin is ~ pH 1 6 at this stage The supernatant is decanted off and discarded and the resin washed with - 12 litres of water and, again, allowed to settle out Again, the supernatant is discarded and the resin is poured onto a sintered Buchner funnel filter and nnsed with a futher - 4 51 of water (to remove most of the residual N-acetyl-phosphinothncin) The major D-phosphmothncin-contammg fraction is eluted from the resm with 15 1 of 0 4M aminonium hydroxide, followed by a 1 41 water nnse of the resin The pH of this D-phosphonothncin-contaimng fraction is ~ 11 4 Optionally, this is reduced to - pH 10 by the addition of, for example, ~ 0 13 Moles of acetic acid and - 600ml of cation exchanger resm in the H* form If added, the resm is allowed to settle out The D-phosphmothncin (supernatant) fraction is then loaded on to a 565ml (5 x 28cm) column of Dowex 1X8 - 400 mesh anion exchange resm in the OH- form (preequihbrated with NaOH and washed with water) A 0M - 0 32M aminonium acetate gradient is applied to the column over 17 column bed volumes 55ml fractions are collected throughout The fractions are monitored by UV at 215nm and also by proton and 31P NMR This analysis indicates that highly pure phosphinothncin is eluted between fractions 39 and 78 N-acetyl phosphinothncin is eluted as unbound matenal and early in the gradient and some glutamate elutes later in fractions 79 - 90
Fractions 63 to 78 (corresponding to 6-7 6 bed volumes) constitute the bulk of highly pure phosphinothncin The phosphinothncin fractions are freeze dned and found to be pure by proton and phosphorous NMR (no other peaks visible apart from acetate, > 95% of the organic matenal is phosphinothncin), although, based upon discrepancies between calculated and observed dry weights it is found that, typically, some residue of inorganic salts (for example aminonium chlonde) remain in the phosphinothncin samples For practical purposes, when the D-phosphmothncin is used (for example to spray on plants) the inorganics can be taken to be inert and only need to be taken account to adjust calculated concentrations when D-phosphmothncin solutions are made of from weighed dry samples
It is expected that the phosphinothncin isolated according to the above method should be substantially enantiomencally pure D-phosphmothncin This is venfied according to the
fluorescent HPLC analysis method of Hon et al (2002) J Chrom B 776, 191 - 198 For example, 50ul of either commercial DL phosphmothncin (0 01-10 ug/ ml) or of sample is dissolved in 0 1M Borate buffer pH8 5 and mixed with 200ul of the same Borate buffer 50ul of 18mM FLEC ((+)-l-(9-fluorenyl) ethyl chloroformate) is then added and the mixtures further incubated for 30 nuns at 40°C Excess FLEC is removed by shaking for 3mins with 500ul of ethyl acetate 100ul of the bottom aqueous layer is removed for FJPLC analysis
An Inertsil ODS2 (15 x 4 6) 5uM partical HPLC column is equilibrated with 77% 10mM aqueous aminonium acetate (pH5 0) 23% Acetonitnle at a flow rate of 0 8ml/mm A 2ul sample is injected onto the column and run isocratically over 60mms and is monitored using fluorescence detection with excitation at 260nm and emission wavelength at 305nm It is observed that the D & L isomers of phosphmothncin are clearly separated and elute at 12 4 and 13 4 mins respectively A sample of the D phosphmothncin isolated according to the current method is run and is estimated to be at a better than 99% enantiomenc excess This is estimated on the basis of spiking with known quantities of commercial DL phosphmothncin and observing how small an increase in the nght-hand, 13 4 minute peak is detectable against the background of the apparently single, 12 4 mm peak yielded by the sample
In addition, the HPLC method is used to estimate the amount of phosphmothncin on the basis of peak integration and companson with a standard curve Additionally total amounts of phosphmothncin are estimated by integration of NMR signals
It is estimated that, in total, from the startmg ~ 9 91g of DL racemate, ~ 1 9 g (38% yield) of pure D phosphmothncin aminonium salt in an enantiomenc excess of > 99% is produced 50-70% of the dry weight of the sample compnses inorganic salts which are earned through Optionally these are removed by further steps of ion exchange and freeze drying (following exchange to volatile salts)
Example 15 Production of enantiomerically pure S-Fluazifop and S-fluazifop butyl
S-Fluazifop acid and its esteis are produced using methods, analogous to those well-known for R-Fluazifop, and as described in the literature (for example D Cartwright, in Proceedings of the Brighton Crop Protection Conference-Weeds (1989) 2, 707-716 and references therein) Similarly, methods for producing the RS racemate are well known Optionally, pure S-Fluazifop is produced via preparative chromatographic resolution from the RS racemate (for example as described by Bewick (1986) in Pesticide Sci, 17, 349-356) From an RS mixture of fluazifop butyl, the S enantiomer is isolated in an enantiomeric excess better than 97% using the HPLC method described by Bewick Alternatively, S Fluazifop is directly resolved from the RS mixture of acids by chromatography down a suitable cyclodextnn column (Journal of Chromatography (1993), 634(2), 197-204) or by using other column chromatographic methods (Biomedical Chromatography (1998), 12(6), 309-316, Journal of Chromatography, A (2001), 937(1-2), 135-138) A further method of general preparative utility for isolating enantiomencally pure S aryloxyphenoxypropionic acids and their esters is described in Chimiques Des Pays-Bas (1991) 110 (05), 185-188 In this case a carboxylesterase NP enzyme is produced and used for enantio-selective hydrolysis of racemic esters of aryloxyphenoxypropionates (the resulting acids being readily separable from the remaining ester)
In a preferred method, enantiomencally pure S- Fluazifop is produced by a direct synthetic method In the first step the intermediate 4-(5-tnfluoromethyl-oyndin-2-yloxy)-phenol is synthesised
Preparation of 4-(5-tnfluoromethyl-oyndm-2-yloxv)-phenol
To a suspension of potassium carbonate (13 81g, 99 mmol) in dry DMF (200 mis), at room temperature is added hydroquinone (10 0g, 91 mmol) and the mixture is stirred for 30 mins 2-Chloro-5-tnfluoromefhyl pyridine (16 49g, 91 mmol) is added and the mixture warmed to 90°C for 16 hours The reaction mixture is poured into water, acidified with dilute HC1 and then extracted with ethyl acetate The combined organic layers are washed with water, dned over magnesium sulphate, filtered and the solvent removed under reduced pressure Column chromatography on silica gel using 10-20% ethyl acetate/hexane as eluent yields, for example, -10 22g of 4-(5-tnfluoromethyl-oyndm-2-yloxy)-phenol in ~ 44% yield
5H (400MHz, CDCl3) 8 45, s, 1H, 7 9, dd, 1H, 7 0, m, 1H, 7 0, d, 2H, 6 8, d, 2H, 5 75, s,
1H
In a further step the intermediate (R)- 2-hydroxypropionic acid benzylester
is synthesised
Preparation of (R)- 2-hydroxvpropionic acid benzylester
To a suspension of sodium D-lactate in DMF at 0°C under nitrogen benzyl bromide is added dropwise The mixture is stirred at 0°C for 16 hours The solvent is then removed under reduced pressure, and the residue partitioned between diethyl ether and water The layers are separated, and the organic phase is washed with saturated sodium bicarbonate, brine, then dned over magnesium sulphate, filtered and the solvent removed under reduced pressure to give (R)- 2-hydroxypropionic acid benzylester as a colourless oil For example 2 81g are made in 88% yield
5H (400MHz, CDCl3) 7 4, m, 5H, 5 23, s, 2H, 4 35, q, 1H, 2 85, d, 1H, 1 45, d, 3H
In a further step the intermediate (S)- 2-[4-(5-Tnfluoromethyl-pyndin-2-yIoxy)-phenoxy]-propiomc acid benzyl ester is synthesised
Preparation of (S)- 2-[4-(5-Tnfluoromethyl-pyridin-2-yloxy)-phenoxyl-propiomc acid benzyl ester
To a solution of 4-(5-tnfluoromethyl-oyndm-2-yloxy)-phenol (2 90g, 114 mmol) and (R)- 2-hydroxypropionic acid benzyl ester (2 25g, 12 5 mmol) in dry THF (100mls) at 0°C under nitrogen is added tnphenylphosphine followed by dropwise addition of diisopropylazodicarboxylate (3 36 mis, 17 mmol) The resulting yellow mixture is stirred for lhr then left the stand for 16 hours The reaction mixture is partitioned between water and ethyl acetate and the layers separated The aqueous is further extracted with ethyl acetate and the combined organic layers dned over magnesium sulphate, filtered and the solvent removed under reduced pressure Column chromatography on silica gel using 10% ethyl acetate/hexane as eluent yielded (S)- 2-[4-(5-Tnfluoromethyl-pyndm-2-yloxy)-phenoxy]-propionic acid benzyl ester as a colourless oil In one example 3 25g are made representing 69% yield and in >99%ee (as determined by nmr)
5H (400MHz, CDCl3) 8 42, s, 1H, 7 89, dd, 1H, 7 35, m, 5H, 7 25, d, 2H, 6 95, d, 1H, 6 9, d, 2H, 5 22, s, 2H, 4 78, q, 1H, 1 65, d, 3H In a final step the S acid is made
Preparation of (S)-2-[4-[5-tnfluoromethyl-pyndin-s-yloxy) phenoxyl-propionic acid
A mixture of (S)- 2-[4-(5-Tnfluoromethyl-pyridm-2-yloxy)-phenoxy]-propionic acid benzylester (3 12g) and Pd/C (5%, 0 lg) in ethyl acetate (20mls) is stirred under a hydrogen atmosphere at 2 5 bar for 2 5hours The reaction mixture is filtered through celite, and the solvent removed under reduced pressure Column chromatography on silica gel using 25% ethyl acetate/ hexane 1% acetic acid as eluent gave (S)-2-[4-[5-tnfluoromethyl-pyndm-s-yloxy) phenoxyj-propiomc acid as a colourless oil For example, 2 41g is made in 99% yield at 99%ee +/- 0 5% (as determined by nmr)
DH (400MHz, CDC13) 8 42, s, 1H, 7 9, dd, 1H, 7 1, m, 2H, 6 9, m, 3H, 4 8, q, 1H, 1 7, d, 3H
Similar methods to those above are used to produce S-enantiomers of other aryloxyphenoxypropionate herbicides (for example, fenoxaprop, haloxyfop, fluozifop and quizalofop and their esters)
The skilled man will appreciate that, while illustrative of the invention, the above examples do not limit its scope

WE CLAIMS
1. A method of producing male or female sterile plants comprising the steps of
transforming plant material with a polynucleotide which encodes at least one enzyme which reacts with a non-phytotoxic substance to produce a phytotoxic one, and regenerating the thus transformed material into a plant, wherein the said non-phytotoxic substance is applied to the plant up to the time of male or female gamete formation and/ or maturation, so that the non-phytotoxic substance provides for the production of a phytotoxic one which selectively prevents the formation of or otherwise renders the said gametes non-functional, wherein the enzyme is expressed preferentially in either male or female reproductive structures, characterised in that (i) the non-phytotoxic substance is selected from the group consisting of ester derivatives of non-phosphonate herbicides which herbicides are directly phytotoxic to non-green tissue, D-alpha amino acids, peptide derivatives of non-protein D-alpha amino acids, S-enantiomers of aryloxyphenoxypropionates and S-enantiomers of ester derivatives of aryloxyphenoxypropionates and (ii) the enzyme is selected from the . group consisting of carboxylesterases, D-amino acid oxidases, D-amino acid dehydrogenases, D-amino acid racemases, 2-arylpropionyl-CoA epimerases, alpha-methylacyl-CoA racemases, thioesterases and acyl-CoA synthetases.
2. A method according to claim 1, wherein the said non-phytotoxic substance is applied in mixture along with at least one further substance which is selected from the group consisting of safeners, gametocides, glutathione-S-transferase inducers, cytochrome P450 inducers, herbicides, fertilizers, nematocides, synergists, insecticides, fungicides, hormones, plant-growth regulators and cytochrome P450 inhibitors.
3. A method according to claim 1 or 2, wherein the non-phytotoxic substance is applied foliarly and is a phloem mobile and metabolically stable oxidiseable substrate of the enzyme, wherein the enzyme provides the phytotoxic product, as a direct or indirect one from the non-phytotoxic substance.
4. A method according to the preceding claim, wherein the phytotoxic product is an indirect one produced in the form of peroxide and/or a super oxide anion.
5. A method according to either of claims 3 or 4, wherein the non-phytotoxic substance
is a D-alpha amino acid selected from the group consisting of D-alanine, D-valine, D-
methionine, D-serine, D-leucine and D-isoleucine and the said enzyme is a D-amino
acid oxidase or oxido reductase which oxidises the said amino acid to a 2-keto acid
with concomitant reduction of oxygen to a peroxide anion.
6. A method according to either of claims 3 or 4, wherein the non-phytotoxic substance
is D-aspartate or D-glutamate and the said enzyme is a D-amino acid oxidase or
oxido reductase which oxidises the said amino acid to a 2-keto acid with concomitant
reduction of oxygen to a peroxide anion.
7. A method according to the preceding claim wherein the enzyme is a mutant D-amino acid oxidase obtainable from Rhodotorula gracilis which oxidase comprises substitutions at positions 213 and/or 238 when compared to the wild type sequence, or is a D-aspartate oxidase.
8. A method according to the preceding claim, wherein the oxidase obtainable from Rhodotorula has at position 213 an amino acid selected from the group consisting of Arg, Lys, Ser, Cys, Asn and Ala, and/or at position 238 an amino acid selected from the group consisting of His, Ser, Cys, Asn and Ala.
9. A method according to any one of claims 3-8, wherein the enzyme is targeted to other than the peroxisome.
10. A method according to claim 1 or 2, wherein the enzyme is a carboxylesterase and the non-phytotoxic substance is either an ester of imazamethabenz or of flamprop.
11. A method according to the preceding claim, wherein the non-phytotoxic substance is imazamethabenz methyl, flamprop methyl or flamprop isopropyl, and the plant is wheat or a plant likewise substantially insensitive per se to imazamethabenz methyl, flamprop methyl or flamprop isopropyl.
12. A method according to claim 1 or 2, wherein the enzyme is a D-amino acid oxidase, a
D-amino acid dehydrogenase or a D-amino acid racemase and the non-phytotoxic
substance is either the D enantiomer of phosphinothricin or a D enantiomer of
bialaphos.
13. A method according to either of claims 1 or 2, wherein the non-phytotoxic substance
is comprised within a mixture, which contains a phytotoxic substance and wherein
the enzyme is a D-amino acid oxidase or oxido reductase which is capable of
oxidising an amino acid to a 2-keto acid with concomitant reduction of oxygen to a
peroxide anion.
14. A method according to the preceding claim wherein the enzyme is a mutant D-amino acid oxidase obtainable from Rhodotorula gracilis which oxidase comprises substitutions at positions 213 and/or 238 when compared to the wild type sequence, or is a D-aspartate oxidase. . -
15. A method according to the preceding claim, wherein the oxidase obtainable from Rhodotorula has at position 213 an amino acid selected from the group consisting of Arg, Lys, Ser, Cys, Asn and Ala, and/or at position 238 an amino acid selected from the group consisting of His, Ser, Cys, Asn and Ala.
16. A method according to any one of claims 13-15, wherein the mixture comprises both D and L phosphinothricin and the plant material expresses a PAT gene substantially only in green tissues and in floral tissue which produce gametes being other than those that are rendered non-functional.
17. A method according to claim 1 or 2, wherein the enzyme is selected from the group consisting of 2-arylpropionyl-CoA epimerases, alpha-methylacyl-CoA racemases, thioesterases and acyl-CoA synthetases and where the non-phytotoxic substance is an S enantiomer of an aryloxyphenoxypropionate or an S enantiomer of an aryloxyphenoxypropionate ester.
18. A method according to the preceding claim, wherein the said S enantiomers of aryloxyphenoxypropionates are selected from the group consisting of S-Fluazifop, S-Quizalofop, S-Propaquizafop, S-Haloxyfop, S-Fenoxaprop, S-Diclofop, S-Cyhalofop, S-Clodinafop.
19. A method of producing enantiomerically pure D-Phosphinothricin (D-PPT), comprising the steps of:
(a) Providing cells which contain an enzyme capable of selectively N-
acylating PPT;
(b) Growing said cells in a medium which contains D-L PPT to produce
conditioned medium;
(c) Separating the cells from the conditioned medium of (b);
(d) Optionally extracting the conditioned medium with a non-aqueous, non miscible solvent, at various pHs, so that the PPT containing fraction is separated from the fraction that contains molecules more water soluble than is PPT;
(e) Optionally admixing with the conditioned or PPT-containing extracted media of step (d) a cation exchange resin in its protonated form, in an amount, and at pH, sufficient to absorb a substantial proportion of the cations - other than PTT, from the medium;
(f) Admixing with the conditioned medium, extracted medium or medium to result from step (e) a cation exchange resin in its protonated form, in an amount, and at a pH, sufficient to bind the bulk of the PPT in the medium;
(g) Harvesting the cation ion exchange resin from step (f) to which the PPT is bound and selectively eluting PPT from it using an eluting medium having a sufficient pH and ionic strength, with the proviso that the pH of the said medium is not so low as to cause racemisation of the thus eluted PPT.
20. D-PPT having an enantiomeric excess of at least 98.5%.