WO1999022011A1

WO1999022011A1 - Reduction of chlorophyll content in oil plant seeds

Info

Publication number: WO1999022011A1
Application number: PCT/EP1998/006852
Authority: WO
Inventors: Bernhard Grimm
Original assignee: Institut für Pflanzengenetik und Kulturpflanzenforschung
Priority date: 1997-10-29
Filing date: 1998-10-29
Publication date: 1999-05-06
Also published as: EP1025248A1; CA2306205A1; AU1156399A

Abstract

The invention relates to a method for the reduction of chlorophyll content in oil plant seeds, especially rape-seeds, based on the expression of chlorophyll synthesis antisense genes. The invention also relates to oil plant seeds which have a reduced chlorophyll content in relation to wild species of seeds. The invention further relates to the use of said seeds to obtain vegetable oil.

Description

Reduction of the chlorophyll content in oil plant seeds

The present invention relates to methods for reducing the chlorophyll content in seeds of oil plants, in particular rapeseed, based on the expression of antisense genes in chlorophyll synthesis. The invention further relates to seeds of oil plants which have a reduced chlorophyll content compared to wild type plants, and to the use of these seeds for the production of vegetable oils.

The cruciferous oilseed rape (Brassica napus) and turnip rape (Brassica rapa) are among the most important oil plants alongside soybeans and cottonseed. The seeds of rapeseed contain around 40% fatty oil, the so-called rapeseed or turnip oil, which is usually obtained from the crushed seeds by pressing or extraction in a yield of approx. 40% and then subjected to refining. The rapeseed oil obtained can then be used as cooking oil, mineral lubricant oil additive, after fat hardening for margarine production and as a raw material in the production of tree wax, plasters, leather greasing agents, etc. Rapeseed oil is also known as a good source of C ₂₀ and C ₂₂ fatty acids, which are important as plastic processing and washing aids. In addition to their use as industrial raw materials, vegetable oils, and in particular rapeseed oil, are also becoming increasingly important as biodiesel fuels. In general, the areas of application of vegetable oils have been significantly expanded in recent years. With increasing environmental awareness, increasingly more environmentally friendly industrial products, such as lubricants and hydraulic oils, were developed.

The problem of high chlorophyll contents in rapeseed is well known, especially in rapeseed cultivation with short growing seasons. With every harvest, breeders and seed farms face the risk of freezing due to frost weigh against the further necessary maturation of the seeds, during which a breakdown of pigments is also observed. For this reason, the rapeseed is often left to ripen for several days after harvesting before the harvest is started.

To make matters worse, low temperatures, also for the seeds sublethal frost temperatures, promote the chlorophyll biosynthesis in rapeseed and thus additionally counteract the degradation of the pigments during ripening, which can result in undesirably high chlorophyll contents in rapeseed, especially in cultivation areas with relatively short growing periods .

During the refining of the rapeseed oil, the pigments contained in the seeds, especially the chlorophylls, and their photosensitive precursors have to be extracted in a complex manner. Apart from the fact that these extractions are time and cost intensive, they always mean a loss of the rapeseed oil yield. Although turnips have somewhat lower requirements than rapeseed in terms of vegetation duration and location, similar problems arise for these Brassicaceae in terms of seed ripening and excess chlorophyll content. The extraction and use of rapeseed oil largely corresponds to that for rapeseed oil.

With the help of molecular biotechnology, the synthesis of chlorophyll in rapeseed and turnip plants and their seeds could be influenced by reducing the amount of excess chlorophyll in rapeseed or producing completely chlorophyll-free seeds. Tetrapyrrole biosynthesis, which occurs mainly in plastids in plants, proceeds, as is currently assumed, according to the following metabolic pathway (see also Figure 1). 5-aminolevulinic acid (ALA) is produced from glutamate via three enzyme activities (glutamyl tRNA synthetase, glutamyl tRNA reductase and glutamate 1-semialdehyde aminotransferase). Two ALA molecules condense to form the cyclic compound, porphobilinogen, which is converted into the first tetrapyrrole, the hydroxymethylbilane, by chaining 4 units. The protopophyrin IX is formed from hydroxymethylbilane by oxidation and side chain modifications via uroporphyrinogen III, coproporphyrinogen and protopophyrinogen IX. The incorporation of a divalent metal cation produces magnesium protopophyrin IX, which is converted into chlorophyll a by further modifications, which include the incorporation of an additional isocyclic ring on ring B and the particularly important esterification of a propionate with a phytol chain.

The knowledge of plant genes which code for enzymes of the chlorophyll synthesis path indicated above and the possibility of transferring such genes in a targeted manner to plants form an essential basis of the present invention.

European patent application 0 779 364 A2 describes an approach to reducing the chlorophyll content in transgenic plants, in which the transcript and protein content of the chlorophyll-binding proteins (chlorophyll a / b binding (CAB) proteins) of the antenna complex of Photosystem II (light harvesting complex associated with photosystem II, LHCII) is reduced. This approach, which is based on the expression of antisense genes for LHCII, therefore relates to proteins which bind already synthesized chlorophyll in Photosystem II, but not to enzymes which are directly involved in chlorophyll synthesis. In addition, experimental data from Flachmann and Kühlbrandt (Plant Cell (1995) 7, 149-160) indicate that the leaves of transgenic tobacco plants, as a result of the expression of antisense genes for LHCII, have RNA contents down to 5% of the control values in wild-type plants are reduced, but neither a decrease in the LHC protein content nor a chlorophyll reduction can be observed. A correlation between the decrease in the amount of LHC-RNA and the decrease in the protein and chlorophyll content could not be found by Flachmann and Kühlbrandt.

In contrast to the strategy pursued in the prior art, it should be possible to achieve a successful and reliable reduction in the chlorophyll content in transgenic plants by means of a direct intervention in the tetrapyrrole synthesis.

It is therefore an object of the invention to show ways in which the chlorophyll synthesis in transgenic oil plant seeds can be reduced or blocked with the aid of genetic engineering methods and the chlorophyll content can thereby be reduced.

Furthermore, it is an important object of the invention to provide transgenic plant seeds with a reduced chlorophyll content compared to seeds of wild-type plants.

Further objects of the invention will become apparent from the following description. These objects are achieved by the subject matter of the independent claims, in particular based on the provision of the methods according to the invention for reducing the chlorophyll content in plant seeds and the plants and parts and products thereof according to the invention with a chlorophyll content reduced compared to wild type plants. The present invention thus relates to the use of DNA sequences which code for enzymes in chlorophyll synthesis and whose targeted transfer to and expression in transgenic plant cells results in a reduction in chlorophyll synthesis. More specifically, the invention relates to the transfer of suitable antisense gene constructs to plants and their expression in the seed tissue.

The invention is based on experiments in which the amount of chlorophyll in plant seeds could be significantly reduced by the seed-specific expression of antisense genes against certain enzyme steps in tetrapyrrole synthesis and the resultant seed-specific inhibition of the activity of defined enzymes in chlorophyll synthesis.

The antisense technique elegantly exploits the complementarity of nucleic acid molecules. Antisense genes, which code complementary to the endogenous RNA for enzymes of the tetrapyrrole metabolism, reduce the endogenous RNA contents or the number of RNA molecules available for the subsequent protein biosynthesis. A reduced content of endogenous RNA inevitably leads to a reduced translation, that is to say a reduced amount of protein, which in turn manifests itself in a reduced activity of the target enzyme.

The invention relates to all genes whose gene products catalyze steps of tetrapyrrole synthesis. Since the expression and activity of any enzyme involved in tetrapyrrole synthesis can be reduced or inhibited by targeted antisense RNA synthesis, the method according to the invention for reducing the chlorophyll content in sperm cells can use all genes of this metabolic pathway, ie also individually or in combination , be performed. These genes are primarily genes which are responsible for glutamyl tRNA synthetase, glutamyl tRNA reductase, glutamate 1 semialdehyde aminotransferase, magnesium chelatase or their subunits CHL I, Code CHL D, CHL H, chlorophyll synthetase and Mg protopophyrin monomethyl ester transferase.

The invention also relates to fragments of such genes involved in chlorophyll synthesis, the use of which within an antisense construct results in reduced activity of the target enzyme. Such fragments could be referred to in the context of this invention as "antisense active" fragments, i.e. their transfer in the form of a suitable construct reduces the corresponding endogenous enzyme activity.

Furthermore, the invention relates to the use of alleles and derivatives of the genes according to the invention for reducing the chlorophyll content in plant seeds, thus also the use of nucleic acid molecules whose sequences differ from the genes according to the invention due to the degeneration of the genetic code and their transmission to plant cells in one go the

Antisense gene-related reduction in the content of the desired enzyme of chlorophyll metabolism results.

Furthermore, the invention relates to the use according to the invention of nucleic acid molecules which contain the antisense genes according to the invention or which have arisen from, or have been derived from, naturally occurring or by genetic engineering or chemical processes and synthesis processes. This can be, for example, DNA or RNA molecules, cDNA, genomic DNA, mRNA etc.

In the context of the invention, the following genes of chlorophyll synthesis are preferably used to reduce the chlorophyll content in seed tissue. Genes coding for glutamate 1 semialdehyde aminotransferase (GSA-AT). This enzyme catalyzes the conversion of glutamate 1-semialdehyde (GSA) to aminolevulinic acid (ALA) through the net transfer of an amino group from C2 to Cl. The expression of a tobacco antisense RNA for GSA-AT has so far only been investigated in leaves of tobacco plants (Höfgen et al. (1994) Proc. Natl. Acad. Sei. USA 91, 1726-1730). DNA sequences from two tobacco full-length cDNA clones are in the GenBank, Accession Nos. X65973 and X65974 are available.

Genes encoding the CHL I and CHL H subunits of magnesium chelatase. The subunits of Mg chelatase are involved in the incorporation of magnesium into Protopoφhyrin IX. Suitable DNA sequences of full-length cDNA clones which code for CHL I and CHL H are described in Kruse et al. (1997) Plant Mol. Biol. 35, 1053-1056 and under Accession Nos. AF014053 (Chl I) or AF014051 and AF14052 (Chl H) are described in the GenBank.

Genes encoding plastid glutamyl tRNA synthetase. This enzyme catalyzes the formation of glutamyl tRNA from glutamic acid.

Genes that code for glutamyl tRNA reductase. This enzyme catalyzes the reduction of activated glutamate to glutamate 1-semialdehyde. Suitable DNA sequences of full-length cDNA clones from a barley cDNA library, which code for the reductase, are shown in

Bougri and Grimm (1996) Plant J. 9, 7867-878 and under Accession Nos. X86101, X86102 and X92403 are described in the gene bank. In an antisense gene construct according to the invention, a gene of chlorophyll synthesis or a fragment thereof, preferably the coding region of such a gene or fragment, is in the antisense orientation, ie 3 '->5' orientation, with the 3 'end a promoter, i.e. a regulatory element that ensures the transcription of the linked gene in plant cells.

For example, the genes according to the invention can be expressed in plant cells under the control of constitutive, but also inducible or tissue or development-specific promoters. These are preferably seed-specific promoters, the use of which enables the targeted inhibition of chlorophyll synthesis in sperm cells.

As examples of seed-specific promoters that can be used in connection with the invention, mention should be made of the USP promoter (described, inter alia, in: Bäumlein et al. (1991) Mol. Gen. Genet. 459-467; Fiedler et al. ( 1993) Plant Mol. Biol. 22, 669-679; DE-C2-39 20 034), the napin promoter (Ericson et al. (1991) Eur. J. Biochem. 197, 741-746; Accession No. X 58142), the 2S albumin promoter (Krebbers et al. (1988) Plant Physiol. 87, 859-866; Accession No. Z 24745), the legumin promoter (Bäumlein et al. (1986) Nucl. Acids Res. 14, 2707-2720; Accession No. X 03677) and the Hordein promoter (Entwistle et al. (1991) Plant Mol. Biol. 17, 1217-1231; Accession No. X 60037).

Optionally, the antisense constructs used according to the invention can additionally comprise enhancer sequences or other regulatory sequences. It is also an object of the invention to provide new plants, plant cells, parts or products which are distinguished by a reduced chlorophyll content compared to wild type plants.

These objects are achieved by the transfer of the antisense nucleic acid molecules according to the invention and their expression in plants. By providing the nucleic acid molecules according to the invention, it is now possible to use genetic engineering methods to modify plant cells to the extent that they have a reduced chlorophyll biosynthesis capacity compared to wild-type cells. According to the invention, these are seeds of oil plants, in particular oilseed rape and turnips, which have a significantly reduced chlorophyll content compared to seeds of wild type plants. The transgenic oilseed rape plants are particularly preferably summer oilseed rape plants. OO rapeseed plants are also suitable for the use of the processes according to the invention, i.e. Rapeseed plants that are free of erucic acid and low in glucosinolate.

The plants that are transformed with the nucleic acid molecules according to the invention and in which a smaller amount of chlorophylls are synthesized due to the integration of such a molecule into their genome can in principle be any plant. It is preferably an oil plant, such as oilseed rape and turnips, from which vegetable oil is obtained, in the production of which high chlorophyll contents are undesirable.

The invention relates in particular to propagation material from plants according to the invention, for example seeds, fruits. Cuttings, tubers, rhizomes etc., this material may be above contains described transgenic plant cells, and parts of these plants such as protoplasts, plant cells and calli; seeds are particularly preferred.

The present invention is also based on the object of providing processes for producing plant cells and plants and parts thereof, in particular seeds, which are distinguished by a reduced chlorophyll content.

This object is achieved by methods by means of which the production of new plant cells and plants which, owing to the transfer and expression of antisense genes which are directed against endogenous genes which code for enzymes of chlorophyll synthesis, are characterized by a reduced chlorophyll content compared to wild-type cells, is possible.

Various genetic engineering transformation methods are available for generating such new plant cells and plants. According to the invention, plant cells which have a reduced chlorophyll content due to the expression of an antisense gene construct according to the invention are produced by a process which comprises the following steps:

a) Production of an expression cassette which comprises the following DNA sequences: a promoter which ensures transcription in plant cells; at least one nucleic acid sequence encoding an enzyme or a fragment thereof that is involved in chlorophyll synthesis, the nucleic acid sequence being coupled in antisense orientation to the 3 'end of the promoter; and optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript which is coupled to the 5 'end of the nucleic acid sequence.

b) Transformation of plant cells with the expression cassette produced in step a).

c) Regeneration of transgenic plants and, if appropriate, the multiplication of the plants.

Furthermore, the invention relates to the use of the antisense constructions according to the invention for producing plants, in particular plant seeds, which have a reduced chlorophyll content. The invention preferably relates to the use of the antisense constructions according to the invention for the production of seeds from oil plants, particularly preferably from rapeseed and turnips, which have a reduced chlorophyll content.

Another object of the invention is to record the possibilities of using the plants according to the invention or their cells, parts and products, in particular their seeds.

The invention relates in particular to the use of the plants according to the invention, in particular their seeds, for obtaining vegetable oils as raw materials for the chemical, cosmetic, pharmaceutical and food industries and as an energy source. The plants according to the invention thus represent an important source for the production of vegetable oils, in particular rapeseed and turnip oils for a broad spectrum of commercial purposes.

For the promoter mentioned, everyone comes in principle for the

Transformation selected plant functional promoter into consideration, which fulfills the condition that the expression regulated by it leads to a reduced chlorophyll synthesis performance in plant cells. With regard to the use of the transgenic plants as a supplier of vegetable oils, promoters which ensure seed-specific expression appear particularly useful for this purpose. Examples of such promoters are the above-mentioned USP, napin, 2S-albumin, legumin and hordein promoters.

If such promoters are not known or are not available, the concept for isolating such promoters is in any case known to the person skilled in the art. In a first step, the poly (A) ⁺ RNA is isolated from seed tissue and a cDNA library is created. In a second step, cDNA clones based on poly (A) ^* RNA molecules from a non-seed tissue are used to identify those clones from the first bank whose hybrid poly (A) ⁺ RNA Molecules are only expressed in the seed tissue. Promoters are then isolated with the help of these cDNAs identified in this way, which can then be used for the expression of the antisense.

To prepare the introduction of foreign genes into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be matched to a suitable one - 1:

Restriction interface to be introduced into the vector. The plasmid obtained is used for the transformation of E. co // cells. Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.

A large number of known techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts, the direct gene transfer of isolated DNA to protoplasts, the electroporation of DNA, the introduction of DNA using the biolistic method as well as other options.

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids such as pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The usual selection markers are known to the person skilled in the art and it is no problem for him to select a suitable marker. Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti and Ri plasmid, must be connected as a flank region to the genes to be introduced .

If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens using a helper phasmid (conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. (1978) Molecular and General Genetics 163, 181-187). Serving as host cell should contain a plasmid of Agrobacterium carrying a vz ^'R region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.

The use of T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanters BV, Alblasserdam (1985) Chapter V; Fraley et al. (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al. (1985) EMBO J. 4, 277-287.

For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells. The plants are regenerated using conventional regeneration methods using known nutrient media. The plants thus obtained can then be examined for the presence of the introduced DNA. Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf., for example, Willmitzer L. (1993) Transgenic Plants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (H. Rehm, G. Reed, A. Pühler, P. Stadler, eds.) Vol. 2, 627-659, VCH Weinheim - New York - Basel - Cambridge).

Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and others. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA. The transformed cells grow within the plant in the usual way (see also McCormick et al. (1986) Plant Cell Reports 5, 81-84). The resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties. Seeds can be obtained from the plants.

Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.

Likewise, transgenic lines can be determined using conventional methods, which are homozygous for the new nucleic acid molecules and which investigate their phenotypic behavior with regard to a changed chlorophyll content and compare them with that of hemizygotic lines.

The transmission and expression of the antisense gene constructs according to the invention can be carried out with the aid of conventional molecular biological and biochemical methods. These techniques are known to the person skilled in the art and he is easily able to choose a suitable detection method, for example a Northern blot analysis for the qualitative and quantitative detection of RNA which is specific for the coding region of the respective antisense gene, or a Southern -Blot analysis to identify the transferred DNA sequences.

The transgenic plant cells or plants as well as parts and products thereof can then be examined for their chlorophyll content. Here For example, the following analysis methods are available:

Determination of chlorophyll and carotenoid contents according to Porra et al. (1989) Biochem. Biophys. Acta 975, 384-394; Determination of ALA synthesis performance (e.g. as described in Zavgorodnyaya et al. (1997) The Plant J. 12, 169-178);

Determination of glutamate 1 semialdehyde aminotransferase activity (see also Zavgorodnyaya et al. (1997) supr).

A reduced chlorophyll content of the transgenic plants or their parts and products compared to wild-type plants can often also be seen with the naked eye or with the aid of optical aids.

For DNA, RNA isolation, sequence analysis, restriction, cloning, gel electrophoresis, radioactive labeling, Southern, Northen and Western blot analyzes, hybridization and the like, common methods can be used, as described in relevant laboratory manuals such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

The following examples serve to explain the invention.

EXAMPLES

Example 1: Preparation of an antisense construct based on DNA sequences which code for a glutamate 1 semialdehyde aminotransferase from tobacco

To construct a GSA-AT antisense mRNA expression vector, the complete 1714 bp. Tobacco GSA-AT cDNA fragment (GenBank Accession No. X65974; see. Höfgen et al, supra) as an EcoRV / Xbal restriction fragment in the binary vector BinHyg-Tx, a plasmid derivative of the vector BinAR (Höfgen and Willmitzer (1990) Plant Sei. 66, 221-230), which previously cloned with the restriction enzymes Smal and Xbal was cut.

To produce a binary vector with a fusion of a USP promoter and the GSA-AT cDNA sequence in antisense orientation, the vector pP30T, which contains the USP promoter from Viceafaba, was a pUC18 derivative (see also Bäumlein et al. (1991) supra), cut with PstI and Bglll. The isolated USP promoter fragment was then cloned into a BamHI / Pstl-cut pBluescript vector (-> pUSPblue). This vector construct pUSPblue was then cut with EcoRI and Xbal to obtain an EcoRI / Xbal promoter fragment. The 35S CaMV promoter was removed from the binary vector BinAR by restriction digestion with EcoRI and Xbal and replaced by this seed-specific promoter by ligation of the EcoRI / Xbal vector fragment with the USP promoter fragment (-> pUSPbin). The complete cDNA sequence (Xbal - 3 '- GSA-AT-cDNA - 5' -Sall) of the tobacco-GSA aminotransferase cut from the pBluescript with Xbal and Sall was used in the Xbal-Sall-cut pUSPbin. The resulting binary vector was named pUSPASGSAT (see also Figure 2).

Instead of the binary vectors BinAR or BinHyg-Tx mentioned, which contains a tetracycline-inducible CaMV 35S promoter, any vector suitable for plant transformation can be used to produce an antisense gene consisting of a fusion of a promoter, preferably a seed-specific promoter, which ensures transcription and translation in plant cells, and DNA sequences which code for enzymes involved in chlorophyll synthesis are used. Example 2:

Transformation of rapeseed plants and regeneration of intact plants

The transformation into summer rape was carried out according to the method of De Block et al. (1989, Plant Physiol. 91, 694-701) and Damgaard et al. (1997, Transgenic Research 6, 279-288).

For this purpose, a recombinant culture of Agrobacterium tumefaciens (strain GV 3101) was washed and in medium 1 (MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473), 2.5 mM MES pH 5.5, 1 mg / 1 benzylaminopurine (BAP), 0.1 mg / 1 naphthylacetic acid (NAA), 0.01 mg / 1 gibberelic acid (GA ₃ ), 200 μM acetosyringone).

The hypocotyl of 14-day-old Brassica "αpw ^ seedlings was cut into 0.5 to 1 cm long segments, with the bacterial suspension for 30 min. incubated and then stored for 5 days in the dark at 21 ° C. on medium 1 containing 0.7% agar. For callus induction, the hypocotyl was at 25 ° C on medium 2 (MS medium, 2.5 mM MES pH 5.7, 30 g / 1 sucrose, 1 mg / 1 kinetin, 1 mg / 1 2,4-dichloropheneoxyacetic acid (2nd , 4-D), 0.01 mg / 1 GA ₃ , 500 mg / 1 polyvinylpyrrolidone (PVP), 5 mg / 1 AgNO ₃ , 5 g / 1 agarose, 250 mg / 1 carbenicillin, 100 mg / 1 kanamycin; at 150 μM photon / πr / sec).

After formation of the callus, shoot induction was carried out on medium 3 (MS medium, 2.5 mM MES pH 5.5, 20 g / 1 sucrose, 40 mg / 1 adenine, 1 mg / 1 BAP, 0.1 mg / 1 NAA , 0.01 mg / 1 GA ₃ , 500 mg / 1 PVP, 5 mg / 1 AgNO ₃ , 100 mg / 1 kanamycin, 5 g / 1 agarose, 250 mg / 1 carbenicillin).

After the shoot had formed, the calli, which showed shoots, were placed on shoot extension medium (medium 4) in glass vessels (MS medium, 2.5 mM MES pH 5.7, 10 g / 1 sucrose, 0.0025 mg / 1 BAP, 100 mg / 1 kanamycin, 7 g / 1 agarose, 250 mg / 1 carbenicillin). After about 2-3 weeks, the shoots were transferred to medium 5 (MS medium, 2.5 mM MES pH 5.5, 7 g / 1 agarose) to form roots.

Alternatively, transgenic oilseed rape plants were produced according to the following protocol: A recombinant culture of Agrobacterium tumefaciens (strain GV 3101) was washed and resuspended in MS medium with 2.5 mM MES pH 5.5. The hypocotyl of rape seedlings 5-7 days old was cut into explants approximately 7 mm long and precultivated in liquid CIM medium for 24 hours. The explants were then co-cultivated in 10 ml of the CIM medium with 50 μl of an overnight culture of the recombinant Agrobacterium strain for 2-3 days in the dark. The CIM medium consists (per liter) of MS medium, 30 g sucrose, 500 mg MES pH 5.8, 1 mg 2.4 D, 1 mg kinetin. The hypocotyl pieces were then washed and cultured on CIM medium with 5 g / 1 agarose, 20 mg kanamycin, 250 mg betabactyl, 250 mg carbenicillin for 7-10 days for callus induction. The slightly swollen explants were then placed on SIM medium for shoot induction. The medium was renewed every 10-14 days. The SIM medium consists (per liter) of MS medium, vitamins, 20 g sucrose, 500 mg MES pH 5.6-5.8, 2 mg zeatin, 2 mg BAP, 100 mg myo-inositol, 5 g agarose, 20 mg kanamycin, 500 mg betabactyl or carbenicillin. After the shoots had formed, the calli, which showed shoots, were placed in glass vessels with MS medium, 500 mg / 1 MES pH 5.7, 20 g / 1 sucrose, 20 mg / 1 kanamycin, 5 g / 1 agarose, 500 mg / 1 Carbenicillin transferred.

In addition to the transformation methods described, rapeseed plants can also be transformed using other techniques. For example, the Moloney et al. (1989, Plant Cell Rep. 8. 238-242), in which an agrobacterial-mediated DNA transfer to cotyledons from 7 day old seedlings via the cut at the petiole. Furthermore, the transformation of protoplasts using cells of different tissues is suitable (e.g. described in Thomzik (1993) In: Biotechnology in agriculture and forestry, Vol. 23, Plant protoplasts and genetic engineering IV (Bajaj, ed.) Springer-Verlag, Berlin, 170-182).

Example 3:

Analysis of transgenic plants that express antisense RNA against glutamate 1-semialdehyde aminotransferase under the control of the USP promoter

Plants that were transformed with the vector construct pUSPASGSAT (see Example 1 and Figure 2) were then examined for the expression of antisense RNA against GSA-AT.

For this purpose, i.a. the chlorophyll contents and synthesis rates in the transgenic plants were determined. For this purpose, chlorophylls were extracted from 100 mg of seed material ground in liquid nitrogen with buffered, ice-cold 80% acetone until the pellet had become colorless. The samples were diluted accordingly and the absorbance at 663, 646 and 750 nm was measured on the spectrophotometer. The formulas by Porra et al. (1989, supra).

For the determination of chlorophyll synthesis rates seed tissue was homogenized in 20 mM K ₂ HPO ₄ / KH ₂ PO ₄ (pH 7.1) were incubated with 34 kBq D- [4- ¹⁴ C] -labeled ALA (5-aminolevulinic acid) for 8 h in the light, then one

Chlorophyll extraction carried out and separated by HPLC. The extraction and separation was carried out according to the method of Gilmore and Yamamoto (1991, J. Chromatography 543, 137-145), modified by Kruse et al. (1995, EMBO J. 14, 3712-3720) as follows: 100 mg of seed material ground in liquid nitrogen were weighed and extracted with 100% acetone and 10 μM KOH until the pellet had become colorless (1 × 400 μl, 3 × 200 μl). The extracts were diluted 4: 1 with H ₂ O for the HPLC runs in order to achieve sharper separations.

The chlorophylls were measured using a LiChrospher 100 HPLC RP 18 column (5 μm, Merck) at a flow of 1 ml / min. eluted with the following gradient: 100% mobile solvent A (780 ml acetonitrile; 80 ml MeOH; 30 ml Tris / HCl 0.1 M pH

8.0) for 7 min., In a linear increase in 6 min. to 100% solvent B (800 ml MeOH; 200 ml hexane), 14 min. 100% solvent B. The eluate was analyzed with a "photodiode array" (PDA) detector and a radioactivity monitor connected downstream. Radioactive labeled chlorophyll could

Retention times that are known for the HPLC system can be assigned.

Furthermore, the 5-aminolevulinic acid synthesis capacity in the transgenic plants was determined. Since enzyme activities in the C5 pathway cannot be determined without purifying the enzymes, indirect methods were chosen to measure the ability of ALA formation from glutamate. On the one hand, the accumulation of ALA after incubation of LA was determined according to the following protocol: 100-300 mg of seed tissue were mixed with 40 mM levulinic acid, a potent inhibitor (substrate analog) of ALA dehydratase (ALAD), in 20 mM K ₂ per batch HPO ₄ / KH ₂ PO ₄ (pH 7.1) incubated in the light for 2-4 h. The plant material was frozen in liquid nitrogen, homogenized and mixed well after adding 1 ml of 20 mM K ₂ HPO ₄ / KH ₂ PO ₄ (pH 7.1). The ALA determination was carried out according to Mauzerall and Granick (1956, J. Biol. Chem. 219, 435-446). After centrifugation for 20 minutes at 15,000 g at 4 ° C., the same volume of 20 mM K ₂ HPO ₄ / KH ₂ PO ₄ (pH 7.1) and 100 μl of ethyl acetoacetate were pipetted into 250 μl of the supernatant. Samples that had been extracted without levulinic acid incubation at time t ₀ served as a control. All samples were for exactly 10 min. heated at 100 ° C, then on ice for 5 min. cooled, with 500 μl modified Ehrlich's reagent (373 ml glacial acetic acid; 90 ml 70% (w / v) perchloric acid; 1.55 g HgCl ₂ ; made up to 500 ml with H ₂ O; in 110 ml of this solution, 2 g of p-dimethylaminobenzaldehyde dissolved) mixed well and for 5 min. Centrifuged at 15,000 g. The absorbance was measured at 525, 553 and 600 nm. The non-specific turbidity, measured at 600 nm, was subtracted from all values; the ratio E ₅₅₃ / E ₅₂₅ should be between 1.3 and 1.5. A calibration series was created to quantify the ALA contents.

On the other hand, the accumulation of ALA after incubation in glutamate and levulinic acid was determined. For this purpose, 100-300 mg of seeds in 92.5 kBq L- [U- ¹⁴ C] -glutamate, 2 mM glutamate, 20 mM LA, 880 μl 20 mM K ₂ HPO ₄ /

KH ₂ PO ₄ (pH 7.2) incubated for 8 h at RT in the light. The plant material was homogenized in 0.5 ml of 1N TCA and 1% SDS, centrifuged and the supernatant was treated with 1 volume of eluent (7.8 g / 1 NaH ₂ PO ₄ ; 1.74 g / 1 SDS; 5 ml / 1 tertiary Amyl alcohol) diluted. [ ¹⁴ C] -labeled ALA was detected according to Pontoppidan and Kannangara (1994, Eur. J. Biochem. 225, 529-537) via HPLC (flow 1 ml / min., Isocratic, RP 8 column, Novapak C8 , 4 μm particle size, 3.9 mm x 150 mm). The amount of radioactively labeled ALA was determined using a connected radioactivity monitor. The peak was identified by co-chromatography with D- [4- ¹⁴ C] -labeled ALA, the quantification using calibration series of the same substance.

Furthermore, Southern and Northern blot experiments were carried out to detect the integration of the transgenes and the influencing of the RNA contents for GSA aminotransferase according to standard methods (for example Sambrook et al. (1989) supra). The amount of GSA aminotransferase was also determined immunologically in classic Western blot analyzes, the antibodies being obtained by immunizing rabbits or mice with Freund's adjuvant. Analysis of transgenic rapeseed expressing the antisense construct contained in the vector pUSPASGSAT showed that the method according to the invention results in a significant reduction in the chlorophyll content.

If molecular biological work has not been adequately described in any way, this was done using standard methods, as described by Sambrook et al. (1989) supra. With regard to the transformation of plants, reference is made to generally known review articles and to the publications mentioned above.

Description of the pictures:

Fig. 1 shows the metabolic pathway of tetrapyrrole biosynthesis

Fig. 2 shows a restriction map of the binary described in Example 1

Vector pUSP-ASGSAT, which contains a fusion of the USP promoter and the region coding for GSA aminotransferase in antisense orientation. The vector pUSP-ASGSAT carries a kanamycin resistance gene as a plant selection marker.

Claims

EXPECTATIONS

1. A method for reducing the chlorophyll content in seeds of oil plants, comprising the following steps:

a) Preparation of an antisense expression vector comprising the following

DNA sequences comprises: a promoter which is functional in plants, in particular seed-specific, at least one nucleic acid sequence which codes for an enzyme or a fragment thereof which is involved in chlorophyll synthesis, the nucleic acid sequence being in the antisense orientation at the 3 'end of the Promoter is coupled; and - if necessary, a termination signal for the termination of the

Transcription and the addition of a poly-A tail to the corresponding transcript which is coupled to the 5 'end of the nucleic acid sequence.

b) transfer of the expression vector from a) to plant cells and

Integration of the nucleic acid sequence into the plant genome,

c) Regeneration of transgenic plants and, if appropriate, multiplication of these

Plants.

2. The method of claim 1, wherein the coding sequence for a glutamate 1-semialdehyde aminotransferase or at least a part thereof.

3. The method according to claim 1, wherein the coding sequence encodes the subunit CHL I of magnesium chelatase or at least a part thereof.

4. The method of claim 1, wherein the coding sequence for the

Coded subunit CHL H of magnesium chelatase or at least a part thereof.

5. The method of claim 1, wherein the coding sequence encodes the plastid glutamyl tRNA synthetase or at least a portion thereof.

6. The method of claim 1, wherein the coding sequence encodes a glutamyl tRNA reductase or at least a portion thereof.

7. The method according to any one of the preceding claims, wherein the

Promoter is a USP promoter.

8. The method according to any one of the preceding claims, wherein the promoter is a napin promoter.

9. The method according to any one of the preceding claims, wherein the promoter is a 2S albumin promoter.

10. The method according to any one of the preceding claims, wherein the promoter is a legumin promoter.

1 1. The method according to any one of the preceding claims, wherein the promoter is a hordein promoter.

12. The method according to any one of the preceding claims, wherein the oil plant belongs to the Brassicacea family.

13. The method according to any one of the preceding claims, wherein the oil plant is rapeseed.

14. The method according to any one of claims 1-12, wherein the oil plant is turnips.

15. Transgenic plant seeds with reduced chlorophyll content compared to seeds of wild type plants, which are obtained from a process according to one of the preceding claims.

16. Use of the seeds according to claim 15 for the production of vegetable oils.