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Title:
TRANSFORMATION OF TAGETES USING THE SELDA SELECTION MARKER
Document Type and Number:
WIPO Patent Application WO/2008/077570
Kind Code:
A1
Abstract:
The present invention relates to improved methods for transformation of Marigold (Tagetes) based on a D-amino acid selection. The method for generating transgenic Tagetes plants or cells comprises the introduction into a Tagetes cell or tissue of a nucleic acid construct comprising a nucleic acid sequence which encodes an enzyme capable of metabolizing D-amino acids, the incubation of said cell or tissue in a selection medium comprising a D-amino acid, and the subsequent incubation in a regeneration medium in order to regenerate transgenic Tagetes 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 or has an effect on the carotenoid biosynthesis pathway of Tagetes, like ketolase. The generated plants may be used for the production of carotenoids, especially of astaxanthin.

Inventors:
FLACHMANN, Ralf (Halberstädter Strasse 20a, Quedlinburg, 06484, DE)
BRIDG-GIANNAKOPOULOS, Hannia (Breite Strasse 17, Quedlinburg, 06484, DE)
KUNZE, Irene (Mühlenweg 11, Gatersleben, 06466, DE)
LEPS, Michael (Juri-Gagarin-Strasse 12, Halberstadt, 38820, DE)
SAUER, George, Mather (Wallstrasse 30, Quedlinburg, 06484, DE)
SCHOPFER, Christel, Renate (Beethovenstrasse 8, Ludwigshafen, 67061, DE)
WENDEROTH, Irina (Nelkenstrasse 5, Mutterstadt, 67112, DE)
EISENREICH, Robert (105 Hickory Street, North Aurora, IL, 60542, US)
Application Number:
EP2007/011217
Publication Date:
July 03, 2008
Filing Date:
December 19, 2007
Export Citation:
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Assignee:
BASF PLANT SCIENCE GMBH (BPS-A30, Ludwigshafen, 67056, DE)
FLACHMANN, Ralf (Halberstädter Strasse 20a, Quedlinburg, 06484, DE)
BRIDG-GIANNAKOPOULOS, Hannia (Breite Strasse 17, Quedlinburg, 06484, DE)
KUNZE, Irene (Mühlenweg 11, Gatersleben, 06466, DE)
LEPS, Michael (Juri-Gagarin-Strasse 12, Halberstadt, 38820, DE)
SAUER, George, Mather (Wallstrasse 30, Quedlinburg, 06484, DE)
SCHOPFER, Christel, Renate (Beethovenstrasse 8, Ludwigshafen, 67061, DE)
WENDEROTH, Irina (Nelkenstrasse 5, Mutterstadt, 67112, DE)
EISENREICH, Robert (105 Hickory Street, North Aurora, IL, 60542, US)
International Classes:
C12N15/82
Attorney, Agent or Firm:
NEUEFEIND, Regina (Maiwald Patentanwalts GmbH, ElisenhofElisenstrasse 3, München, 80335, DE)
Download PDF:
Claims:

C l a i m s

1. A method for generating transgenic Tagetes plants or cells comprising the steps of a) introducing into a Tagetes cell or tissue a nucleic acid construct comprising a promoter active in Tagetes cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid and/or a derivative thereof; b) incubating the Tagetes cell or tissue of step a) on or in a selection medium comprising a D-amino acid and/or a derivative thereof; and optionally c) transferring the Tagetes cell or tissue of step b) to a regeneration medium and regenerating Tagetes plants comprising the nucleic acid construct.

2. The method of claim 1, wherein the promoter is a constitutive promoter, an inducible promoter and/or a tissue-specific promoter.

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

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

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

6. The method of any one of claims 1 to 5, wherein the nucleic acid construct further comprises a selection marker gene with growth delaying or growth inhibiting function; and the selection medium further comprises an antibiotic, a herbicide and/or another selective compound.

7. The method of claim 1, wherein step a) comprises the following steps: providing cotyledonary segments of a Tagetes seedling; - co-cultivating the tissue with an Agrobacterium comprising a nucleic acid construct comprising a promoter active in the tissue and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid and/or a derivative thereof.

8. The method of claim 7, wherein step b) comprises transferring the cotyledonary segments on or in a selection medium comprising at least one plant growth factor in a concentration suitable to induce de novo shoot induction from the tissue; at least one D-amino acid and/or a derivative thereof and cultivating the tissue on the medium until shoots have been induced and developed therefrom.

9. The method of claim 8, wherein step c) comprises isolating the developed shoots and transferring the shoots to a rooting medium and cultivating the shoots on the rooting medium until the shoots have formed roots, and further regenerating the so derived plantlets into mature plants, which have inserted into

their genome the nucleic acid construct.

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

11. The method of any one of claims 1 or 10, wherein the selection medium comprises at least one D-amino acid and/or a derivative thereof in a concentration of 0.05 mM to 100 mM, preferably 0.1 mM to 50 mM, and more preferably 0.3 mM to 5 mM.

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

13. The method of any one of claims 1 to 12, wherein the D-amino acid is selected from the group consisting of D-alanine and D-serine.

14. The method of any one of claims 1 to 13, wherein the enzyme capable of metabolizing a D-amino acid is selected from the group consisting of D-serine ammonialyases, D-amino acid oxidases and D-alanine transaminases.

15. The method of claim 14, wherein the D-serine ammonialyase is encoded 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 acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence from a), b) or c), or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase.

16. The method of claim 14, wherein the D-amino acid oxidase is encoded 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. 3; b) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of the

nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence from a), b) or c), or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase.

17. A transgenic Tagetes plant or cell comprising a nucleic acid construct comprising a promoter active in Tagetes cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid and/or a derivative thereof; or generated in a method according to any one of claims 1 to 16.

18. The transgenic Tagetes plant or cell of claim 17, wherein the comprised nucleic acid construct further comprises at least one gene of interest.

19. The transgenic Tagetes plant or cell of claim 18, wherein the at least one gene of interest is a nucleic acid sequence encoding a protein which is involved in or has an effect on the carotenoid

biosynthesis pathway.

20. The transgenic Tagetes plant or cell of claim 19, wherein the at least one gene of interest is a nucleic acid sequence encoding a ketolase.

21. The transgenic Tagetes plant or cell of any one of claims 18 to 21, wherein 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.

22. Use of a transgenic Tagetes plant of any one of claims 17 to 21 for the production of carotenoids.

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

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

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

26. A heterologous nucleic acid construct comprising a) a promotor active in Tagetes cells selected from the group consisting of a constitutive promoter, an inducible promoter and a tissue-specific promoter;

b) a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid 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 D-amino acid is encoded 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 acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the nucleotide sequence from a) or b) under stringent conditions, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence from a), b) or c), or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase.

e) a nucleic acid sequence comprising a nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQ ID No. 3; f) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; g) a nucleic acid sequence 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 the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; h) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence from e), f) or g), or comprising a fragment of the nucleotide sequence, wherein the fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase.

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

Tagetes plants, preferably from the Tagetes flower petals.

Description:

Transformation of Tagetes using the SELDA selection marker

FIELD OF THE INVENTION The present invention relates to improved methods for transformation of Marigold (Tagetes) based on a D-amino acid selection.

BACKGROUND OF THE INVENTION

The plant genus Tagetes, which belongs to the family of the Asteraceae, exhibits quite a few interesting properties, which explains its use both as a crop plant and as an ornamental. Many species of this genus produce bioactive compounds with a nematicidal, fungicidal and insecticidal action, accumulate flavonoids and carotenoids with antioxidant and coloring activities, are salt resistent, and serve decorative purposes owing to their wide range of flower colors and flower shapes.

The nematicidal action of the Tagetes roots is based on the synthesis of thiophene derivatives. Thiophene derivatives are heterocyclic, sulfur-containing compounds which accumulate mainly in roots and hypocotyls. They are characterized by a 5- membered ring which contains an S atom. A sulfhydryl group, which originates from the amino acid cysteine, participates in the formation of the ring. Important representatives of the thiophenes which are also formed in Tagetes are, for example, BBT (5-(but-3-en-l-ynyl)-2,2'-bi-thienyl), BBTOAc (5-(4-acetoxy-l-butynyl)-2,2'- bithienyl), BBTOH (5-(4-hydroxy-l-butynyl)-2,2'-bithienyl) and [alpha]-T (2,2':5',2"-terthienyl).

The petals of Tagetes contain 9-22% of flavonoids and approx. 27% of carotenoids, in which [beta] -carotene accounts for 0.4%, cryptoxanthin esters for 1.5% and xanthophyll esters for 86.1% (Benk et al., Riechst, Aromen und Koerpen, 26 (1976), 216-221). According to an estimate from 1975, over 2000 different flavonoids exist. Some important representatives are, for example, the anthocyanins with 250 known structures, the chalcones with 60, the aurones with 20 and the flavones with 350

known structures, all of which have coloring properties. Flavonols with 350 and the isoflavonoids with 15 representatives impart to the plants properties such as phagoprotection or have a toxic effect on fungi.

Carotenoids belong to the large group of the terpenoids. Most of them are tetraterpenes. Carotenoid-hydrocarbons are also termed carotenes, and their oxidized derivatives are the xanthophylls. Carotenoids are essential components in photosynthetically active membranes of all plants, algae and cyanobacteria. They are found in the pigment systems (light traps) of the chloroplasts and participate in the process of the primary light absorption and photon channeling of photosynthesis. Moreover, they also act as light receptors in a series of other light-induced processes in the plant. The yellow color of many flowers is due to carotenoid-containing, usually with low concentrations of chlorophyll or chlorophyll-free, chromoplasts. Plant carotenoids also act as precursors for the biosynthesis of the plant growth regulator abscisic acid and of vitamin A, which is important for human and animal nutrition. It is increasingly of interest as a potential anticancer agent and is employed as colorant in the cosmetics and food industries. Carotenoids from dried and pulverized Tagetes petals are already being 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, the carotenoid content and the carotenoid composition in plants. However, the limited genetic variability in known Tagetes varieties greatly limits the possibilities of improving these varieties with the aid of traditional biolological plant breeding methods.

Modern biotechnological research and development has provided useful techniques for the improvement of agricultural products by plant genetic engineering. Plant

genetic engineering involves the transfer of a desired gene or genes into the inheritable germline of crop plants such that those genes can be bred into or among the elite varieties used in modern agriculture. Gene transfer techniques allow the development of new classes of elite crop varieties with improved disease resistance, herbicide tolerance, and increased nutritional value. Various methods have been developed for transferring genes into plant tissues including high velocity microprojection, microinjection, electroporation, direct DNA uptake, and Agrobacteήum-mediated gene transformation. Although widely used for dicotyledonous plants, DNA delivery using particle bombardment, electroporation, or Agrobαcterium-mediated delivery into Tagetes has proven to be difficult. Two methods routinely used are an Agrobαcterium-based method targeting the cotyledonary-node axillary meristems (Hinchee 1988) and a method using particle bombardment of mature zygotic embryos (Finer 1991).

The lack of effective selective agents is one of the bottlenecks in the efficiency of different Tagetes transformation methods. The efficiency of tissue culture selection systems depends on many factors including tissue type, size of explant, chemical characteristics of the selectable agent and concentrations and time of application. The most used method of selection is known as negative selection, which employs selection markers that confer resistance against a phytotoxic agent (such as an herbicide or antibiotic). The negative selection markers employed so far are mainly limited to neomycin 3'-O-phosphotransferase (nptlϊ), phosphinothricin acetyltransferases (PAT; also named Bialophos ® resistance; bar; de Block 1987; EP 0 333 033; US 4,975,374), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; conferring resistance to Glyphosate ® (N-(phosphonomethyl) glycine); and hygromycin B. Alternative selection marker systems, such as a system based on D- amino acid metabolizing enzymes (e.g., D-amino acid dehydratases or oxidases), has been recently described on a general basis (WO 03/060133; Erikson 2004).

Although some of the problems linked to the transformation of Tagetes have been overcome by the methods described in the art, there is still a significant need for improvement, since all methods known so far have only a low to moderate transformation and — especially - regeneration efficiency. Although significant advances have been made in the field of Agrobacterium-mediated transformation methods, 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 D-amino acid metabolizing enzymes has been recently described on a general basis (WO 03/060133; Erikson 2004), this D-amino acid based selection method cannot be easiliy adopted and optimised on every plant system or the achievable transformation efficiency is too low, thus the system is not suitable. Therefore, it could not have been expected that this selection system provides an improved method having higher overall efficiency in the process of generation of transgenic Tαgetes plants. This objective is solved by the present invention by providing a (for humans) non-toxic selection method based on D-amino acids to generate transgenic Tαgetes cells or plants, e.g. for the production of chemicals, e.g. astaxanthin.

SUMMARY OF THE INVENTION

A first embodiment of the invention relates to the method for generating transgenic Tαgetes plants or cells comprising the steps of a) introducing into a Tαgetes cell or tissue a nucleic acid construct comprising a promoter active in Tαgetes cells and operably linked thereto a nucleic acid sequence encoding an

enzyme capable of metabolizing a D-amino acid and/or a derivative thereof; b) incubating the Tagetes cell or tissue of step a) on or in a selection medium comprising a D-amino acid and/or a derivative thereof; and optionally c) transferring said Tagetes cell or tissue of step b) to a regeneration medium and regenerating Tagetes plants comprising the nucleic acid construct.

Various promoters are known to be functional in Tagetes and are suitable to carry out the method of the invention. A promoter active in Tagetes plant can be a constitutive promoter, an inducible promoter and/or a tissue-specific promoter.

In a preferred embodiment, the nucleic acid construct further comprises at least one gene of interest. More preferably, the at least one gene of interest encodes a protein which is involved in or has an effect on the carotenoid biosynthesis pathway. Most preferably, the one gene of interest encodes a ketolase (e.g. from Scenedesmus vacuolatus (see EP 06124146.9) or from Haematococcus pluvialis). It is also preferred that the nucleic acid construct further comprises a selection marker gene with growth delaying or growth inhibiting function.

In a preferred embodiment of the invention, the introduction of a nucleic acid construct into a Tagetes cell or tissue comprises: (i) providing cotyledonary segments of a Tagetes seedling; and (ii) co-cultivating said cotyledonary segments with an Agrobacterium comprising a nucleic acid construct comprising a promoter active in said tissue and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a

D-amino acid and/or a derivative thereof. Preferably, the cotyledons are provided from seedlings derived from mature seeds of a regenerable Tagetes line.

Preferably, the selection medium comprises at least one plant growth factor in a concentration suitable to induce de novo shoot induction from said tissue and at least one D-amino acid and/or a derivative thereof. In another preferred embodiment, the selection medium further comprises an antibiotic, a herbicide and/or another selective compound. Preferably, the selection medium comprises a D-amino acid and/or a derivative thereof in a concentration of 0.05 mM to 100 mM, 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 medium comprises transferring the co-cultivated cotyledonary segments on or in a selection medium comprising at least one plant growth factor, more preferably at least one plant growth factor in a concentration suitable to induce de novo shoot induction from said tissue, and a D-amino acid and/or a derivative thereof; and and cultivating said tissue until shoots have been induced and developed therefrom.

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

In one preferred embodiment introduction of the nucleic acid construct is mediated by Agrobacterium mediated transformation. Preferably, the Agrobacterium is a disarmed Agrobacterium tumefaciens or Agrobacterium rhizogenes bacterium.

Preferably, the the D-amino acid is D-alanine, D-serine and/or a derivative thereof.

There are various ways to conduct the selection scheme based on D-amino acids or related compounds hereunder.

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 D-amino acid is selected from the group consisting of D-serine ammonialyases, D-amino acid oxidases and D-alanine transaminases.

In a preferred embodiment, the D-serine ammonialyase is encoded 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 acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the 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 D-serine ammonialyase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 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 D-serine ammonialyase.

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

In another preferred embodiment, the D-amino acid oxidase is encoded 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. 3; b) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the 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 D-amino acid oxidase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 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 D-amino acid oxidase.

For these enzymes selection is preferably done on a medium comprising a D-amino acid in a concentration from about 0.05 mM to about 100 mM, more preferably from

about 0.1 mM to about 50 mM, and most preferably 0.3 mM to about 5 mM. Preferably, in this embodiment, D-serine and/or D-alanine are employed in a concentration of about 0.05 mM to about 100 mM, more preferably about 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 the invention relates to a Tagetes plant or cell comprising a nucleic acid construct comprising a promoter active in Tagetes cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid. Preferably, said Tagetes plant or cell further comprises at least one gene of interest. More preferably, the at least one gene of 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 acid construct 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, more preferably for the production of astaxanthin. Synthesis of novel carotenoids, naturally not present in Tagetes, shall take place in the flowers, preferentially in the petals of Tagetes. However, accumulation of carotenoids in other tissues is also acceptable. 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, preferably from the Tagetes petals.

The constructs provided hereunder are novel and especially useful for carrying out the invention. In consequence, another embodiment of the invention relates to a heterologous nucleic acid construct comprising a promotor active in Tagetes cells selected from the group consisting of a constitutive promotor, an inducible promotor and a tissue-specific promotor; a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid and being operably linked 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 D-amino acid is selected from the group consisting of D-serine ammonialyases, D-amino acid oxidases and D-alanine transaminases.

In another preferred embodiment, the enzyme capable of metabolizing a D-amino acid comprised by the constructs above is encoded 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 acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the 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 D-serine ammonialyase;

d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 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 D-serine ammonialyase. e) a nucleic acid sequence comprising a nucleotide sequence encoding a protein with the amino acid sequence depicted in SEQ ID No. 3; f) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase; g) a nucleic acid sequence 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 to code for a protein having the enzymatic activity of a D-amino acid oxidase; h) a nucleic acid sequence comprising a nucleotide sequence which shows at least 60% identity to the nucleotide sequence from e), f) or g), or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase.

The Tagetes plants and cells provided above are novel and, hence, also the method for producing cartinoids from these plants is novel. This method comprises generating transgenic Tagetes plants according to the above-described method. Regenerated Tagetes plants may be used for feeding animals or as human diets. Preferably, the carotenoids are extracted from the regenerated Tagetes plants, preferably from the Tagetes petals.

Other objects, advantages, and features of the present 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. The left 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 in marigolds") and WO 2003/060133 ("Selective plant growth using D-amino acids) are hereby incorporated by reference.

Abbreviations: BAP - 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid; MS - Murashige and Skoog medium (Murashige T and Skoog F (1962) Physiol. Plant. 15, 472-497); NAA - 1-naphtaleneacetic acid; MES, 2-(N-morpholino- ethanesulfonic 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 or genera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be 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 so forth.

The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a 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 of that list.

As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The abbreviations used herein are conventional one letter codes for the amino acids: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; 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 (see L. Stryer, Biochemistry, 1988, W. H. Freeman and Company, New York. The letter "x" as used herein within an amino acid sequence can stand for any amino acid residue.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers or hybrids thereof in either single-or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used inter-changeably 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 10 nucleotides 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 sequence-specific manner 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 acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeably herein 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 any reason (e.g., confer improved qualities), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited

to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding 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 of interest 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, for example a messenger RNA (mRNA) sequence the blocking of whose expression is sought to be initiated by hybridization with the target sequence.

The term "sense" is understood to mean a nucleic acid having a sequence which is homologous or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing the expression of the said gene of interest. In another embodiment, the gene of interest is an inhibiting nucleic acid molecule selected from the group consisting of antisense oligonucleotide, antisense DNA, antisense RNA, iRNA, ribozyme, shRNA and siRNA. Preferably, the inhibiting nucleic acid molecule inhibits the expression of an endogenous Tagetes protein. Said endogenous 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 by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete"

complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. A "complement" of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acids show total complementarity to the nucleic acids of the nucleic acid sequence.

The term "genome" or "genomic DNA" is referring to the heritable genetic information of a host organism. Said genomic DNA 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 to the chromosomal DNA of the nucleus.

The term "chromosomal DNA" or "chromosomal DNA-sequence" is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chromatids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated and analyzed 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, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be

isolated in that such a vector or composition is not part of its original environment. Preferably, the term "isolated" when used in relation to a nucleic acid refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with 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 their natural 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% free from 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 acid construct" 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 to obtain one of a Nucleic acid construct. Constructs can be prepared by means of customary recombination and cloning techniques as are described, for example, in Maniatis 1989, Silhavy 1984, and in Ausubel 1987.

The term "wild-type", "natural" or of "natural origin" means with respect to an organism, polypeptide, or nucleic acid sequence, 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 by experimental manipulations and may include

gene sequences found in that cell so long as the introduced gene contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring gene.

The terms "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. A promoter, transcription regulating sequence or other genetic element is considered to be "heterologous" in relation to another sequence (e.g., encoding a marker sequence or a gene of interest) if said two sequences are not combined or differently operably linked their natural environment. 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 its natural environment.

The term "trans gene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations by man. Preferably, said sequence is resulting in a genome which is different from a naturally occurring organism (e.g., said sequence, if endogenous to said organism, is introduced into 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 is introduced so long as it

does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.

The term "transgenic" or "recombinant" when used in reference to a cell or an organism (e.g., with regard to a Tagetes plant or cell) refers to a cell or organism which contains a transgene, or whose genome has been altered by the introduction of a transgene. A transgenic organism or tissue may comprise one or more transgenic cells. Preferably, the organism or tissue is substantially 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 the nucleic 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 are modified, changed, mutated or otherwise manipulated by man.

A "recombinant polypeptide" is a non-naturally occurring polypeptide that differs in sequence from a naturally occurring polypeptide by at least one amino acid residue. Preferred methods for producing said recombinant polypeptide and/or nucleic acid may comprise directed or non-directed 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 of sequence relation ship or complementarity. The following terms are used to describe the sequence relationships between two or more

nucleic acids or amino acid sequences: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (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 reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b)As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, 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 two sequences. 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 to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al. 1981; the homology alignment algorithm of Needleman and Wunsch 1970; the search-for-similarity-method of Pearson and Lipman 1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993. For comparing sequences hereunder, preferably the algorithms

BLASTN for nucleotide sequences, BLASTX for proteins with their respective default parameters are used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & 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 default settings (gap opening penalty 15/19, gap extension penalty 6.66/0.05; gap separation penalty range 8; % identity for alignment delay 40; using residue specific gaps and hydrophilic residue gaps). Comparison is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In addition to calculating percent sequence 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 sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the

test nucleic acid sequence to the reference nucleic acid sequence is less than 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 complementary strand through base pairing." (Coombs 1994). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young, 1985)]. Other references include more sophisticated computations, which take structural 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. An example of stringent wash conditions is a 0.2 X SSC wash at 65°C for 15 minutes (see, Maniatis, infra, for a description of SSC 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 45 0 C for 15 minutes. An example 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. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 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 6O 0 C for long probes (e.g., >50 nucleotides). Stringent conditions may also be 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 that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon 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 conditions for 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 37 0 C, and a wash in 0.1 x SSC 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 M NaCl/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 60 0 C.

The term "equivalent" when made in reference to a hybridization condition as it relates to a hybridization condition of interest means that the hybridization condition and the hybridization condition of interest result in hybridization of nucleic acid sequences which have the same range of percent (%) homology. For example, if a hybridization condition of interest results in hybridization of a first nucleic acid

sequence with other nucleic acid sequences that have from 80% to 90% homology to the first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization condition of interest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the other nucleic 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 conditions may 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 high stringency hybridization different from, but equivalent to, the above-listed conditions. Those skilled in the art know that whereas higher stringencies may be preferred to reduce or eliminate non-specific binding, lower stringencies may be preferred 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 the expression 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 then translated 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 sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 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 are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5'-flanking region may contain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene. The 3'-flanking region may contain sequences, which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

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

The term "genetically-modified organism" or "GMO" refers to any organism that comprises transgene DNA. Exemplary organisms 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 is present at any stage of a plant's development. Such structures include one or more plant organs including, but are not limited 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. The population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise. The cells may be synchronized or not synchronized. A plant cell within the meaning of this invention may be isolated (e.g., in suspension 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 differentiated and 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, protocorm-like bodies, etc.). Plant tissue may be inplanta, in organ culture, tissue culture, or cell culture.

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

The term "structural gene" as used herein is intended to mean a DNA sequence that is 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 mRNA into one or more polypeptides.

The term "expression cassette" or "expression construct" as used herein is intended to mean the combination of any nucleic acid 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 of a nucleotide sequence which direct the initiation of transcription (i.e., is capable of controlling the transcription of the nucleotide sequence into mRNA). A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest (e.g., proximal to the transcriptional start site of a structural gene) whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. 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'-TATAAT-3' (TATA) box about 10-30 bp 5' to the transcription start (cap) site, which, by convention, is numbered +1. Bases 3' to the cap site are given

positive numbers, whereas bases 5' to the cap site receive negative numbers, reflecting their distance from the cap site. Another promoter component, the CAAT box, is often found about 30 to 70 bp 5' to the TATA box and has homology to the canonical form 5'-CCAAT-3' (Breathnach 1981). In plants the CAAT box is sometimes replaced by a sequence known as the AGGA box, a region having adenine residues symmetrically flanking the triplet G(orT)NG (Messing 1983). Other sequences conferring regulatory influences on transcription can be found within the promoter region and extending as far as 1000 bp or more 5' from the cap site. The term "constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene 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 all-or-nothing response to environmental stimuli, or it may result in variations in the level of gene expression. In this invention, the heat shock regulatory elements function to enhance transiently the level of downstream gene expression in response to sudden temperature elevation.

Polyadenylation signal refers to any nucleic acid sequence capable of effecting mRNA processing, usually characterized by the addition of polyadenylic acid tracts to the 3'-ends of the mRNA precursors. The polyadenylation signal DNA segment may itself be a composite of segments derived from several sources, naturally occurring or synthetic, and may be from a genomic DNA or an RNA-derived cDNA. Polyadenylation signals are commonly recognized by the presence of homology to

the canonical form 5'-AATAA-3', although variation of distance, partial "readthrough", and multiple tandem canonical sequences are not uncommon (Messing 1983). It should be recognized that a canonical "polyadenylation signal" may in fact cause 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 sudden temperature elevations. The response is seen as an immediate albeit transitory enhancement in level of expression of a downstream 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'- CTGGAATNTTCTAGA-3 1 (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 (Pelham 1982). A chemically synthesized oligonucleotide copy of this consensus sequence can replace the natural sequence in conferring heat shock inducibility.

Leader sequence refers to a DNA sequence comprising about 100 nucleotides located between the transcription start site and 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 the coding sequences (exons) but are then removed in the formation of the mature mRNA. Introns may occur anywhere within a transcribed 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 in the primary

transcript are excised and the coding sequences are simultaneously and precisely ligated to form the mature mRNA. The junctions of introns and exons form the splice sites. The base sequence of an intron begins with GU and ends with 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 of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfill its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. The expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions, which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 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 cloning techniques 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 be positioned 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, can exist in a vector-integrated 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 to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g., β-glucuronidase) encoded by the transgene (e.g., the uid Agene) as demonstrated herein [e.g., histochemical assay of GUS enzyme activity by staining with X-gluc which gives a blue precipitate in the presence of the GUS enzyme; and a chemiluminescent assay of GUS enzyme activity using the GUS-Light kit (Tropix)]. The term "transient transformant" refers to a cell which has transiently incorporated one or more transgenes. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more transgenes into the genome of a cell, preferably resulting in chromosomal integration and stable heritability through meiosis. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. The term "stable transformant" refers to a cell, which has stably integrated one or more transgenes into the genomic DNA (including the DNA of the plastids and the nucleus), preferably integration into the chromosomal DNA of the nucleus. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from 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 viral vectors involving epichromosomal replication and gene expression, which may exhibit variable properties with respect to meiotic stability. Transformation also includes introduction of genetic material into plant cells in the form of plant viral vectors involving epichromosomal replication and gene expression, which may exhibit variable properties with respect to meiotic stability. Preferably, the term "transformation" includes introduction of genetic material into plant cells resulting in chromosomal integration and stable heritability through meiosis.

The terms "infecting" and "infection" with a bacterium refer to co-incubation 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 introduced into one or more cells of the target biological sample.

The term "Agrobacterium" refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium, which causes crown gall. The term "Agrobacterium" includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in infected plants), and Agrobacterium rhizogenes (which causes hairy root disease in infected host plants). 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 "nopaline-type" Agrobacteria; Agrobacterium strains which cause production of octopine (e.g., strain LBA4404, Ach5, B6) are referred to as "octopine-type" Agrobacteria; and Agrobacterium strains which cause production of agropine (e.g., strain EHA 105, EHAlOl, A281) are referred to as "agropine-type" Agrobacteria.

The terms "bombarding, "bombardment," and "Holistic bombardment" refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/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 helium gas-driven microprojectile accelerator (PDS-1000/He) (BioRad).

The term "microwounding" when made in reference to plant tissue refers to the introduction of microscopic wounds in that tissue. 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 of transformed cells (or transgenic organisms grown from individual transformed cells) that are recovered under standard experimental conditions (i.e. standardized or normalized with respect to amount of cells contacted with foreign DNA, amount of delivered DNA, type and conditions of DNA delivery, general culture conditions etc.) For example, when isolated explants of cotyledons are used as starting material for transformation, the frequency of transformation can be expressed as the number 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 mean undifferentiated 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 the stalk by which a leaf is attached to a stem, also called a leaf-stalk. The term "axillary bud" is intended to mean a small protuberance 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 is borne. 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. The embryo 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 obtaining cell clusters, such as callus, that show unorganized growth by culturing differentiated cells of plant tissues on a dedifferentiation medium. More specifically, the term "dedifferentiation" as used herein is intended to mean the process of formation of rapidly dividing cells without particular function in the scope of the plant body. These cells often possess an increased potency with regard to its ability to develop into various plant tissues. Preferably the term is intended to mean the reversion of a differentiated or specialized tissues to a more pluripotent or totipotent (e.g., embryonic) form. Dedifferentiation may lead to reprogramming of a plant tissue (revert first to undifferentiated, non-specialized cells, then to new and different paths). The term "totipotency" as used herein is intended to mean a plant cell containing all the genetic and/or cellular information required to form an entire plant. Dedifferentiation can be initiated by certain plant growth regulators (e.g., auxin and/or cytokinin compounds), especially by certain combinations and/or concentrations thereof.

"Carotenoids" which comprise the most important group of 40-carbon terpenes and terpenoids are pigments that have a variety of commercial applications. Carotenoids are a class of hydrocarbons (carotenes) and their hydroxylated derivatives (xanthophylls) which comprise 40-carbon (C40) terpenoids consisting of eight isoprenoid (C5) units joined together. The terpenoids are joined in such a manner that the arrangement of the isoprenoid units is reversed at the center of the molecule placing the terminal methyl groups in a 1,6 relationship and the non-terminal methyl groups in a 1,5 relationship. "Carotenoids" can be monocyclic, bicyclic or acyclic. The carotenoids of the most value are intermediates in the carotenoid biosynthetic pathway and consist of lycopene, beta-carotene, zeaxanthin and astaxanthin.

"Astaxanthin" supplied from biological sources, such as crustaceans, yeast and green algae is limited by low yield and costly extraction methods when compared with that obtained by organic synthetic methods. Usual synthetic methods however, 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 carotenoid biosynthesis in marigolds") is herein incorported by reference.

Carotenoids are synthesized by serveral organisms like bacteria, yeast, algae and plants. They are deposited and accumulated either as free carotenoids or as

carotenoid derivatives, e.g. glycosides or esters. Those carotenoids and their carotenoid esters like lycopene, beta-carotene, zeaxanthin, astaxanthin, adonirubin or other xanthophylls and ketocarotenoids can be extracted from the target tissues by organic solvents. The carotenoid-containing material can be processed 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 a few possible solvents and solvent mixtures) releases the carotenoids of interest. The extraction effect and efficiency can be adjusted and modified via different solvent ratios which take into account the different polarity of various solvents. Via evaporation of the used solvents, high concentrations of carotenoids and carotenoid esters can be achieved. Subsequent chromatographic separation can further improve the purity of individual carotenoids.Further isolating procedures for carotenoids are described for example in Egger and Kleinig (Phytochemistry (1967) 6.437-440) and Egger (Phytochemistry (1965) 4.609-618).

Examples of nucleic acids encoding a "ketolase" and the corresponding ketolases which can be used in the process according to the invention are, for example, sequences from Haematoccus pluvialis. US 2006/0194274 and US 2006/0112451 are herein incorported by reference. Ketolase activity is understood as meaning the enzyme activity of a ketolase. A ketolase is understood as meaning a protein which has the enzymatic activity to introduce a keto group on the optionally 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 the steps of a) introducing into a Tagetes cell or tissue a nucleic acid construct comprising a promoter active in Tagetes cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid and/or a derivative thereof; b) incubating said Tagetes cell or tissue of step a) on a selection medium comprising a D-amino acid and/or a derivative thereof; and optionally c) transferring said Tagetes cell or tissue of step b) to a regeneration medium and regenerating 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 sequence encoding an enzyme capable of metabolizing a D-amino acid. Preferably said promoter is heterologous in relation to said enzyme encoding sequence. The promoter active in Tagetes plants and the D- amino acid metabolizing enzyme are defined below in detail.

1.1.1 The enzyme capable of metabolizing a D-amino acid

The person skilled in the art is aware of numerous sequences suitable to metabolize a D-amino acid. The term "enzyme capable of metabolizing a D-amino acid" means preferably an enzyme, which converts and/or metabolizes a D-amino acid with an activity that is at least two times (at least 100% higher), preferably at least three times, more preferably at least five times, even more preferably at least 10

times, most preferably at least 50 times or 100 times the activity for the conversion of the corresponding L-amino acid and - more preferably - also of any other D- and/or L- or achiral amino acid.

Preferably, the enzyme capable of metabolizing a D-amino acid is selected from the group consisting of D-serine ammonia-lyase (D-Serine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14), D-Amino acid oxidases (EC 1.4.3.3), and D-Alanine transaminases (EC 2.6.1.21). More preferably, the enzyme capable of metabolizing D-alanine or D-serine is selected from the group consisting of D-serine ammonia- lyase (D-Serine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14), and D-Amino acid oxidases (EC 1.4.3.3).

The term " D-serine ammonia-lyase" (D-Serine dehydratases; EC 4.3.1.18; formerly EC 4. 2.1.14) means enzymes catalyzing the conversion of D-serine to pyruvate and ammonia. The reaction catalyzed probably involves initial elimination of water (hence the enzyme's original classification as EC 4.2.1.14), followed by isomerization and hydrolysis of the product with C-N 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 2-oxoglutarate to pyruvate and D-glutamate. D-glutamate is much less toxic to plants than D- Alanine. http://www.expasy.Org/enzyme/2.6.l.21.

The term D-amino acid oxidase (EC 1.4.3.3; abbreviated DAAO, DAMOX, or DAO) is referring to the enzyme converting a D-amino acid into a 2-oxo acid, by - preferably - employing Oxygen (O 2 ) as a substrate and producing hydrogen peroxide (H 2 O 2 ) as a co-product (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 the oxidation of neutral and basic D-amino acids into their corresponding keto acids. DAAOs have been characterized and sequenced in fungi and vertebrates where they are known to be located in the peroxisomes. In DAAO, a conserved histidine has been shown (Miyano 1991) to be important for the enzyme's catalytic activity. In a preferred embodiment of the invention a DAAO is referring to a protein comprising the following consensus motif:

[LIVM]-[LIVM]-H + -[NHA]-Y-G-X-[GSA]-[GSA]-X-G-X 5 -G-X-A

wherein amino acid residues given in brackets represent alternative residues for the respective position, x represents any amino acid residue, and indices numbers indicate the respective number of consecutive amino acid residues. The abbreviation for the individual amino acid residues have their standard IUPAC meaning as defined above. D- Amino acid oxidase (EC-number 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 from a yeast (e.g. Rhodotorula gracilis), fungus, or animal or it may be a prokaryotic enzyme, for example, from a bacterium such as Escherichia coli. For examples of suitable enzyme see http://www.expasy.Org/enzyme/l.4.3.3. Examples of suitable polypeptides, which metabolise D-amino acids are shown in Table 1. The nucleic acid sequences encoding said enzymes are 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 have been characterized

and shown to differ slightly in substrate affinities (Gabler 2000), but in general they display broad substrate specificity, oxidative Iy deaminating all D-amino acids.

Table 1: Enzymes suitable for metabolizing D-serine and/or D-alanine. Especially preferred enzymes are presented in bold letters

Enzyme EC number Example Source organism Substrate

D-Serine dehydratase EC 4.3.1.18 P54555 Bacillus subtilis D-Ser

(D-Serine ammonia (originally EC P00926 Escherichia coli. DSDA D-Thr lyase, D-Serine 4.2 1.14) Q9KL72 Vibrio cholera. VCA0875 D-allothreonine deaminiase) Q9KC12 Bacillus halodurans.

D-Amino acid oxidase EC 1.4.3.3 JX0152 Fusarium solani Most D-amino

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 Rhodotorula gracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serine dehydratase (D-serine 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 (see international patent application PCT/EP 2005/002734; WO 2005/090581).

Suitable D-amino acid metabolizing enzymes also include fragments, mutants, derivatives, variants and alleles of the polypeptides exemplified above. Suitable fragments, mutants, derivatives, variants and alleles are those, which retain the functional characteristics of the D-amino acid metabolizing enzyme as defined above. Changes to a sequence, to produce a mutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid that make no difference to the encoded amino acid sequence are included.

More preferably for the method of the invention, the D-serine ammonialyase is 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 acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 2, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-serine ammonialyase; c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the 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 D-serine ammonialyase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 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 D-serine ammonialyase

and wherein selection is done on a medium comprising D-serine 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 under dedifferentiating conditions is preferably from about 1 to 10 weeks, preferably from 2 to 8 weeks, more preferably from 3 to 4 weeks. During selection, different D-amino acids in different concentrations may be used to select Tagetes plants or cells.

"Same activity" in the context of a D-serine ammonia-lyase means the capability to metabolize D-serine, preferably as the most preferred substrate. Metabolization means the lyase reaction specified above.

Also more preferably for the method of the invention, the enzyme capable of metabolizing D-serine and D-alanine is 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. 3; b) a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID No. 4, or comprising a fragment of said nucleotide sequence, wherein said fragment is sufficient to code for a protein having the enzymatic activity of a D-amino acid oxidase;

c) a nucleic acid sequence comprising a nucleotide sequence which hybridizes to a complementary strand of the 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 D-amino acid oxidase; d) a nucleic acid sequence comprising a nucleotide sequence which shows at least 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 D-amino acid oxidase.

and wherein selection is done on a medium comprising D-alanine and/or D- serine 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, D-alanine (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, D-serine (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. The total selection time under dedifferentiating conditions is preferably from about 1 to 10 weeks, preferably from 2 to 8 weeks, more preferably from 3 to 4 weeks. During selection, different D- amino acids in different concentrations may be used to select Tagetes plants or cells.

"Same activity" in the context of a D-amino acid oxidase means the capability to metabolize a broad spectrum of D-amino acids (preferably at least D- serine and/or D-alanine). Metabolization means the oxidase reaction specified above.

Mutants and 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 D-serine and or D-alanine. Such sequences and proteins also encompass, sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Polynucleotides encoding a candidate enzyme can, for example, be modulated with DNA shuffling protocols. DNA shuffling is a method to rapidly, easily and efficiently introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA. DNA shuffling can be based on a process of recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by a polymerase chain reaction-like process. See, e.g., Stemmer 1994 a,b; Crameri 1997; Moore 1997; Zhang 1997; Crameri 1998; US 5,605,793, US 5,837,458, US 5,830,721 and US 5,811,238. The resulting dsdA- or dao-like enzyme encoded by the shuffled DNA may possess different amino acid sequences from the original version of enzyme. Exemplary ranges for sequence identity are specified above.

The D-amino acid metabolizing enzyme of the invention may be expressed in the cytosol, peroxisome, or other intracellular compartment of the plant cell. Compartmentalisation of the D-amino acid metabolizing enzyme may be achieved by fusing the nucleic acid sequence encoding the DAAO polypeptide to a sequence encoding a transit peptide to generate a fusion protein. Gene products expressed without such transit peptides generally accumulate 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 of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. 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 tissue-specific or tissue preferred manner such that it is only active in transcribing the 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 induce transcription of an operably linked nucleotide sequence in at least one Tagetes cell, tissue, organ or plant at at least one time point in development or under dedifferentiated conditions. Such promoter may be a non-plant promoter (e.g., derived from a plant virus or Agrobacterium) or a plant promoter, preferably a dicotyledonous plant promoter. The person skilled in the 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 development-dependent. 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 over a 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 CaMV promoter (US 5,352,605; WO 84/02913;

Benfey 1989), the Rubisco small subunit (SSU) promoter (US 4,962,028), the legumin B promoter (GenBank Ace. No. X03677), the promoter of the nopaline synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the vacuolar ATPase subunits, the pEMU promoter (Last 1991); the MAS promoter (Velten 1984), the promoter of the Arabidopsis thalicma nitrilase-1 gene (GenBank Ace. No.: U38846, nucleotides 3862 to 5325 or else 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 plants are 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 {Gossypium hirsutum Actin 2 gene); AFl 11812 {Brassica napus Actin gene)). Use of their promoters in heterologous expression is described for the Banana actin promoter (US20050102711). An et al. [Plant J 1996 10(l):107- 121] reported that Act! and ActS mRNA were expressed strongly in leaves, roots, stems, flowers, pollen, and siliques. Chimeric GUS constructs expressed most of the vegetative tissues but almost no expression was detected in seed coates, hypocotyls, gynoecia, or pollen sacs.

b) Tissue-specific or tissue-preferred promoters

Furthermore preferred are promoters with specificities for seeds, such as, for example, the phaseolin promoter (US 5,504,200; Bustos 1989; Murai 1983; Sengupta-Gopalan 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 leaf-specific and light-induced promoter such as that from cab or Rubisco (Simpson 1985; Timko 1985); an anther-specific promoter such as that from LAT52 (T well 1989b); and a microspore-preferred promoter such as that from apg (T well 1983). Flower-specific and especially petal-specific promotors are preferred for Tagetes. Relevant flower- and petal specific promotors are known from the art. WO 04/27070, WO 05/019460, WO 06/117381 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 which the expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters such as, for example, the PRPl promoter (Ward 1993), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracyclin- inducible promoter (Gatz 1991; Gatz 1992), an abscisic acid-inducible promoter EP 0 335 528) or an ethanol-cyclohexanone-inducible promoter (WO 93/21334) can likewise be used. Also suitable is the promoter of the glutathione-S transferase isoform II gene (GST-II-27), which can be activated by exogenously applied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide (WO 93/01294) and which is operable in a large number of tissues of both monocots and dicots. Further exemplary inducible promoters that can be utilized in the instant invention include that from the ACEl system which responds to copper (Mett 1993); or the In2 promoter from maize which responds to benzenesulfonamide herbicide safeners (Hershey 1991; Gatz 1994). A promoter that responds to an inducing agent to which plants do not normally respond can be utilized. An examplary inducible promoter is

the inducible promoter from a steroid hormone 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 acid sequence 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 acid sequence in at least one cell or tissue of a Tagetes plant or derived from a Tagetes plant. Preferably it means a constitutive transcription 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 a large 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) may comprise further functional elements and genetic control sequences in addition to the promoter active in Tagetes plants. The terms "functional elements" or "genetic control sequences" are to be understood in the broad sense and refer to all those sequences, 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 the invention encompass a promoter active in Tagetes plants 5 '-upstream of the nucleic acid sequence (e.g., encoding the D-amino acid metabolizing enzyme), and 3 '-downstream a terminator sequence and polyadenylation signals and, if appropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed.

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

Polyadenylation signals which are suitable as genetic control sequences are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens. Examples of particularly suitable 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. Functional elements may include for example (but shall not be limited to) selectable or screenable marker genes (in addition to the D-amino 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., 2-deoxyglucose, 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 a transformed plant in comparison with a non-transformed one such as the genes and methods described by Ebinuma et al. 2000a,b, and in EP-A 0 601 092. c) Counter selection markers; i.e. markers suitable to select organisms with defined deleted sequences comprising said marker (Koprek 1999). Examples comprise the cytosine deaminase codA (Schlaman 1997). 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 beta-glucuronidase (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 ensure replication of the expression cassettes or vectors according to the invention in, for example, E. coli. Examples which may be 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)Sequences which make possible homologous recombination, marker deletion, or insertion into the genome of a host organism. Methods based on the cre/lox (Sauer 1998; Odell 1990; Dale 1991), FLP/FRT (Lysnik 1993), or Ac/Ds system (Wader

1987; US 5,225,341; Baker 1987; Lawson 1994) permit a - if appropriate tissue- specific and/or inducible - removal of a specific DNA sequence from the genome of the host organism. Control sequences may in this context mean the specific flanking sequences (e.g., lox sequences), which later allow removal (e.g., by means of ere recombinase) (see also see international patent application PCT/EP

2005/002734; WO 2005/090581)), iv)Elements, for example border sequences, which make possible the Agrobacterium-mediated transfer in plant cells for the transfer and integration into the plant genome, such as, for example, the right or left border of the T-DNA 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. The person 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) may be enhanced. Advantageous properties may be conferred either by over-expressing proteins or by decreasing expression of endogenous proteins by e.g., expressing a corresponding antisense (Sheehy 1988; US 4,801,340; MoI 1990) or double-stranded RNA (Matzke 2000; Fire 1998; Waterhouse 1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; 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 D-amino acid metabolizing enzyme. Expression can be, for example, constitutive, inducible or development-

dependent. Various promoters 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 include but 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. Especially preferred are Tagetes plants selected from the 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.

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 of cotyledons, 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 tissue comprises: (i) providing cotyledonary segments of a Tagetes plant; and (ii) co-cultivating said tissue with an Agrobacterium comprising a nucleic acid construct comprising a promoter active in said tissue and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid

and/or a derivative thereof. Preferably, the cotyledons 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 a regenerable 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 Agrobacterium-mediated transformation protocols. 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 - optionally- soaked 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 26-24 °C day/night, 16 hours light/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 which said 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 known to 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 transformation of 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 non-mature seeds, or petals.

For example, the nucleic acid constructs can be introduced directly to plant cells using ballistic methods, such as DNA particle bombardment, or the nucleic acid construct can be introduced using techniques such as electroporation and microinjection of a cell. Particle-mediated transformation techniques (also known as "biolistics") are described in, e.g., EP-Al 270,356; US 5,100,792, EP-A-444 882, EP-A-434 616; Klein 1987; Vasil 1993; and Becker 1994). These methods involve penetration of cells by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface. The biolistic PDS-1000 Gene Gun (Biorad, Hercules, CA) uses helium pressure to accelerate DNA-coated gold or tungsten 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, EP-A 331 083, EP-A 175 966, Green 1987), polyethylene glycol (PEG) mediated transformation (Paszkowski 1984; Lazzeri 1995), liposome-based gene delivery (WO

93/24640; Freeman 1984), electroporation (EP-A 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 to meet any particular requirement (in fact the ,,naked" expression cassettes can be utilized). Simple plasmids such as those of the pUC series may be used.

2.2.2 Soil-borne bacteria mediated transformation (co-cultivation) In addition and preferred to these "direct" transformation techniques, transformation can also be carried out by bacterial infection by means of soil born bacteria such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.

2.2.2.1 Choice of strains, vectors, and co-cultivation conditions The soil-borne bacterium employed for transfer of a DNA (e.g., T-DNA) into Tagetes genome can be any specie of the Rhizobiaceae family. The Rhizobiaceae family comprises the genera Agrobacterium, Rhizobium, Sinorhizobium, and Allorhizobium are genera within the bacterial family and have been included in the alpha-2 subclass of Proteobacteria on the basis of ribosomal characteristics. Members of this family are aerobic, Gram-negative. The cells are normally rod-shaped (0.6-1.0 μm by 1.5-3.0 μm), occur singly or in pairs, without endospore, and are motile by one to six peritrichous flagella. Considerable extracellular polysaccharide slime is usually produced during growth on carbohydrate-containing media. Especially preferred are Rhizobiaceae such as Sinorhizobium meliloti, Sinorhizobium medicae, Sinorhizobium fredii, Rhizobium sp. NGR234, Rhizobium sp. BR816, Rhizobium sp. N33, Rhizobium sp. GRH2, Sinorhizobium saheli, Sinorhizobium terangae, Rhizobium leguminosarum biovar trifolii, Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar phaseoli, Rhizobium tropici, Rhizobium etli,

Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium mongolense, Rhizobium lupini, Mesorhizobium loti, Mesorhizobium huakuii, Mesorhizobium ciceri, Mesorhizobium mediterraneium, Mesorhizobium ticmshanense, 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 described in Broothaerts (2005).

The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium. The classification and characterization of Agrobacterium strains including differentiation of Agrobacterium tumefaciens and Agrobacterium rhizogenes and their various opine-type classes is a practice well known in the art (see for example Laboratory guide for identification of plant pathogenic bacteria, 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 plant-pathogenic properties may not be justified. Accordingly more advanced methods based on genome analysis and comparison (such as 16S rRNA sequencing; RFLP, Rep-PCR, etc.) are employed to elucidate the relationship of the various strains (see for example Young 2003, Farrand 2003, de Bruijn 1996, Vinuesa 1998). The phylogenetic relationships of members of the genus Agrobacterium by two methods 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 soil-borne bacteria are capable to mediate T-DNA transfer provided that they the relevant functional

elements for the T-DNA transfer of a Ti- or Ri-plasmid (Klein & Klein 1953; Hooykaas 1977; van Veen 1988).

Preferably, the soil-born bacterium is of the genus Agrobacterium. The term "Agrobacterium" as used herein refers to a soil-borne, Gram-negative, rod-shaped 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 all Rhizobium species, based on comparative 16S rDNA analyses (Sawada 1993, Young 2003). Agrobacterium is an artificial genus comprising plant-pathogenic species.

The term Ti-plasmid 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 Ti-plasmid of 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 "nopaline-type" Agrobacteria; Agrobacterium strains which cause production of octopine {e.g., strain LBA4404, Ach5, B6) are referred to as "octopine-type" Agrobacteria; and Agrobacterium strains which cause production of agropine {e.g., strain EHA105, EHAlOl, A281) are referred to as "agropine-type" Agrobacteria. A disarmed Ti-plasmid is understood as a Ti-plasmid lacking its crown gall mediating properties but otherwise providing the functions for plant infection. Preferably, the T-DNA region of said "disarmed" plasmid was modified in a way, that beside the border sequences no functional internal Ti-sequences can be transferred into the plant genome. In a preferred embodiment - when used with a binary vector system - the entire T-DNA region (including the T-DNA borders) is deleted.

The term Ri-plasmid as used herein is referring to a plasmid, which is replicable in Agrobacteήum and is in its natural, "armed" form mediating hairy-root disease in Agrobacterium infected plants. Infection of a plant cell with a natural, "armed" form of an Ri-plasmid of Agrobacterium generally results in the production of opines (specific amino sugar derivatives produced in transformed plant cells such as e.g., agropine, cucumopine, octopine, mikimopine etc.) by the infected cell. Agrobacterium rhizogenes strains are traditionally distinguished into subclasses in the same way A. tumefaciens strains are. The most common strains are agropine-type strains {e.g., characterized by the Ri-plasmid pRi-A4), mannopine-type strains {e.g., characterized by the Ri-plasmid pRi8196) and cucumopine-type strains {e.g., characterized by the Ri-plasmid pRi2659). Some other strains are of the mikimopine- type {e.g., characterized by the Ri-plasmid pRil723). Mikimopine and cucumopine are stereo isomers but no homology was found between the pRi plasmids on the nucleotide level (Suzuki 2001). A disarmed Ri-plasmid is understood as a Ri-plasmid lacking its hairy-root disease mediating properties but otherwise providing the functions for plant infection. Preferably, the T-DNA region of said "disarmed" Ri plasmid was modified in a way, that beside the border sequences no functional internal Ri-sequences could be transferred into the plant genome. In a preferred embodiment - when used with a binary vector system - the entire T-DNA region (including the T-DNA borders) is deleted.

The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant (Kado 1991). Vectors are based on the Agrobacterium Ti- or Ri-plasmid and utilize a natural system of DNA transfer into the plant genome. As part of this highly developed parasitism Agrobacterium transfers a defined part of its genomic information (the T-DNA; flanked by about 25 bp repeats, named left and right border) into the chromosomal

DNA of the plant cell (Zupan 2000). By combined action of the so called vir genes (part of the original Ti-plasmids) said DNA-transfer is mediated. For utilization of this natural system, Ti-plasmids were developed which lack the original tumor inducing genes ("disarmed vectors"). In a further improvement, the so called "binary vector systems", the T-DNA was physically separated from the other functional elements of the Ti-plasmid (e.g., the vir genes), by being incorporated into a shuttle vector, which allowed easier handling (EP-A 120 516; US 4,940,838). These binary vectors comprise (beside the disarmed T-DNA with its border sequences), prokaryotic sequences for replication both in Agrobacterium and E. coli. It is an advantage of Agrobacterium-mediated transformation that in general only the DNA flanked by the borders is transferred into the genome and that preferentially only one copy is inserted. Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated 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 Agrobαcteriα-mediated transformation the genetic composition (e.g., comprising an expression cassette) is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid T-DNA is linked to the expression cassette to be introduced in the form of a flanking region. Binary vectors are preferably used. Binary vectors are capable of replication both in E. coli and in Agrobacterium. They may comprise a selection marker gene and a linker or polylinker (for insertion of e.g. the expression cassette to be transferred) flanked by the right and left T-DNA border sequence. They can be transferred directly into Agrobacterium (Holsters 1978). The selection marker gene permits the selection of transformed Agrobacteria and is, for example, the nptll gene, which confers resistance to kanamycin. The Agrobacterium which acts as the host organism in this case should already contain a plasmid with

the vir region. The latter is required for transferring the T-DNA to the plant cell. An Agrobacterium transformed in this way can be used for transforming plant cells. The use of T-DNA 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 P-type plasmid RK2. Most of these vetors are derivatives of pB INl 9 (Bevan 1984). Various binary vectors are 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). Improved vector systems are described also in WO 02/00900.

Preferably the soil-borne bacterium is a bacterium belonging to family Agrobacterium, more preferably a disarmed Agrobacterium tumefaciens or rhizogenes strain. In a preferred embodiment, Agrobacterium strains for use in the practice of the 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). Other suitable 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 soil-borne bacterium 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 2 nd , 2004, and international application PCT/EP2005/009366 hereby incorporated entirely by reference.

A binary vector or any other vector can be modified by common DNA recombination techniques, multiplied in E. coli, and introduced into Agrobacterium by e.g., electroporation or other transformation techniques (Mozo 1991).

Agrobacteria are grown and used in a manner as known in the art. The vector comprising Agrobacterium strain may, for example, 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; see Example 3) supplemented with the appropriate antibiotic (e.g., 50 mg/1 kanamycin sulfate). Bacteria are collected with a loop from the solid medium and resuspended. In a preferred embodiment of the invention, Agrobacterium cultures are started by use of aliquots frozen at -80°C. For Agrobacterium treatment of the Tagetes cotyledon explants, the bacteria are preferably resuspended in a co-cultivation medium. The concentration of Agrobacterium used for infection, direct contact time, and co -cultivation may need to be varied. Thus, generally a range of Agrobacterium concentrations from OD 6O0 0.1 to 3.0. Preferably for the explants the following concentrations of Agrobacterium suspensions are employed from about OD 60O = 0.1 to about 3, preferably from about OD 600 = 0.5 to 2, more preferably from about OD 600 = 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 minutes to 3 hours, preferably about 0.5 hours to 1 hour. The excess media is drained and the Agrobacterium are permitted to co- cultivate with the meristem tissue for about 1 to about 6 days, preferably about 3 to about 5 days for Agrobacterium tumefaciens strains, and about 2 to about 3 days for Agrobacterium rhizogenes strains, preferably in the dark at about 26°C during the day and at about 24°C during the night. During this step, the Agrobacterium transfers

the foreign genetic construct into 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 co-culture medium with ethylene inhibitors (e.g., silver nitrate), phenol-absorbing compounds (like polyvinylpyrrolidone, Perl 1996) or antioxidants (such as thiol compounds, e.g., dithiothreitol, L-cysteine, Olhoft 2001) which can decrease tissue necrosis due to plant defense responses (like phenolic oxidation) may further improve the efficiency of Agrobacterium-mcdiated transformation.

Supplementation of the co-cultivation medium with antioxidants (e.g., dithiothreitol), or thiol compounds (e.g., L-cysteine, Olhoft 2001; US2001034888) which can decrease tissue necrosis due to plant defense responses (like phenolic oxidation) may further improve the efficiency of Agrobαcterium-mediated transformation. In another preferred embodiment, the co-cultivation medium of comprises least one thiol compound, preferably selected from the group consisting of sodium thiolsulfate, dithiotrietol (DTT) and cysteine. Preferably the concentration is between about 1 mM and 1OmM of L-Cysteine, 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 co-cultivation. "Plant phenolic compounds" or "plant phenolics" suitable within the scope of the invention are those isolated substituted phenolic molecules which are capable to induce a positive chemotactic response, particularly those who are capable to induce increased vir gene expression in a Ti-plasmid containing Agrobαcterium sp., particularly a Ti-plasmid containing Agrobαcterium tumefαciens. A preferred plant phenolic compound is acetosyringone

(3,5-dimethoxy-4-hydroxyacetophenone). Certain compounds, such as osmoprotectants (e.g. L-proline preferably at a concentration of about 200-1000 mg/L or betaine), phytohormes (inter alia NAA), opines, or sugars, act synergistically when added in combination with plant phenolic compounds.

Particularly suited induction conditions for Agrobacterium tumefaciens have been described (Vernade 1988). Efficiency of transformation with Agrobacterium can be enhanced by numerous other methods known in the art like for example vacuum infiltration (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 co-cultivation, and (bl) transferring said co-cultivated explants after step (b) to a medium comprising at least one antibiotic suitable to inhibit Agrobacterium growth, and - optionally - at least one plant growth factor, wherein said medium is preferably lacking D-alanine and/or D-serine or a derivative thereof in a phytotoxic concentration, and, (b2) further incubating said explant after step (b ) and - optionally (bl) - on a shoot induction medium comprising at least one plant growth factor, wherein said shoot induction medium is preferably lacking D-alanine and/or D-serine 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 D-amino acid or a derivative thereof in a total concentration from about 0.05 mM to about 100 mM, and cultivating said transferred shoots on said 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 (co-cultivation), during inoculation or after inoculation with Agrobacterium. For achieving both beneficial effects wounding is preferably done prior to or during co-cultivation, more preferably prior to co-cultivation. Many methods of wounding can be used, including, for example, cutting, abrading, piercing, poking, penetration with fine particles or pressurized fluids, plasma wounding, application of hyperbaric pressure, or sonication. Wounding can be 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 co-cultivation step is vacuum infiltration (Bechtold 1998; Trieu 2000).

2.3 Post co-cultivation treatment

After the co-cultivation it is preferred to remove the soil-borne bacteria by washing and/or treatment with appropriate antibiotics. In consequence, the medium employed after the co-cultivation step e.g., the medium employed in additional steps (bl), (b2), and/or (cl) preferably contains a bacteriocide (antibiotic). This step is intended to terminate or at least retard the growth of the non-transformed 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 least one antibiotic suitable to inhibit Agrobacterium growth, and - optionally - at least one plant growth factor, wherein said medium is preferably

lacking D-alanine and/or D-serine 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 250-500 mg/L; Timentin™ is a mixture of ticarcillin disodium and clavulanate potassium; 0.8 g Timentin™ contains 50 mg clavulanic acid with 750 mg ticarcillin. Chemically, ticarcillin disodium is N-(2-Carboxy-3,3- dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]hept-6-yl)-3-thio-p henemalonamic acid disodium salt. Chemically, clavulanate potassium is potassium (Z)-(2R, 5R)-3-(2- hydroxyethylidene)-7-oxo-4-oxa-l-azabicyclo [3.2.0] heptane-2-carboxylate).

2.4 Selection

Agrobacterium-mediated 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 by the selection protocol based on D-amino acid metabolizing enzymes provided herein.

Thus, after co-cultivation and - optionally - a recovery step (see below) the target tissue (e.g., the cotyledonary tissue) is transferred to and incubated on a selection medium.

It is preferred that freshly transformed (co-cultivated) explants are incubated for a certain time from about 1 hour to about 10 days, preferably from 1 day to 8 days, more preferably from about 4 to about 7 days after co-cultivation (step (b) or (bl)) on a medium lacking the selection compound (e.g. D-alanine and/or D-serine or a derivative thereof in a phytotoxic concentration). Establishment of a reliable resistance level against said selection compound needs some time to prevent unintended damage by the selection compound even to the transformed cells and

tissue. Accordingly, the method of the invention 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 D-amino acid or a derivative thereof in a phytotoxic concentration (i.e., in a concentration which either terminates or at least retard the growth of the non-transformed cells). The term "phytotoxic", "phytotoxicity" or "phytotoxic effect" as used herein is intended to mean any measurable, negative effect on the physiology of a plant or plant cell resulting in symptoms including (but not limited to) for example reduced or impaired growth, reduced or impaired photosynthesis, reduced or impaired cell division, reduced or impaired regeneration (e.g., of a mature plant from a cell 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%, preferably at least 80%, more preferably at least 90% in comparison with a plant which was not treated with said phytotoxic compound.

The specific compound employed for selection is chosen depending on which marker protein is expressed. For example in cases where the E.coli D-serine ammonia-lyase is employed, selection is done on a medium comprising D-serine. In cases where the Rhodotorula gracilis D-amino acid oxidase is employed, selection is done on a medium comprising D-alanine and/or D-serine.

The fact that D-amino acids are employed does not rule out the presence of L-amino acid structures or L-amino acids. For some applications it may be preferred (e.g., for cost reasons) to apply a racemic mixture of D- and L-amino acids (or a mixture with enriched content of D-amino acids). Preferably, the ratio of the D-amino acid to the

corresponding L-enantiomer is at least 1 :1, preferably 2:1, more preferably 5:1, most preferably 10:1 or 100:1. The use of D-alanine has the advantage that racemic mixtures of D- and L-alanine can be applied without disturbing or detrimental effects of the L-enantiomer. Therefore, in an improved embodiment a racemic mixture of D/L-alanine is employed as compound.

The term "derivative" with respect to D-amino acids means chemical compound, which comprise the respective D-amino acid structure, but are chemically modified. As used herein the term a "D-amino acid structure" (such as a "D-serine structure") is intended to include the D-amino acid, as well as analogues, derivatives and mimetics of the D-amino acid that maintain the functional activity of the compound. As used herein, a "derivative" also refers to a form of a D-amino acid in which one or more reaction groups on the compound have been derivatized with a substituent group. The D-amino acid employed may be modified by an amino-terminal or a carboxy- terminal modifying group or by modification of the side-chain. The amino-terminal modifying group may be - for example - selected from the group consisting of phenylacetyl, diphenylacetyl, triphenylacetyl, butanoyl, isobutanoyl hexanoyl, propionyl, 3-hydroxybutanoyl, 4-hydroxybutanoyl, 3-hydroxypropionoyl, 2,4- dihydroxybutyroyl, 1-Adamantanecarbonyl, 4-methylvaleryl, 2- hydroxyphenylacetyl, 3-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, 3,5- dihydroxy-2-naphthoyl, 3,7-dihydroxy-2-napthoyl, 2-hydroxycinnamoyl, 3- hydroxycinnamoyl, 4-hydroxycinnamoyl, hydrocinnamoyl, 4-formylcinnamoyl, 3- hydroxy-4-methoxycinnamoyl, 4-hydroxy-3-methoxycinnamoyl, 2- carboxycinnamoyl, 3,4,-dihydroxyhydrocinnamoyl, 3,4-dihydroxycinnamoyl, trans- Cinnamoyl, (±)-mandelyl, (±)-mandelyl-(±)-mandelyl, glycolyl, 3-formylbenzoyl, 4- formylbenzoyl, 2-formylphenoxyacetyl, 8-formyl-l-napthoyl, 4-

(hydroxymethyl)benzoyl, 3-hydroxybenzoyl, 4-hydroxybenzoyl, 5-hydantoinacetyl, L-hydroorotyl, 2,4-dihydroxybenzoyl, 3-benzoylpropanoyl, (±)-2,4-dihydroxy-3,3-

dimethylbutanoyl, DL-3-(4-hydroxyphenyl)lactyl, 3-(2-hydroxyphenyl)propionyl, 4- (2-hydroxyphenyl)propionyl, D-3-phenyllactyl, 3-(4-hydroxyphenyl)propionyl, L-3- phenyllactyl, 3-pyridylacetyl, 4-pyridylacetyl, isonicotinoyl, 4-quinolinecarboxyl, 1- isoquinolinecarboxyl and 3-isoquinolinecarboxyl. The carboxy-terminal modifying group may be - for example - selected from the group consisting of an amide group, an alkyl amide group, an aryl amide group and a hydroxy group. The "derivative" as used herein is intended to include molecules which, mimic the chemical structure of a respective D-amino acid structure and retain the functional properties of the D- amino acid structure. Approaches to designing 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 modifications include N-alkyl (or aryl) substitutions, or backbone crosslinking to construct lactams and other cyclic structures. Other derivatives include C-terminal hydroxymethyl derivatives, O-modifϊed derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides. Furthermore, D- amino acid structure comprising herbicidal compounds may be employed. Such compounds are for example described in US 5,059,239, and may include (but shall not be limited to) N-benzoyl-N-(3-chloro-4-fluorophenyl)-DL-alanine, N-benzoyl-N- (3-chloro-4-fluorophenyl) -DL-alanine methyl ester, N-benzoyl-N-(3-chloro-4- fluorophenyl)-DL-alanine ethyl ester, N-benzoyl-N-(3-chloro-4-fluorophenyl)-/J)- alanine, N-benzoyl-N-(3-chloro-4-fluorophenyl)-Z)-α/ύf«/«e methyl ester, or N- benzoyl-N-(3-chloro-4-fluorophenyl)-D-α/α«me isopropyl ester.

The selection compound (D-amino acids or derivatives thereof in a phytotoxic concentration) may be used in combination with other substances. For the purpose of application, the selection compound may also be used together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in

known manner, e.g. into emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations in e.g. polymer substances. As with the nature of the compositions to be used, the methods of application, such as spraying, atomising, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances. However, more preferably the selection compound is directly applied to the medium. It is an advantage that stock solutions of the selection compound can be made and stored at room temperature for an extended period without a loss of selection efficiency.

The optimal concentration of the selection compound (i.e. D-amino acids, derivatives thereof or any combination thereof) may vary depending on the target tissue employed for transformation but in general (and preferably for transformation of cotyledonary tissue) the total concentration (i.e. the sum in case of a mixture) of D- amino acids or derivatives thereof is in the range from about 0.05 mM to about 100 mM. For example in cases where the E.coli D-serine ammonia-lyase is employed, selection is done on a medium comprising D-serine (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, more preferably about 0.3 mM to about 5 mM. In cases where the Rhodotorula gracilis D-amino acid oxidase is employed, selection is done on a medium comprising D-alanine and/or D-serine (e.g., incorporated into agar-solidified MS media plates), 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, D-alanine (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, D-serine (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.

Also the selection time may vary depending on the target tissue used and the regeneration protocol employed. The selection pressure (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 selection time under dedifferentiating conditions (i.e., callus or shoot induction) is from about 1 to about 10 weeks, preferably, about 3 to 7 weeks, more preferably about 3 to 4 weeks. However, it is preferred that the selection under the dedifferentiating conditions is employed for not longer than 70 days. Preferably, wherein selection is done using about 0.05 to about 100 mM D-alanine and/or D-serine for about 3 to 4 weeks under dedifferentiating conditions. In between the selection period the explants may be transferred to fresh selection medium one or more times. For the specific protocol provided herein it is preferred that two selection medium steps (e.g., one transfer to new selection medium) is employed. Preferably, the selection of step is done in two steps, using a first selection step for about 14 to 20 days, then transferring the surviving cells or tissue to a second selection medium with essentially the same composition than the first selection medium for additional 14 to 20 days. However, it is also possible to apply a single step selection. The presence of the D-amino acid metabolizing enzymes does not rule out that additional markers are employed.

After the co-cultivation step (and an optional recovery step) the co-cultivated explants are incubated on a shoot induction medium comprising at least one plant growth factor. Said incubation on shoot induction medium can be started

immediately after the co-cultivation step (i.e. in parallel with step (bl) for inhibiting growth of the Agrobacteria) or after other intermediate steps 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 growth regulator, like e.g., cytokinin compounds (e.g., 6-benzylaminopurine) and/or auxin compounds (e.g., 2,4-D). The term "plant growth regulator" (PGR) as used herein means naturally occurring or synthetic (not naturally occurring) compounds that can regulate 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 elongation and 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 is indoleacetic acid (IAA), which is transported polarly in roots and stems. Synthetic auxins are used extensively in modern agriculture. Synthetic auxin compounds comprise indole-3-butyric acid (IBA), naphthylacetic acid (NAA), and 2,4-dichlorphenoxyacetic acid (2,4-D). 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" comprises compounds, which stimulate cellular division, expansion of cotyledons, and growth of lateral buds. They delay senescence of detached leaves and, in combination with auxins (e.g. IAA), may influence formation of roots and shoots. Cytokinin compounds may comprise, for example, 6- isopentenyladenine (IPA) and 6-benzyladenine/6-benzylaminopurine (BAP).

In one preferred embodiment, inoculated cotyledon segments are incubated in the presence of 0.1 to 5 mg/L IAA and 1 to 10 mg/L BAP to induce shoot formation (Selection Medium SMl + selective agent). Preferably 1 and 5 mg/L, respectively, are used.

Preferably, after 2 to 3 weeks the explants are transferred to fresh SM2 medium containing both PGRs at reduced concentration and preferably the selection compound. During this period, buds enlarge and small shoots begin to form. After this period shoots, buds and bud clusters are cut from original plant tissues and subcultured for another 2 weeks on SM3 medium which is preferably free of PGRs, but contains the selective agent. Within the next two weeks individual, putative transgenic shoots began to establish themselves under selection pressure and occasionally began to form roots while non-transgenic 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 for continued development (elongation and root formation). After additional 1 to 6 weeks (preferably 2 x 2 weeks), leaves and putative transgenic shoots are sampled for molecular analysis to determine transgenicity. Acoording to the analysis result transgenic 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 before transferring to the greenhouse. Regenerated plants obtained using this method are fertile and have produced on average 2 to 1O g seeds per plant.

The T 0 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 the presence of concentrations of a corresponding selection compound which suppresses growth of an untransformed wild type tissue. The resulting plants can be bred and hybridized in the customary fashion. Two or more generations should be grown in 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 in said Tagetes plants or cells and operably linked thereto a nucleic acid sequence encoding an enzyme capable of metabolizing a D-amino acid, wherein said promoter is heterologous in relation to said enzyme encoding sequence. Preferably, the promoter and/or the enzyme capable of metabolizing a D-amino 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 to parts 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 and stored properly with adequate label on the

seed bags. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary. For example transgenic events in Tl or T2 generations could be involved in pre breeding hybridization program for combining different transgenes (gene stacking). Other important aspects of the invention include the progeny of the transgenic plants prepared by the disclosed methods, as well as the cells derived from such progeny, and the seeds obtained from such progeny.

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

Descendants can be generated by sexual or non-sexual propagation. Non-sexual propagation can be realized by introduction of somatic embryogenesis by techniques well known in the art. Preferably, descendants are generated by sexual propagation / fertilization. Fertilization can be realized either by selfing (self-pollination) or crossing with other transgenic or non-transgenic plants. The transgenic plant of the invention can herein function either as maternal or paternal plant. After the fertilization process, seeds are harvested, germinated and grown into mature plants. Isolation and identification of descendants, which underwent the excision process can be done at any stage of plant development. Methods for said identification are well known in the art and may comprise - for example - PCR analysis, Northern blot, Southern blot, or phenotypic screening (e.g., for a negative selection marker).

Descendants may comprise one or more copies of the at least one 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 plant organisms, roots, leaves and the like - derived from the above-described transgenic organisms, and transgenic propagation material (such as petals).

A further subject matter of the invention relates to the use of the above-described transgenic organisms according to the invention and the cells, cell cultures, parts - such as, for example, in the case of transgenic plant organisms, petals and the like - derived from them, for the production of pharmaceuticals or fine chemicals. Fine chemicals is understood as meaning enzymes, 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. The production 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 those novel carotenoids, naturally not present in Tagetes erecta, shall take place in the flowers, preferentially in the petals of Tagetes. However, accumulation of carotenoids in other tissues is also acceptable.

The flowers of Tagetes erecta naturally accumulate significant amounts of alpha- carotenoids, usually about 90%, and only low amounts of beta-carotenoids. However, beta-carotenoids, especially beta-carotene and zeaxanthin, serve as substrates for an enzymatic activity which introduces keto-groups. This enzyme activity, the beta-carotene ketolase, synthesizes echinenone and canthaxanthin which can 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 also known the farming of shrimps.

Astaxanthin is used in those diets as it contributes to the reddish pigmentation of salmon and shrimps which are requested by 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 of substrates (.e.g. beta-carotene).

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

Lutein-depleted Tagetes plants are transformed by a genetic construct carrying the ketolase gene from Haematococcus pluvialis. Those transgenic plants show a red flower phenotype, in contrast to the yellow flower type of untransformed control plants.

Examples

Example 1: Generation of lutein-depleted Tagetes plant.

The generation of a lutein-depleted Tagetes plant has been disclosed in US 6,784,351 which is herewith completely incorporated by reference.

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

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

Example 2: Breeding of 31360-2-09-08 and 31360-2-09.

Tagetes erecta selection 124-257, described in U.S. Patent No. 6,784,351, was found to have a low transformation rate using the identified tissue culture regeneration medium and Agrobacterium transformation technique. Using a standardized method, different plant selections can be transformed at different rates; therefore, to recover a target number of transformed plants, it can be expected that a selection having a low transformation rate would require use of a higher number of explants than a selection

having a high transformation rate. A selection having a low transformation rate would require at least about 200 explants to recover about 1 transformed plant.

Instead of further optimizing the transformation protocol, a plant breeding backcross technique well known to those skilled in the art was used to transfer the mutation resulting in the increased zeaxanthin to lutein ratio of selection 124-257 to a selection having a higher transformation rate. Several Tagetes erecta marigold plants were identified as having acceptable transformation rates, and from these Tagetes erecta marigold plant named 13819 was selected. Tagetes erecta 13819 is a proprietary breeding selection of PanAmerican Seed located at 622 Town Road, West Chicago,

Illinois 60185.

In the backcross program, selection 124-257 was used as the female parent in a cross with a selection of 13819 as the male parent. The resulting population was identified as 11754, and from this population a plant identified as 11754-2F was selected based on its hybrid characteristics. Plant 11754-2F was selfed and from this population plant identified as 11754-2F-1 was selected based on carotenoid profile and plant habit. Plant 11754-2F-1 was used as male parent in a cross with 13819 as the female parent. The resulting population was identified as 31360, and from this population a plant identified as 31360-2 was selected based on carotenoid profile and plant habit. Plant 31360-2 was selfed and from the resulting population, plants 31360-2-08 and 31360-2-09 were selected based on carotenoid profile, total carotenoid concentration, and plant habit. Both selections were selfed and seed from the cross was used to test transformation rates, and the seedlings from both selections were found to have acceptable transformation rates. In addition, the resulting plants from the selfed 31360-2-08 plant were found to be uniform for carotenoid profile, carotenoid concentration, and plant habit. The resulting plants from the selfed 31360-2-09 plant were found to segregate for total carotenoid concentration and plant habit

characteristics. From the selfed 31360-2-09 population, a plant identified as 31360-2- 09-08 was selected based on carotenoid profile, total carotenoid concentration, and plant habit. The selfed population from the 31360-2-09-08 plant was found to be uniform for carotenoid profile, carotenoid concentration, and to have an acceptable transformation rate.

Example 3: Agrobacterium-mediated transformation of Tagetes.

In vitro seed germination:

Approximately 200 to 600 (preferably 400) mature seeds of a regenerable marigold line (31360-2-09-08 or 31360-2-09) were placed 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 Tween-20 per 100 ml solution. The flask was placed in a vacuum chamber and stirred (under vacuum 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 an additional 15-minutes. After the final bleach treatment, the seeds were rinsed 3-4 times with sterile water and plated onto Murashigi and Skoog basal medium [MS medium = Murashigi and Skoog basal salts & vitamins] + 30 grams/L sucrose, 7.0 grams/L agar (Sigma-Aldrich Corp. St. Louis MO 63178) in 100 x 15 mm Petri dishes (10-15 seeds/plate). All media were adjusted to pH 5,75. After plating the seeds, cultures were sealed with Parafilm and germinated for 3 to 14 days (preferably 7 days) under standard culture conditions (i.e. approximately 26-24° 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, adaxial side down, onto Marigold Regeneration Medium #1 (MRMl = Vi strength MS macronutrients, full strength MS mironutrients

and 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 to pH 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 50 ml of liquid YEP medium (YEP: 10 grams/L yeast extract, 10 grams/L peptone, 5 grams/L NaCl ) + 50 mg/L kanamycin sulfate, 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 28 0 C. After overnight incubation, the bacterial suspension was transferred to a 50 ml conical, screw-cap centrifuge tube and centrifuged for 10 minutes at 4000 rpm, at a temperature of 4 0 C. After centrifugation the supernatant was discarded and the bacterial pellet re-suspended in fresh YEP medium with no antibiotics (re-suspension is done in variable volumes, dependent on the number of inoculations planned). The cultures were returned 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 2-3 minutes prior to inoculation, ImI of Silwet stock solution (Stock solution = 1.5μl Silwet L-77 wetting agent, per ImI YEP medium) was added per 30 ml of bacterial suspension.

Agrobacterium inoculation and co-cultivation:

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 at setting #6). After vortexing, the explants were damped dry with sterile filter paper and transferred, adaxial side down, to MRMl medium + 150 μM acetosyringone. The

lids were placed on the plates but left unsealed, and cultures (bacteria/explants) were allowed to co-cultivate in the dark for 1-6 days (preferably 3 days) at about 26 0 C day/24°C night temperatures.

Selection of transgenic lines:

After co-cultivation, explants were transferred (9 explants per plate), adaxial side down, onto Selection Medium #1 [SMl = MRMl + 500mg/L Timentin and selective agent (e.g. D-serine, D-alanine, phosphinothricin etc.)]. Plates were sealed with air- permeable venting tape and incubated for 2 weeks under standard culture conditions (i.e. approximately 26° C day/24°C night temperature, 16 hours light/8 hours dark). After 2 weeks of culture, obvious shoot-bud regeneration was visible on responding 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. D-serine, D-alanine, phosphinothricin etc.)] and incubated for 2 weeks under standard culture conditions (i.e. approximately 26° C day/24°C night temperature, 16 hours light/8 hours dark). During this period, buds enlarge and small shoots begin to form. After 2 weeks of incubation on SM2, shoots, buds and bud clusters were cut from original explant tissues and subcultured to Selection Medium #3 [SM3 = MS medium, 8 grams/L agar, 500mg/L Timentin and selective agent (e.g. D-serine, D-alanine, phosphinothricin etc.)] and incubated for 2 weeks under standard conditions (i.e. -26° C day/24°C night temperature, 16 hours light/8 hours dark). After 2 weeks on SM3, individual, putative transgenic shoots began to establish themselves under selection pressure and occasionally began to form roots while non-transgenic 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 SM3 for continued development. After an additional 2 weeks of culture, putative transgenic shoots were, normally, easily distinguishable from non-

transgenic shoots by their vigorous root and shoot growth compared to non- transgenic shoots. At this time, individual (independent lines) putative transgenic shoots were subcultured one final time to fresh 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 backbone integration.

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 were placed 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 #P2893 containing 3 mM MgCl 2 ) plus 110 ul IM MgCl 2 (with a final concentration of 11 mM) and 40 ul 30OuM Sulforhodamine 101 (Sigma Catalog #S7635, final concentraion of 60 nM).

2. The Q-PCR 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 #1402-9700): 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 (Al-Fl) are a two fold dilution series of a known one copy 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 dao-gene detection, the set "EcDsdA" was used for the dsdA-gene detection. As endogenous probe the set "Ecyc" was used. The set "Ecyc" was used to detect an endogenous single-copy gene of Tagetes.

Legend: BHQ-I 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 D-serine as the selective agent.

AU transformations were done using the marigold transformation protocol described above. After a 3-day co-cultivation period, continuous selection pressure was applied through culture media containing D-serine 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 analysis detecting the gene dsdA.

In the first four weeks of selection pressure, explant growth and shoot regeneration vigor clearly declined with increasing D-serine concentrations. Explants grown on media containing 0.1 and 0.33 mM D-serine appeared to be unaffected and looked virtually identical to controls grown on media without D-serine. A modest reduction in growth and vigor was seen as D-serine was increased to 1.0 mM with more dramatic effects at concentrations of 3.3 mM and higher. All explants on 33.3 mM D-serine died after approximately 6 weeks of selection pressure.

Although D-serine clearly affected the growth and vigor of these cultures, after 4-6 weeks of selection pressure, there were no obvious differences in the growth of developing shoots derived from control explants (non-inoculated) compared to those from explants inoculated with RETO 17 {dsdA gene), grown under the same selection pressure. At this point, individual developing shoots from both control and RETO 17 inoculated cultures were subcultured to hormone free, selection medium and incubated under standard conditions. After an additional two weeks of culture, numerous shoots continued to develop on all D-serine concentrations except 33.3 mM. In addition, several RETO 17 derived shoots were observed growing more vigorously than respective control shoots cultured on 1.0, 3.3 and 10.0 mM D-serine selection medium. The most pronounced growth differences were in root proliferation and this was most obvious at 3.3 mM D-serine.

At this time individual plantlets were classified with a ranking of 1-3 based on difference in growth compared to non-transgenic 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 longer and more vigorous roots but growth is not as good as plants from Class 1. The differences are not as obvious and some plants 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 on selection.

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 various D-serine selection levels.

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

If 1.0, 3.3 or 10 mM D-serine was used as the selection pressure, ~90% (10 /11) of the lines labeled as "Class 1" tested positive for the dsdA gene (see Table 2 below) indicating that transgenic plants can be identified at these levels using the dsdA gene. Since the original number of explants inoculated for each selection level was 100 this results in a preliminary transformation 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 is most 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 to allow 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 D-serine 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 D-serine as the selective agent.

All transformations were done using the marigold transformation protocol described above. After a 3-day co-cultivation period, continuous selection pressure was applied through culture media containing D-serine 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 analysis detecting the gene daol.

Early in this experiment, results for daol were similar to those for dsdA on D-serine selection. In the first four weeks of selection pressure, explant growth and shoot regeneration vigor clearly declined with increasing D-serine concentrations. Explants grown on media containing 0.1 and 0.33 mM D-serine appeared to be unaffected and looked virtually identical to controls grown on media without D-serine. A modest reduction in growth and vigor was seen as D-serine was increased to 1.0 mM with more dramatic effects at concentrations of 3.3 mM and higher. After approximately 6 weeks of selection pressure relatively few shoots were seen developing on 10.0 mM and all explants on 33.3 mM D-serine had died.

During the first 4-6 weeks of selection pressure, there were no obvious differences in the growth of developing shoots derived from control explants (non-inoculated) compared to those from explants inoculated with RETO 16 {daol gene), grown under the same selection pressure. At this point, individual developing shoots from both control and RETO 16 inoculated cultures were subcultured to hormone free, selection medium and incubated under standard conditions. After an additional two weeks of culture, shoots continued to develop on all D-serine concentrations except 33.3 mM. In addition, several RETO 16 derived shoots were observed growing more vigorously

than respective control shoots cultured on 0.33, 1.0, and 3.3 mM D-serine selection medium. The most pronounced growth differences were in root proliferation and this was most obvious at 3.3 mM D-serine.

At this time individual plantlets were classified with a ranking of 1-3 based on difference in growth compared to non-transgenic control cultures (for definition see earlier example). A summary of the numbers of shoots recovered in each class is shown in Table 3.

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

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

The best treatment for selection of transgenic lines was 3.3 mM D-serine. In this treatment 57% of the lines labeled "Class 1" tested positive for the daol gene, indicating that selection of transgenic lines is possible, although selection is not as clean as observed with dsdA selection (previous example). The rate of escapes for the

D-serine levels 0.3 and 1.0 mM (also labeled "Class 1") was even higher. It is also interesting to note that no "Class 1" transgenic lines were recovered at 10.0 mM. This is in contrast to the dsdA gene where transgenic lines were recovered. Perhaps the daol gene is not as effective at de-toxifying the D-serine (in marigold) as the dsdA gene. However, some transgenics were recovered and (as suggested for 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 D-serine selection level. These data are from one experiment and 100 explants were inoculated for each selection level (except for 0.33 mM for which there were 81).

Example 7: Marigold transformation with the daol (SELDA) selectable marker construct (RET016) using D-alanine as the selective agent.

All transformations were done using the marigold transformation protocol described above. After a 3-day co-cultivation period, continuous selection pressure was applied through culture media containing D-alanine 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 analysis detecting the gene dao\.

As with D-serine selection, in the first four weeks of selection pressure, explants grown on media containing 0.1 and 0.33 mM D-alanine appeared to be unaffected and looked virtually identical to controls grown on media without D-alanine. The first obvious reduction in growth and vigor was seen at 1.0 mM D-alanine with more dramatic effects at concentrations of 3.3 mM and higher. At concentrations above

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

After 4-6 weeks on D-alanine selection, there were no obvious differences in the growth of developing shoots derived from control explants (non-inoculated) compared to those from explants inoculated with RETO 16 (dao\ gene), grown under the same selection pressure. Again, at this point, individual developing shoots from both control and RETO 16 inoculated cultures were subcultured to fresh, hormone- free, selection medium and incubated under standard conditions. After an additional two weeks of culture, shoots continued to develop on D-alanine concentrations of 10.0 mM and lower. Consistent with D-serine selection, several RETO 16 derived shoots were observed growing more vigorously than respective control shoots cultured on 1.0 and 3.3 mM D-alanine selection medium. The most pronounced growth differences were in root proliferation and this was most obvious at 3.3 mM D-serine. No roots ever developed on shoots recovered from 10 mM D-alanine, but some shoots were observed with slightly more vigorous growth than control shoots.

At this time individual plantlets were classified with a ranking of 1-3 based on differences in growth compared to non-transgenic 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 D-alanine 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 lines were randomly selected for analysis.

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

The best treatment for selection of transgenic lines was 3.3 mM D-alanine. 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 mM D- alanine where 70% (7/10) of lines identified as "Class 1" tested positive for the dao\

gene, but at this D-alanine concentration, 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 D-serine selection, the dao\ gene did allow effective selection of transgenic marigold lines using D-alanine as the selective agent (especially at 3.3 mM). The most distinctive phenotype 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 D-alanine selection level. These data are from two experiments and 200 explants were inoculated for each selection level.

Example 8: Varying D-serine 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 3-day co- cultivation period all explants were subcultured to SMl medium containing D-serine

at concentrations of 1.0, 3.3, 5.0, 7.0 and, 9.0 mM D-serine. After approximately 9 weeks of continuous selection pressure leaf samples of each putative transgenic shoot identified as Class 1 or Class 2 (as previously described) were analysed by TaqMan molecular analysis. The combined results from two experiments are shown below.

Consistent with previous examples, explant growth and shoot regeneration vigor declined with increasing D-serine concentrations. Also, within 4-6 weeks of selection pressure, there were no obvious differences in the growth of developing shoots derived from control explants (non-inoculated) compared to those from explants inoculated with RETO 17 (dsdA gene), grown under the same selection pressure. At this point, individual developing shoots from both control and RETO 17 inoculated cultures were subcultured to hormone free, selection medium and incubated under standard conditions. After an additional two weeks of culture, shoots continued to develop on all D-serine concentrations.

Clear differences in both root and shoot growth were seen at D-serine concentrations 3.3mM and higher, although the numbers of putative transgenic shoots recovered were considerably lower from 7.0 mM and 9.0 mM D-serine treatments. At 1.0 mM D-serine, differences in growth and vigor between putative transgenic and control shoots were more difficult to see and 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 Taqman molecular 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 D-serine, although 3.3 mM D- serine produced comparable results. Under both of these concentrations transgenic

shoots were effectively identified with minimal escapes. D-serine concentrations of 7.0 and 9.0 mM also provided clean selection with minimal escapes; however, the number of transgenic lines recovered was considerably lower indicating excessive selection pressure. At the other end of the spectrum, 1.0 mM D-serine 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 D-serine 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 either pBHX 925 (UBQ10L::<&<£4::nos) or RET017 (NOS::dsdA::nos) and were allowed to co-cultivate for 3-days under standard conditions. After co-cultivation, explants were subcultured to SMl medium containing D-serine at concentrations of either 4.0 or 5.0 mM D-serine. After approximately 9 weeks of continuous selection pressure, leaf samples of each

putative transgenic shoot were taken for TaqMan molecular analysis. This experiment was run once with a selection pressure of 4.0 mM D-serine and twice with a selection pressure of 5.0 mM D-serine. Combined results are described below.

Leaf samples from 146 (81,pBHX925 and 65, RETO 17) putative transgenic lines were evaluated for gene copy number and vector backbone integration via TaqMan molecular analysis. Regardless of the D-serine concentration used for selection, preliminary transformation rates (PTE) always tended to be higher for pBHX925 than for RETO 17. At 4 mM D-serine, the PTE 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 D-serine, 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 by either the UBQlOL (pBHX925) or the NOS (RET017) promoters. Each shoot was evaluated for the presence of the dsdA gene.

Although population sizes were relatively low, transgenic lines from each construct were also compared for gene copy number. These lines were categorized as 1 gene copy, 2 gene copies and everything with greater than 2 gene copies (as determined by TaqMan analysis). Regardless of the construct used, over 50% of the transgenic

lines recovered had a single gene 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 copy number.

Table 9: Comparison of gene copy number in transgenic lines produced with either pBHX925 (UBQ10L::cfr<i4::nos) or RET017 (NOSwdsdAv.nos). Transgenic lines were categorized as 1 gene copy, 2 gene copies and everything with greater than 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 various promoters for driving the dsdA (SELDA) selectable marker gene, and determine their effects on marigold transformation efficiency. 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 (PcUbi4-2::<ft<£4::nos). All explants were inoculated with Agrobacterium using standard procedures and after co-cultivation, explants were subcultured to SMl medium containing 5.0 mM D-serine. After approximately 9 weeks of continuous selection pressure, leaf samples of each putative transgenic shoot were analysed by

TaqMan molecular analysis. The combined results from two experiments are shown below.

Leaf samples from 110 putative transgenic lines (57, RET017; 41, RLM407; 5, RET063 and 7, RLM407) were evaluated for gene 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% PTE respectively). The PTE for RLM 407 was approximately 10% which is similar to that of RETO 17 indicating the PcUbi4-2 promoter shows activity comparable to the NOS promoter for driving the dsdA (SELDA) selection. However, the PTE's for both 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 compared the NOS (RETO 17) promoter to the AtAct2i (RET063) and STPT (RLM254) promoters, while experiment #2 compared the NOS (RETO 17) promoter to the PcUbi4-2 (RLM407) promoter.

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

Example 11: Description of the plant transformation vectors.

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

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

The ubqlOL promoter [Norris et al., Plant MoI. Biol., 21 :895-906 (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. The nopaline synthase poly-adenylation signal (nos terminator) is synthesized by PCR using the plasmid pBIN19 as template and the primers nos-Forward (5' CCT GGT ACC GAT CGT TCA AAC ATT TGG CAA TA 3'; SEQ ID No. 24) and nos- Reverse (5' GGG GAA TTC GAT ATC CGA TCT AGT AAC ATA GAT G 3'; SEQ ID No. 25). The PCR product is digested with EcoR I and Kpn I then ligated

into pUC19 digested with the same enzymes. The resulting plasmid is designated pUCnos.

The ubqlOL promoteτ-dsdA coding region is removed from pUCdsdlOL by digestion with Hind III and BsrG I then ligated into pUCnos 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 the plasmid pBIN19 digested with Dr a III (made blunt by treatment with DNA polymerase Klenow fragment and dNTPs) and Hind III. The resulting plasmid is designated pBIN-dsdA 1.

A portion of pUC19 including the multiple cloning site is synthesized by PCR using the primers mcs-Forward (5' GCC AAG CTT GCA TGC CTG CAG GTC 3'; SEQ ID No. 26) and mcs-Reverse (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 pBIN-dsdAl is removed by digestion with Hind III and Pme I, and the pUC19 mcs PCR product (digested with Hind III and Pme I) is inserted by ligation. The resulting plasmid is designated 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 encoding dsdA was replaced by a sequence encoding dao.

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