Patent WO2008077570A1 - Transformation of tagetes using the selda selection marker - Google Patents

22.9.2015 Patent WO2008077570A1 Transformation of tagetes using the selda selection marker Google Patents

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Publication number WO2008077570 A1Publication type ApplicationApplication number PCT/EP2007/011217Publication date Jul 3, 2008Filing date Dec 19, 2007Priority date Dec 22, 2006

Inventors Ralf Flachmann, 7 More »

Applicant Basf Plant Science Gmbh, 8 More »

Export Citation BiBTeX, EndNote, RefMan

Patent Citations (5), Classifications (7), Legal Events (3)

External Links: Patentscope, Espacenet

CLAIMS (1)

1. C l a i m s

1. A method for generating transgenic Tagetes plants orcells comprising the steps of a) introducing into a Tagetescell or tissue a nucleic acid construct comprising a promoteractive in Tagetes cells and operably linked thereto a nucleicacid sequence encoding an enzyme capable of metabolizinga Damino acid and/or a derivative thereof; b) incubating theTagetes cell or tissue of step a) on or in a selection mediumcomprising a Damino acid and/or a derivative thereof; andoptionally c) transferring the Tagetes cell or tissue of step b)to a regeneration medium and regenerating Tagetes plantscomprising the nucleic acid construct.

2. The method of claim 1, wherein the promoter is aconstitutive promoter, an inducible promoter and/or a tissuespecific promoter.

3. The method of claims 1 or 2, wherein the nucleic acidconstruct further comprises at least one gene of interest.

4. The method of claim 3, wherein the at least one gene ofinterest codes for a protein which is involved in or has aneffect on the carotenoid biosynthesis pathway.

5. The method of claims 3 or 4, wherein the at least onegene of interest codes for a ketolase.

6. The method of any one of claims 1 to 5, wherein thenucleic acid construct further comprises a selection markergene with growth delaying or growth inhibiting function; andthe selection medium further comprises an antibiotic, aherbicide and/or another selective compound.

7. The method of claim 1, wherein step a) comprises thefollowing steps: providing cotyledonary segments of aTagetes seedling; cocultivating the tissue with anAgrobacterium comprising a nucleic acid constructcomprising a promoter active in the tissue and operablylinked thereto a nucleic acid sequence encoding an enzymecapable of metabolizing a Damino acid and/or a derivativethereof.

Transformation of tagetes using the seldaselection marker WO 2008077570 A1

ABSTRACT

The present invention relates to improved methods for transformation of Marigold(Tagetes) based on a Damino acid selection. The method for generatingtransgenic Tagetes plants or cells comprises the introduction into a Tagetes cellor tissue of a nucleic acid construct comprising a nucleic acid sequence whichencodes an enzyme capable of metabolizing Damino acids, the incubation ofsaid cell or tissue in a selection medium comprising a Damino acid, and thesubsequent incubation in a regeneration medium in order to regenerate transgenicTagetes plants. Preferably, the nucleic acid construct further comprises at least one gene of interest which, for example, codes for a protein which is involved in orhas an effect on the carotenoid biosynthesis pathway of Tagetes, like ketolase. The generated plants may be used for the production of carotenoids, especially ofastaxanthin.

DESCRIPTION

Transformation of Tagetes using the SELDA selection marker

FIELD OF THE INVENTION The present invention relates to improved methodsfor transformation of Marigold (Tagetes) based on a Damino acid selection.

BACKGROUND OF THE INVENTION

The plant genus Tagetes, which belongs to the family of the Asteraceae, exhibitsquite a few interesting properties, which explains its use both as a crop plant andas an ornamental. Many species of this genus produce bioactive compoundswith a nematicidal, fungicidal and insecticidal action, accumulate flavonoids andcarotenoids with antioxidant and coloring activities, are salt resistent, and servedecorative purposes owing to their wide range of flower colors and flowershapes.

The nematicidal action of the Tagetes roots is based on the synthesis ofthiophene derivatives. Thiophene derivatives are heterocyclic, sulfurcontainingcompounds which accumulate mainly in roots and hypocotyls. They arecharacterized by a 5 membered ring which contains an S atom. A sulfhydrylgroup, which originates from the amino acid cysteine, participates in theformation of the ring. Important representatives of the thiophenes which are alsoformed in Tagetes are, for example, BBT (5(but3enlynyl)2,2'bithienyl),BBTOAc (5(4acetoxylbutynyl)2,2' bithienyl), BBTOH (5(4hydroxylbutynyl)2,2'bithienyl) and [alpha]T (2,2':5',2"terthienyl).

The petals of Tagetes contain 922% of flavonoids and approx. 27% ofcarotenoids, in which [beta] carotene accounts for 0.4%, cryptoxanthin estersfor 1.5% and xanthophyll esters for 86.1% (Benk et al., Riechst, Aromen undKoerpen, 26 (1976), 216221). According to an estimate from 1975, over 2000different flavonoids exist. Some important representatives are, for example, theanthocyanins with 250 known structures, the chalcones with 60, the auroneswith 20 and the flavones with 350 known structures, all of which have coloringproperties. Flavonols with 350 and the isoflavonoids with 15 representativesimpart to the plants properties such as phagoprotection or have a toxic effect onfungi.

Carotenoids belong to the large group of the terpenoids. Most of them aretetraterpenes. Carotenoidhydrocarbons are also termed carotenes, and theiroxidized derivatives are the xanthophylls. Carotenoids are essential componentsin photosynthetically active membranes of all plants, algae and cyanobacteria.They are found in the pigment systems (light traps) of the chloroplasts andparticipate in the process of the primary light absorption and photon channeling of

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8. The method of claim 7, wherein step b) comprisestransferring the cotyledonary segments on or in a selectionmedium comprising at least one plant growth factor in aconcentration suitable to induce de novo shoot inductionfrom the tissue; at least one Damino acid and/or aderivative thereof and cultivating the tissue on the mediumuntil shoots have been induced and developed therefrom.

9. The method of claim 8, wherein step c) comprisesisolating the developed shoots and transferring the shoots toa rooting medium and cultivating the shoots on the rootingmedium until the shoots have formed roots, and furtherregenerating the so derived plantlets into mature plants,which have inserted into their genome the nucleic acidconstruct.

10. The method of any one of claims 7 to 9, wherein thecotyledonary segments are provided from seedlings derivedfrom mature seeds of a regenerable Tagetes line.

11. The method of any one of claims 1 or 10, wherein theselection medium comprises at least one Damino acidand/or a derivative thereof in a concentration of 0.05 mM to100 mM, preferably 0.1 mM to 50 mM, and more preferably0.3 mM to 5 mM.

12. The method of any one of claims 7 to 11, wherein theAgrobacterium is a disarmed Agrobacterium tumefaciens ora Agrobacterium rhizogenes bacterium.

13. The method of any one of claims 1 to 12, wherein the Damino acid is selected from the group consisting of Dalanine and Dserine.

14. The method of any one of claims 1 to 13, wherein theenzyme capable of metabolizing a Damino acid is selectedfrom the group consisting of Dserine ammonialyases, Damino acid oxidases and Dalanine transaminases.

15. The method of claim 14, wherein the Dserineammonialyase is encoded by a nucleic acid sequenceselected from the group consisting of: a) a nucleic acidsequence comprising a nucleotide sequence encoding aprotein with the amino acid sequence depicted in SEQ IDNo. 1; b) a nucleic acid sequence comprising the nucleotidesequence depicted in SEQ ID No. 2, or comprising afragment of the nucleotide sequence, wherein the fragmentis sufficient to code for a protein having the enzymaticactivity of a Dserine ammonialyase; c) a nucleic acidsequence comprising a nucleotide sequence whichhybridizes to a complementary strand of the nucleotidesequence from a) or b) under stringent conditions, orcomprising a fragment of the nucleotide sequence, whereinthe fragment is sufficient to code for a protein having theenzymatic activity of a Dserine ammonialyase; d) a nucleicacid sequence comprising a nucleotide sequence whichshows at least 60% identity to the nucleotide sequence froma), b) or c), or comprising a fragment of the nucleotidesequence, wherein the fragment is sufficient to code for aprotein having the enzymatic activity of a Dserineammonialyase.

16. The method of claim 14, wherein the Damino acidoxidase is encoded by a nucleic acid sequence selectedfrom the group consisting of: a) a nucleic acid sequencecomprising a nucleotide sequence encoding a protein withthe amino acid sequence depicted in SEQ ID No. 3; b) anucleic acid sequence comprising the nucleotide sequencedepicted in SEQ ID No. 4, or comprising a fragment of thenucleotide sequence, wherein the fragment is sufficient to

photosynthesis. Moreover, they also act as light receptors in a series of otherlightinduced processes in the plant. The yellow color of many flowers is due tocarotenoidcontaining, usually with low concentrations of chlorophyll orchlorophyllfree, chromoplasts. Plant carotenoids also act as precursors for thebiosynthesis of the plant growth regulator abscisic acid and of vitamin A, whichis important for human and animal nutrition. It is increasingly of interest as apotential anticancer agent and is employed as colorant in the cosmetics and foodindustries. Carotenoids from dried and pulverized Tagetes petals are alreadybeing used as poultry feed additive for intensifying the color of chicken egg yolk.

Thus, there is currently a great economic interest in increasing, or improving, thecarotenoid content and the carotenoid composition in plants. However, thelimited genetic variability in known Tagetes varieties greatly limits thepossibilities of improving these varieties with the aid of traditional biolologicalplant breeding methods.

Modern biotechnological research and development has provided usefultechniques for the improvement of agricultural products by plant geneticengineering. Plant genetic engineering involves the transfer of a desired gene orgenes into the inheritable germline of crop plants such that those genes can bebred into or among the elite varieties used in modern agriculture. Gene transfertechniques allow the development of new classes of elite crop varieties withimproved disease resistance, herbicide tolerance, and increased nutritional value.Various methods have been developed for transferring genes into plant tissuesincluding high velocity microprojection, microinjection, electroporation, directDNA uptake, and Agrobacteήummediated gene transformation. Although widelyused for dicotyledonous plants, DNA delivery using particle bombardment,electroporation, or Agrobαcteriummediated delivery into Tagetes has proven tobe difficult. Two methods routinely used are an Agrobαcteriumbased methodtargeting the cotyledonarynode axillary meristems (Hinchee 1988) and a methodusing particle bombardment of mature zygotic embryos (Finer 1991).

The lack of effective selective agents is one of the bottlenecks in the efficiencyof different Tagetes transformation methods. The efficiency of tissue cultureselection systems depends on many factors including tissue type, size ofexplant, chemical characteristics of the selectable agent and concentrations andtime of application. The most used method of selection is known as negativeselection, which employs selection markers that confer resistance against aphytotoxic agent (such as an herbicide or antibiotic). The negative selectionmarkers employed so far are mainly limited to neomycin 3'Ophosphotransferase

(nptlϊ), phosphinothricin acetyltransferases (PAT; also named Bialophos®

resistance; bar; de Block 1987; EP 0 333 033; US 4,975,374), 5enolpyruvylshikimate3phosphate synthase (EPSPS; conferring resistance to

Glyphosate® (N(phosphonomethyl) glycine); and hygromycin B. Alternativeselection marker systems, such as a system based on D amino acidmetabolizing enzymes (e.g., Damino acid dehydratases or oxidases), has beenrecently described on a general basis (WO 03/060133; Erikson 2004). Althoughsome of the problems linked to the transformation of Tagetes have beenovercome by the methods described in the art, there is still a significant need forimprovement, since all methods known so far have only a low to moderatetransformation and — especially regeneration efficiency. Although significantadvances have been made in the field of Agrobacteriummediated transformationmethods, a need continues to exist for improved methods to facilitate the ease,speed and efficiency of such methods for transformation of Tαgetes plants.

Although alternative selection marker systems, such as a system based on Damino acid metabolizing enzymes has been recently described on a generalbasis (WO 03/060133; Erikson 2004), this Damino acid based selection methodcannot be easiliy adopted and optimised on every plant system or the achievabletransformation efficiency is too low, thus the system is not suitable. Therefore, itcould not have been expected that this selection system provides an improvedmethod having higher overall efficiency in the process of generation of transgenicTαgetes plants. This objective is solved by the present invention by providing a(for humans) nontoxic selection method based on Damino acids to generatetransgenic Tαgetes cells or plants, e.g. for the production of chemicals, e.g.astaxanthin.

SUMMARY OF THE INVENTION



code for a protein having the enzymatic activity of a Damino acid oxidase; c) a nucleic acid sequence comprisinga nucleotide sequence which hybridizes to a complementarystrand of the nucleotide sequence from a) or b) understringent conditions, or comprising a fragment of thenucleotide sequence, wherein the fragment is sufficient tocode for a protein having the enzymatic activity of a Damino acid oxidase; d) a nucleic acid sequence comprisinga nucleotide sequence which shows at least 60% identity tothe nucleotide sequence from a), b) or c), or comprising afragment of the nucleotide sequence, wherein the fragmentis sufficient to code for a protein having the enzymaticactivity of a Damino acid oxidase.

17. A transgenic Tagetes plant or cell comprising a nucleicacid construct comprising a promoter active in Tagetes cellsand operably linked thereto a nucleic acid sequenceencoding an enzyme capable of metabolizing a Daminoacid and/or a derivative thereof; or generated in a methodaccording to any one of claims 1 to 16.

18. The transgenic Tagetes plant or cell of claim 17, whereinthe comprised nucleic acid construct further comprises atleast one gene of interest.

19. The transgenic Tagetes plant or cell of claim 18, whereinthe at least one gene of interest is a nucleic acid sequenceencoding a protein which is involved in or has an effect onthe carotenoid biosynthesis pathway.

20. The transgenic Tagetes plant or cell of claim 19, whereinthe at least one gene of interest is a nucleic acid sequenceencoding a ketolase.

21. The transgenic Tagetes plant or cell of any one of claims18 to 21, wherein the Tagetes plant or cell is selected fromthe group consisting of Tagetes (T.)patula, T. erecta, T.laxa, T. minuta, T. lucida, T. argentina cabrera, T. tenuifolia,T. lemmonii and T. bipinata.

22. Use of a transgenic Tagetes plant of any one of claims17 to 21 for the production of carotenoids.

23. Use of claim 22, wherein the carotenoids accumulate inTagetes plant material, preferably the petals, which can bedried for use in feed or human diets.

24. Use of claim 23, wherein the carotenoids are extractedfrom Tagetes plant material.

25. Use of any one of claims claims 22 to 24, wherein thecarotenoid is astaxanthin.

26. A heterologous nucleic acid construct comprising a) apromotor active in Tagetes cells selected from the groupconsisting of a constitutive promoter, an inducible promoterand a tissuespecific promoter; b) a nucleic acid sequenceencoding an enzyme capable of metabolizing a Daminoacid and being operably linked to the promotor of item a);and c) a nucleic acid sequence encoding a ketolase.

27. The heterologous nucleic acid construct of claim 26,wherein the enzyme capable of metabolizing a Damino acidis encoded by a nucleic acid sequence selected from thegroup consisting of: a) a nucleic acid sequence comprising anucleotide sequence encoding a protein with the amino acidsequence depicted in

SEQ ID No. 1; b) a nucleic acid sequence comprising thenucleotide sequence depicted in SEQ ID No. 2, orcomprising a fragment of the nucleotide sequence, whereinthe fragment is sufficient to code for a protein having the

A first embodiment of the invention relates to the method for generatingtransgenic Tαgetes plants or cells comprising the steps of a) introducing into aTαgetes cell or tissue a nucleic acid construct comprising a promoter active inTαgetes cells and operably linked thereto a nucleic acid sequence encoding anenzyme capable of metabolizing a Damino acid and/or a derivative thereof; b)incubating the Tagetes cell or tissue of step a) on or in a selection mediumcomprising a Damino acid and/or a derivative thereof; and optionally c)transferring said Tagetes cell or tissue of step b) to a regeneration medium andregenerating Tagetes plants comprising the nucleic acid construct.

Various promoters are known to be functional in Tagetes and are suitable to carryout the method of the invention. A promoter active in Tagetes plant can be aconstitutive promoter, an inducible promoter and/or a tissuespecific promoter.

In a preferred embodiment, the nucleic acid construct further comprises at leastone gene of interest. More preferably, the at least one gene of interest encodes aprotein which is involved in or has an effect on the carotenoid biosynthesispathway. Most preferably, the one gene of interest encodes a ketolase (e.g. fromScenedesmus vacuolatus (see EP 06124146.9) or from Haematococcuspluvialis). It is also preferred that the nucleic acid construct further comprises aselection marker gene with growth delaying or growth inhibiting function.

In a preferred embodiment of the invention, the introduction of a nucleic acidconstruct into a Tagetes cell or tissue comprises: (i) providing cotyledonarysegments of a Tagetes seedling; and (ii) cocultivating said cotyledonarysegments with an Agrobacterium comprising a nucleic acid construct comprisinga promoter active in said tissue and operably linked thereto a nucleic acidsequence encoding an enzyme capable of metabolizing a Damino acid and/or aderivative thereof. Preferably, the cotyledons are provided from seedlings derivedfrom mature seeds of a regenerable Tagetes line.

Preferably, the selection medium comprises at least one plant growth factor in aconcentration suitable to induce de novo shoot induction from said tissue and atleast one Damino acid and/or a derivative thereof. In another preferredembodiment, the selection medium further comprises an antibiotic, a herbicideand/or another selective compound. Preferably, the selection medium comprisesa Damino acid and/or a derivative thereof in a concentration of 0.05 mM to 100mM, more preferably 0.1 mM to 50 mM, and most preferably 0.3 mM to 5 mM.

Preferably, incubating the Tagetes cell or tissue on or in a selection mediumcomprises transferring the cocultivated cotyledonary segments on or in aselection medium comprising at least one plant growth factor, more preferably atleast one plant growth factor in a concentration suitable to induce de novo shootinduction from said tissue, and a Damino acid and/or a derivative thereof; andand cultivating said tissue until shoots have been induced and developedtherefrom.

In an optional embodiment of the invention, the Tagetes cell or tissue aretransferred to a regenerating medium which comprises isolating the developedshoots and transferring said shoots to a rooting medium and cultivating saidshoots on said rooting medium until said shoots have formed roots, and furtherregenerating the so derived plantlets into mature plants, which have inserted intotheir genome the nucleic acid construct.

In one preferred embodiment introduction of the nucleic acid construct ismediated by Agrobacterium mediated transformation. Preferably, theAgrobacterium is a disarmed Agrobacterium tumefaciens or Agrobacteriumrhizogenes bacterium. Preferably, the the Damino acid is Dalanine, Dserineand/or a derivative thereof.

There are various ways to conduct the selection scheme based on Daminoacids or related compounds hereunder.

Various enzymes are known to the person skilled in the art, which can be usedas D amino acid metabolizing enzymes. Preferably, the the enzyme capable ofmetabolizing a Damino acid is selected from the group consisting of Dserineammonialyases, Damino acid oxidases and Dalanine transaminases.

In a preferred embodiment, the Dserine ammonialyase is encoded by a nucleicacid sequence selected from the group consisting of: a) a nucleic acid sequencecomprising a nucleotide sequence encoding a protein with the amino acid



enzymatic activity of a Dserine ammonialyase; c) a nucleicacid sequence comprising a nucleotide sequence whichhybridizes to a complementary strand of the nucleotidesequence from a) or b) under stringent conditions, orcomprising a fragment of the nucleotide sequence, whereinthe fragment is sufficient to code for a protein having theenzymatic activity of a Dserine ammonialyase; d) a nucleicacid sequence comprising a nucleotide sequence whichshows at least 60% identity to the nucleotide sequence froma), b) or c), or comprising a fragment of the nucleotidesequence, wherein the fragment is sufficient to code for aprotein having the enzymatic activity of a Dserineammonialyase. e) a nucleic acid sequence comprising anucleotide sequence encoding a protein with the amino acidsequence depicted in SEQ ID No. 3; f) a nucleic acidsequence comprising the nucleotide sequence depicted inSEQ ID No. 4, or comprising a fragment of the nucleotidesequence, wherein the fragment is sufficient to code for aprotein having the enzymatic activity of a Damino acidoxidase; g) a nucleic acid sequence comprising a nucleotidesequence which hybridizes to a complementary strand ofthe nucleotide sequence from e) or f) under stringentconditions, or comprising a fragment of the nucleotidesequence, wherein the fragment is sufficient to code for aprotein having the enzymatic activity of a Damino acidoxidase; h) a nucleic acid sequence comprising a nucleotidesequence which shows at least 60% identity to thenucleotide sequence from e), f) or g), or comprising afragment of the nucleotide sequence, wherein the fragmentis sufficient to code for a protein having the enzymaticactivity of a Damino acid oxidase.

28. A method for producing carotenoids comprising a)generating transgenic Tagetes plants according to any oneof claims 1 to 16; and b) extracting the carotenoids from theregenerated

Tagetes plants, preferably from the Tagetes flower petals.

sequence depicted in SEQ ID No. 1 ; b) a nucleic acid sequence comprising thenucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of saidnucleotide sequence, wherein said fragment is sufficient to code for a proteinhaving the enzymatic activity of a Dserine ammonialyase; c) a nucleic acidsequence comprising a nucleotide sequence which hybridizes to acomplementary strand of the nucleotide sequence from a) or b) under stringentconditions, or comprising a fragment of said nucleotide sequence, wherein saidfragment is sufficient to code for a protein having the enzymatic activity of a Dserine ammonialyase; d) a nucleic acid sequence comprising a nucleotidesequence which shows at least 60% identity to the nucleotide sequence from a),b) or c), or comprising a fragment of said nucleotide sequence, wherein saidfragment is sufficient to code for a protein having the enzymatic activity of a Dserine ammonialyase.

For these enzymes selection is preferably done on a medium comprising Dserine in a concentration from about 0.05 mM to about 100 mM, more preferablyfrom about 0.1 mM to about 50 mM, and most preferably 0.3 mM to about 5 mM.

In another preferred embodiment, the Damino acid oxidase is encoded by anucleic acid sequence selected from the group consisting of: a) a nucleic acidsequence comprising a nucleotide sequence encoding a protein with the aminoacid sequence depicted in SEQ ID No. 3; b) a nucleic acid sequence comprisingthe nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment ofsaid nucleotide sequence, wherein said fragment is sufficient to code for aprotein having the enzymatic activity of a Damino acid oxidase; c) a nucleic acidsequence comprising a nucleotide sequence which hybridizes to acomplementary strand of the nucleotide sequence from a) or b) under stringentconditions, or comprising a fragment of said nucleotide sequence, wherein saidfragment is sufficient to code for a protein having the enzymatic activity of a Damino acid oxidase; d) a nucleic acid sequence comprising a nucleotidesequence which shows at least 60% identity to the nucleotide sequence from a),b) or c), or comprising a fragment of said nucleotide sequence, wherein saidfragment is sufficient to code for a protein having the enzymatic activity of a Damino acid oxidase.

For these enzymes selection is preferably done on a medium comprising a Damino acid in a concentration from about 0.05 mM to about 100 mM, morepreferably from about 0.1 mM to about 50 mM, and most preferably 0.3 mM toabout 5 mM. Preferably, in this embodiment, Dserine and/or Dalanine areemployed in a concentration of about 0.05 mM to about 100 mM, more preferablyabout 0.1 mM to about 50 mM, most preferably about 0.3 mM to about 5 mM.

Also the products of said method are considered to be new and inventive over the art. Thus, another embodiment of theinvention relates to a Tagetes plant or cell comprising a nucleic acid construct comprising a promoter active in Tagetes cellsand operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a Damino acid.Preferably, said Tagetes plant or cell further comprises at least one gene of interest. More preferably, the at least one geneof interest codes for a protein which is involved in or has an effect on the carotenoid biosynthesis pathway. Most preferably,the one gene of interest codes for a ketolase (eg. from Haematococcus pluvialis). It is also preferred that the nucleic acidconstruct further comprises an antibiotic resistance gene and/or a herbicide resistance gene.

The Tagetes plant or cell is selected from the group consisting of Tagetes (T.) patula, T. erecta, T. laxa, T. minuta, T.lucida, T. argentina cabrera, T. tenuifolia, T. lemmonii and T. bipinata.

In a preferred embodiment of the present invention, the transgenic Tagetes are used for the production of carotenoids, morepreferably for the production of astaxanthin. Synthesis of novel carotenoids, naturally not present in Tagetes, shall takeplace in the flowers, preferentially in the petals of Tagetes. However, accumulation of carotenoids in other tissues is alsoacceptable. Plant material, preferably petals, with accumulated carotenoids may be used for the production of cartenoids,especially of astaxanthin. Various methods are known to extract the carotenoids from Tagetes plant material, preferablyfrom the Tagetes petals. The constructs provided hereunder are novel and especially useful for carrying out the invention. Inconsequence, another embodiment of the invention relates to a heterologous nucleic acid construct comprising a promotoractive in Tagetes cells selected from the group consisting of a constitutive promotor, an inducible promotor and a tissuespecific promotor; a nucleic acid sequence encoding an enzyme capable of metabolizing a Damino acid and being operablylinked to the promotor; and a nucleic acid sequence encoding a ketolase.

Various enzymes are known to the person skilled in the art, which can be used as D amino acid metabolizing enzymes.Preferably, the the enzyme capable of metabolizing a Damino acid is selected from the group consisting of Dserineammonialyases, Damino acid oxidases and Dalanine transaminases.

In another preferred embodiment, the enzyme capable of metabolizing a Damino acid comprised by the constructs above isencoded by a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence comprising a



nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQ ID No. 1 ; b) a nucleic acidsequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of said nucleotidesequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a Dserineammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand ofthe nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of said nucleotide sequence,wherein said fragment is sufficient to code for a protein having the enzymatic activity of a Dserine ammonialyase; d) anucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence froma), b) or c), or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a proteinhaving the enzymatic activity of a Dserine ammonialyase. e) a nucleic acid sequence comprising a nucleotide sequenceencoding a protein with the amino acid sequence depicted in SEQ ID No. 3; f) a nucleic acid sequence comprising thenucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of said nucleotide sequence, wherein saidfragment is sufficient to code for a protein having the enzymatic activity of a Damino acid oxidase; g) a nucleic acidsequence comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from e)or f) under stringent conditions, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient tocode for a protein having the enzymatic activity of a Damino acid oxidase; h) a nucleic acid sequence comprising anucleotide sequence which shows at least 60% identity to the nucleotide sequence from e), f) or g), or comprising afragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activityof a Damino acid oxidase.

The Tagetes plants and cells provided above are novel and, hence, also the method for producing cartinoids from theseplants is novel. This method comprises generating transgenic Tagetes plants according to the abovedescribed method.Regenerated Tagetes plants may be used for feeding animals or as human diets. Preferably, the carotenoids are extractedfrom the regenerated Tagetes plants, preferably from the Tagetes petals. Other objects, advantages, and features of thepresent invention will become apparent from the following specification.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Binary expression vector pBHX925 containing the E. coli dsdA gene driven by the A. thaliana ubqlO promoter. Theleft border (LB), right border (RB) and representative unique restriction sites are indicated.

GENERAL DEFINITIONS

The teachings, methods, sequences etc. employed and described in the international patent applications WO 01/46445("Production of transgenic plants of the Tagetes species"), WO 00/32788 ("Method for regulating carotenoid biosynthesis inmarigolds") and WO 2003/060133 ("Selective plant growth using Damino acids) are hereby incorporated by reference.

Abbreviations: BAP 6benzylaminopurine; 2,4D 2,4dichlorophenoxyacetic acid; MS Murashige and Skoog medium(Murashige T and Skoog F (1962) Physiol. Plant. 15, 472497); NAA 1naphtaleneacetic acid; MES, 2(Nmorpholinoethanesulfonic acid, IAA indole acetic acid; IBA: indole butyric acid; Kan: Kanamycin sulfate; GA3 Gibberellic acid;Timentin™: ticarcillin disodium i clavulanate potassium.

It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species orgenera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which willbe limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms"a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to"a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and soforth.

The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is usedin conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numericalvalues set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value bya variance of 20 percent, preferably 10 percent, more preferably 5 percent up or down (higher or lower).

As used herein, the word "or" means any one member of a particular list and also includes any combination of members ofthat list.

As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representingamino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by theoneletter symbols recommended by the IUPACIUB Biochemical Nomenclature Commission. Nucleotides, likewise, maybe referred to by their commonly accepted singleletter codes. The abbreviations used herein are conventional one lettercodes for the amino acids: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamicacid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline;Q, glutamine; R, arginine ; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid (seeL. Stryer, Biochemistry, 1988, W. H. Freeman and Company, New York. The letter "x" as used herein within an amino acidsequence can stand for any amino acid residue. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotidesand polymers or hybrids thereof in either singleor doublestranded, sense or antisense form. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degeneratecodon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" isused interchangeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide".



The phrase "nucleic acid sequence" as used herein refers to a consecutive list of abbreviations, letters, characters or words,which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid,usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A"coding region" of a nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a sequencespecificmanner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences.The coding region is said to encode such a polypeptide or protein. Unless otherwise indicated, a particular nucleic acidsequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeablyherein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide".

The term "gene of interest" refers to any nucleotide sequence, the manipulation of which may be deemed desirable for anyreason (e.g., confer improved qualities), by one of ordinary skill in the art. Such nucleotide sequences include, but are notlimited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistancegenes, growth factors, etc.), and noncoding regulatory sequences which do not encode an mRNA or protein product, (e.g.,promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.). For example, a gene ofinterest could code for a protein which is involved in or has an effect on the plant biosynthesis pathway.

The term "antisense" is understood to mean a nucleic acid having a sequence complementary to a target sequence, forexample a messenger RNA (mRNA) sequence the blocking of whose expression is sought to be initiated by hybridizationwith the target sequence.

The term "sense" is understood to mean a nucleic acid having a sequence which is homologous or identical to a targetsequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of agiven gene. According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing theexpression of the said gene of interest. In another embodiment, the gene of interest is an inhibiting nucleic acid moleculeselected from the group consisting of antisense oligonucleotide, antisense DNA, antisense RNA, iRNA, ribozyme, shRNAand siRNA. Preferably, the inhibiting nucleic acid molecule inhibits the expression of an endogenous Tagetes protein. Saidendogenous protein preferably is involved in or has an effect on the plant biosynthesis pathway.

As used herein, the terms "complementary" or "complementarity" are used in reference to nucleotide sequences related bythe basepairing rules. For example, the sequence 5'AGT3' is complementary to the sequence 5'ACT3'. Complementaritycan be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to thebase pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid baseis matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridization between nucleic acid strands. A "complement" of a nucleicacid sequence as used herein refers to a nucleotide sequence whose nucleic acids show total complementarity to thenucleic acids of the nucleic acid sequence.

The term "genome" or "genomic DNA" is referring to the heritable genetic information of a host organism. Said genomicDNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g.,chloroplasts) and other cellular organelles (e.g., mitochondria). Preferably the terms genome or genomic DNA is referring tothe chromosomal DNA of the nucleus.

The term "chromosomal DNA" or "chromosomal DNAsequence" is to be understood as the genomic DNA of the cellularnucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes orchromatids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated andanalyzed by various methods known in the art like e.g., polymerase chain reaction (PCR) analysis, Southern blot analysis,fluorescence in situ hybridization (FISH), and in situ PCR.

The term "isolated" as used herein means that a material has been removed from its original environment. For example, anaturallyoccurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotidescan be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be isolated inthat such a vector or composition is not part of its original environment. Preferably, the term "isolated" when used in relationto a nucleic acid refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acidwith which it is ordinarily associated in its natural source.

As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences that are removed from theirnatural environment, isolated or separated. An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence."Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% freefrom other components with which they are naturally associated.

A "polynucleotide construct" refers to a nucleic acid at least partly created by recombinant methods. The term "nucleic acidconstruct" is referring to a polynucleotide construct consisting of deoxyribonucleotides. The construct may be single or preferably double stranded. The construct may be circular or linear. The skilled worker is familiar with a variety of ways toobtain one of a Nucleic acid construct. Constructs can be prepared by means of customary recombination and cloningtechniques as are described, for example, in Maniatis 1989, Silhavy 1984, and in Ausubel 1987.

The term "wildtype", "natural" or of "natural origin" means with respect to an organism, polypeptide, or nucleic acidsequence, that said organism is naturally occurring or available in at least one naturally occurring organism which is not



changed, mutated, or otherwise manipulated by man.

The term "foreign gene" refers to any nucleic acid (e.g., gene sequence) which is introduced into the genome of a cell byexperimental manipulations and may include gene sequences found in that cell so long as the introduced gene containssome modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturallyoccurringgene.

The terms "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotidesequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated innature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which itis introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodesRNA and proteins that are not normally produced by the cell into which it is expressed. A promoter, transcription regulatingsequence or other genetic element is considered to be "heterologous" in relation to another sequence (e.g., encoding amarker sequence or a gene of interest) if said two sequences are not combined or differently operably linked their naturalenvironment. Preferably, said sequences are not operably linked in their natural environment (i.e. come from different genes).Most preferably, said regulatory sequence is covalently joined and adjacent to a nucleic acid to which it is not adjacent in itsnatural environment.

The term "trans gene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell orwhich has been manipulated by experimental manipulations by man. Preferably, said sequence is resulting in a genomewhich is different from a naturally occurring organism (e.g., said sequence, if endogenous to said organism, is introducedinto a location different from its natural location, or its copy number is increased or decreased). A transgene may be an"endogenous DNA sequence", "an "exogenous DNA sequence" (e.g., a foreign gene), or a "heterologous DNA sequence".The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it isintroduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable markergene, etc.) relative to the naturallyoccurring sequence.

The term "transgenic" or "recombinant" when used in reference to a cell or an organism (e.g., with regard to a Tagetes plantor cell) refers to a cell or organism which contains a transgene, or whose genome has been altered by the introduction of atransgene. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue issubstantially consisting of transgenic cells (i.e., more than 80%, preferably 90%, more preferably 95%, most preferably 99%of the cells in said organism or tissue are transgenic). The term "recombinant" with respect to nucleic acids means that thenucleic acid is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment."Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques, i.e.produced from cells transformed by an recombinant nucleic acid construct encoding the desired polypeptide or protein.Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but aremodified, changed, mutated or otherwise manipulated by man.

A "recombinant polypeptide" is a nonnaturally occurring polypeptide that differs in sequence from a naturally occurringpolypeptide by at least one amino acid residue. Preferred methods for producing said recombinant polypeptide and/or nucleicacid may comprise directed or nondirected mutagenesis, DNA shuffling or other methods of recursive recombination.

The terms "homology" or "identity" when used in relation to nucleic acids or amino acid sequences refers to a degree ofsequence relation ship or complementarity. The following terms are used to describe the sequence relationships betweentwo or more nucleic acids or amino acid sequences: (a) "reference sequence", (b) "comparison window", (c) "sequenceidentity", (d) "percentage of sequence identity", and (e) "substantial identity".

(a)As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A referencesequence may be a subset or the entirety of a specified sequence; for example, as a segment of a fulllength cDNA or genesequence, or the complete cDNA or gene sequence.

(b)As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotidesequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30,

40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due toinclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identitybetween any two sequences can be accomplished using a mathematical algorithm. Preferred, nonlimiting examples of suchmathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al. 1981; thehomology alignment algorithm of Needleman and Wunsch 1970; the searchforsimilaritymethod of Pearson and Lipman1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993. For comparing sequenceshereunder, preferably the algorithms BLASTN for nucleotide sequences, BLASTX for proteins with their respective defaultparameters are used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, 1989). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.Multiple augments (i.e. of more than 2 sequences) are preferably performed using the Clustal W algorithm (Thompson 1994;



e.g., in the software VectorNTI™, version 9; Invitrogen Inc.) with the scoring matrix BLOSUM62MT2 with the defaultsettings (gap opening penalty 15/19, gap extension penalty 6.66/0.05; gap separation penalty range 8; % identity foralignment delay 40; using residue specific gaps and hydrophilic residue gaps). Comparison is preferably made using theBlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" isintended any sequence comparison program that, for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In addition to calculating percentsequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see,e.g., Karlin & Altschul (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequenceswould occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is lessthan about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The term "hybridization" as used herein includes "any process by which a strand of nucleic acid joins with a complementarystrand through base pairing." (Coombs 1994). Hybridization and the strength of hybridization (i.e., the strength of theassociation between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleicacids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. Asused herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature atwhich a population of doublestranded nucleic acid molecules becomes half dissociated into single strands. The equation forcalculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of theTm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 MNaCl [see e.g., Anderson and Young, 1985)]. Other references include more sophisticated computations, which takestructural as well as sequence characteristics into account for the calculation of Tm.

An example of conditions for "hybridization under stringent conditions" is 0.15 M NaCl at 72°C for about 15 minutes. Anexample of stringent wash conditions is a 0.2 X SSC wash at 65°C for 15 minutes (see, Maniatis, infra, for a description ofSSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An

example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 X SSC at 450C for 15 minutes. Anexample low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4 to 6 X SSC at 40°C for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is

typically at least about 30°C and at least about 6O0C for long probes (e.g., >50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 X (or higher)than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins thatthey encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of highly stringent conditionsfor hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern

or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 370C, and a wash in 0.1 xSSC at 60 to 65°C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35%formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX to 2X SSC (20 X SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45%

formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5 X to 1 X SSC at 55 to 600C.

The term "equivalent" when made in reference to a hybridization condition as it relates to a hybridization condition of interestmeans that the hybridization condition and the hybridization condition of interest result in hybridization of nucleic acidsequences which have the same range of percent (%) homology. For example, if a hybridization condition of interest resultsin hybridization of a first nucleic acid sequence with other nucleic acid sequences that have from 80% to 90% homology tothe first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the othernucleic acid sequences that have from 80% to 90% homology to the first nucleic acid sequence.

When used in reference to nucleic acid hybridization one skilled in the art knows well that numerous equivalent conditionsmay be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA,base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or highstringency hybridization different from, but equivalent to, the abovelisted conditions. Those skilled in the art know thatwhereas higher stringencies may be preferred to reduce or eliminate nonspecific binding, lower stringencies may bepreferred to detect a larger number of nucleic acid sequences having different homologies.

The term "gene" refers to a coding region operably joined to appropriate regulatory sequences capable of regulating theexpression of the polypeptide in some manner. A gene includes untranslated regulatory regions of DNA (e. g., promoters,enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF)as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term



"structural gene" as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is thentranslated into a sequence of amino acids characteristic of a specific polypeptide.

As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequenceswhich encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The codingregion is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and onthe 3 'side by one of the three triplets, which specify stop codons (i.e., TAA, TAG, TGA). In addition to containing introns,genomic forms of a gene may also include sequences located on both the 5' and 3'end of the sequences, which arepresent on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flankingsequences are located 5' or 3' to the nontranslated sequences present on the mRNA transcript). The 5'flanking region maycontain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene. The3'flanking region may contain sequences, which direct the termination of transcription, posttranscriptional cleavage andpolyadenylation.

The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are usedinterchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.

The term "geneticallymodified organism" or "GMO" refers to any organism that comprises transgene DNA. Exemplaryorganisms include plants, animals and microorganisms.

The term "plant" as used herein refers to a plurality of plant cells, which are largely differentiated into a structure that ispresent at any stage of a plant's development. Such structures include one or more plant organs including, but are notlimited to, fruit, shoot, stem, leaf, flower petal, etc.

The term "cell" or "plant cell" as used herein refers to a single cell. The term "cells" refers to a population of cells. Thepopulation may be a pure population comprising one cell type. Likewise, the population may comprise more than one celltype. In the present invention, there is no limit on the number of cell types that a cell population may comprise. The cellsmay be synchronized or not synchronized. A plant cell within the meaning of this invention may be isolated (e.g., insuspension culture) or comprised in a plant tissue, plant organ or plant at any developmental stage.

The term "organ" with respect to a plant (or "plant organ") means parts of a plant and may include (but shall not limited to)for example roots, fruits, shoots, stem, leaves, anthers, sepals, petals, pollen, seeds, etc.

The term "tissue" with respect to a plant (or "plant tissue") means arrangement of multiple plant cells including differentiatedand undifferentiated tissues of plants. Plant tissues may constitute part of a plant organ e.g., the epidermis of a plant leaf)but may also constitute tumor tissues (e.g., callus tissue) and various types of cells in culture e.g., single cells, protoplasts,embryos, calli, protocormlike bodies, etc.). Plant tissue may be inplanta, in organ culture, tissue culture, or cell culture.

The term "chromosomal DNA" or "chromosomal DNAsequence" is to be understood as the genomic DNA of the cellularnucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes orchromatids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated andanalyzed by various methods known in the art like e.g., PCR analysis, Southern blot analysis, fluorescence in situhybridization (FISH), and in situ PCR. The term "structural gene" as used herein is intended to mean a DNA sequence thatis transcribed into mRNA, which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene,expression involves transcription of the structural gene into mRNA and optionally the subsequent translation of mRNAinto one or more polypeptides.

The term "expression cassette" or "expression construct" as used herein is intended to mean the combination of any nucleicacid sequence to be expressed in operable linkage with a promoter sequence and optionally additional elements (like e.g.,terminator and/or polyadenylation sequences) which facilitate expression of said nucleic acid sequence.

"Promoter", "promoter element," or "promoter sequence" as used herein, refers to the nucleotide sequences at the 5' end ofa nucleotide sequence which direct the initiation of transcription (i.e., is capable of controlling the transcription of thenucleotide sequence into mRNA). A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotidesequence of interest (e.g., proximal to the transcriptional start site of a structural gene) whose transcription into mRNA itcontrols, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation oftranscription. Promoter sequences are necessary, but not always sufficient, to drive the expression of a downstream gene.In general, eukaryotic promoters include a characteristic DNA sequence homologous to the consensus 5'TATAAT3'(TATA) box about 1030 bp 5' to the transcription start (cap) site, which, by convention, is numbered +1. Bases 3' to the capsite are given positive numbers, whereas bases 5' to the cap site receive negative numbers, reflecting their distance fromthe cap site. Another promoter component, the CAAT box, is often found about 30 to 70 bp 5' to the TATA box and hashomology to the canonical form 5'CCAAT3' (Breathnach 1981). In plants the CAAT box is sometimes replaced by asequence known as the AGGA box, a region having adenine residues symmetrically flanking the triplet G(orT)NG (Messing1983). Other sequences conferring regulatory influences on transcription can be found within the promoter region andextending as far as 1000 bp or more 5' from the cap site. The term "constitutive" when made in reference to a promotermeans that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of astimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of atransgene in substantially any cell and any tissue.

Regulatory Control refers to the modulation of gene expression induced by DNA sequence elements located primarily, but



not exclusively, upstream of (5' to) the transcription start site. Regulation may result in an allornothing response toenvironmental stimuli, or it may result in variations in the level of gene expression. In this invention, the heat shockregulatory elements function to enhance transiently the level of downstream gene expression in response to suddentemperature elevation.

Polyadenylation signal refers to any nucleic acid sequence capable of effecting mRNA processing, usually characterized bythe addition of polyadenylic acid tracts to the 3'ends of the mRNA precursors. The polyadenylation signal DNA segmentmay itself be a composite of segments derived from several sources, naturally occurring or synthetic, and may be from agenomic DNA or an RNAderived cDNA. Polyadenylation signals are commonly recognized by the presence of homology tothe canonical form 5'AATAA3', although variation of distance, partial "readthrough", and multiple tandem canonicalsequences are not uncommon (Messing 1983). It should be recognized that a canonical "polyadenylation signal" may in factcause transcriptional termination and not polyadenylation per se (Montell 1983).

Heat shock elements refer to DNA sequences that regulate gene expression in response to the stress of suddentemperature elevations. The response is seen as an immediate albeit transitory enhancement in level of expression of adownstream gene. The original work on heat shock genes was done with Drosophila but many other species including plants(Barnett 1980) exhibited analogous responses to stress. The essential primary component of the heat shock element was

described in Drosophila to have the consensus sequence (SEQ ID No. 28) 5' CTGGAATNTTCTAGA31 (where N=A, T, C,or G) and to be located in the region between residues 66 through 47 bp upstream to the transcriptional start site (Pelham1982). A chemically synthesized oligonucleotide copy of this consensus sequence can replace the natural sequence inconferring heat shock inducibility.

Leader sequence refers to a DNA sequence comprising about 100 nucleotides located between the transcription start siteand the translation start site. Embodied within the leader sequence is a region that specifies the ribosome binding site.

Introns or intervening sequences refer in this work to those regions of DNA sequence that are transcribed along with thecoding sequences (exons) but are then removed in the formation of the mature mRNA. Introns may occur anywhere within atranscribed sequence— between coding sequences of the same or different genes, within the coding sequence of a gene,interrupting and splitting its amino acid sequences, and within the promoter region (5' to the translation start site). Introns inthe primary transcript are excised and the coding sequences are simultaneously and precisely ligated to form the maturemRNA. The junctions of introns and exons form the splice sites. The base sequence of an intron begins with GU and endswith AG. The same splicing signal is found in many higher eukaryotes.

The term "operable linkage" or "operably linked" is to be understood as meaning, for example, the sequential arrangement ofa regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatoryelements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfill its intended function toallow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. The expression may resultdepending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, directlinkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancersequences, can also exert their function on the target sequence from positions, which are further away, or indeed from otherDNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly ispositioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. Thedistance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably lessthan 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.Operable linkage, and an expression cassette, can be generated by means of customary recombination and cloningtechniques as described (e.g., in Maniatis 1989; Silhavy 1984; Ausubel 1987; Gelvin 1990). However, further sequences,which for example act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also bepositioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins.Preferably, the expression cassette, consisting of a linkage of promoter and nucleic acid sequence to be expressed, canexist in a vectorintegrated form and be inserted into a plant genome, for example by transformation.

The term "transformation" as used herein refers to the introduction of genetic material (e.g., a transgene) into a cell.Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers tothe introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell'sgenome. Transient transformation may be detected by, for example, enzymelinked immunosorbent assay (ELISA) whichdetects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation maybe detected by detecting the activity of the protein (e.g., βglucuronidase) encoded by the transgene (e.g., the uid Agene) asdemonstrated herein [e.g., histochemical assay of GUS enzyme activity by staining with Xgluc which gives a blueprecipitate in the presence of the GUS enzyme; and a chemiluminescent assay of GUS enzyme activity using the GUSLight kit (Tropix)]. The term "transient transformant" refers to a cell which has transiently incorporated one or moretransgenes. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration ofone or more transgenes into the genome of a cell, preferably resulting in chromosomal integration and stable heritabilitythrough meiosis. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cellwith nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, stabletransformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplifytransgene sequences. The term "stable transformant" refers to a cell, which has stably integrated one or more transgenesinto the genomic DNA (including the DNA of the plastids and the nucleus), preferably integration into the chromosomal DNAof the nucleus. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNAfrom the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not



contain a transgene. Transformation also includes introduction of genetic material into plant cells in the form of plant viralvectors involving epichromosomal replication and gene expression, which may exhibit variable properties with respect tomeiotic stability. Transformation also includes introduction of genetic material into plant cells in the form of plant viralvectors involving epichromosomal replication and gene expression, which may exhibit variable properties with respect tomeiotic stability. Preferably, the term "transformation" includes introduction of genetic material into plant cells resulting inchromosomal integration and stable heritability through meiosis.

The terms "infecting" and "infection" with a bacterium refer to coincubation of a target biological sample, (e.g., cell, tissue,etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introducedinto one or more cells of the target biological sample.

The term "Agrobacterium" refers to a soilborne, Gramnegative, rodshaped phytopathogenic bacterium, which causescrown gall. The term "Agrobacterium" includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typicallycauses crown gall in infected plants), and Agrobacterium rhizogenes (which causes hairy root disease in infected hostplants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopaline, agropine,octopine etc.) by the infected cell. Thus, Agrobacterium strains which cause production of nopaline (e.g., strain LBA4301,C58, A208) are referred to as "nopalinetype" Agrobacteria; Agrobacterium strains which cause production of octopine (e.g.,strain LBA4404, Ach5, B6) are referred to as "octopinetype" Agrobacteria; and Agrobacterium strains which causeproduction of agropine (e.g., strain EHA 105, EHAlOl, A281) are referred to as "agropinetype" Agrobacteria. The terms"bombarding, "bombardment," and "Holistic bombardment" refer to the process of accelerating particles towards a targetbiological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sampleand/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g.,US 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the heliumgasdriven microprojectile accelerator (PDS1000/He) (BioRad).

The term "microwounding" when made in reference to plant tissue refers to the introduction of microscopic wounds in thattissue. Microwounding may be achieved by, for example, particle bombardment as described herein.

The "efficiency of transformation" or "frequency of transformation" as used herein can be measured by the number oftransformed cells (or transgenic organisms grown from individual transformed cells) that are recovered under standardexperimental conditions (i.e. standardized or normalized with respect to amount of cells contacted with foreign DNA, amountof delivered DNA, type and conditions of DNA delivery, general culture conditions etc.) For example, when isolated explantsof cotyledons are used as starting material for transformation, the frequency of transformation can be expressed as thenumber of transgenic plant lines obtained per 100 isolated explants transformed.

The terms "meristem" or "meristematic cells" or meristematic tissue" can be used interchangeable and are intended to meanundifferentiated plant tissue, which continually divides, forming new cells, as that found at the tip of a stem or root. The term"node" or "leaf node" is intended to mean the point on a stem where a leaf is attached or has been attached. The term"internode" is intended to mean the section or part between two nodes on a stem. The term "petiole" is intended to mean thestalk by which a leaf is attached to a stem, also called a leafstalk. The term "axillary bud" is intended to mean a smallprotuberance along a stem or branch, sometimes enclosed in protective scales and containing an undeveloped shoot, leaf,or flower; also called a lateral bud. The term "hypocotyl" is intended to mean the part of the stem between the seed leaves(the cotyledons) and the root. The term "leaf axil" is intended to mean the angle between a leaf and the stem on which it isborne. The axillary bud occurs at the leaf axil. The term "cotyledon" is intended to mean a leaf of the embryo of a seed plant,which upon germination either remains in the seed or emerges, enlarges, and becomes green; also called a seed leaf. Theembryo axis is located between the cotyledons and is attached to them near the end closest to the micropyle.

The term "dedifferentiation", "dedifferentiation treatment" or "dedifferentiation pretreatment" means a process of obtainingcell clusters, such as callus, that show unorganized growth by culturing differentiated cells of plant tissues on adedifferentiation medium. More specifically, the term "dedifferentiation" as used herein is intended to mean the process offormation of rapidly dividing cells without particular function in the scope of the plant body. These cells often possess anincreased potency with regard to its ability to develop into various plant tissues. Preferably the term is intended to mean thereversion of a differentiated or specialized tissues to a more pluripotent or totipotent (e.g., embryonic) form. Dedifferentiationmay lead to reprogramming of a plant tissue (revert first to undifferentiated, nonspecialized cells, then to new and differentpaths). The term "totipotency" as used herein is intended to mean a plant cell containing all the genetic and/or cellularinformation required to form an entire plant. Dedifferentiation can be initiated by certain plant growth regulators (e.g., auxinand/or cytokinin compounds), especially by certain combinations and/or concentrations thereof. "Carotenoids" whichcomprise the most important group of 40carbon terpenes and terpenoids are pigments that have a variety of commercialapplications. Carotenoids are a class of hydrocarbons (carotenes) and their hydroxylated derivatives (xanthophylls) whichcomprise 40carbon (C40) terpenoids consisting of eight isoprenoid (C5) units joined together. The terpenoids are joined insuch a manner that the arrangement of the isoprenoid units is reversed at the center of the molecule placing the terminalmethyl groups in a 1,6 relationship and the nonterminal methyl groups in a 1,5 relationship. "Carotenoids" can bemonocyclic, bicyclic or acyclic. The carotenoids of the most value are intermediates in the carotenoid biosynthetic pathwayand consist of lycopene, betacarotene, zeaxanthin and astaxanthin.

"Astaxanthin" supplied from biological sources, such as crustaceans, yeast and green algae is limited by low yield andcostly extraction methods when compared with that obtained by organic synthetic methods. Usual synthetic methodshowever, are time consuming and, hence, very expensive. It is therefore desirable to find a relatively inexpensive source of(3S, 3'S) astaxanthin to be used as a feed supplement in aquaculture and as a valuable chemical for other industrial uses.One approach to increase the productivity of astaxanthin production in a biological system is to use genetic engineering



technology. Genes suitable for this conversion have been reported.

The "carotenoid biosynthesis pathway" is known from the art. For example, WO 00/32788 ("Method for regulating carotenoidbiosynthesis in marigolds") is herein incorported by reference.

Carotenoids are synthesized by serveral organisms like bacteria, yeast, algae and plants. They are deposited andaccumulated either as free carotenoids or as carotenoid derivatives, e.g. glycosides or esters. Those carotenoids and theircarotenoid esters like lycopene, betacarotene, zeaxanthin, astaxanthin, adonirubin or other xanthophylls andketocarotenoids can be extracted from the target tissues by organic solvents. The carotenoidcontaining material can beprocessed as fresh or dried material. After a suitable homogenization, repeated extraction with organic solvents like aceton,hexane, methylenchloride, tert.butylmethylether or solvent mixtures like ethanol/hexane or acetone/hexane (to name only afew possible solvents and solvent mixtures) releases the carotenoids of interest. The extraction effect and efficiency can beadjusted and modified via different solvent ratios which take into account the different polarity of various solvents. Viaevaporation of the used solvents, high concentrations of carotenoids and carotenoid esters can be achieved. Subsequentchromatographic separation can further improve the purity of individual carotenoids.Further isolating procedures forcarotenoids are described for example in Egger and Kleinig (Phytochemistry (1967) 6.437440) and Egger (Phytochemistry(1965) 4.609618).

Examples of nucleic acids encoding a "ketolase" and the corresponding ketolases which can be used in the processaccording to the invention are, for example, sequences from Haematoccus pluvialis. US 2006/0194274 and US2006/0112451 are herein incorported by reference. Ketolase activity is understood as meaning the enzyme activity of aketolase. A ketolase is understood as meaning a protein which has the enzymatic activity to introduce a keto group on theoptionally substituted, [beta]ionone ring of carotenoids. DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention relates to the method for generating transgenic Tagetes plants or cells comprising thesteps of a) introducing into a Tagetes cell or tissue a nucleic acid construct comprising a promoter active in Tagetes cellsand operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a Damino acid and/or aderivative thereof; b) incubating said Tagetes cell or tissue of step a) on a selection medium comprising a Damino acidand/or a derivative thereof; and optionally c) transferring said Tagetes cell or tissue of step b) to a regeneration medium andregenerating Tagetes plants comprising said nucleic acid construct.

1. The Nucleic acid construct of the invention 1.1 The first expression construct of the invention

The first expression construct comprises a promoter active in Tagetes and operably linked thereto a nucleic acid sequenceencoding an enzyme capable of metabolizing a Damino acid. Preferably said promoter is heterologous in relation to saidenzyme encoding sequence. The promoter active in Tagetes plants and the D amino acid metabolizing enzyme are definedbelow in detail.

1.1.1 The enzyme capable of metabolizing a Damino acid

The person skilled in the art is aware of numerous sequences suitable to metabolize a Damino acid. The term "enzymecapable of metabolizing a Damino acid" means preferably an enzyme, which converts and/or metabolizes a Damino acidwith an activity that is at least two times (at least 100% higher), preferably at least three times, more preferably at least fivetimes, even more preferably at least 10 times, most preferably at least 50 times or 100 times the activity for the conversionof the corresponding Lamino acid and more preferably also of any other D and/or L or achiral amino acid.

Preferably, the enzyme capable of metabolizing a Damino acid is selected from the group consisting of Dserine ammonialyase (DSerine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14), DAmino acid oxidases (EC 1.4.3.3), and DAlaninetransaminases (EC 2.6.1.21). More preferably, the enzyme capable of metabolizing Dalanine or Dserine is selected fromthe group consisting of Dserine ammonia lyase (DSerine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14), and DAminoacid oxidases (EC 1.4.3.3).

The term " Dserine ammonialyase" (DSerine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14) means enzymescatalyzing the conversion of Dserine to pyruvate and ammonia. The reaction catalyzed probably involves initial eliminationof water (hence the enzyme's original classification as EC 4.2.1.14), followed by isomerization and hydrolysis of the productwith CN bond breakage. For examples of suitable enzyme see http://www.expasy.Org/enzyme/4.3.l.18.

The term "D Alanine transaminases" (EC 2.6.1.21) means enzymes catalyzing the reaction of D Alanine with 2oxoglutarate to pyruvate and Dglutamate. Dglutamate is much less toxic to plants than D Alanine.http://www.expasy.Org/enzyme/2.6.l.21.

The term Damino acid oxidase (EC 1.4.3.3; abbreviated DAAO, DAMOX, or DAO) is referring to the enzyme converting aDamino acid into a 2oxo acid, by preferably employing Oxygen (O2) as a substrate and producing hydrogen peroxide

(H2O2) as a coproduct (Dixon 1965a,b,c; Massey 1961; Meister 1963). DAAO can be described by the Nomenclature

Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) with the EC (Enzyme Commission)number EC 1.4.3.3. Generally an DAAO enzyme of the EC 1.4.3.3. class is an FAD flavoenzyme that catalyzes theoxidation of neutral and basic Damino acids into their corresponding keto acids. DAAOs have been characterized andsequenced in fungi and vertebrates where they are known to be located in the peroxisomes. In DAAO, a conserved histidinehas been shown (Miyano 1991) to be important for the enzyme's catalytic activity. In a preferred embodiment of theinvention a DAAO is referring to a protein comprising the following consensus motif:



[LIVM][LIVM]H+[NHA]YGX[GSA][GSA]XGX5GXA

wherein amino acid residues given in brackets represent alternative residues for the respective position, x represents anyamino acid residue, and indices numbers indicate the respective number of consecutive amino acid residues. Theabbreviation for the individual amino acid residues have their standard IUPAC meaning as defined above. D Amino acidoxidase (ECnumber 1.4.3.3) can be isolated from various organisms, including but not limited to pig, human, rat, yeast,bacteria or fungi. Example organisms are Candida tropicalis, Trigonopsis variabilis, Neurospora crassa, Chlorella vulgaris,and Rhodotorula gracilis. A suitable D amino acid metabolising polypeptide may be an eukaryotic enzyme, for example froma yeast (e.g. Rhodotorula gracilis), fungus, or animal or it may be a prokaryotic enzyme, for example, from a bacterium suchas Escherichia coli. For examples of suitable enzyme see http://www.expasy.Org/enzyme/l.4.3.3. Examples of suitablepolypeptides, which metabolise Damino acids are shown in Table 1. The nucleic acid sequences encoding said enzymesare available form databases (e.g., under Genbank Acc.No. U60066, A56901, AF003339, Z71657, AF003340, U63139,D00809, Z50019, NC_003421, AL939129, AB042032). As demonstrated above, DAAO from several different species havebeen characterized and shown to differ slightly in substrate affinities (Gabler 2000), but in general they display broadsubstrate specificity, oxidative Iy deaminating all Damino acids.

Table 1: Enzymes suitable for metabolizing Dserine and/or Dalanine. Especially preferred enzymes are presented in boldletters

Enzyme EC number Example Source organism Substrate

DSerine dehydratase EC 4.3.1.18 P54555 Bacillus subtilis DSer

(DSerine ammonia (originally EC P00926 Escherichia coli. DSDA DThr lyase, DSerine 4.2 1.14) Q9KL72 Vibrio cholera.VCA0875 Dallothreonine deaminiase) Q9KC12 Bacillus halodurans.

DAmino acid oxidase EC 1.4.3.3 JX0152 Fusarium solani Most Damino

001739 Caenorhabditis elegans. acid

033145 Mycobacterium leprae. AAO.

035078 Rattus norvegicus (Rat)

045307 Caenorhabditis elegans

P00371 Sus scrofa (Pig)

P14920 Homo sapiens (Human)

P14920 Homo sapiens (Human)

P18894 Mus musculus (Mouse)

P22942 Oryctolagus cuniculus (Rabbit)

P24552 Fusarium solani (subsp. pisi)

(Nectria haematococca)

P80324 Rhodosporidium toruloides

(Yeast) (Rhodotorula gracilis)

Q 19564 Caenorhabditis elegans

Q28382 Sus scrofa (pig)

Q7SFW4 Neurospora crassa

Q7Z312 Homo sapiens (Human)

Q82MI8 Streptomyces avermitilis

Q8P4M9 Xanthomonas campestris



Especially preferred in this context are the dao\ gene (EC: 1.4. 3.3: GenBank Acc.No.: U60066) from the yeast Rhodotorulagracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (Dserine dehydratase (Dserine deaminase) [EC: 4.3. 1.18;GenBank Acc.No.: J01603). The dao\ gene is of special advantage since it can be employed as a dual function marker (seeinternational patent application PCT/EP 2005/002734; WO 2005/090581).

Suitable Damino acid metabolizing enzymes also include fragments, mutants, derivatives, variants and alleles of thepolypeptides exemplified above. Suitable fragments, mutants, derivatives, variants and alleles are those, which retain thefunctional characteristics of the Damino acid metabolizing enzyme as defined above. Changes to a sequence, to produce amutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotidesin the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encodedpolypeptide. Of course, changes to the nucleic acid that make no difference to the encoded amino acid sequence areincluded.

More preferably for the method of the invention, the Dserine ammonialyase is selected from the group consisting of a) anucleic acid sequence comprising a nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQID No. 1 ; b) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising afragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activityof a Dserine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to acomplementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of saidnucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a Dserineammonialyase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to thenucleotide sequence from a), b) or c), or comprising a fragment of said nucleotide sequence, wherein said fragment issufficient to code for a protein having the enzymatic activity of a Dserine ammonialyase

and wherein selection is done on a medium comprising Dserine in a concentration from about 0.05 mM to about 100 mM,preferably about 0.1 mM to about 50 mM, more preferably about 0.3 mM to about 5 mM. The total selection time underdedifferentiating conditions is preferably from about 1 to 10 weeks, preferably from 2 to 8 weeks, more preferably from 3 to 4weeks. During selection, different Damino acids in different concentrations may be used to select Tagetes plants or cells.

"Same activity" in the context of a Dserine ammonialyase means the capability to metabolize Dserine, preferably as themost preferred substrate. Metabolization means the lyase reaction specified above.

Also more preferably for the method of the invention, the enzyme capable of metabolizing Dserine and Dalanine is selectedfrom the group consisting of a) a nucleic acid sequence comprising a nucleotide sequence encoding a protein with the aminoacid sequence depicted in SEQ ID No. 3; b) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQID No. 4, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a proteinhaving the enzymatic activity of a Damino acid oxidase; c) a nucleic acid sequence comprising a nucleotide sequencewhich hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, orcomprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having theenzymatic activity of a Damino acid oxidase; d) a nucleic acid sequence comprising a nucleotide sequence which shows atleast 60% identity to the nucleotide sequence from a), b) or c), or comprising a fragment of said nucleotide sequence,wherein said fragment is sufficient to code for a protein having the enzymatic activity of a Damino acid oxidase.

and wherein selection is done on a medium comprising Dalanine and/or D serine in a total concentration from about 0.05mM to about 100 mM, preferably about 0.1 mM to about 50 mM, more preferably about 0.3 mM to about 5 mM. Preferably,Dalanine (e.g., if employed as only selection compound) is employed in a concentration of about 0.05 mM to about 100 mM,preferably about 0.1 mM to about 50 mM, more preferably about 0.3 mM to about 5 mM. Preferably, Dserine (e.g., if

https://patentimages.storage.googleapis.com/WO2008077570A1/imgf000042_0001.png



employed as only selection compound) is employed in a concentration of about 0.05 mM to about 100 mM, preferably about0.1 mM to about 50 mM, more preferably about 0.3 mM to about 5 mM. The total selection time under dedifferentiatingconditions is preferably from about 1 to 10 weeks, preferably from 2 to 8 weeks, more preferably from 3 to 4 weeks. Duringselection, different D amino acids in different concentrations may be used to select Tagetes plants or cells.

"Same activity" in the context of a Damino acid oxidase means the capability to metabolize a broad spectrum of Daminoacids (preferably at least D serine and/or Dalanine). Metabolization means the oxidase reaction specified above. Mutantsand derivatives of the specified sequences can also comprise enzymes, which are improved in one or more characteristics(Ki, substrate specificity etc.) but still comprise the metabolizing activity regarding Dserine and or Dalanine. Suchsequences and proteins also encompass, sequences and protein derived from a mutagenic and recombinogenic proceduresuch as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a newpolypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. Polynucleotides encoding a candidate enzyme can, for example, bemodulated with DNA shuffling protocols. DNA shuffling is a method to rapidly, easily and efficiently introduce mutations orrearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or moreDNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is anonnaturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes anenzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activitywith respect to the enzyme encoded by the template DNA. DNA shuffling can be based on a process of recursiverecombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of thefragments by a polymerase chain reactionlike process. See, e.g., Stemmer 1994 a,b; Crameri 1997; Moore 1997; Zhang1997; Crameri 1998; US 5,605,793, US 5,837,458, US 5,830,721 and US 5,811,238. The resulting dsdA or daolike enzymeencoded by the shuffled DNA may possess different amino acid sequences from the original version of enzyme. Exemplaryranges for sequence identity are specified above. The Damino acid metabolizing enzyme of the invention may be expressedin the cytosol, peroxisome, or other intracellular compartment of the plant cell. Compartmentalisation of the Damino acidmetabolizing enzyme may be achieved by fusing the nucleic acid sequence encoding the DAAO polypeptide to a sequenceencoding a transit peptide to generate a fusion protein. Gene products expressed without such transit peptides generallyaccumulate in the cytosol.

1.1.2 Promoters for Tagetes plants 1.1.2.1 General promoter

The term "promoter" as used herein is intended to mean a DNA sequence that directs the transcription of a DNA sequence(e.g., a structural gene). Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site ofa structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducingagent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.Also, the promoter may be regulated in a tissuespecific or tissue preferred manner such that it is only active in transcribingthe associated coding region in a specific tissue type(s) such as leaves, roots or meristem.

The term "promoter active in Tagetes cells" means any promoter, whether plant derived or not, which is capable to inducetranscription of an operably linked nucleotide sequence in at least one Tagetes cell, tissue, organ or plant at at least onetime point in development or under dedifferentiated conditions. Such promoter may be a nonplant promoter (e.g., derivedfrom a plant virus or Agrobacterium) or a plant promoter, preferably a dicotyledonous plant promoter. The person skilled inthe art is aware of several promoters which, might be suitable for use in Tagetes plants. In this context, expression can be,for example, constitutive, inducible or developmentdependent. The following promoters are preferred:

a) Constitutive promoters

"Constitutive" promoters refers to those promoters which ensure expression in a large number of, preferably all, tissues overa substantial period of plant development, preferably at all times during plant development. Examples include the CaMV(cauliflower mosaic virus) 35S promoter (Franck 1980; Shewmaker 1985; Gardner 1986; Odell 1985), the 19S CaMVpromoter (US 5,352,605; WO 84/02913; Benfey 1989), the Rubisco small subunit (SSU) promoter (US 4,962,028), thelegumin B promoter (GenBank Ace. No. X03677), the promoter of the nopaline synthase from Agrobacterium, the TR dualpromoter, the OCS (octopine synthase) promoter from Agrobacterium, the cinnamyl alcohol dehydrogenase promoter (US5,683,439), the promoters of the vacuolar ATPase subunits, the pEMU promoter (Last 1991); the MAS promoter (Velten1984), the promoter of the Arabidopsis thalicma nitrilase1 gene (GenBank Ace. No.: U38846, nucleotides 3862 to 5325 orelse 5342), and further promoters of genes with constitutive expression in plants.

Other suitable constitutive promoters are actin promoters. Sequences for several actin promoters from dicotyledonous plantsare available by the genomic sequences disclosed in Genbank (for example: AY063089 Arabidopsis thaliana Actinδ gene);AY096381 (Arabidopsis thaliana Actin 2 gene; AY305730: Gossypium hirsutum Actin 8 gene); AY305724 Gossypiumhirsutum Actin 2 gene); AFl 11812 Brassica napus Actin gene)). Use of their promoters in heterologous expression isdescribed for the Banana actin promoter (US20050102711). An et al. [Plant J 1996 10(l):107 121] reported that Act! andActS mRNA were expressed strongly in leaves, roots, stems, flowers, pollen, and siliques. Chimeric GUS constructsexpressed most of the vegetative tissues but almost no expression was detected in seed coates, hypocotyls, gynoecia, orpollen sacs.

b) Tissuespecific or tissuepreferred promoters

Furthermore preferred are promoters with specificities for seeds, such as, for example, the phaseolin promoter (US5,504,200; Bustos 1989; Murai 1983; SenguptaGopalan 1985), the promoter of the 2S albumin gene (Joseffson 1987), the



legumine promoter (Shirsat 1989), the USP (unknown seed protein) promoter (Baumlein 1991a), the napin gene promoter(US 5,608,152; Stalberg 1996), the promoter of the sucrose binding proteins (WO 00/26388) or the legumin B4 promoter(LeB4; Baumlein 1991b; Becker 1992), the Arabidopsis oleosin promoter (WO 98/45461), and the Brassica Bce4 promoter(WO 91/13980). Further preferred are a leafspecific and lightinduced promoter such as that from cab or Rubisco (Simpson1985; Timko 1985); an antherspecific promoter such as that from LAT52 (T well 1989b); and a microsporepreferredpromoter such as that from apg (T well 1983). Flowerspecific and especially petalspecific promotors are preferred forTagetes. Relevant flower and petal specific promotors are known from the art. WO 04/27070, WO 05/019460, WO06/117381 and EP 06115339.1 are incorporated herein with reference.

c) Inducible promoters

The expression cassettes may also contain a (chemically) inducible promoter (review article: Gatz 1997), by means of whichthe expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters such as, forexample, the PRPl promoter (Ward 1993), a salicylic acidinducible promoter (WO 95/19443), a benzenesulfonamideinducible promoter (EP 0 388 186), a tetracyclin inducible promoter (Gatz 1991; Gatz 1992), an abscisic acidinduciblepromoter EP 0 335 528) or an ethanolcyclohexanoneinducible promoter (WO 93/21334) can likewise be used. Also suitableis the promoter of the glutathioneS transferase isoform II gene (GSTII27), which can be activated by exogenously appliedsafeners such as, for example, N,Ndiallyl2,2dichloroacetamide (WO 93/01294) and which is operable in a large number oftissues of both monocots and dicots. Further exemplary inducible promoters that can be utilized in the instant inventioninclude that from the ACEl system which responds to copper (Mett 1993); or the In2 promoter from maize which responds tobenzenesulfonamide herbicide safeners (Hershey 1991; Gatz 1994). A promoter that responds to an inducing agent to whichplants do not normally respond can be utilized. An examplary inducible promoter is the inducible promoter from a steroidhormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena 1991).

Particularly preferred are constitutive promoters. Furthermore, promoters may be linked operably to the nucleic acidsequence to be expressed, which promoters make possible the expression in further plant tissues or in other organisms,such as, for example, E. coli bacteria. Suitable plant promoters are, in principle, all of the above described promoters.

"Promoter activity" in Tagetes plants means the capability to realize transcription of an operably linked nucleic acidsequence in at least one cell or tissue of a Tagetes plant or derived from a Tagetes plant. Preferably it means a constitutivetranscription activity allowing for expression in most tissues and most developmental stages.

It is known to the person skilled in the art that promoter sequences can be modified (e.g., truncated, fused, mutated) to alarge extent without significantly modifying their transcription properties.

1.1.3 Additional elements The expression cassettes of the invention (or the vectors in which these are comprised) maycomprise further functional elements and genetic control sequences in addition to the promoter active in Tagetes plants. Theterms "functional elements" or "genetic control sequences" are to be understood in the broad sense and refer to all thosesequences, which have an effect on the materialization or the function of the expression cassette according to the invention.For example, genetic control sequences modify the transcription and translation. Genetic control sequences are described(e.g., Goeddel 1990; Gruber 1993 and the references cited therein). Preferably, the expression cassettes according to theinvention encompass a promoter active in Tagetes plants 5 'upstream of the nucleic acid sequence (e.g., encoding the Damino acid metabolizing enzyme), and 3 'downstream a terminator sequence and polyadenylation signals and, ifappropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to beexpressed.

Genetic control sequences and functional elements furthermore also encompass the 5 'untranslated regions, introns or noncoding 3 'region of genes, such as, for example, the actin1 intron, or the AdhlS introns 1, 2 and 6 (general reference: TheMaize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been demonstrated that theymay play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5 'untranslatedsequences can enhance the transient expression of heterologous genes. Examples of translation enhancers which may bementioned are the tobacco mosaic virus 5' leader sequence (Gallie 1987) and the like. Furthermore, they may promote tissuespecificity (Rouster 1998).

Polyadenylation signals which are suitable as genetic control sequences are plant polyadenylation signals, preferably thosewhich correspond essentially to TDNA polyadenylation signals from Agrobacterium tumefaciens. Examples of particularlysuitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator.

The genetic component and/or expression cassette of the invention may comprise further functional elements. Functionalelements may include for example (but shall not be limited to) selectable or screenable marker genes (in addition to the Damino acid metabolizing enzymes). Selectable and screenable markers may include a) negative selection markers; i.e.,markers conferring a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2deoxyglucose, WO 98/45456),antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or glyphosate).Especially preferred negative selection markers are those which confer resistance to herbicides (see below in the

Cotransformation section for details). b) Positive selection markers; i.e. markers conferring a growth advantage to atransformed plant in comparison with a nontransformed one such as the genes and methods described by Ebinuma et al.2000a,b, and in EPA 0 601 092. c) Counter selection markers; i.e. markers suitable to select organisms with defineddeleted sequences comprising said marker (Koprek 1999). Examples comprise the cytosine deaminase codA (Schlaman1997). d) Reporter genes; i.e. markers encoding readily quantifiable proteins (via color or enzyme activity; Schenborn 1999).Preferred are green fluorescent protein (GFP) (Sheen 1995; Haseloff 1997; Reichel 1996; Tian 1997; WO 97/41228; Chui



1996; Leffel 1997), and betaglucuronidase (GUS) being very especially preferred (Jefferson 1987a,b).

Functional elements which may be comprised in a vector of the invention include i) Origins of replication which ensurereplication of the expression cassettes or vectors according to the invention in, for example, E. coli. Examples which maybe mentioned are ORI (origin of DNA replication), the pBR322 ori or the Pl 5 A ori (Maniatis, 1989), ii) Multiple cloning sites(MCS) to enable and facilitate the insertion of one or more nucleic acid sequences (e.g. a gene of interest), iii)Sequenceswhich make possible homologous recombination, marker deletion, or insertion into the genome of a host organism. Methodsbased on the cre/lox (Sauer 1998; Odell 1990; Dale 1991), FLP/FRT (Lysnik 1993), or Ac/Ds system (Wader 1987; US5,225,341; Baker 1987; Lawson 1994) permit a if appropriate tissue specific and/or inducible removal of a specific DNAsequence from the genome of the host organism. Control sequences may in this context mean the specific flankingsequences (e.g., lox sequences), which later allow removal (e.g., by means of ere recombinase) (see also see internationalpatent application PCT/EP

2005/002734; WO 2005/090581)), iv)Elements, for example border sequences, which make possible the Agrobacteriummediated transfer in plant cells for the transfer and integration into the plant genome, such as, for example, the right or leftborder of the TDNA or the vir region.

1.2. The gene of interest

The nucleic acid construct inserted into the genome of the target plant may comprise at least one gene of interest. Theperson skilled in the art is aware of numerous sequences which may be utilized in this context, e.g. to produce chemicals,fine chemicals or pharmaceuticals (e.g. carotenoids), conferring resistance to herbicides, or conferring male sterility.Furthermore, growth, yield, and resistance against abiotic and biotic stress factors (like e.g., fungi, viruses or insects) maybe enhanced. Advantageous properties may be conferred either by overexpressing proteins or by decreasing expression ofendogenous proteins by e.g., expressing a corresponding antisense (Sheehy 1988; US 4,801,340; MoI 1990) or doublestranded RNA (Matzke 2000; Fire 1998; Waterhouse 1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO00/44895; WO 00/49035; WO 00/63364).

For expression of these sequences all promoters suitable for expression of genes in Tagetes can be employed. Preferably,the gene of interest is operated by a promoter which is not identical to the promoter used to express the Damino acidmetabolizing enzyme. Expression can be, for example, constitutive, inducible or development dependent. Variouspromoters are known for expression in Tagetes are known in the art (see above for details).

2. The transformation and selection method of the invention 2.1 Source and preparation of the plant material

Various plant materials can be employed for the transformation procedure disclosed herein. Such plant material may includebut is not limited to for example leaf, root, immature and mature embryos, pollen, meristematic tissues, inflorescences,callus, protoplasts or suspensions of plant cells.

The plant material for transformation can be obtained or isolated from virtually any Tagetes variety or plant. Especiallypreferred are Tagetes plants selected from the the group consisting of Tagetes (T.) patula, T. erecta, T. laxa, T. minuta, Tlucida, T. argentina cabrera, T. tenuifolia, T. lemmonii and T. bipinata.

Although several transformation and regeneration methods based on different Tagetes explants are described in the art,which are all well known to the person skilled in the art, the method of the invention is preferably based on segments ofcotyledons, which more preferably is derived from the mature seeds of a regenerable Tagetes plant.

In a preferred embodiment of the invention, the introduction of a nucleic acid construct into a Tagetes cell or tissuecomprises: (i) providing cotyledonary segments of a Tagetes plant; and (ii) cocultivating said tissue with an Agrobacteriumcomprising a nucleic acid construct comprising a promoter active in said tissue and operably linked thereto a nucleic acidsequence encoding an enzyme capable of metabolizing a Damino acid and/or a derivative thereof. Preferably, thecotyledons are provided by mature seeds of a regenerable Tagetes line.

The method based on cotyledonary segments, preferably from a seedling which is derived from a mature seed of aregenerable Tagetes plant. Segments are cut in 2 to 3 segments, each of a size of 0.3 0.5 cm in width and length.

The time period required for this method is greatly reduced compared to other Agrobacteriummediated transformationprotocols. Viable phenotypically positive Tagetes shoots can be collected 4 to 6 weeks from the initiation of the procedure.Furthermore, the method of the invention is highly genotype and cultivar independent.

The starting material for the transformation process is normally a Tagetes seed. Seeds are first sterilized optionallysoaked for softening. The seeds are then put on germination medium and germinated for a time period of about 3 to 14 days,preferably for about 5 to 8 days, and most preferably for about 7 days. The epicotyl is preferably about 0.5 cm at this time.Preferably germination is carried out under standard culture conditions (i.e. approximately 2624 °C day/night, 16 hourslight/8 hours dark).

2.2 Transformation Procedures 2.2.1 General Techniques

A nucleic acid construct according to the invention may advantageously be introduced into cells using vectors into whichsaid nucleic acid construct is inserted.

Examples of vectors may be plasmids, cosmids, phages, viruses, retroviruses or

Agrobacteria. In an advantageous embodiment, a nucleic acid construct is introduced by means of plasmid vectors.



Preferred vectors are those, which enable the stable integration of the expression cassette into the host genome.

The nucleic acid construct can be introduced into the target plant cells and/or organisms by any of the several means knownto those of skill in the art, a procedure which is termed transformation. Various transformation procedures suitable for

Tagetes have been described. Direct gene transfer als well as indirect gene transfer are suitable methods for transformationof Tagetes tissue or cells. Different tissues may be used as a starting material for transformation, such as cotyledons,leaves, hypocotyls, scions, roots, callus, mature and nonmature seeds, or petals.

For example, the nucleic acid constructs can be introduced directly to plant cells using ballistic methods, such as DNAparticle bombardment, or the nucleic acid construct can be introduced using techniques such as electroporation andmicroinjection of a cell. Particlemediated transformation techniques (also known as "biolistics") are described in, e.g., EPAl270,356; US 5,100,792, EPA444 882, EPA434 616; Klein 1987; Vasil 1993; and Becker 1994). These methods involvepenetration of cells by small particles with the nucleic acid either within the matrix of small beads or particles, or on thesurface. The biolistic PDS1000 Gene Gun (Biorad, Hercules, CA) uses helium pressure to accelerate DNAcoated gold ortungsten microcarriers toward target cells. The process is applicable to a wide range of tissues and cells from organisms,including plants. Other transformation methods are also known to those skilled in the art.

Other techniques include microinjection (WO 92/09696, WO 94/00583, EPA 331 083, EPA 175 966, Green 1987),polyethylene glycol (PEG) mediated transformation (Paszkowski 1984; Lazzeri 1995), liposomebased gene delivery (WO93/24640; Freeman 1984), electroporation (EPA 290 395, WO 87/06614; Fromm 1985; Shimamoto 1992).

In the case of injection or electroporation of DNA into plant cells, the nucleic acid construct to be transformed not need tomeet any particular requirement (in fact the ,,naked" expression cassettes can be utilized). Simple plasmids such as thoseof the pUC series may be used.

2.2.2 Soilborne bacteria mediated transformation (cocultivation) In addition and preferred to these "direct" transformationtechniques, transformation can also be carried out by bacterial infection by means of soil born bacteria such asAgrobacterium tumefaciens or Agrobacterium rhizogenes.

2.2.2.1 Choice of strains, vectors, and cocultivation conditions The soilborne bacterium employed for transfer of a DNA(e.g., TDNA) into Tagetes genome can be any specie of the Rhizobiaceae family. The Rhizobiaceae family comprises thegenera Agrobacterium, Rhizobium, Sinorhizobium, and Allorhizobium are genera within the bacterial family and have beenincluded in the alpha2 subclass of Proteobacteria on the basis of ribosomal characteristics. Members of this family areaerobic, Gramnegative. The cells are normally rodshaped (0.61.0 μm by 1.53.0 μm), occur singly or in pairs, withoutendospore, and are motile by one to six peritrichous flagella. Considerable extracellular polysaccharide slime is usuallyproduced during growth on carbohydratecontaining media. Especially preferred are Rhizobiaceae such as Sinorhizobiummeliloti, Sinorhizobium medicae, Sinorhizobium fredii, Rhizobium sp. NGR234, Rhizobium sp. BR816, Rhizobium sp. N33,Rhizobium sp. GRH2, Sinorhizobium saheli, Sinorhizobium terangae, Rhizobium leguminosarum biovar trifolii, Rhizobiumleguminosarum biovar viciae, Rhizobium leguminosarum biovar phaseoli, Rhizobium tropici, Rhizobium etli, Rhizobiumgalegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium mongolense, Rhizobium lupini,Mesorhizobium loti, Mesorhizobium huakuii, Mesorhizobium ciceri, Mesorhizobium mediterraneium, Mesorhizobiumticmshanense, Bradyrhizobium elkanni, Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Azorhizobium caulinodans,Allobacterium undicola, Phyllobacterium myrsinacearum, Agrobacterium tumefaciens, Agrobacterium radiobacter,Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Preferred are also the strains and method describedin Broothaerts (2005).

The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic genericcircumscription support their amalgamation into a single genus, Rhizobium. The classification and characterization ofAgrobacterium strains including differentiation of Agrobacterium tumefaciens and Agrobacterium rhizogenes and their variousopinetype classes is a practice well known in the art (see for example Laboratory guide for identification of plant pathogenicbacteria, 3rd edition. (2001) Schaad, Jones, and Chun (eds.) ISBN 0890542635; for example the article of Moore et al.published therein). Recent analyses demonstrate that classification by its plantpathogenic properties may not be justified.Accordingly more advanced methods based on genome analysis and comparison (such as 16S rRNA sequencing; RFLP,RepPCR, etc.) are employed to elucidate the relationship of the various strains (see for example Young 2003, Farrand2003, de Bruijn 1996, Vinuesa 1998). The phylogenetic relationships of members of the genus Agrobacterium by twomethods demonstrating the relationship of Agrobacterium strains K599 are presented in Llob 2003.

It is known in the art that not only Agrobacterium but also other soilborne bacteria are capable to mediate TDNA transferprovided that they the relevant functional elements for the TDNA transfer of a Ti or Riplasmid (Klein & Klein 1953;Hooykaas 1977; van Veen 1988).

Preferably, the soilborn bacterium is of the genus Agrobacterium. The term "Agrobacterium" as used herein refers to a soilborne, Gramnegative, rodshaped phytopathogenic bacterium. The species of Agrobacterium, Agrobacterium tumefaciens(syn. Agrobacterium radiobacter), Agrobacterium rhizogenes,

Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with allRhizobium species, based on comparative 16S rDNA analyses (Sawada 1993, Young 2003). Agrobacterium is an artificialgenus comprising plantpathogenic species.

The term Tiplasmid as used herein is referring to a plasmid, which is replicable in Agrobacterium and is in its natural,"armed" form mediating crown gall in Agrobacterium infected plants. Infection of a plant cell with a natural, "armed" form of a



Tiplasmid of Agrobacterium generally results in the production of opines e.g., nopaline, agropine, octopine etc.) by theinfected cell. Thus, Agrobacterium strains which cause production of nopaline e.g., strain LBA4301, C58, A208) are referredto as "nopalinetype" Agrobacteria; Agrobacterium strains which cause production of octopine e.g., strain LBA4404, Ach5,B6) are referred to as "octopinetype" Agrobacteria; and Agrobacterium strains which cause production of agropine e.g.,strain EHA105, EHAlOl, A281) are referred to as "agropinetype" Agrobacteria. A disarmed Tiplasmid is understood as a Tiplasmid lacking its crown gall mediating properties but otherwise providing the functions for plant infection. Preferably, the TDNA region of said "disarmed" plasmid was modified in a way, that beside the border sequences no functional internal Tisequences can be transferred into the plant genome. In a preferred embodiment when used with a binary vector system the entire TDNA region (including the TDNA borders) is deleted. The term Riplasmid as used herein is referring to aplasmid, which is replicable in Agrobacteήum and is in its natural, "armed" form mediating hairyroot disease inAgrobacterium infected plants. Infection of a plant cell with a natural, "armed" form of an Riplasmid of Agrobacteriumgenerally results in the production of opines (specific amino sugar derivatives produced in transformed plant cells such ase.g., agropine, cucumopine, octopine, mikimopine etc.) by the infected cell. Agrobacterium rhizogenes strains aretraditionally distinguished into subclasses in the same way A. tumefaciens strains are. The most common strains areagropinetype strains e.g., characterized by the Riplasmid pRiA4), mannopinetype strains e.g., characterized by the Riplasmid pRi8196) and cucumopinetype strains e.g., characterized by the Riplasmid pRi2659). Some other strains are ofthe mikimopine type e.g., characterized by the Riplasmid pRil723). Mikimopine and cucumopine are stereo isomers but nohomology was found between the pRi plasmids on the nucleotide level (Suzuki 2001). A disarmed Riplasmid is understoodas a Riplasmid lacking its hairyroot disease mediating properties but otherwise providing the functions for plant infection.Preferably, the TDNA region of said "disarmed" Ri plasmid was modified in a way, that beside the border sequences nofunctional internal Risequences could be transferred into the plant genome. In a preferred embodiment when used with abinary vector system the entire TDNA region (including the TDNA borders) is deleted.

The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetictransformation of the plant (Kado 1991). Vectors are based on the Agrobacterium Ti or Riplasmid and utilize a naturalsystem of DNA transfer into the plant genome. As part of this highly developed parasitism Agrobacterium transfers a definedpart of its genomic information (the TDNA; flanked by about 25 bp repeats, named left and right border) into thechromosomal DNA of the plant cell (Zupan 2000). By combined action of the so called vir genes (part of the original Tiplasmids) said DNAtransfer is mediated. For utilization of this natural system, Tiplasmids were developed which lack theoriginal tumor inducing genes ("disarmed vectors"). In a further improvement, the so called "binary vector systems", the TDNA was physically separated from the other functional elements of the Tiplasmid (e.g., the vir genes), by beingincorporated into a shuttle vector, which allowed easier handling (EPA 120 516; US 4,940,838). These binary vectorscomprise (beside the disarmed TDNA with its border sequences), prokaryotic sequences for replication both inAgrobacterium and E. coli. It is an advantage of Agrobacteriummediated transformation that in general only the DNAflanked by the borders is transferred into the genome and that preferentially only one copy is inserted. Descriptions ofAgrobacterium vector systems and methods for Agrobacteriummediated gene transfer are known in the art (Miki 1993;Gruber 1993; Moloney 1989). New and small binary vectors are mentioned in EP 1 294 907.

Hence, for Agrobαcteriαmediated transformation the genetic composition (e.g., comprising an expression cassette) isintegrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is tobe used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid TDNA is linked to the expression cassette to be introduced in the form of a flanking region. Binary vectors are preferablyused. Binary vectors are capable of replication both in E. coli and in Agrobacterium. They may comprise a selection markergene and a linker or polylinker (for insertion of e.g. the expression cassette to be transferred) flanked by the right and left TDNA border sequence. They can be transferred directly into Agrobacterium (Holsters 1978). The selection marker genepermits the selection of transformed Agrobacteria and is, for example, the nptll gene, which confers resistance tokanamycin. The Agrobacterium which acts as the host organism in this case should already contain a plasmid with the virregion. The latter is required for transferring the TDNA to the plant cell. An Agrobacterium transformed in this way can beused for transforming plant cells. The use of TDNA for transforming plant cells has been studied and described intensively(EP 120 516; Hoekema 1985).

Common binary vectors are based on "broad host range"plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson 1985)derived from the Ptype plasmid RK2. Most of these vetors are derivatives of pB INl 9 (Bevan 1984). Various binary vectorsare known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories,Inc. USA). Additional vectors were improved with regard to size and handling (e.g. pPZP; Hajdukiewicz 1994). Improvedvector systems are described also in WO 02/00900.

Preferably the soilborne bacterium is a bacterium belonging to family Agrobacterium, more preferably a disarmedAgrobacterium tumefaciens or rhizogenes strain. In a preferred embodiment, Agrobacterium strains for use in the practice ofthe invention include octopine strains, e.g., LBA4404 or agropine strains, e.g., EHAlOl or EHAl 05. Suitable strains of A.tumefaciens for DNA transfer are for example EHAlOl [pEHAlOl] (Hood 1986), EHA105[pEHA105] (Li 1992),LBA4404[pAL4404] (Hoekema 1983), C58Cl [pMP90] (Koncz & Schell 1986), and C58Cl[pGV2260] (Deblaere 1985). Othersuitable strains are Agrobacterium tumefaciens C58, a nopaline strain. Other suitable strains are A. tumefaciens C58C1(Van Larebeke 1974), A136 (Watson 1975) or LBA4011 (Klapwijk 1980). In another preferred embodiment the soilbornebacterium is a disarmed strain variant of Agrobacterium rhizogenes strain K599 (NCPPB 2659). Such strains are described

in US provisional application Application No. 60/606,789, filed September 2nd, 2004, and international applicationPCT/EP2005/009366 hereby incorporated entirely by reference. A binary vector or any other vector can be modified bycommon DNA recombination techniques, multiplied in E. coli, and introduced into Agrobacterium by e.g., electroporation orother transformation techniques (Mozo 1991).



Agrobacteria are grown and used in a manner as known in the art. The vector comprising Agrobacterium strain may, forexample, be grown for 3 days on YEP medium (10 g/1 yeast extract, 10 g/1 peptone, 5 g/1 NaCl, 8 g/1 agar, pH 6.8; seeExample 3) supplemented with the appropriate antibiotic (e.g., 50 mg/1 kanamycin sulfate). Bacteria are collected with aloop from the solid medium and resuspended. In a preferred embodiment of the invention, Agrobacterium cultures are startedby use of aliquots frozen at 80°C. For Agrobacterium treatment of the Tagetes cotyledon explants, the bacteria arepreferably resuspended in a cocultivation medium. The concentration of Agrobacterium used for infection, direct contacttime, and co cultivation may need to be varied. Thus, generally a range of Agrobacterium concentrations from OD6O0 0.1 to

3.0. Preferably for the explants the following concentrations of Agrobacterium suspensions are employed from about OD60O= 0.1 to about 3, preferably from about OD600 = 0.5 to 2, more preferably from about OD600 = 0.7 to 1.2.

The explants are then inoculated with the Agrobacterium culture for a few minutes to a few hours, typically about 10 minutesto 3 hours, preferably about 0.5 hours to 1 hour. The excess media is drained and the Agrobacterium are permitted to cocultivate with the meristem tissue for about 1 to about 6 days, preferably about 3 to about 5 days for Agrobacteriumtumefaciens strains, and about 2 to about 3 days for Agrobacterium rhizogenes strains, preferably in the dark at about 26°Cduring the day and at about 24°C during the night. During this step, the Agrobacterium transfers the foreign genetic constructinto some cells in the Tagetes explants. Normally no selection compound is present during this step.

2.2.2.2 Modifications for enhancing transformation efficiency Supplementation of the coculture medium with ethyleneinhibitors (e.g., silver nitrate), phenolabsorbing compounds (like polyvinylpyrrolidone, Perl 1996) or antioxidants (such asthiol compounds, e.g., dithiothreitol, Lcysteine, Olhoft 2001) which can decrease tissue necrosis due to plant defenseresponses (like phenolic oxidation) may further improve the efficiency of Agrobacteriummcdiated transformation.

Supplementation of the cocultivation medium with antioxidants (e.g., dithiothreitol), or thiol compounds (e.g., Lcysteine,Olhoft 2001; US2001034888) which can decrease tissue necrosis due to plant defense responses (like phenolic oxidation)may further improve the efficiency of Agrobαcteriummediated transformation. In another preferred embodiment, the cocultivation medium of comprises least one thiol compound, preferably selected from the group consisting of sodiumthiolsulfate, dithiotrietol (DTT) and cysteine. Preferably the concentration is between about 1 mM and 1OmM of LCysteine,0.1 mM to 5 mM DTT, and/or 0.1 mM to 5 mM sodium thiolsulfate.

The target tissue and/or the Agrobαcteriα may be treated with a phenolic compound prior to or during the Agrobαcterium cocultivation. "Plant phenolic compounds" or "plant phenolics" suitable within the scope of the invention are those isolatedsubstituted phenolic molecules which are capable to induce a positive chemotactic response, particularly those who arecapable to induce increased vir gene expression in a Tiplasmid containing Agrobαcterium sp., particularly a Tiplasmidcontaining Agrobαcterium tumefαciens. A preferred plant phenolic compound is acetosyringone (3,5dimethoxy4hydroxyacetophenone). Certain compounds, such as osmoprotectants (e.g. Lproline preferably at a concentration of about2001000 mg/L or betaine), phytohormes (inter alia NAA), opines, or sugars, act synergistically when added in combinationwith plant phenolic compounds.

Particularly suited induction conditions for Agrobacterium tumefaciens have been described (Vernade 1988). Efficiency oftransformation with Agrobacterium can be enhanced by numerous other methods known in the art like for example vacuuminfiltration (WO 00/58484), heat shock and/or centrifugation, addition of silver nitrate, sonication etc.

Preferably the method of the invention comprises one or more additional steps selected from the group of:

(al) wounding the explant prior to, during or immediately after cocultivation, and (bl) transferring said cocultivated explantsafter step (b) to a medium comprising at least one antibiotic suitable to inhibit Agrobacterium growth, and optionally atleast one plant growth factor, wherein said medium is preferably lacking Dalanine and/or Dserine or a derivative thereof in aphytotoxic concentration, and, (b2) further incubating said explant after step (b ) and optionally (bl) on a shoot inductionmedium comprising at least one plant growth factor, wherein said shoot induction medium is preferably lacking Dalanineand/or Dserine or a derivative thereof in a phytotoxic concentration, and

(cl) transferring said shoots after step (c or b2) to a shoot elongation medium comprising

(i) at least one plant growth factor in a concentration suitable to allow shoot elongation, and (ii) optionally a Damino acid or aderivative thereof in a total concentration from about 0.05 mM to about 100 mM, and cultivating said transferred shoots onsaid shoot elongation medium until said shoots have elongated to a length of at least about 2 cm.

Wounding by cutting can be prior to inoculation (cocultivation), during inoculation or after inoculation with Agrobacterium.For achieving both beneficial effects wounding is preferably done prior to or during cocultivation, more preferably prior to cocultivation. Many methods of wounding can be used, including, for example, cutting, abrading, piercing, poking, penetrationwith fine particles or pressurized fluids, plasma wounding, application of hyperbaric pressure, or sonication. Wounding canbe performed using objects such as, but not limited to, scalpels, scissors, needles, abrasive objects, airbrush, particles,electric gene guns, or sound waves. Another alternative to enhance efficiency of the cocultivation step is vacuum infiltration(Bechtold 1998; Trieu 2000).

2.3 Post cocultivation treatment

After the cocultivation it is preferred to remove the soilborne bacteria by washing and/or treatment with appropriateantibiotics. In consequence, the medium employed after the cocultivation step e.g., the medium employed in additionalsteps (bl), (b2), and/or (cl) preferably contains a bacteriocide (antibiotic). This step is intended to terminate or at least retardthe growth of the nontransformed cells and kill the remaining Agrobacterium cells. Accordingly, the method of the invention



comprises preferably the step of: (bl) transferring said cotyledonary segments after step (b) to a medium comprising at leastone antibiotic suitable to inhibit Agrobacterium growth, and optionally at least one plant growth factor, wherein saidmedium is preferably lacking Dalanine and/or Dserine or a derivative thereof in a phytotoxic concentration, and,

Preferred antibiotics to be employed are e.g., carbenicillin (500 mg/L or preferably 100 mg/L) or Timentin™(GlaxoSmithKline; used preferably at a concentration of about 250500 mg/L; Timentin™ is a mixture of ticarcillin disodiumand clavulanate potassium; 0.8 g Timentin™ contains 50 mg clavulanic acid with 750 mg ticarcillin. Chemically, ticarcillindisodium is N(2Carboxy3,3 dimethyl7oxo4thialazabicyclo[3.2.0]hept6yl)3thiophenemalonamic acid disodium salt.Chemically, clavulanate potassium is potassium (Z)(2R, 5R)3(2 hydroxyethylidene)7oxo4oxalazabicyclo [3.2.0]heptane2carboxylate).

2.4 Selection

Agrobacteriummediated techniques typically result in gene delivery into a very limited number of cells in the targeted tissue.Especially for Tαgetes transformation efficiencies (without selection) are in general very low. This problem is overcome bythe selection protocol based on Damino acid metabolizing enzymes provided herein.

Thus, after cocultivation and optionally a recovery step (see below) the target tissue (e.g., the cotyledonary tissue) istransferred to and incubated on a selection medium.

It is preferred that freshly transformed (cocultivated) explants are incubated for a certain time from about 1 hour to about 10days, preferably from 1 day to 8 days, more preferably from about 4 to about 7 days after cocultivation (step (b) or (bl)) on amedium lacking the selection compound (e.g. Dalanine and/or Dserine or a derivative thereof in a phytotoxicconcentration). Establishment of a reliable resistance level against said selection compound needs some time to preventunintended damage by the selection compound even to the transformed cells and tissue. Accordingly, the method of theinvention may comprise a step between co cultivation and selection, which is carried out without a selection compound.During this recovery period shoot induction (see below) may already be initiated.

The selection medium comprises at least one Damino acid or a derivative thereof in a phytotoxic concentration (i.e., in aconcentration which either terminates or at least retard the growth of the nontransformed cells). The term "phytotoxic","phytotoxicity" or "phytotoxic effect" as used herein is intended to mean any measurable, negative effect on the physiologyof a plant or plant cell resulting in symptoms including (but not limited to) for example reduced or impaired growth, reduced orimpaired photosynthesis, reduced or impaired cell division, reduced or impaired regeneration (e.g., of a mature plant from acell culture, callus, or shoot etc.), reduced or impaired fertility etc. Phytotoxicity may further include effects like e.g.,necrosis or apoptosis. In a preferred embodiment results in a reduction of growth or regenerability of at least 50%, preferablyat least 80%, more preferably at least 90% in comparison with a plant which was not treated with said phytotoxiccompound.

The specific compound employed for selection is chosen depending on which marker protein is expressed. For example incases where the E.coli Dserine ammonialyase is employed, selection is done on a medium comprising Dserine. In caseswhere the Rhodotorula gracilis Damino acid oxidase is employed, selection is done on a medium comprising Dalanineand/or Dserine.

The fact that Damino acids are employed does not rule out the presence of Lamino acid structures or Lamino acids. Forsome applications it may be preferred (e.g., for cost reasons) to apply a racemic mixture of D and Lamino acids (or amixture with enriched content of Damino acids). Preferably, the ratio of the Damino acid to the corresponding Lenantiomeris at least 1 :1, preferably 2:1, more preferably 5:1, most preferably 10:1 or 100:1. The use of Dalanine has the advantagethat racemic mixtures of D and Lalanine can be applied without disturbing or detrimental effects of the Lenantiomer.Therefore, in an improved embodiment a racemic mixture of D/Lalanine is employed as compound.

The term "derivative" with respect to Damino acids means chemical compound, which comprise the respective Daminoacid structure, but are chemically modified. As used herein the term a "Damino acid structure" (such as a "Dserinestructure") is intended to include the Damino acid, as well as analogues, derivatives and mimetics of the Damino acid thatmaintain the functional activity of the compound. As used herein, a "derivative" also refers to a form of a Damino acid inwhich one or more reaction groups on the compound have been derivatized with a substituent group. The Damino acidemployed may be modified by an aminoterminal or a carboxy terminal modifying group or by modification of the sidechain.The aminoterminal modifying group may be for example selected from the group consisting of phenylacetyl,diphenylacetyl, triphenylacetyl, butanoyl, isobutanoyl hexanoyl, propionyl, 3hydroxybutanoyl, 4hydroxybutanoyl, 3hydroxypropionoyl, 2,4 dihydroxybutyroyl, 1Adamantanecarbonyl, 4methylvaleryl, 2 hydroxyphenylacetyl, 3hydroxyphenylacetyl, 4hydroxyphenylacetyl, 3,5 dihydroxy2naphthoyl, 3,7dihydroxy2napthoyl, 2hydroxycinnamoyl, 3hydroxycinnamoyl, 4hydroxycinnamoyl, hydrocinnamoyl, 4formylcinnamoyl, 3 hydroxy4methoxycinnamoyl, 4hydroxy3methoxycinnamoyl, 2 carboxycinnamoyl, 3,4,dihydroxyhydrocinnamoyl, 3,4dihydroxycinnamoyl, trans Cinnamoyl, (±)mandelyl, (±)mandelyl(±)mandelyl, glycolyl, 3formylbenzoyl, 4 formylbenzoyl, 2formylphenoxyacetyl, 8formyllnapthoyl,4

(hydroxymethyl)benzoyl, 3hydroxybenzoyl, 4hydroxybenzoyl, 5hydantoinacetyl, Lhydroorotyl, 2,4dihydroxybenzoyl, 3benzoylpropanoyl, (±)2,4dihydroxy3,3 dimethylbutanoyl, DL3(4hydroxyphenyl)lactyl, 3(2hydroxyphenyl)propionyl, 4 (2hydroxyphenyl)propionyl, D3phenyllactyl, 3(4hydroxyphenyl)propionyl, L3 phenyllactyl, 3pyridylacetyl, 4pyridylacetyl,isonicotinoyl, 4quinolinecarboxyl, 1 isoquinolinecarboxyl and 3isoquinolinecarboxyl. The carboxyterminal modifying groupmay be for example selected from the group consisting of an amide group, an alkyl amide group, an aryl amide group anda hydroxy group. The "derivative" as used herein is intended to include molecules which, mimic the chemical structure of a



respective Damino acid structure and retain the functional properties of the D amino acid structure. Approaches todesigning amino acid or peptide analogs, derivatives and mimetics are known in the art (e.g., see Farmer 1980; Ball 1990;Morgan 1989; Freidinger 1989; Sawyer 1995; Smith 1995; Smith 1994; Hirschman 1993). Other possible modificationsinclude Nalkyl (or aryl) substitutions, or backbone crosslinking to construct lactams and other cyclic structures. Otherderivatives include Cterminal hydroxymethyl derivatives, Omodifϊed derivatives (e.g., Cterminal hydroxymethyl benzylether), Nterminally modified derivatives including substituted amides such as alkylamides and hydrazides. Furthermore, Damino acid structure comprising herbicidal compounds may be employed. Such compounds are for example described in US5,059,239, and may include (but shall not be limited to) NbenzoylN(3chloro4fluorophenyl)DLalanine, NbenzoylN (3chloro4fluorophenyl) DLalanine methyl ester, NbenzoylN(3chloro4 fluorophenyl)DLalanine ethyl ester, NbenzoylN(3chloro4fluorophenyl)/J) alanine, NbenzoylN(3chloro4fluorophenyl)Z)α/ύf«/«e methyl ester, or N benzoylN(3chloro4fluorophenyl)Dα/α«me isopropyl ester.

The selection compound (Damino acids or derivatives thereof in a phytotoxic concentration) may be used in combinationwith other substances. For the purpose of application, the selection compound may also be used together with the adjuvantsconventionally employed in the art of formulation, and are therefore formulated in known manner, e.g. into emulsifiableconcentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, solublepowders, dusts, granulates, and also encapsulations in e.g. polymer substances. As with the nature of the compositions tobe used, the methods of application, such as spraying, atomising, dusting, scattering, coating or pouring, are chosen inaccordance with the intended objectives and the prevailing circumstances. However, more preferably the selectioncompound is directly applied to the medium. It is an advantage that stock solutions of the selection compound can be madeand stored at room temperature for an extended period without a loss of selection efficiency.

The optimal concentration of the selection compound (i.e. Damino acids, derivatives thereof or any combination thereof)may vary depending on the target tissue employed for transformation but in general (and preferably for transformation ofcotyledonary tissue) the total concentration (i.e. the sum in case of a mixture) of D amino acids or derivatives thereof is inthe range from about 0.05 mM to about 100 mM. For example in cases where the E.coli Dserine ammonialyase isemployed, selection is done on a medium comprising Dserine (e.g., incorporated into agar solidified MS media plates),preferably in a concentration from about 0.05 mM to about 100 mM, preferably about 0.1 mM to about 50 mM, morepreferably about 0.3 mM to about 5 mM. In cases where the Rhodotorula gracilis Damino acid oxidase is employed,selection is done on a medium comprising Dalanine and/or Dserine (e.g., incorporated into agarsolidified MS mediaplates), preferably in a total concentration from about 0.05 mM to about 100 mM, preferably about 0.1 mM to about 50 mM,more preferably about 0.3 mM to about 5 mM. Preferably, Dalanine (e.g., if employed as only selection compound) isemployed in a concentration of about 0.05 mM to about 100 mM, preferably about 0.1 mM to about 50 mM, more preferablyabout 0.3 mM to about 5 mM. Preferably, Dserine (e.g., if employed as only selection compound) is employed in aconcentration of about 0.05 mM to about 100 mM, preferably about 0.1 mM to about 50 mM, more preferably about 0.3 mMto about 5 mM.

Also the selection time may vary depending on the target tissue used and the regeneration protocol employed. The selectionpressure (by presence of the selection compound) by be hold for the entire regeneration process including shoot induction,shoot elongation, and rooting.

In general a selection time is at least about 5 days, preferably at least about 14 days. More specifically the total selectiontime under dedifferentiating conditions (i.e., callus or shoot induction) is from about 1 to about 10 weeks, preferably, about 3to 7 weeks, more preferably about 3 to 4 weeks. However, it is preferred that the selection under the dedifferentiatingconditions is employed for not longer than 70 days. Preferably, wherein selection is done using about 0.05 to about 100 mMDalanine and/or Dserine for about 3 to 4 weeks under dedifferentiating conditions. In between the selection period theexplants may be transferred to fresh selection medium one or more times. For the specific protocol provided herein it ispreferred that two selection medium steps (e.g., one transfer to new selection medium) is employed. Preferably, theselection of step is done in two steps, using a first selection step for about 14 to 20 days, then transferring the survivingcells or tissue to a second selection medium with essentially the same composition than the first selection medium foradditional 14 to 20 days. However, it is also possible to apply a single step selection. The presence of the Damino acidmetabolizing enzymes does not rule out that additional markers are employed.

After the cocultivation step (and an optional recovery step) the cocultivated explants are incubated on a shoot inductionmedium comprising at least one plant growth factor. Said incubation on shoot induction medium can be started immediatelyafter the cocultivation step (i.e. in parallel with step (bl) for inhibiting growth of the Agrobacteria) or after other intermediatesteps such as (bl) (inhibiting growth of the Agrobacteria) and/or (b2) (regeneration without selection compound; see below).

2.5 Regeneration of fertile Tagetes

The media employed for shoot induction (and/or shoot elongation) are preferably supplemented with one or more plant growthregulator, like e.g., cytokinin compounds (e.g., 6benzylaminopurine) and/or auxin compounds (e.g., 2,4D). The term "plantgrowth regulator" (PGR) as used herein means naturally occurring or synthetic (not naturally occurring) compounds that canregulate plant growth and development. PGRs may act singly or in consort with one another or with other compounds (e.g.,sugars, amino acids). The term "auxin" or "auxin compounds" comprises compounds, which stimulate cellular elongationand division, differentiation of vascular tissue, fruit development, formation of adventitious roots, production of ethylene, and in high concentrations induce dedifferentiation (callus formation). The most common naturally occurring auxin isindoleacetic acid (IAA), which is transported polarly in roots and stems. Synthetic auxins are used extensively in modernagriculture. Synthetic auxin compounds comprise indole3butyric acid (IBA), naphthylacetic acid (NAA), and 2,4dichlorphenoxyacetic acid (2,4D). Compounds that induce shoot formation may include, but not limited to, IAA, NAA, IBA,



cytokinins, auxins, kinetins, glyphosate, and thiadiazuron. The term "cytokinin" or "cytokinin compound" comprisescompounds, which stimulate cellular division, expansion of cotyledons, and growth of lateral buds. They delay senescenceof detached leaves and, in combination with auxins (e.g. IAA), may influence formation of roots and shoots. Cytokinincompounds may comprise, for example, 6 isopentenyladenine (IPA) and 6benzyladenine/6benzylaminopurine (BAP). Inone preferred embodiment, inoculated cotyledon segments are incubated in the presence of 0.1 to 5 mg/L IAA and 1 to 10mg/L BAP to induce shoot formation (Selection Medium SMl + selective agent). Preferably 1 and 5 mg/L, respectively, areused.

Preferably, after 2 to 3 weeks the explants are transferred to fresh SM2 medium containing both PGRs at reducedconcentration and preferably the selection compound. During this period, buds enlarge and small shoots begin to form. Afterthis period shoots, buds and bud clusters are cut from original plant tissues and subcultured for another 2 weeks on SM3medium which is preferably free of PGRs, but contains the selective agent. Within the next two weeks individual, putativetransgenic shoots began to establish themselves under selection pressure and occasionally began to form roots while nontransgenic shoots grew more slowly or start to deteriorate depending on selective agent.

These individual, putative transgenic shoots were then cut at the bases of their stems and subcultured to fresh medium forcontinued development (elongation and root formation). After additional 1 to 6 weeks (preferably 2 x 2 weeks), leaves andputative transgenic shoots are sampled for molecular analysis to determine transgenicity. Acoording to the analysis resulttransgenic plants are chosen and transferred directly into soil.

Rooted shoots are hardened in a tenth with adjusted humidity (80 100%, relative humidity) for 2 to 3 weeks beforetransferring to the greenhouse. Regenerated plants obtained using this method are fertile and have produced on average 2 to1O g seeds per plant. The T0 plants created by this technique are transgenic plants and are regularly recovered with quite

reasonable yields.

Transformed plant material (e.g., cells, tissues or plantlets), which express marker genes, are capable of developing in thepresence of concentrations of a corresponding selection compound which suppresses growth of an untransformed wild typetissue. The resulting plants can be bred and hybridized in the customary fashion. Two or more generations should be grownin order to ensure that the genomic integration is stable and hereditary.

Another embodiment of the invention relates to the Tagetes cells and plants made by the method provided hereunder. Thus,another embodiment relates to a Tagetes plant or cell comprising a nucleic acid construct comprising a promoter active insaid Tagetes plants or cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable ofmetabolizing a Damino acid, wherein said promoter is heterologous in relation to said enzyme encoding sequence.Preferably, the promoter and/or the enzyme capable of metabolizing a Damino acid are defined as above. More preferably,said Tagetes plant or cell is further comprising at least one gene of interest. Other embodiments of the invention relate toparts of said Tagetes plant including but not limited to Tagetes petals and their use for production of carotenoids.

In one preferred embodiment the Tagetes plant selected from the group consisting of Tagetes (T.) patula, T. erecta, T laxa,T minuta, T. lucida, T. argentina cabrera, T. tenuifolia, T. lemmonii and T. bipinata.

The resulting transgenic plants can be self pollinated or crossed with other Tagetes plants. Tl seeds are harvested, dried andstored properly with adequate label on the seed bags. Two or more generations should be grown in order to ensure that thegenomic integration is stable and hereditary. For example transgenic events in Tl or T2 generations could be involved in prebreeding hybridization program for combining different transgenes (gene stacking). Other important aspects of the inventioninclude the progeny of the transgenic plants prepared by the disclosed methods, as well as the cells derived from suchprogeny, and the seeds obtained from such progeny.

2.6 Generation of descendants After transformation, selection and regeneration of a transgenic plant (comprising the nucleicacid construct of the invention) descendants are generated, which either i) carry the marker sequence or ii) because of theactivity of the excision promoter underwent excision and do not comprise the marker sequence(s) and expression cassettefor the endonuclease.

Descendants can be generated by sexual or nonsexual propagation. Nonsexual propagation can be realized by introductionof somatic embryogenesis by techniques well known in the art. Preferably, descendants are generated by sexualpropagation / fertilization. Fertilization can be realized either by selfing (selfpollination) or crossing with other transgenic ornontransgenic plants. The transgenic plant of the invention can herein function either as maternal or paternal plant. After thefertilization process, seeds are harvested, germinated and grown into mature plants. Isolation and identification ofdescendants, which underwent the excision process can be done at any stage of plant development. Methods for saididentification are well known in the art and may comprise for example PCR analysis, Northern blot, Southern blot, orphenotypic screening (e.g., for a negative selection marker). Descendants may comprise one or more copies of the at leastone gene of interest. Preferably, descendants are isolated which only comprise one copy of said gene of interest.

Also in accordance with the invention are cells, cell cultures, parts such as, for example, in the case of transgenic plantorganisms, roots, leaves and the like derived from the abovedescribed transgenic organisms, and transgenic propagationmaterial (such as petals).

A further subject matter of the invention relates to the use of the abovedescribed transgenic organisms according to theinvention and the cells, cell cultures, parts such as, for example, in the case of transgenic plant organisms, petals and thelike derived from them, for the production of pharmaceuticals or fine chemicals. Fine chemicals is understood as meaningenzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavors, aromas and colorants. Especially



preferred is the production of tocopherols and tocotrienols, and of carotenoids. Culturing the transformed host organisms,and isolation from the host organisms or from the culture medium, is performed by methods known to the skilled worker. Theproduction of pharmaceuticals such as, for example, antibodies or vaccines, is described (e.g., by Hood 1999; Ma 1999).

3. Further modifications

It is intended to use plants of Tagetes erecta for the production of carotenoids, especially of astaxanthin. Synthesis of thosenovel carotenoids, naturally not present in Tagetes erecta, shall take place in the flowers, preferentially in the petals ofTagetes. However, accumulation of carotenoids in other tissues is also acceptable. The flowers of Tagetes erecta naturallyaccumulate significant amounts of alpha carotenoids, usually about 90%, and only low amounts of betacarotenoids.However, betacarotenoids, especially betacarotene and zeaxanthin, serve as substrates for an enzymatic activity whichintroduces ketogroups. This enzyme activity, the betacarotene ketolase, synthesizes echinenone and canthaxanthin whichcan be converted to astaxanthin, hydroxyechinenone, adonirubin and adonixanthin by a hydroxylase activity.

Currently, crystalline astaxanthin is used in fish diets for the pigmentation of salmonids. Application of astaxanthin is alsoknown the farming of shrimps.

Astaxanthin is used in those diets as it contributes to the reddish pigmentation of salmon and shrimps which are requestedby the customers. It is undesirable to have additional carotenoids present which are known to add yellow pigmentation, e.g.lutein and zeaxanthin.

Prerequisite for the economical synthesis of astaxanthin is therefore to i) avoid lutein and ii) to increase the levels ofsubstrates (.e.g. betacarotene).

A procedure to create Tagetes plants is described which are i) devoid of lutein due to a knockout mutation in the alphacarotenoid pathway, ii) transformable and therefore accessible to genetic engineering.

Luteindepleted Tagetes plants are transformed by a genetic construct carrying the ketolase gene from Haematococcuspluvialis. Those transgenic plants show a red flower phenotype, in contrast to the yellow flower type of untransformedcontrol plants. Examples

Example 1: Generation of luteindepleted Tagetes plant.

The generation of a luteindepleted Tagetes plant has been disclosed in US 6,784,351 which is herewith completelyincorporated by reference.

Naturally occurring Tagetes plants which accumulate relatively high concentrations of betacarotenoids while most of thealphacarotenoids do not accumulate, are not known. Therefore, it was the task to develop a Tagetes plant which fulfills therequirements of i) being largely devoid of lutein and associated alphacarotenoids in its flower petals and ii) beingcharacterized with relatively high levels of total carotenoids .

The process for creating Tagetes plants with luteindepleted flowers is described in the US patent No. 6,784,351. Thispatent describes in detail i) the EMS mutagenesis of Tagetes erecta "Scarletade" and "13819", and ii) the HPLC screeningprocedure to identify certain abnormal carotenoid profiles in flowers of "Scarletade" and "13819". The especially interestingmutant of Scarletade, 124 257, is described by its changed carotenoid profile in petals and leaves.

Example 2: Breeding of 3136020908 and 31360209.

Tagetes erecta selection 124257, described in U.S. Patent No. 6,784,351, was found to have a low transformation rateusing the identified tissue culture regeneration medium and Agrobacterium transformation technique. Using a standardizedmethod, different plant selections can be transformed at different rates; therefore, to recover a target number of transformedplants, it can be expected that a selection having a low transformation rate would require use of a higher number of explantsthan a selection having a high transformation rate. A selection having a low transformation rate would require at least about200 explants to recover about 1 transformed plant.

Instead of further optimizing the transformation protocol, a plant breeding backcross technique well known to those skilled inthe art was used to transfer the mutation resulting in the increased zeaxanthin to lutein ratio of selection 124257 to aselection having a higher transformation rate. Several Tagetes erecta marigold plants were identified as having acceptabletransformation rates, and from these Tagetes erecta marigold plant named 13819 was selected. Tagetes erecta 13819 is aproprietary breeding selection of PanAmerican Seed located at 622 Town Road, West Chicago,

Illinois 60185.

In the backcross program, selection 124257 was used as the female parent in a cross with a selection of 13819 as the maleparent. The resulting population was identified as 11754, and from this population a plant identified as 117542F wasselected based on its hybrid characteristics. Plant 117542F was selfed and from this population plant identified as 117542F1 was selected based on carotenoid profile and plant habit. Plant 117542F1 was used as male parent in a cross with13819 as the female parent. The resulting population was identified as 31360, and from this population a plant identified as313602 was selected based on carotenoid profile and plant habit. Plant 313602 was selfed and from the resultingpopulation, plants 31360208 and 31360209 were selected based on carotenoid profile, total carotenoid concentration, andplant habit. Both selections were selfed and seed from the cross was used to test transformation rates, and the seedlingsfrom both selections were found to have acceptable transformation rates. In addition, the resulting plants from the selfed31360208 plant were found to be uniform for carotenoid profile, carotenoid concentration, and plant habit. The resulting



plants from the selfed 31360209 plant were found to segregate for total carotenoid concentration and plant habitcharacteristics. From the selfed 31360209 population, a plant identified as 313602 0908 was selected based oncarotenoid profile, total carotenoid concentration, and plant habit. The selfed population from the 3136020908 plant wasfound to be uniform for carotenoid profile, carotenoid concentration, and to have an acceptable transformation rate.

Example 3: Agrobacteriummediated transformation of Tagetes.

In vitro seed germination:

Approximately 200 to 600 (preferably 400) mature seeds of a regenerable marigold line (3136020908 or 31360209) wereplaced in a 125 ml Erlenmeyer flask along with a magnetic stir bar and 100 ml of a 15% Clorox bleach solution (0.6%NaOCl), containing 1 drop of Tween20 per 100 ml solution. The flask was placed in a vacuum chamber and stirred (undervacuum pressure of 625 mmHg) for 15 minutes with constant agitation (stir plate setting of 6). After the vacuum treatment,the spent bleach was removed, replaced with 100 ml of fresh bleach solution and seeds were stirred (without vacuum) for anadditional 15minutes. After the final bleach treatment, the seeds were rinsed 34 times with sterile water and plated ontoMurashigi and Skoog basal medium [MS medium = Murashigi and Skoog basal salts & vitamins] + 30 grams/L sucrose, 7.0grams/L agar (SigmaAldrich Corp. St. Louis MO 63178) in 100 x 15 mm Petri dishes (1015 seeds/plate). All media wereadjusted to pH 5,75. After plating the seeds, cultures were sealed with Parafilm and germinated for 3 to 14 days (preferably7 days) under standard culture conditions (i.e. approximately 2624° C day/night, 16 hours light/8 hours dark).

Explant preparation:

At least 2 hours prior to Agrobacterium inoculation, individual cotyledons were cut into 2 or 3 segments and cultured, adaxialside down, onto Marigold Regeneration Medium #1 (MRMl = Vi strength MS macronutrients, full strength MS mironutrientsand vitamins, 1.0 mg/L IAA, 5.0 mg/L BAP, 2.0 mg/L glycine, 250 mg/L peptone, 2 grams/L glucose, 30 grams/L sucrose, &8 grams/L Agar) + 150 μM acetosyringone, in 100 x 15 mm Petri plates (50 explants per plate). All media were adjusted topH 5,75.

Agrobacterium tumefaciens preparation:

Agrobacterium glycerol stock cultures are removed from a 80° C freezer, allowed to thaw and 200 μl were transferred to 50ml of liquid YEP medium (YEP: 10 grams/L yeast extract, 10 grams/L peptone, 5 grams/L NaCl ) + 50 mg/L kanamycinsulfate, in a 250 ml Erlenmeyer flask. This culture was placed on a rotary shaker set at 225 rpm and incubated overnight

(preferably 16 hours), in the dark at a temperature of 280C. After overnight incubation, the bacterial suspension wastransferred to a 50 ml conical, screwcap centrifuge tube and centrifuged for 10 minutes at 4000 rpm, at a temperature of

40C. After centrifugation the supernatant was discarded and the bacterial pellet resuspended in fresh YEP medium with noantibiotics (resuspension is done in variable volumes, dependent on the number of inoculations planned). The cultures werereturned to the shaker and incubated until the titer reached an optical density (OD) ranging from 0.1 1 (preferably an OD =1) measured at 600 nm by a Spectronic Genesys5, spectrophotometer. Approximately 23 minutes prior to inoculation, ImI ofSilwet stock solution (Stock solution = 1.5μl Silwet L77 wetting agent, per ImI YEP medium) was added per 30 ml ofbacterial suspension.

Agrobacterium inoculation and cocultivation:

Fifty explants (contents of one entire plate) were transferred to approximately 5 ml of Agrobacterium suspension in a 15 ml,sterile screw cap, conical, centrifuge tube. This tube was vortexed for approximately 10 seconds (constant vortexing atsetting #6). After vortexing, the explants were damped dry with sterile filter paper and transferred, adaxial side down, toMRMl medium + 150 μM acetosyringone. The lids were placed on the plates but left unsealed, and cultures

(bacteria/explants) were allowed to cocultivate in the dark for 16 days (preferably 3 days) at about 260C day/24°C nighttemperatures.

Selection of transgenic lines:

After cocultivation, explants were transferred (9 explants per plate), adaxial side down, onto Selection Medium #1 [SMl =MRMl + 500mg/L Timentin and selective agent (e.g. Dserine, Dalanine, phosphinothricin etc.)]. Plates were sealed with airpermeable venting tape and incubated for 2 weeks under standard culture conditions (i.e. approximately 26° C day/24°Cnight temperature, 16 hours light/8 hours dark). After 2 weeks of culture, obvious shootbud regeneration was visible onresponding explants. Whole explants, having shoot/bud regeneration were then transferred to Selection Medium #2 [SM2 =MS medium, 0.1 mg/L IAA, 0.5 mg/L BAP, 8 grams/L agar, 500mg/L Timentin and selective agent (e.g. Dserine, Dalanine,phosphinothricin etc.)] and incubated for 2 weeks under standard culture conditions (i.e. approximately 26° C day/24°C nighttemperature, 16 hours light/8 hours dark). During this period, buds enlarge and small shoots begin to form. After 2 weeks ofincubation on SM2, shoots, buds and bud clusters were cut from original explant tissues and subcultured to SelectionMedium #3 [SM3 = MS medium, 8 grams/L agar, 500mg/L Timentin and selective agent (e.g. Dserine, Dalanine,phosphinothricin etc.)] and incubated for 2 weeks under standard conditions (i.e. 26° C day/24°C night temperature, 16hours light/8 hours dark). After 2 weeks on SM3, individual, putative transgenic shoots began to establish themselves underselection pressure and occasionally began to form roots while nontransgenic shoots grew more slowly or start to deterioratedepending on selective agent. These individual, putative transgenic shoots were then cut at the bases of their stems andsubcultured to fresh SM3 for continued development. After an additional 2 weeks of culture, putative transgenic shootswere, normally, easily distinguishable from non transgenic shoots by their vigorous root and shoot growth compared to nontransgenic shoots. At this time, individual (independent lines) putative transgenic shoots were subcultured one final time tofresh SM3 medium, each line in a separate vessel. After a final week of culture, tissue samples (young healthy leaves) were



collected from each line and evaluated via TaqMan molecular analysis for gene copy number and vector backboneintegration.

Example 4: Test for transgenicity via TaqMan Copy Assay.

Tissue samples were taken during tissue culture, approximately 9 weeks after start of transformation. Leaf segments wereplaced into 96 well plates and DNA was extracted using MagAttract 96 DNA Plant Kit (Qiagen Catalog #67163)

Setup for reactions 1. The supplemented 2x JumpStart Mix contained 10 ml of 2x JumpStart Mix (Sigma Catalog #P2893containing 3 mM MgCl2) plus 110 ul IM MgCl2 (with a final concentration of 11 mM) and 40 ul 30OuM Sulforhodamine 101

(Sigma Catalog #S7635, final concentraion of 60 nM).

2. The QPCR Working Master Mix for the required number of samples is described below.

For duplex reactions

3. Dispense Working Master Mix into wells of PCR reaction plate (USA Scientific Catalog #14029700): 15 μl per well (e.g.96 well plate)

4. Transfer DNA sample to the appropriate wells: 5 μl per well (e.g. 96 well plate)

• The first column on the plate is used for controls the first six wells (AlFl) are a two fold dilution series of a known onecopy control

the seventh well (Gl) is blank

the last well (H 1 ) is plasmid DNA

• The concentration of the plasmid DNA should be in the range to yield a Ct value around 25 in the TaqMan assay.

5. Seal plates immediately using the optical sealing tapes (Corning Catalog #6575) and analyze or store appropriately.

6. Run plate on ABI7500 (Applied Biosystems) under the cycling conditions:

Ramp times = Auto

Cycle Temp Time Repeat

Hold 95 5:00 0

Cycle 95 0:15 35 60 1 :00

Total time is about 1 hour and 30 minutes. Primer information

To detect transgenic events, 2 different primer pairs were used. The set "daol" was used for daogene detection, the set"EcDsdA" was used for the dsdAgene detection. As endogenous probe the set "Ecyc" was used. The set "Ecyc" was usedto detect an endogenous singlecopy gene of Tagetes.

Legend: BHQI stands for "black hole quencher"

FAM stands for succinimidyl ester of carboxyfluorescein

Example 5: Marigold transformation with the dsdA (SELDA) selectable marker construct (RETO 17) using Dserine as theselective agent.

AU transformations were done using the marigold transformation protocol described above. After a 3day cocultivation





period, continuous selection pressure was applied through culture media containing Dserine at concentrations of 0.1, 0.33,1.0, 3.3, 10.0 and 33.3 mM for approximately 9 weeks, after which samples were taken for TaqMan molecular analysisdetecting the gene dsdA. In the first four weeks of selection pressure, explant growth and shoot regeneration vigor clearlydeclined with increasing Dserine concentrations. Explants grown on media containing 0.1 and 0.33 mM Dserine appearedto be unaffected and looked virtually identical to controls grown on media without Dserine. A modest reduction in growthand vigor was seen as Dserine was increased to 1.0 mM with more dramatic effects at concentrations of 3.3 mM andhigher. All explants on 33.3 mM Dserine died after approximately 6 weeks of selection pressure.

Although Dserine clearly affected the growth and vigor of these cultures, after 46 weeks of selection pressure, there wereno obvious differences in the growth of developing shoots derived from control explants (noninoculated) compared to thosefrom explants inoculated with RETO 17 dsdA gene), grown under the same selection pressure. At this point, individualdeveloping shoots from both control and RETO 17 inoculated cultures were subcultured to hormone free, selection mediumand incubated under standard conditions. After an additional two weeks of culture, numerous shoots continued to develop onall Dserine concentrations except 33.3 mM. In addition, several RETO 17 derived shoots were observed growing morevigorously than respective control shoots cultured on 1.0, 3.3 and 10.0 mM Dserine selection medium. The mostpronounced growth differences were in root proliferation and this was most obvious at 3.3 mM Dserine.

At this time individual plantlets were classified with a ranking of 13 based on difference in growth compared to nontransgenic control cultures (see definitions below).

Definition for Class 1 : Plantlets having obviously more vigorous growth than control shoots (on the same selection media).These shoots usually had healthier roots (both in number and vigor). Also, shoot development was generally more vigorous,with no yellowing of upper leaves and shoot apex.

Definition for Class 2: Plantlets appear slightly more vigorous than control shoots (on the same selection media), with longerand more vigorous roots but growth is not as good as plants from Class 1. The differences are not as obvious and someplants have yellowing of upper leaves and shoot apex.

Definition for Class 3: Plant morphology, shoot and root development are essentially the same as control shoots onselection.

A summary of the numbers of shoots recovered in each class is shown in Table 2.

Table 1: Summary of plantlets recovered from inoculated explants (RETO 17, dsdA) after 10 weeks of selection on variousDserine selection levels.

Leaf samples from 314 in vitro, putative transgenic lines were analysed via TaqMan molecular analysis for detection of thedsdA. A summary of the data is shown in Table 2 below.

If 1.0, 3.3 or 10 mM Dserine was used as the selection pressure, ~90% (10 /11) of the lines labeled as "Class 1" testedpositive for the dsdA gene (see Table 2 below) indicating that transgenic plants can be identified at these levels using thedsdA gene. Since the original number of explants inoculated for each selection level was 100 this results in a preliminarytransformation rate of 4% for (1.0 and 3.3 mM).

For the lines labeled Class 2, only the 3.3 mM selection level appeared to successfully pick out transgenic lines. This ismost likely because below this level, shoot growth is not inhibited enough to make the proper subjective selection.

It also appears that some transgenic lines were recovered even though they looked identical to control shoots (labeled"Class 3"). This indicates that transformation is taking place but the expression level of the dsdA gene is not high enough toallow selection based on a growth difference.

Table 2: Summary of TaqMan molecular data for detection of the dsdA gene for each class of shoot recovered at each Dserine selection level. These data are from one experiment and 100 explants were inoculated for each selection level.

Example 6: Marigold transformation with the daol (SELDA) selectable marker construct (RETO 16) using Dserine as theselective agent.

All transformations were done using the marigold transformation protocol described above. After a 3day cocultivation





period, continuous selection pressure was applied through culture media containing Dserine at concentrations of 0.1, 0.33,1.0, 3.3, 10.0 and 33.3 mM for approximately 9 weeks, after which samples were taken for TaqMan molecular analysisdetecting the gene daol.

Early in this experiment, results for daol were similar to those for dsdA on Dserine selection. In the first four weeks ofselection pressure, explant growth and shoot regeneration vigor clearly declined with increasing Dserine concentrations.Explants grown on media containing 0.1 and 0.33 mM Dserine appeared to be unaffected and looked virtually identical tocontrols grown on media without Dserine. A modest reduction in growth and vigor was seen as Dserine was increased to1.0 mM with more dramatic effects at concentrations of 3.3 mM and higher. After approximately 6 weeks of selectionpressure relatively few shoots were seen developing on 10.0 mM and all explants on 33.3 mM Dserine had died.

During the first 46 weeks of selection pressure, there were no obvious differences in the growth of developing shootsderived from control explants (noninoculated) compared to those from explants inoculated with RETO 16 daol gene), grownunder the same selection pressure. At this point, individual developing shoots from both control and RETO 16 inoculatedcultures were subcultured to hormone free, selection medium and incubated under standard conditions. After an additionaltwo weeks of culture, shoots continued to develop on all Dserine concentrations except 33.3 mM. In addition, severalRETO 16 derived shoots were observed growing more vigorously than respective control shoots cultured on 0.33, 1.0, and3.3 mM Dserine selection medium. The most pronounced growth differences were in root proliferation and this was mostobvious at 3.3 mM Dserine.

At this time individual plantlets were classified with a ranking of 13 based on difference in growth compared to nontransgenic control cultures (for definition see earlier example). A summary of the numbers of shoots recovered in each classis shown in Table 3.

Table 3: Summary of plantlets recovered from inoculated explants (RETO 16, daol) after 8 weeks of selection on various Dserine selection levels.

Leaf samples from 203 in vitro, putative transgenic lines were analyzed by TaqMan molecular analysis for detection of thedaol. A summary of the data is shown in Table 4.

The best treatment for selection of transgenic lines was 3.3 mM Dserine. In this treatment 57% of the lines labeled "Class1" tested positive for the daol gene, indicating that selection of transgenic lines is possible, although selection is not asclean as observed with dsdA selection (previous example). The rate of escapes for the Dserine levels 0.3 and 1.0 mM (alsolabeled "Class 1") was even higher. It is also interesting to note that no "Class 1" transgenic lines were recovered at 10.0mM. This is in contrast to the dsdA gene where transgenic lines were recovered. Perhaps the daol gene is not as effectiveat detoxifying the Dserine (in marigold) as the dsdA gene. However, some transgenics were recovered and (as suggestedfor dsdA) a stronger promoter may also help the effectiveness of dao\.

Table 4: Summary of TaqMan molecular data for detection of the daolgene for each class of shoot recovered at each Dserine selection level. These data are from one experiment and 100 explants were inoculated for each selection level (exceptfor 0.33 mM for which there were 81).

Example 7: Marigold transformation with the daol (SELDA) selectable marker construct (RET016) using Dalanine as theselective agent.

All transformations were done using the marigold transformation protocol described above. After a 3day cocultivationperiod, continuous selection pressure was applied through culture media containing Dalanine at concentrations of 0.1, 0.33,1.0, 3.3, 10.0 and 33.3 mM for approximately 9 weeks, after which samples were taken for TaqMan molecular analysisdetecting the gene dao\.

As with Dserine selection, in the first four weeks of selection pressure, explants grown on media containing 0.1 and 0.33mM Dalanine appeared to be unaffected and looked virtually identical to controls grown on media without Dalanine. Thefirst obvious reduction in growth and vigor was seen at 1.0 mM Dalanine with more dramatic effects at concentrations of3.3 mM and higher. At concentrations above





3.3 mM, explants were quite sensitive. At 10 mM Dalanine many explants were completely brown and dead after twoweeks of selection pressure. At 33.3 mM D alanine, all explants were dead after 2 weeks of selection.

After 46 weeks on Dalanine selection, there were no obvious differences in the growth of developing shoots derived fromcontrol explants (noninoculated) compared to those from explants inoculated with RETO 16 (dao\ gene), grown under thesame selection pressure. Again, at this point, individual developing shoots from both control and RETO 16 inoculatedcultures were subcultured to fresh, hormone free, selection medium and incubated under standard conditions. After anadditional two weeks of culture, shoots continued to develop on Dalanine concentrations of 10.0 mM and lower. Consistentwith Dserine selection, several RETO 16 derived shoots were observed growing more vigorously than respective controlshoots cultured on 1.0 and 3.3 mM Dalanine selection medium. The most pronounced growth differences were in rootproliferation and this was most obvious at 3.3 mM Dserine. No roots ever developed on shoots recovered from 10 mM Dalanine, but some shoots were observed with slightly more vigorous growth than control shoots. At this time individualplantlets were classified with a ranking of 13 based on differences in growth compared to nontransgenic control cultures(for definition see earlier example).

A summary of the numbers of shoots recovered in each class is shown in Table 5.

Table 5: Summary of plantlets recovered from inoculated explants (RET016, dao\) after 8 weeks of selection on various Dalanine selection levels. AU of the 33.3 mM material died. For 0.1 and 0.3 mM all plantlets were class 3 and so 36 lineswere randomly selected for analysis.

Leaf samples from 144 in vitro, putative transgenic lines from various Dalanine selection levels were analysed via Taqmanmolecular analysis. A summary of the data is shown in Table 6 below.

The best treatment for selection of transgenic lines was 3.3 mM Dalanine. In this treatment, 86% (6/7) of lines identified as"Class 1" and 50% (2/4) lines identified as "Class 2" tested positive for the dao\ gene. The next best treatment was 1.0 mMD alanine where 70% (7/10) of lines identified as "Class 1" tested positive for the dao\ gene, but at this Dalanineconcentration, only 10% (2/20) of the lines identified as "Class 2" tested positive for the dao\ gene.

Although phenotypes were not as distinct as those observed in Dserine selection, the dao\ gene did allow effectiveselection of transgenic marigold lines using Dalanine as the selective agent (especially at 3.3 mM). The most distinctivephenotype was observed in root growth, which is consistent with results seen in earlier examples.

Table 6: Summary of TaqMan molecular data for detection of the dao\ gene for each class of shoot recovered at each Dalanine selection level. These data are from two experiments and 200 explants were inoculated for each selection level.

Example 8: Varying Dserine selection levels to optimize selection pressure for marigold transformation with the dsdA(SELDA) selectable marker construct (RET017).

Explant preparation and inoculation was done as described above. After a 3day co cultivation period all explants weresubcultured to SMl medium containing Dserine at concentrations of 1.0, 3.3, 5.0, 7.0 and, 9.0 mM Dserine. Afterapproximately 9 weeks of continuous selection pressure leaf samples of each putative transgenic shoot identified as Class1 or Class 2 (as previously described) were analysed by TaqMan molecular analysis. The combined results from twoexperiments are shown below.

Consistent with previous examples, explant growth and shoot regeneration vigor declined with increasing Dserineconcentrations. Also, within 46 weeks of selection pressure, there were no obvious differences in the growth of developingshoots derived from control explants (noninoculated) compared to those from explants inoculated with RETO 17 (dsdAgene), grown under the same selection pressure. At this point, individual developing shoots from both control and RETO 17inoculated cultures were subcultured to hormone free, selection medium and incubated under standard conditions. After anadditional two weeks of culture, shoots continued to develop on all Dserine concentrations.

Clear differences in both root and shoot growth were seen at Dserine concentrations 3.3mM and higher, although thenumbers of putative transgenic shoots recovered were considerably lower from 7.0 mM and 9.0 mM Dserine treatments. At





1.0 mM Dserine, differences in growth and vigor between putative transgenic and control shoots were more difficult to seeand most recovered shoots fell under the Class 2 designation.

Leaf samples from 119 in vitro, putative transgenic lines designated either Class 1 or Class 2 were analyzed via Taqmanmolecular analysis for detection of the dsdA. A summary of molecular data is given in Table 7.

The best overall selection was obtained with 5.0 mM Dserine, although 3.3 mM D serine produced comparable results.Under both of these concentrations transgenic shoots were effectively identified with minimal escapes. Dserineconcentrations of 7.0 and 9.0 mM also provided clean selection with minimal escapes; however, the number of transgeniclines recovered was considerably lower indicating excessive selection pressure. At the other end of the spectrum, 1.0 mMDserine was not high enough to provide effective selection as evidenced by the high number of escapes.

Table 7: Summary of TaqMan molecular data for detection of the dsdA gene for each class of shoot recovered at each Dserine selection level.

Example 9: Comparing the effect of different promoters for driving dsdA (SELDA).

Using standard transformation procedures described above, explants were inoculated with Agrobacterium containing eitherpBHX 925 (UBQ10L::<&<£4::nos) or RET017 (NOS::dsdA::nos) and were allowed to cocultivate for 3days under standardconditions. After cocultivation, explants were subcultured to SMl medium containing Dserine at concentrations of either 4.0or 5.0 mM Dserine. After approximately 9 weeks of continuous selection pressure, leaf samples of each putative transgenicshoot were taken for TaqMan molecular analysis. This experiment was run once with a selection pressure of 4.0 mM Dserine and twice with a selection pressure of 5.0 mM Dserine. Combined results are described below.

Leaf samples from 146 (81,pBHX925 and 65, RETO 17) putative transgenic lines were evaluated for gene copy number andvector backbone integration via TaqMan molecular analysis. Regardless of the Dserine concentration used for selection,preliminary transformation rates (PTE) always tended to be higher for pBHX925 than for RETO 17. At 4 mM Dserine, thePTE for pBHX925 was 11% with approximately 3% escapes compared to a PTE of 7% with 10% excapes for RETO 17.Although transformation rates were lower using 5.0 mM Dserine, the trends were the same (see Table 8).

Table 8: Summary of TaqMan molecular data for 146 putative transgenic shoots produced using the dsdA gene driven byeither the UBQlOL (pBHX925) or the NOS (RET017) promoters. Each shoot was evaluated for the presence of the dsdAgene.

Although population sizes were relatively low, transgenic lines from each construct were also compared for gene copynumber. These lines were categorized as 1 gene copy, 2 gene copies and everything with greater than 2 gene copies (asdetermined by TaqMan analysis). Regardless of the construct used, over 50% of the transgenic lines recovered had a singlegene copy (see Table 9). Results for two copy and multiple copy events were also similar.

Indicating that either the UBQlOL or NOS promoter could be used to drive dsdA expression with little effect on gene copynumber.

Table 9: Comparison of gene copy number in transgenic lines produced with either pBHX925 (UBQ10L::cfr<i4::nos) orRET017 (NOSwdsdAv.nos). Transgenic lines were categorized as 1 gene copy, 2 gene copies and everything with greaterthan 2 gene copies (as determined by TaqMan analysis).

Example 10: Comparing the effect of promoters for driving dsdA (SELDA).

Using standard transformation procedures (described above), two separate experiments were run to compare variouspromoters for driving the dsdA (SELDA) selectable marker gene, and determine their effects on marigold transformationefficiency. The first experiment compared the standard RETO 17 (NQSv.dsdAv.nos) construct to RET063

and RLM254 (STPT: :dsdA::nos), while the second experiment compared RETO 17 (NOS::dsdA::nos) to construct RLM407







(PcUbi42::<ft<£4::nos). All explants were inoculated with Agrobacterium using standard procedures and after cocultivation,explants were subcultured to SMl medium containing 5.0 mM Dserine. After approximately 9 weeks of continuous selectionpressure, leaf samples of each putative transgenic shoot were analysed by TaqMan molecular analysis. The combinedresults from two experiments are shown below.

Leaf samples from 110 putative transgenic lines (57, RET017; 41, RLM407; 5, RET063 and 7, RLM407) were evaluated forgene copy number via TaqMan molecular analysis. Although transgenic lines were recovered from all constructs tested,there appeared to be differences in transformation efficiencies between promoters used to drive dsdA (see Table 10).Preliminary transformation rates were similar for RETO 17 in both experiment 1 and experiment 2 (7% and 8% PTErespectively). The PTE for RLM 407 was approximately 10% which is similar to that of RETO 17 indicating the PcUbi42promoter shows activity comparable to the NOS promoter for driving the dsdA (SELDA) selection. However, the PTE's forboth RET063 and RLM254 were considerably lower (1% and 2% respectively) when compared to RETl 07.

Each shoot was evaluated for the presence of the dsdA gene using TaqMan molecular analysis. Experiment #1 comparedthe NOS (RETO 17) promoter to the AtAct2i (RET063) and STPT (RLM254) promoters, while experiment #2 compared theNOS (RETO 17) promoter to the PcUbi42 (RLM407) promoter.

Table 10: Summary of TaqMan molecular data for 1 10 putative transgenic shoots produced using the dsdA gene driven byvarious promoters.

Example 11: Description of the plant transformation vectors.

Construction of the binary vector pBHX925 pUC19::ubqlOL::dsdA::nos

The coding region of the Dserine ammonia lyase gene (dsdA) from Escherichia coli is synthesized by PCR using theplasmid RLM407 as template and the primers dsdA Forward (5' AGC GCA TCT AGA GAT ATC ATG GAA AAC GCT AAAATG AA 3'; SEQ ID No. 22) and dsdAReverse (5' CCT GCA ATT GTG TAC ATT ATT AAC GGC CTT TTG CCA GAT A3'; SEQ ID No. 23). The PCR product is digested with Xba I and Mfe I then ligated into pUC19 digested with Xba I andEcoR I. The resulting plasmid is designated pUCdsdA.

The ubqlOL promoter [Norris et al., Plant MoI. Biol., 21 :895906 (1993)] from pSAN282 is digested with Hind III and Sma I,and ligated into plasmid pUCdsdA digested with the same enzymes. The resulting plasmid is designated pUCdsdl OL. Thenopaline synthase polyadenylation signal (nos terminator) is synthesized by PCR using the plasmid pBIN19 as templateand the primers nosForward (5' CCT GGT ACC GAT CGT TCA AAC ATT TGG CAA TA 3'; SEQ ID No. 24) and nosReverse (5' GGG GAA TTC GAT ATC CGA TCT AGT AAC ATA GAT G 3'; SEQ ID No. 25). The PCR product is digestedwith EcoR I and Kpn I then ligated into pUC19 digested with the same enzymes. The resulting plasmid is designatedpUCnos.

The ubqlOL promoteτdsdA coding region is removed from pUCdsdlOL by digestion with Hind III and BsrG I then ligated intopUCnos digested with Hind III and Acc65 I. The resulting plasmid is designated pDSDA.

pBIN19::ubqlOL::ύfe<£4::nos

The ubqlOL::<&ύiL4::nos cassette is removed from pDSDA by digestion with EcoR V and Hind III then ligated into theplasmid pBIN19 digested with Dr a III (made blunt by treatment with DNA polymerase Klenow fragment and dNTPs) andHind III. The resulting plasmid is designated pBINdsdA 1.

A portion of pUC19 including the multiple cloning site is synthesized by PCR using the primers mcsForward (5' GCC AAGCTT GCA TGC CTG CAG GTC 3'; SEQ ID No. 26) and mcsReverse (5' CAC GTT TAA ACT ACC GCA CAG ATG 3';SEQ ID No. 27) , adding a Pme I site at the 3 ' .

The nos promoter/nptll gene/nos terminator selectable marker in pBINdsdAl is removed by digestion with Hind III and PmeI, and the pUC19 mcs PCR product (digested with Hind III and Pme I) is inserted by ligation. The resulting plasmid isdesignated ppBHX925.

With respect to the sequence listing, it is noted that SEQ ID NO: 6 is equal to SEQ ID NO: 5, wherein a sequence encodingdsdA was replaced by a sequence encoding dao. References

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PATENT CITATIONS

Cited Patent Filing date Publication date Applicant Title

WO2000032788A2 * Nov 30, 1999 Jun 8, 2000ChrHansenAs

Method for regulating carotenoid biosynthesis in marigolds

WO2001046445A2 * Dec 13, 2000 Jun 28, 2001 Heim Ute Production of transgenic plants of the tagetes species

WO2003060133A2 * Jan 13, 2003 Jul 24, 2003 OskarErikson Selective plant growth using damino acids

WO2005090581A1 * Mar 15, 2005 Sep 29, 2005BasfPlantScienceGmbh

Improved constructs for marker excision based on dualfunction selectionmarker

DE10258971A1 * Dec 16, 2002 Jul 1, 2004SungeneGmbh &Co. Kgaa

Use of astaxanthincontaining plant material, or extracts, from Tagetes for oraladministration to animals, particularly for pigmentation of fish, crustacea, birdsand their products

* Cited by examiner

CLASSIFICATIONS

International Classification C12N15/82Cooperative Classification C12N15/821, C12N15/8205, C12N15/825European Classification C12N15/82C4B8, C12N15/82A8D, C12N15/82A4B

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Patent WO2008077570A1 - Transformation of tagetes using the selda selection marker - Google Patents