CA2306205A1

CA2306205A1 - Reduction of chlorophyll content in oil plant seeds

Info

Publication number: CA2306205A1
Application number: CA002306205A
Authority: CA
Inventors: Bernhard Grimm
Original assignee: Individual
Current assignee: Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Priority date: 1997-10-29
Filing date: 1998-10-29
Publication date: 1999-05-06
Also published as: EP1025248A1; WO1999022011A1; 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

This invention concerns methods for reducing the chlorophyll content the seeds of oil plants, in particular rapeseed, based on the expression of chlorophyll synthesis antisense genes.
In addition, the method concerns seeds of oil plants that have reduced chlorophyll content compared to the wild types, as well as the use of these seeds to produce vegetable oils.
Next to soy and cottonseeds, the crucifers rape (Brassica napus) and turnip (Brassica rapa) are among the most important oil plants. The seeds of the rape plant contain about 40%
fatty oil, the so called rapeseed oil or turnipseed oil, which as a rule is obtained from the crushed seeds by pressing or extraction in a yield of about 40% and then is refined.
The rapeseed oil that is obtained can then be used as food oil, mineral lubricant oil additive, for production of margarine after hydrogenation, and as raw material in the production of grafting wax, plasters, leather dressings, and so forth. Rapeseed oil is also known as a good source for, among other things, C2o and C22 fatty acids, which are important as agents in plastics processing and detergents. In addition to their use as industrial raw materials, vegetable oils, especially rapeseed oil, are becoming increasingly important as biodiesel fuels. Generally speaking, the ranges of use of vegetable oils have become considerably broader in recent years. With rising environmental awareness, environmentally compatible industrial products, for example, lubricants and hydraulic fluids, have increasingly been developed.
The problem of high chlorophyll levels in rapeseeds is generally known, especially in rape cultivation with short growing seasons. With every harvest growers and seed concerns must weigh the risk of losing the harvest to frost against the additionally necessary ripening of the seeds, during which, moreover, decomposition of pigments occurs. For this reason rape is frequently allowed to lie for several days after harvesting for further ripening before the harvest is brought in.
A further complication is that low temperatures, frost temperatures that are sublethal for the seeds promote chlorophyll biosynthesis in rapeseeds and thus additionally counteract the decomposition of pigments that takes place during ripening, due to which undesirably high chlorophyll levels in rapeseeds can occur in particular in areas of cultivation with relatively short growing seasons.
In refining the rapeseed oil the pigments, especially the chlorophylls contained in the seeds and their photosensitive precursors, must be extracted at high cost.
Apart from the fact that these extractions are costly from the standpoint of time and money, they also always mean a loss of the rapeseed oil yield. Although turnips make somewhat lesser demands than rape with regard to growing time and location, these Brassicaceae have similar problems with regard to the ripening of the seeds and excess chlorophyll content. The recovery and use of turnipseed oil largely correspond to those of rapeseed oil.

'L.
Chlorophyll synthesis in rape and turnip plants and their seeds could be affected by means of molecular biotechnology to the extent that the amount of excess chlorophyll in the rapeseeds could be reduced or completely chlorophyll-free seeds could be produced.
Tetrapyrrole biosynthesis, which in plants takes place chiefly in plastids, progresses, as is currently assumed, according to the following metabolic pathway (see also in this regard 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 a cyclic compound, porphobilinogen, which is converted to the first tetrapyrrole, hydroxymethylbilane by concatenation of 4 units.
Protoporphyrin IX results from hydroxymethylbilane through oxidations and side chain modifications via uroporphyrinogen III, coproporphyrinogen and protoporphyrinogen IX.
Incorporation of a bivalent metal cation results in magnesium protoporphyrin IX, which converts to chlorophyll a through additional modifications, which involve, among other things, 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 that code for enzymes of the above indicated chlorophyll synthesis path and the possibility of transfernng such genes in a targeted way to plants is an important basis of this invention.
European Patent Application No. 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 alb binding (CAB) proteins) of the aerial complex of the 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
thus concerns proteins that already bind synthesized chlorophyll in photosystem II, but not enzymes that are directly involved in chlorophyll synthesis.
In addition, experimental data from Flachmann and Kiihlbrandt (Plant Cell (1995) 7, 149-160) indicate that as a consequence of the expression of antisense genes for LHCII the RNA
contents in the leaves of transgenic tobacco plants are reduced by up to 5% of the control values in wild types, but neither a reduction of the LHC protein content nor a chlorophyll reduction is observed. Flachmann and Kiihlbrandt were unable to establish a correlation between the decrease of the LHC-RNA level and the reduction of the protein and chlorophyll content.
In contrast to the strategy pursued in the prior art, it ought to be possible to achieve a successful and reliable reduction of the chlorophyll content in transgenic plants by means of direct intervention in the tetrapyrrole synthesis.

R
Therefore, a task of this invention is to point out ways by which chlorophyll synthesis in transgenic oil plant seeds can be reduced or blocked and in this way the chlorophyll content reduced by means of genetic engineering methods.
In addition, an important task of the invention is to make available transgenic oil seeds with chlorophyll content that is lower than that of seeds from wild species of plants.
Other tasks of the invention arise from the following description. These tasks are solved through the objects of the independent claims, in particular on the basis of making available of the methods for reduction of the chlorine content in plant seeds in accordance with the invention as well as the plants and parts and products of plants with chlorophyll content reduced compared to wild species of plants in accordance with the invention.
This invention thus concerns the use of DNA sequences that code for chlorophyll synthesis enzymes and whose targeted transfer to and expression in transgenic plant cells result in a reduction of chlorophyll synthesis. More precisely, the invention concerns the transfer of suitable antisense gene constructs to plants and their expression in the plant tissue.
The invention is based on experiments in which it was possible to reduce significantly the amount of chlorophyll in plant seeds by the seed-specific expression of antisense genes to certain enzyme steps of tetrapyrrole synthesis and the resulting seed-specific inhibition of the activity of specific enzymes of chlorophyll synthesis.
The antisense technique employs the complementarity of nucleic acid molecules in an elegant way. Antisense genes that code for tetrapyrrole metabolism enzymes complementarily to endogenous RNA reduce the endogenous RNA contents or the number of RNA
molecules available for subsequent protein biosynthesis. A reduced content of endogenous RNA necessarily results in reduced translation, and thus a reduced amount of protein, which again is expressed in a reduced activity of the target enzyme.
The invention concerns 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 for reducing the chlorophyll content of seed cells uses all of the genes of this metabolic pathway, i.e., individually or in combination. These genes are above all genes that code for glutamyl tRNA
synthetase, glutamyl tRNA reductase, glutamate 1-semialdehyde aminotransferase, magnesium chelatase or its subunits CHL I, CHL D, CHL H, chlorophyll synthetase and Mg protoporphyrin monomethyl ester transferase.
The invention also concerns fragments of genes that are involved in chlorophyll synthesis and whose use within an antisense construct results in reduced activity of the target enzyme.
Such fragments could in connection with this invention be called "antisense-active" fragments, g, i.e., their transfer in the form of a suitable construct brings about a reduction of the corresponding endogenous enzyme activity.
In addition, the invention concerns the use of alleles and derivatives of the genes in accordance with the invention to reduce the chlorophyll content of plant seeds, thus the use of nucleic acid molecules whose sequences, due to degeneration of the genetic code, differ from the genes in accordance with the invention and whose transfer to plant cells results in a reduction of the content of the desired enzyme of chlorophyll metabolism caused by the antisense gene.
In addition, the invention concerns the use in accordance with the invention of nucleic acid molecules that contain the antisense genes in accordance with the invention or that result from them by naturally occurring or by genetic engineering or chemical processes and synthesis methods or derived from them. These can be, for example, DNA or RNA molecules, cDNA, genomic DNA, mRNA, etc.
The following genes of chlorophyll synthesis are preferably used for reduction of the chlorophyll content of seed tissue within the scope of the invention.
Genes that code 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 C 1. The expression of a tobacco antisense RNA for GSA-AT up to now has been investigated only in the leaves of tobacco plants (Hofgen et al. (1994) Proc. Natl. Acad. Sci. USA 91, 1726-1730). DNA sequences of two tobacco full length cDNA clones are available in the Gene Bank, Accession Nos. X65973 and X65974.
Genes that code for the subunits CHL I and CHL H of magnesium chelatase. The subunits of Mg chelatase are involved in the incorporation of magnesium into protoporphyrin IX.
Suitable DNA sequences of full length cDNA clones that 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 AF 14052 (Chl H) in the gene bank.
Genes that code for the 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 bank that code for the reductase are described in Bougri and Grimm (1996) Plant J. 9, 7867-878 and under Accession Nos. X86101, X86102 and X92403 in the gene bank.
In an antisense gene construct in accordance with the invention a gene of chlorophyll synthesis or a fragment thereof, preferably the coding region of such a gene or a fragment, is bonded in antisense orientation, i.e., 3' -~ 5' orientation, with the 3' end of a promoter, thus a regulator element that guarantees the transcription of the coupled gene into plant cells.

The genes in accordance with the invention can be expressed in plant cells, for example, under control of constitutive, but also inducible or tissue- or development-specific promoters.
These are preferably seed-specific promoters whose use enables the targeted inhibition of chlorophyll synthesis in seed cells.
Examples of seed-specific promoters that can be used in connection with the invention that may be mentioned are the USP promoter (described, among other places, in:
Baumlein 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 (Baumlein et al. (1986) Nucl.
Acids Res. 14, 2707-2720; Accession No. X 03677) and the hordeine promoter (Entwistle et al.
(1991) Plant Mol. Biol. 17, 1217-1231; Accession No. X60037).
Optionally, the antisense constructs used in accordance with the invention can additionally include enhancer sequences or other regulator sequences.
It is likewise a task of the invention to make available new plants, plant cells, plant parts or plant products that are characterized by a reduced chlorophyll content compared to the wild species.
These tasks are solved through the transfer of the antisense nucleic acid molecules in accordance with the invention and their expression in plants. The making available of the nucleic acid molecules in accordance with the invention now presents the possibility of altering plant cells by means of genetic engineering methods to the extent that they have a reduced chlorophyll biosynthesis power compared to wild types. In accordance with the invention these are seeds of oil plants, especially rape and turnip, that have significantly reduced chlorophyll content compared to seeds of the wild species. The transgenic rape plants are especially preferably spring rape plants. Likewise 00 rape plants, i.e., rape plants that are erucic acid-free and low-glucosinolate, are also suitable for use of the methods in accordance with the invention.
The plants that are transformed with the nucleic acid molecules in accordance with the invention and in which a lower amount of chlorophyll is synthesized because of the integration of such a molecule into their genome can in principle be any plants.
Preferably, these are oil plants like rape and turnip from which a vegetable oil is obtained and in the recovery of which high chlorophyll contents are undesirable.
An obj ect of the invention is in particular propagation material of plants in accordance with the invention, for example, seeds, fruits, cuttings, bulbs, root stock, etc., where this propagation material optionally contains the above described transgenic plant cells, as well as parts of these plants like protoplasts, plant cells and calli; especially preferably these are seeds.

~e This invention additionally has the task of making available methods for producing plant cells and plants and parts thereof, especially seeds, that are characterized by a reduced chlorophyll content.
This task is solved by methods with which the generation of new plant cells and plants that are characterized by a reduced chlorophyll content compared to the wild types due to the transfer and expression of antisense genes that are directed toward endogenous genes that code for the enzymes of chlorophyll synthesis is possible.
There are various genetic engineering transformation methods for generation of such new plant cells and plants. In accordance with the invention, plant cells that are characterized by a reduced chlorophyll content due to the expression of an antisense gene construct in accordance with the invention are produced by a method that includes the following steps:
a) Preparation of an expression cassette, which includes the following DNA
sequences:
- a promoter that ensures transcription into plant cells;
- at least one nucleic acid sequence that codes for an enzyme or a fragment thereof that participates in chlorophyll synthesis, where the nucleic acid sequence is coupled in antisense orientation to the 3' end of the promoter, and - optionally a termination signal for termination of transcription and addition of a poly (A) tail to the corresponding transcript that is coupled to the S' end of the nucleic acid sequence.
b) Transformation of plant cells with the expression cassette prepared in step (a).
c) Regeneration of transgenic plants and optionally propagation of the plants.
.
Furthermore, the invention concerns the use of the antisense constructions in accordance with the invention to generate plants, especially plant seeds, that have a reduced chlorophyll content. Preferably the invention concerns the use of the antisense constructions in accordance with the invention to generate seeds of oil plants, especially rape and turnip, that have a reduced chlorophyll content.
Another task of the invention is to point out the possibilities of using the plants in accordance with the invention or their cells, parts and products, especially their seeds.
An object of the invention is in particular the use of the plants in accordance with the invention, especially their seeds, to obtain vegetable oils as raw materials for the chemical, cosmetics, pharmaceuticals and food industry and as energy Garners.
The plants in accordance with the invention thus represent an important source for obtaining vegetable oils, especially rapeseed and turnipseed oil, for a broad spectrum of commercial purposes.
Possibilities for said promoter are in principle any of the functional promoters in the plants chosen for transformation that satisfy the requirement that the expression that they regulate leads to a reduced chlorophyll synthesis power in plant cells. In view of the use of the transgenic plants as providers of vegetable oils promoters that ensure seed-specific expression appear to be particularly meaningful here. Examples of such promoters are the USP, napin, 2S albumin, legumin and hordeine promoters mentioned above.
If such promoters are not known or are not available, in any case the concept of isolating such promoters is well known to the specialist. Here poly(A)+ RNA is isolated from seed tissue and a cDNA bank is set up in a first step. In a second step cDNA clones that are based on poly(A)+ RNA molecules from tissue not deriving from the seeds from the first bank are used to identify by means of hybridization those clones whose corresponding poly(A)+
RNA molecules are expressed only in the plant tissue. Then the cDNAs identified in this way are used to isolate promoters that then can be used for antisense expression.
A large number of cloning vectors that contain a replication signal for E.
coli and a marker gene for selection of transformed bacterial cells is available for preparation for the insertion of foreign genes into higher plants. Examples of such vectors are pBR322, the pUC
series, MI3mp series, pACYC 184, and so forth. The desired sequence can be introduced at a convenient restriction cut site in the vector. The resulting plasmid is used for transformation of E. coli cells. The transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. In general restriction analyses, gel electrophoreses and other biochemical/molecular biological methods are used as analysis methods to characterize the resulting plasmid DNA. After each manipulation the plasmid DNA can be split and the recovered DNA fragments can be bonded to other DNA sequences. Each plasmid DNA
sequence can be cloned in the same or other plasmids.
A larger number of known techniques are available for introduction of DNA into a plant host cell, and the specialist can easily determine the method that is suitable in each case. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, fusion of protoplasts, direct gene transfer of isolated DNA in protoplasts, electroporation of DNA, the insertion of DNA by means of biolistic methods as well as other possibilities.
In the injection and electroporation of DNA in plant cells no special requirements per se are made on the plasmids that are used. This is also true for direct gene transfer. Simple plasmids like pUC derivatives can be used. However, if whole plants are to be regenerated from the cells that are transformed in this way, the presence of a selectable marker gene is necessary. The common selection markers are known to the specialist and selecting a suitable marker will not be problem.
Additional DNA sequences may be necessary, in each case according to the method of insertion of desired genes into the plant cell. If, for example, Ti or Ri plasmid is used for transformation of the plant cell, then at least the right boundary, and frequently the right and left boundary, of the T-DNA contained in the Ti and Ri plasmid must be bonded to the genes to be introduced as a flanking region.
If agrobacteria are used for the transformation, the DNA to be introduced must be cloned in special plasmids, namely either in an intermediary or a binary vector. The intermediary vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination by means of sequences that are homologous to sequences in the T-DNA. The plasma additionally contains the vir region necessary for transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediary vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate both in E.
coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA boundary region. They can be transformed directly to the agrobacteria (Holsters et al. (1978) Molecular and General Genetics 163, 181-187). The agrobacterium that serves as host cell should contain a plasmid that carries a vir region. The vir region is necessary for transfer of the T-DNA to the plant cells. Additional T-DNA can be present. The agrobacterium transformed in this way is used for transformation of plant cells.
The use of T-DNA for transformation of plant cells has been intensively investigated and described extensively in EP 120 515; Hoekema in: the Binary Plant Vector System, Offsetdrokkerij Kanters B.V., Alblasserdam (1985) Chapter V; Fraley et al.
(1993) Crit. Rev.
Plant. Sci., 4, 1-46 and An et al. (1985) EMBO J. 4, 277-387.
For the transfer of the DNA into the plant cells plant, explants can be cultured expediently with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Then from the infected plant material (for example, leaf pieces, stem segments, roots, as well as protoplasts or suspension-cultured plant cells) whole plants can be regenerated in a suitable medium, which can contain antibiotics or biocides for selection of transformed cells. The regeneration of the plants takes place by conventional regeneration methods using known nutrient media.
The plants obtained in this way can then be tested for presence of the introduced DNA.
Other possibilities for the introduction of foreign DNA using the biolistic method or by protoplast transformation are well known (see, for example, Willmitzer L. (1993) Transgenic Plants, in:
Biotechnology, A
Multi-volume Comprehensive Treatise (H.J. Rehm. G. Reed, A. Piihler, P.
Stadler, eds.) Vol. 2, 627-659, V.C.H. Weinheim - New York - Basel - Cambridge).
Once the introduced DNA is integrated into the genome of the plant cell, as a rule it is stable there and continues to remain in the descendants of the original transformed cell. It normally contains a selection marker that confers resistance to a biocide or antibiotic like kanamycin, 6418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin, etc., to the transformed plant cells. The individually selected marker should then allow the selection of the transformed cells compared to cells that lack the introduced 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 in the normal way and crossed with plants that have the same transformed genetic makeup or another 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 in order to guarantee that the phenotypic trait will continue to remain stable and be inherited. In addition, seeds should be harvested in order to guarantee that the corresponding phenotype or other characteristics have remained preserved.
Likewise, conventional methods can be used to determine transgenic lines that are homozygous for the new nucleic acid molecules and their phenotypic behavior with respect to altered chlorophyll content tested and compared with that of hemizygous lines.
The transfer and expression of the antisense gene constructs in accordance with the invention can take place with the aid of traditional molecular biological and biochemical methods. These techniques are known to the specialist and he is capable of easily selecting a suitable detection method, for example, a Northern blot analysis, for qualitative and quantitative detection of RNA that is specific for the coding region of the relevant antisense gene, or a Southern blot analysis for identification of the transferred DNA sequences.
The resulting transgenic plant cells or plants as well as parts and products thereof can then be tested far their chlorophyll content. The following analysis methods, for example, suggest themselves on this regard:
- Determination of the chlorophyll and carotinoid contents after Porra et al.
(1989) Biochem. Biophys. Acta 975, 384-394;
- determination of ALA synthesis power (as described, for example, in Zavgorodnyaya et al. (1997) The Plant J. 12, 169-178);
- determination of the glutamate 1-semialdehyde aminotransferase activity (see also Zavgorodnyaya et al. (1997) supra).
Often a reduced chlorophyll content in transgenic plants or their parts and products compared to the wild types can be detected with the naked eye or with optical aids.
Common methods such as are described in the relative laboratory manuals like Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, can be used for DNA and RNA
isolation, sequence analysis, restriction, cloning, gel electrophoresis, radioactive labeling, Southern, Northern and Western blot analyses, hybridization and the like.
The following examples serve to illustrate the invention.

~b Examples Example 1 .
Preparation of an antisense construct based on DNA sequences that code for a glutamate 1-semialdehyde aminotransferase from tobacco For construction of a GSA-AT antisense mRNA expression vector the complete 1714 by tobacco GSa-AT-cDNA fragment (Gene Bank Accession No. X65974; see Hofgen et al., supra) as EcoRV/XbaI restriction fragment was cloned into the binary vector BinHyg-Tx, a plasmid derivative of the vector BinAR (Hofgen and Willmitzer (1990) Plant Sci. 66, 221-230), which was cut beforehand with the restriction enzymes SmaI and XbaI.
For preparation of a binary vector with a fusion of an USP promoter and the GSA-AT-cDNA sequence in antisense orientation the vector pP30T containing the USP
promoter from Vicea faba, a pUC 18 derivative (see also Baumlein et al. ( 1991 ), supra) was cut with PstI and BgIII. The isolated USP promoter fragment was then cloned in a BamHI/PstI-cut pBluescript vector (-> pUSPblue). This vector construct pUSPblue was then cut with EcoRI
and XbaI in order to obtain an EcoRI/XbaI promoter fragment. The 355 CaMV promoter was removed from the binary vector BinAR by restriction digestion with EcoRI and XbaI and, by ligation of the EcoRI/XbaI vector fragment with the USP promoter fragment replaced by this seed-specific promoter (~ pUSPbin). The complete cDNA sequence (XbaI-3'-GSA-AT-cDNA-S'-SaII) cut from the pBluescript with XbaI and SaII was then inserted in the XbaI-SaII-cut pUSPbin.of tobacco GSA aminotransferase. The resulting binary vector was characterized as pUSPASGSAT
(see also Figure 2).
In place of the said binary vectors BinAR or BinHyg-Tx, which contain a tetracycline-inducible CaMV 355 promoter, any suitable vector for plant transformation can be used to prepare an antisense gene consisting of a fusion of a promoter, preferably a seed-specific promoter that guarantees transcription and translation in plant cells, and DNA
sequences that code for enzymes that participate in chlorophyll synthesis.
Example 2:
Transformation of rape plants and regeneration of intact plants The transformation in summer rape was carried out by 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 a recombinant culture of Agrobacterium tumefaciens (strain GV 3101) was washed and resuspended in medium 1 (MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473), 2.SmM MES, pH 5.5, 1 mg/L benzylaminopurine (BAP), 0.1 mg/L
napthylacetic acid (NAA), 0.01 mg/L gibberellic acid (GA3), and 200 pM acetosyringone).

The hypocotyl of 14 day old Brassica napus seedlings were cut into segments of 0.5 to 1 cm long, incubated for 30 min with the bacterial suspension and then kept for 5 days in the dark at 21 °C in 0.7% agar containing medium 1. For callus induction the hypocotyl was incubated at 25°C on medium 2 (MS medium, 2.SmM MES, pH 5.7, 30 g/L
sucrose, 1 mg/L
kinetin, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.01 mg/L GA3, S00 mg/L
polyvinylpyrrolidone (PVP), 5 mg/L AgN03, 5 g/L agarose, 250 mg/L
carbenicillin, 100 mg/L
kanamycin; at 150 pM photon/m2/sec).
Formation of the callus was followed by induction of shoots on medium 3 (MS
medium, 2.SmM MES, pH 5.5, 20 g/L sucrose, 40 mg/L adenine, 1 mg/L BAP, 0.1 mg/L NAA, 0.01 mg/L
GA3, 500 mg/L PVP, 5 mg/L AgN03, 100 mglL kanamycin, 5 gIL agarose, 250 mg/L
carbenicillin).
After shoot formation has occurred, the calli, which show shoots are transferred to a shoot elongation medium (medium 4) in glass vessels (MS medium, 2.SmM MES, pH
5.7, g/L sucrose, 0.0025 mg/L BAP, 100 mg/L kanamycin, 7 g/L agarose, 250 mg/L
carbenicillin).
After 2-3 weeks the shoots were transferred to medium 5 (MS medium, 2.SmM MES, pH 5.5, 7 g/L agarose) in order to form roots.
Alternatively, rape plants were generated by the following protocol: a recombinant culture ofAgrobacterium tumefaciens (strain GV 3101) was washed and resuspended in MS
medium with 2.SmM MES, pH 5.5. The hypocotyl of 5-7 day old rape seedlings were cut into explants about 7 mm long and precultured in liquid CIM medium for 24 h. Then the exphnts were cocultured for 2-3 days in the dark in 50 pL of an overnight culture of the recombinant agrobacterium strain in 10 mL of the CIM medium. 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. Then the hypocotyl pieces were washed and cultured for 7-10 days to callus induction on CIM
medium containing S
g/L agarose, 20 mg kanamycin, 250 mg betabactyl and 250 mg carbenicillin. Then the slightly swollen explantates were placed on SIM medium for shoot induction. The medium was replaced every 10-14 days. 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 myoinositol, 5 g agarose, mg kanamycin, S00 mg betabactyl or carbenicillin. After the end of shoot formation, the kalli that showed shoots were transferred to glass vessels containing MS medium, 500 mg/L MES, pH 5.7, 20 g/L sucrose, 20 mg/L kanamycin, S/L agarose and S00 mg/L
carbenicillin.
In addition to the described transformation methods, rape plants can also be transformed by means of other techniques. For this purpose, the protocol of Moloney et al.
(1989, Plant Cell Rep. 8, 238-242) suggests itself; in this protocol an agrobacteria-mediated DNA transfer to cotyledons of 7-day-old seedlings takes place via the cut site on the leaf stem. In addition, the transformation of protoplasts using cells of various tissues is suitable (described, for example, in 1~.
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 the antisense RNA to glutamate 1-semialdehyde aminotransferase under control of the USP promoter Plants that were transformed with the vector construct pUSPASGSAT (see Example and Figure 2) were then tested for expression of the antisense RNA to GSA-AT.
For this purpose the chlorophyll contents and synthesis rates in the transgenic plants were determined. For this chlorophylls were extracted from 100 mg plant material ground in liquid nitrogen with buffered, ice-cold 80% acetone until the pellet had become colorless. The samples were appropriately diluted and the extinction measured at 663, 646 and 750 nm on a spectrophotometer. The formulas given by Porra et al. (1989, supra) were employed for the calculation of the chlorophyll contents.
To determine chlorophyll synthesis rates, seed tissue was incubated in 20mM
K2HP04/KH2P04 (pH 7.1) with 34 kBq D-[4-'4C]-labeled ALA (5-aminolevulinic acid) for 8 h in light, then a chlorophyll extraction was carried out and separation was done via HPLC. The extraction and separation followed the technique of Gilmore and Yamamoto (1991, J.
Chromatography 543, 137-145), modified by Kurse et al. (1995, EMBO J. 14, 3712-3720), as follows: 100 mg seed material ground in liquid nitrogen was weighed and extracted with 100%
acetone and 10 , as follows: 100 mg seed material ground in liquid nitrogen was weighed and extracted with 100% acetone and 10 uM KOH until the pellet had become colorless (1 time 400 pL, 3 times 200 pL). The extracts were diluted 4:1 with HZ for the HPLC
run in order to achieve sharper separations. The chlorophylls were eluted by means of a LiChrospher 100 HPLC
RP 18 column (5 um, Merck) at a flow rate of 1 rnL/min, using the following gradients: 100%
eluent A (780 mL acetonitrile; 80 mL MeOH; 30 mL tris/HCI, O.1M, pH 8.0) for 7 min, in a linear increase over 6 min to 100% eluent B (800 mL MeOH; 200 mL hexane), and 14 min 100% eluent B. The eluate was analyzed with a photodiode array (PDA) detector and connected radioactivity monitor. Radioactively labeled chlorophyll could be categorized according to the reaction times, which are known for the HPLC system.
In addition, the S-aminolevulinic acid synthesis capacity in the transgenic plants was determined. Since enzyme activities in the CS pathway cannot be determined without purifying the enzymes, indirect methods were selected to measure the capacity for ALA
formation from glutamate. For one, the accumulation of ALA after incubation of LA was determined by the following protocol: 100-300 mg seed tissue per batch was incubated in light for 2-4 h with 40n~1VI levulinic acid, a potent inhibitor (substrate similarity) of ALA
dehydratase (ALAD), in ~3 1~
20mM K2HP04/KH2P04 (pH 7.1). The plant material was frozen in liquid nitrogen, homogenized and, after adding 1 mL 20mM K2HP04/KH2P04 (pH 7.1), thoroughly mixed. The ALA determination followed Mauzerall and Granick (1956, J. Biol. Chem. 219, 435-446). After centrifuging for 20 min at 15,000 g at 4°C, 250 ~L 20mM K2HP04/KH2P04 (pH 7.1 ) and 100 ~L ethyl acetoacetate was pipetted into 250 uL of the supernatant. Samples that had been extracted without levulenic acidincubation at time to served as control. All samples were heated for exactly 10 min at 100°C, then cooled on ice for 5 min, thoroughly mixed with 500 mL
modified Ehrlich's reagent (373 mL glacial acetic acid; 90 mL 70% (w/v) perchloric acid; 1.55 g HgCl2; volume filled to 500 mL with H20; 2 g p-dimethylaminobenzaldehyde dissolved in 110 mL of this solution) and centrifuged for 5 min at 15,000 g. Extinctions at 525, 553 and 600 nm were measured. The nonspecific turbidity, measured at 600 nm, was subtracted from all of the values; the ratio Ess3~szs should lie between 1.3 and 1.5. A reference series was run in order to be able to quantify the ALA contents.
In addition, the accumulation of ALA was determined after incubation in glutamate and levulinic acid. For this purpose 100-300 mg of seeds are incubated in 92.5 kBq L-[U-'4C]
glutamate, 2mM glutamate, 20mM LA, 880 ~L 20mM K2HP04/KH2P04 (pH 7.2) for 8 h at room temperature in light. The plant material was homogenized in 0.5 mL 1N TCA
and 1%
SDS, centrifuged and the supernatant was diluted with 1 volume of eluent (7.8 g/L NaH2P04;
1.74 g/L SDS, 5 mL/L tert-amyl alcohol). The ['4C]-labeled ALA that formed was detected in accordance with Pontoppidan and Kannangara (1994, Eur. J. Biochem. 225, 529-537) via HPLC
(flow rate 1 mL/min, isocratic mode; RP 8 column, Novapak C8, 4 um particle size, 3.9 mm x 150 mm). The amount of radioactively labeled ALA was determined via an affixed radioactivity monomer. Peak identification was done by Co chromatography with D-[4-'4C]-labeled ALA, and quantification was done by means of reference series of the same substance.
In addition, Southern and Northern blot experiments were carned out to detect the integration of the trans genes and the effect on the RNA content for GSA-aminotransferase following standard methods (for example, (Sambrook et al. (1989) supra). The amount of GSA
aminotransferase was also determined immunologically in the classic Western Blot analysis, with antibodies being obtained by immunization of rabbits or mice with Freund's adjuvant.
The analysis of transgenic rapeseeds that express the antisense construct contained in the vector pUSPASGSAT showed that the method in accordance with the invention results in a significant reduction of the chlorophyll content.
If the molecular biological operations have not in any way been described sufficiently, they were carried out by standard techniques as described in Sambrook et al.
(1989) supra. With regard to the transformation of plants, reference is made to generally known survey articles as well as to the publications mentioned above.

r ' , : . . . , 1$
Description of figures:
Figure 1 shows the metabolic pathway of tetrapyrrole biosynthesis. .
Figure 2 shows a restriction map of the binary vector pUSP-ASGSAT described in Example 1, which contains a fusion of the USP promoter and the region coding for GSA
aminotransferase in antisense orientation. The vector pUSP-ASGSAT bears a kanamycin resistance gene as the phase selection marker.
;.

Claims

1. A method for reducing the chlorophyll content in seeds of oil plants, which includes the following steps:
a) preparation of an antisense expression vector that includes the following DNA
sequences:
- a promoter that is capable of functioning in plants, in particular a seed specific promoter, - at least one nucleic acid sequence that codes for an enzyme or a fragment thereof that participates in chlorophyll synthesis, where the nucleic acid sequence is linked in antisense orientation to the 3' end of the promoter, and - optionally a termination signal for the termination of transcription and the addition of a poly (A) tail to the corresponding transcript, which is linked 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 optionally propagation of these plants.

2. A method as in Claim 1, where the coding sequence codes for a glutamate 1 semialdehyde aminotransferase or at least a part thereof.

3. A method as in Claim 1, where the coding sequence codes for the CHL I
subunit of magnesium chelatase or at least a part thereof.

4. A method as in Claim 1, where the coding sequence codes for the CHL H
subunit of magnesium chelatase or at least a part thereof.

5. A method as in Claim 1, where the coding sequence codes for the plasmid glutamyl tRNA synthetase or at least a part thereof.

6. A method as in Claim 1, where the coding sequence codes for a glutamyl tRNA
reductase or at least a part thereof.

7. A method as in one of the preceding claims, where the promoter is USP
promoter.

8. A method as in one of the preceding claims, where the promoter is a napin promoter.

9. A method as in one of the preceding claims, where the promoter is a 2S-albumin promoter.

10. A method as in one of the preceding claims, where the promoter is a legumin promoter.

11. A method as in one of the preceding claims, where the promoter is a hordeine promoter.

12. A method as in one of the preceding claims, where the oil plant belongs to the family of the Brassicaceae.

13. A method as in one of the preceding claims, where the oil plant is rape.

14. A method as in one of Claims 1-12, where the oil plant is turnip.

15. Transgenic plant seeds with chlorophyll content reduced by comparison with seeds from wild species, which contain a nucleotide sequence that codes for an enzyme, or a fragment thereof, that participates in chlorophyll synthesis.

16. The use of the seeds as in Claim l5 to obtain vegetable oils.