AU1019501A - Process for modifying plants - Google Patents

Description

WO 01/31027 PCT/EPOO/09374 PROCESS FOR MODIFYING PLANTS Field of the invention 5 The invention relates to a process for the modification of plants, more specifically a process for increasing the isoprenoid level in plants. Background of the invention 10 Many approaches have been suggested for modifying the isoprenoid production in plants. Whereas only a few sterols exist in animals, with 15 cholesterol being by far the major one, in plants a wide range of sterols are found. Structural variations between these arise from different substitutions in the side chain and the number and position of double bonds in the tetracyclic skeleton. Plant sterols can be grouped by the 20 presence or absence of one or more functionalities. For example they can be divided into three groups based on methylation levels at C4 as follows: 4-desmethylsterols or end product sterols, 4o-monomethylsterols and 4,4-di methylsterols. Naturally occurring 4-desmethylsterols 25 include sitosterol, stigmasterol, brassicasterol, A7 avenasterol and campesterol. In most higher plants, sterols with a free 3@-hydroxyl group (free sterols) are the major end products. However sterols also occur as conjugates, for example, where the 3-hydroxy group is esterified by a fatty 30 acid chain, phenolic acids or sugar moieties to give steryl esters. For the purpose of this description the term sterol refers both to free sterols and conjugated sterols. However WO 01/31027 PCT/EPOO/09374 2 in this specification references to levels, amounts or percentages of sterol refer to the total weight sterol groups whereby the weight of the conjugating groups such as fatty acid, phenolic acid or sugar groups is excluded. 5 To date most studies aimed at manipulating sterols in plants have involved other than 4-desmethylsterols with the purpose of increasing resistance to pests or to fungicides. 10 WO 98/45457 describes the modulation of phytosterol compositions to confer resistance to insects, nematodes, fungi and/or environmental stresses, and/or to improve the nutritional value of plants by using a double stranded DNA molecule comprising a promoter, a DNA sequence encoding a 15 first enzyme which binds a first sterol and produces a second sterol and a 3' non-translated region which causes polyadenylation at the 3' end of the RNA. Preferably the enzyme is selected from the group consisting of S-adenosyl L-methionine-A 2 4

(

2 5 -sterol methyl transferase, a C-4 20 demethylase, a cycloeucalenol to obtusifoliol-isomerase, a 14-at-demethylase, a A 8 to A 7 - isomerase, a A 7 -C-5-desaturase and a 24,25-reductase. US 5,306,862 describes a method of increasing sterol 25 accumulation in a plant by increasing the copy number of a gene encoding a polypeptide having HMG-CoA reductase activity to increase the resistance of plants to pests. Similarly US 5,349,126 discloses a process to increase the squalene and sterol accumulation in transgenic plants by 30 increasing the amount of a gene encoding a polypeptide having HMG-CoA reductase activity to increase the pest resistance of transgenic plants.

WO 01/31027 PCT/EPOO/09374 3 WO 97/48793 discloses a C-14 sterol reductase polypeptide for the genetic manipulation of a plant sterol biosynthetic pathway. 5 WO 96/09393 discloses a DNA sequence encoding squalene synthetase. WO 97/34003 discloses a process of raising squalene levels 10 in plants by introduction into a genome of a plant a DNA to suppress expression of squalene epoxidase. WO 93/16187 discloses new plants containing in its genome one or more genes involved in the early stages of 15 phytosterol biosynthesis, preferably the genes encode mevanolate kinase. US 5,589,619 discloses accumulation of squalene in plants by introducing a HMG-CoA reductase gene to increase 20 production of sterol and resistance to pests. Example 10 discloses increased squalene levels in the seeds of these plants. In plants, mevalonate synthesis via HMGR is one of the 25 steps in isoprenoid biosynthesis. Gondet et al in Plant Physiology (1994) 105:509-518 has isolated a tobacco mutant showing dramatically altered sterol compositions in leaf tissue with significant 30 increases in the proportion of cyclopropylsterols and HMGR activities increased by approximately 3-fold.

WO 01/31027 PCT/EPOO/09374 4 Re et al in The Plant Journal (1995) 7(5), 771-784 have shown that the over-expression of HMG CoA reductase is not sufficient to alter the bulk synthesis and accumulation of end product of the plant isoprenoid pathway. 5 Applicants believe that the reason for this is that the activity of HMGR in plants is subject to feedback inhibition by sterols. Some HMGR genes, however are non feed back inhibited. Examples of such genes are non-plant 10 HMGR genes lacking the membrane binding domain such as the truncated hamster HMGR genes or the truncated Saccharomyces cerevisiae genes, and HMGR genes (or truncated versions thereof) from high isoprenoid producing plants such as Hevea brasiliensis. 15 A truncated hamster HMGR gene, lacking the membrane binding domain, was expressed in tobacco plants under the control of the CaMV 35S promoter (Chappell et al., Plant Physiology (1995) 109: 1337-1343). This resulted in a 3- to 6- fold 20 increase in total HMGR activity in leaf tissue. Schaller et al in Plant Physiology (1995) 109:761-770 discloses the introduction of a HMGR1 gene from Hevea brasiliensis into tobacco leading to an enhanced sterol 25 production especially of cycloartenol in leaf tissue. Polakowski et al in Applied Microbial Biotechnology (1998) 59:66-71 describes the use of a truncated Saccharomyces cerevisiae hmg 1 gene in yeast, leading to the accumulation 30 of squalene. The present invention aims to increase sterol levels in plants, whereby the sterols are preferably nutritionally attractive 4-desmethylsterols such as sitosterols, WO 01/31027 PCT/EPOO/09374 5 stigmasterols, brassicasterol, A7-avenasterol or campesterols and whereby the sterols are preferably expressed in the seeds. 5 It has been found that genes expressing specific HMG reductase enzymes can advantageously be used to increase the nutritional value of plants especially in the seeds thereof. Surprisingly it has been found that the use of non feedback regulated HMGR leads to the enhancement of 10 nutritionally beneficial sterol for example in the seeds of said plants. Surprisingly it has also been found that particularly high levels of sterols can be obtained by using truncated plant HMGR genes. 15 Statement of the invention Accordingly the invention relates to the use of a gene expressing a non-feed back inhibited HMG-reductase to increase the level of 4-desmethylsterols in the seeds of 20 plants. Preferably the gene expressing a non-feed back inhibited HMG-reductase is a truncated plant HMGR gene. Accordingly in a second aspect the invention relates to a method to produce plants having a modified sterol 25 production by incorporating into the plant genome a heterologous gene whereby said gene expresses a truncated plant HMG-reductase. In a third aspect the present invention relates to modified 30 plants having incorporated in their genome a heterologous gene expressing a truncated plant HMG-reductase.

WO 01/31027 PCT/EPOO/09374 6 Detailed description of the invention In higher plants, isoprenoids are a large family of 5 compounds with diverse roles. They include sterols, the plant hormones gibberellins and abscisic acid, components of photosynthetic pigments, phytoalexins and a variety of other specialised terpenoids. 10 Sterols, especially 4-desmethylsterols are of interest and colour of fruits and vegetable oils. Of particular interest are isoprenoid compounds of nutritional -benefit such as fat soluble sterols. These may be efficacious in reducing coronary heart disease, for example, some phytosterols have 15 been shown to lower serum cholesterol levels when increased in the diet. Expression of such compounds in plant seeds in particular in oilseeds is commercially advantageous as generally the 20 harvesting of such ingredients from seeds is very convenient and in some instances it may be possible to extract the oil in combination with the sterols from the seed, leading to an oil containing elevated levels of sterol without or with the reduced need for separate 25 addition of sterols. Preferred sterols are 4-desmethylsterols, most preferred sitosterol, stigmasterol, brassicasterol, avenasterol and campesterol. Also preferably at least part of the sterols, 30 for example at least 50 wt% based on the total of the sterols in the seed are esters of sterols with C10-24 fatty acids. In a very preferred embodiment the sterols comprise C10-24 esters of 4-desmethylsterols.

WO 01/31027 PCT/EPOO/09374 7 As discussed above, several approaches have been suggested to alter the levels of isoprenoids in plants. It has now been found that for the enhancement of isoprenoid levels in seeds a preferred route is to use a non feedback inhibited 5 HMGR gene. The use of such genes is especially advantageous to enhance the levels of 4-desmethylsterols, even more preferred the level of stigmasterol, sitosterol and campesterol in plant tissue for example seeds. Also the use of such genes is especially advantageous to enhance the 10 levels of isoprenoids in plant tissue such as oilseeds containing more than 10 wt% based on dry weight of triglycerides. In a first embodiment of the invention the non-feed back 15 inhibited HMG reductase is an enzyme which is expressed by a truncated non-plant HMGR gene, said truncation preferably leading to an enzyme lacking the membrane binding domain, but whereby the HMGR functionality of the gene is preferably maintained. Examples of such genes are the 20 truncated hamster or yeast HMGR genes. A second -preferred- embodiment of a non-feedback inhibited HMG reductase is an enzyme expressed by HMGR genes from high isoprenoid producing plants such as Hevea 25 brasiliensis. Especially preferred are truncated versions of HMGR produced by genes from high isoprenoid producing plants such as Hevea brasiliensis, most preferred truncated versions are used whereby said HMGR lacks the membrane binding domain. 30 The intact HMGR enzyme comprises three regions: a catalytic region, containing the active site of the enzyme, a WO 01/31027 PCT/EPOO/09374 8 membrane binding region, anchoring the enzyme to the endoplasmic reticulum and a linker region joining the catalytic and membrane binding regions of the enzyme. The membrane-binding domain occupies the N-terminal region of 5 the enzyme, whereas the catalytic region occupies the C terminal region. It is believed that feedback inhibition in most plants generally requires the presence of the membrane-binding region of the enzyme. Therefore a preferred embodiment of the invention relates to the use of 10 a HMGR gene expressing an enzyme with an inactivated or without a membrane binding domain, whereby said gene is preferably used to increase the level of 4-desmethylsterols in plant tissue such as the seeds of plants. 15 An example of HMG reductase with an inactivated or without a membrane binding domain is the HMG reductase expressed by the truncated hamster HMGR gene as described by Chappell (see above). The truncation is believed to remove the membrane binding domain from the HMG reductase whereafter 20 a significant reduction of feedback inhibition occurs. Other truncated or mutated genes whereby the membrane binding domain is removed or inactivated can equally be used. An example of this is the truncated HMGR gene as used by Polakowski (see above). 25 Preferred examples of HMG reductases are those expressed by HMGR genes obtained from plants which naturally have the tendency to develop high levels of isoprenoids such as for example triterpenes and rubber. Examples of such plants are 30 Asteraceae, especially Euphorbiaceae. Therefore another preferred embodiment of the invention relates to the use of a HMGR gene isolated from Asteraceae to increase the level WO 01/31027 PCT/EPOO/09374 9 of sterols, particularly 4-desmethylsterols in plant tissue, particularly the seeds of plants. Preferably the HMGR gene is isolated from Hevea brasiliensis. Especially preferably truncated versions of such plant genes may be 5 used. The invention also provides a method of transforming a plant by a) transforming a plant cell with a recombinant DNA 10 construct comprising a DNA segment encoding a polypeptide with non feedback inhibited HMGR activity and a promoter for driving the expression of said polypeptide in said plant cell to form a transformed plant cell. 15 b) regenerating the transformed plant cell into the transgenic plant. Preferably this method is using a construct comprising a DNA segment derived from plants, particularly a DNA segment 20 encoding a HMG-reductase derived from Asteraceae, most preferred a truncated plant HMG-reductase for example a truncated HMG-reductase derived from Asteraceae especially Hevea brasiliensis. 25 Furthermore this method preferably involves selecting transgenic plants that have enhanced levels of sterols particularly 4-desmethylsterols in plant tissue particularly in the seeds compared to wild type strains of the same plant. 30 DNA segments encoding non feedback inhibited HMGR for use according to the present invention may suitably be obtained from animals, microbial sources or plants, Alternatively, WO 01/31027 PCT/EPOO/09374 10 equivalent genes could be isolated from gene libraries, for example by hybridisation techniques with DNA probes. The gene sequences of interest will be operably linked (that 5 is, positioned to ensure the functioning of) to one or more suitable promoters which allow the DNA to be transcribed. Suitable promoters, which may be homologous or heterologous to the gene, useful for expression in plants are well known in art, as described, for example, in Weising et al, (1988), 10 Ann. Rev. Genetics, 22, 421-477). Promoters for use according to the invention may be inducible, constitutive or tissue-specific or have various combinations of such characteristics. Useful promoters include, but are not limited to constitutive promoters such as carnation etched 15 ring virus (CERV), cauliflower mosaic virus (CaMV) 355 promoter, or more particularly the double enhanced cauliflower mosaic virus promoter, comprising two CaMV 35S promoters in tandem (referred to as a "Double 35S" promoter). 20 It may be desirable to use a tissue-specific or developmentally regulated promoter instead of a constitutive promoter in certain circumstances. A tissue-specific promoter allows for overexpression in certain tissues 25 without affecting expression in other tissues. By way of illustration, a preferred promoter used in overexpression of enzymes in seed tissue is an ACP promoter as described in W092/18634. 30 The promoter and termination regulatory regions will be functional in the host plant cell and may be heterologous (that is, not naturally occurring) or homologous (derived WO 01/31027 PCT/EPOO/09374 11 from the plant host species) to the plant cell and the gene. Suitable promoters which may be used are described above. The termination regulatory region may be derived from the 3' 5 region of the gene from which the promoter was obtained or from another gene. Suitable termination regions which may be used are well known in the art and include Agrobacterium tumefaciens nopaline synthase terminator (Tnos), Agrobacterium tumefaciens mannopine synthase terminator 10 (Tmas) and the CaMV 35S terminator (T35S). Particularly preferred termination regions for use according to the invention include the pea ribulose bisphosphate carboxylase small subunit termination region (TrbcS) or the Tnos termination region. 15 Such gene constructs may suitably be screened for activity by transformation into a host plant via Agrobacterium and: screening for increased isoprenoid levels. 20 Suitably, the nucleotide sequences for the genes may be extracted from the Genbank nucleotide database and searched for restriction enzymes that do not cut. These restriction sites may be added to the genes by conventional methods such as incorporating these sites in PCR primers or by sub 25 cloning. Preferably the DNA construct according to the invention is comprised within a vector, most suitably an expression vector adapted for expression in an appropriate host (plant) 30 cell. It will be appreciated that any vector which is capable of producing a plant comprising the introduced DNA sequence will be sufficient.

WO 01/31027 PCT/EPOO/09374 12 Suitable vectors are well known to those skilled in the art and are described in general technical references such as Pouwels et al, Cloning Vectors. A laboratory manual, 5 Elsevier, Amsterdam (1986). Particularly suitable vectors include the Ti plasmid vectors. Transformation techniques for introducing the DNA constructs according to the invention into host cells are well known in 10 the art and include such methods as micro-injection, using polyethylene glycol, electroporation, or high velocity ballistic penetration. A preferred method for use according to the present invention relies on agrobacterium - mediated transformation. 15 After transformation of the plant cells or plant, those plant cells or plants into which the desired DNA has been incorporated may be selected by such methods as antibiotic resistance, herbicide resistance, tolerance to amino-acid 20 analogues or using phenotypic markers. Various assays may be used to determine whether the plant cell shows an increase in gene expression, for example, Northern blotting or quantitative reverse transcriptase PCR 25 (RT-PCR). Whole transgenic plants may be regenerated from the transformed cell by conventional methods. Such transgenic plants having improved isoprenoid levels may be propagated and self-pollinated to produce homozygous lines. Such plants produce seeds containing the genes for the 30 introduced trait and can be grown to produce plants that will produce the selected phenotype.

WO 01/31027 PCT/EPOO/09374 13 Preferably the level of sterols, especially the level of 4 desmethyl sterols in the plant and preferably in the seeds of the plants is at least 5wt% more than the level in corresponding plants without the non-feedback inhibited 5 HMGR gene, more preferred more than 10% more, especially preferred more than 15 % more, most preferred more than 25% more. In a very advantageous embodiment the level of desmethyl sterols is at least 2 times the level in unmodified plants, more preferred at least 5 times. 10 Especially preferably the level of sterols in plant tissue e.g. in leaves or seeds is more than 0.500 wt% based on dry weight. Another advantage of the current invention is the enhancement of the level of esterified sterols. Most 15 preferably at least 50% of the sterols are in esterified form, more preferred more than 60%. Suitable plants to be modified may be selected from a wide range. Preferably edible plants are modified, for example 20 plants having edible parts (e.g. vegetables such as cabbage, spinach, lettuce, broccoli, tomato, corn and wheat) or plants having edible fruits (e.g. palm oil trees, tomato plants, fruit trees etc) and plants having edible or extractable seeds (e.g. nut trees, oilseed plants such as 25 soy, rapeseed and sunflower). Preferably the modified plants are oilseed plants such as sunflower, rapeseed and soy or plants having oily fruits such as palm trees or leaf vegetables such as lettuce and spinach. 30 The invention also provides seeds obtained from oil plants with a non-feedback-inhibited HMGR gene, especially preferred oilseeds are tobacco seeds, canola seeds, WO 01/31027 PCT/EPOO/09374 14 rapeseed, sunflower seed. Also provided is a method to extract oil, whereby the oil is extracted from these seeds. Any suitable method can be used for such extraction. 5 The invention also provides plant tissue from plants with a non-feedback inhibited heterlogous plant HMGR gene expressing a truncated HMG-reductase. Suitable plant tissue may be leaves, stems, fruits, seeds, flowers or combinations thereof. 10 The invention will now be further illustrated in the following examples.

WO 01/31027 PCT/EPOO/09374 15 Example 1 Transformation of tobacco with Hevea brasiliensis hmg 1 cDNA A binary plasmid pHEV 36 containing a 2.1 kb cDNA of Hevea 5 brasiliensis hmg 1 (accession number X54659) in pMON 9818 (Cuozzo et al, Biotechnology (1988) 6: 549) was obtained from Nam Chua, Rockefeller University, New York (Figure 1). Binary vector was transformed into Agrobacterium 10 tumefaciens pGV3850 using triparental mating as described in Rogers et al 1988: Use of co-integrating Ti-plasmid vectors in Plant Molecular Biology Manual, eds Galvin & Schilperoort, Kluwer Academic Press. Transformants were analysed for presence of the gene of interest by PCR. 15 PCR positive cultures were used to inoculate a 10 ml Lennox media broth containing kanamycin 50 pg/ml and rifampicin 50 pg/ml. The overnight culture was spun down at 3000g and resuspended in an equal volume of MS media (3% sucrose). 20 Leaf segments were cut from young Nicotiana tabacum L. cv. SRi leaves from plants grown in tissue culture. Segments were placed directly into the agrobacterium solution and left for 10 minutes. The segments were then removed and placed upper surface down on feeder plates (10 per plate) 25 and left for 2 days in low light at 22 0 C. The leaf segments were then placed on tobacco shooting media with hormones containing cefotaxime 500 pg/ml and kanamycin 50 pg/ml with the upper surface up and placed in a growth room at 24 0 C with a 16hrs light 8 hrs dark regime. Three weeks later the 30 callusing segments were transferred to tubs of tobacco shooting media. Once formed shoots were excised and placed on tobacco shooting media without hormones containing WO 01/31027 PCT/EPOO/09374 16 cefotaxime 500 pg/ml and kanamycin 50 pg/ml to root. Rooted plants were then potted up into a 50% perlite 50% compost mixture and placed in a propagator. After 1 week the plants were removed from the propagator and subsequently potted up 5 into 5 inch pots. Once flowering had begun paper bags were placed over the flowers to prevent cross pollination. When flowering had finished and pods formed, the bags were removed and the amount of water supplied reduced. Seed was harvested from dry pods and stored for subsequent analysis. 10 Example 2 Sterol Analysis of transgenic tobacco seeds The plant tissue obtained in accordance to example 1 is 15 freeze dried, then ground to a fine powder. 250pl of 0.2 % w/v dihydrocholesterol dissolved in chloroform is pipetted into a screw-top septum vial. After removal of solvent, an amount of the plant tissue (50 mg) is added to the vial, and total lipid extracted with 5 ml of a 2:1 v/v mixture of 20 chloroform:methanol. The vial is capped and placed in a hot block maintained at 80-85'C. After 30 minutes the contents are filtered and the vial is washed out with a second 5ml aliquot of the chloroform:methanol mixture. The contents of the vial are filtered once more and the filtrates combined. 25 The solvent portion of the filtrate is blown off using a stream of nitrogen gas to isolate the lipid residue. The lipid fraction is then subjected to transmethylation by heating at 80-85 0 C in 1 ml of toluene and 2 ml of 0.5N 30 sodium methoxide in methanol. After 30 minutes, 2 ml of a 14 % boron trifluoride solution in methanol is added and heated for a further 10 minutes at 80-85 0 C. After cooling, 2-3 ml of diethyl ether followed by 5 ml of deionised water WO 01/31027 PCT/EPOO/09374 17 are added. The ether fraction is removed and a further ether extraction carried out. The ether fractions are combined, backwashed with approx. 5 ml of water and dried overnight over anhydrous sodium sulphate. The ether phase 5 is filtered and the solvent removed using a stream of nitrogen gas. Sterols are dissolved in 300-400 pL of toluene and silylated by the addition of 200 pl of 95:5 N,O 10 bis(trimethylsilyl)acetamide:trimethylchlorosilane followed by incubation at 500C for 10 minutes. GC analysis is carried out using a 25 m x 0.32 mm i.d. ~(0.25 pm film thickness) 5% BPX5 column (ex SGE) in a Perkin-Elmer 8420 GC. The temperature program is 180-2400C at 10 0 C/min, 15 followed by 240-3550C at 15 0 C/min. and, finally, 5 min. at 3550C. The FID temperature is 3800C and the helium pressure 10 psi. A volume of 1.0 pl is injected onto the column. A GC response factor of 1.0 for each of the sterols with respect to the dihydrocholesterol internal calibrant is 20 assumed. The five main sterol peaks (cholesterol, campesterol, stigmasterol, B-sitosterol, isofucosterol) and the intermediate compound cycloartenol were identified by 25 comparison with authentic samples and library spectra following GC-MS analysis (Hewlett Packard 5890 Series 2 Plus GC interfaced to a 5972A mass selective detector) using a 30m x 0.25mm i.d. (0.25 pm film thickness) HP5-MS column. The oven temperature program was 100-320*C at 30 10*C/min, then 8 min. at 3200C. Electron impact spectra were recorded at 70 eV and an electron multiplier voltage of 2494 V. A helium flow rate of 1ml/min at constant flow WO 01/31027 PCT/EPOO/09374 18 and a 1.0 pl splitless injection were employed. The MS data range was 65-520 Daltons. The reproducibility of this methodology was confirmed by 5 repeated analysis of a particular batch of wild type tobacco seed. The amount of each sterol in plant tissue is expressed as a percentage of the dry sample weight. Table 1 shows the sterol analysis of mature seeds obtained 10 from tobacco transformed with H. brasiliensis hmgl cDNA. Seeds from 38 independent transgenic plants (HMGR) were analysed along with seeds from 8 independent untransformed plants (SR1) which had been generated via tissue culture. -The total sterol content of the SR1 control seeds ranged 15 from 0.364%-0.386% dry weight with a mean of 0.374 (S.D. 0.0072). The HMGR transgenic seeds contained total sterol contents of up to 0.439% which corresponds to increases of up to 17.4% compared to the mean of control seeds. 25 of the 38 HMGR transgenic plants contained total sterol 20 contents above the control mean.

WO 01/31027 PCT/EPOO/09374 19 Table 1: Hevea (rubber) HMGR cDNA in tobacco - mature seed analysis Total sterols as % of dry weight Sample Choleste Campeste stigmaste Sitoste Isofuco cycloarte Total rol rol rol rol sterol nol sterols HMGR2 49 0.0334 0.0585 0.0420 0.1684 0.0832 0.0539 0.439 HMGR2 16 0.0376 0.0580 0.0365 0.1592 0.0844 0.0551 0.431 HMGR2 43 0.0293 0.0607 0.0395 0.1660 0.0796 0.0540 0.429 HMGR2 36 0.0268 0.0584 0.0419 0.1913 0.0749 0.0291 0.422 HMGR2 11 0.0296 0.0568 0.0382 0.1627 0.0806 0.0540 0.422 HMGR2 48 0.0283 0.0580 0.0403 0.1628 0.0784 0.0474 0.415 HMGR2 14 0.0279 0.0596 0.0401 0.1639 0.0752 0.0455 0.412 HMGR2 25 0.0287 0.0552 0.0368 0.1637 0.0802 0.0469 0.411 HMGR2 23 0.0289 0.0545 0.0367 0.1599 0.0754 0.0535 0.409 HMGR2 27 0.0267 0.0559 0.0388 0.1618 0.0754 0.0494 0.408 HMGR2 10 0.0272 0.0546 0.0398 0.1579 -0.0761 0.0522 0.408 HMGR2 12 0.0255 0.0545 0.0370 0.1625 0.0728 0.0512 0.404 HMGR2 32 0.0309 0.0538 0.0354 0.1532 0.0804 0.0492 0.403 HMGR2 2 0.0363 0.0529 0.0347 0.1562 0.0848 0.0355 0.401 HMGR2 52 0.0295 0.0555 0.0383 0.1593 0.0767 0.0372 0.397 HMGR2 3 0.0266 0.0532 0.0385 0.1562 0.0732 0.0378 0.386 HMGR2 37 0.0253 0.0543 0.0371 0.1544 0.0702 0.0443 0.386 HMGR2 9 0.0264 0.0529 0.0383 0.1557 0.0686 0.0435 0.385 HMGR2 35 0.0262 0.0516 0.0372 0.1565 0.0718 0.0408 0.384 HMGR2 8 0.0253 0.0556 0.0358 0.1549 0.0738 0.0383 0.384 HMGR2 6 0.0291 0.0518 0.0354 0.1576 0.0785 0.0288 0.381 HMGR2 50 0.0278 0.0519 0.0332 0.1531 0.0783 0.0362 0.381 HMGR2 7 0.0288 0.0492 0.0349 0.1532 0.0756 0.0358 0.377 HMGR2 42 0.0266 0.0528 0.0373 0.1607 0.0734 0.0264 0.377 HMGR2 53 0.0299 0.0528 0.0345 0.1528 0.0756 0.0298 0.375 HMGR2 1 0.0285 0.0519 0.0376 0.1490 0.0726 0.0336 0.373 HMGR2 55 0.0289 0.0515 0.0371 0.1532 0.0681 0.0314 0.370 HMGR2 5 0.0320 0.0488 0.0349 0.1452 0.0774 0.0302 0.368 HMGR2 45 0.0274 0.0535 0.0377 0.1500 0.0678 0.0313 0.368 HMGR2 54 0.0291 0.0505 0.0346 0.1493 0.0746 0.0286 0.367 HMGR2 29 0.0220 0.0503 0.0385 0.1494 0.0613 0.0422 0.364 HMGR2 31 0.0261 0.0509 0.0325 0.1530 0.0700 0.0304 0.363 HMGR2 26 0.0309 0.0486 0.0326 0.1475 0.0708 0.0313 0.362 HMGR2 46 0.0293 0.0388 0.0321 0.1533 0.0748 0.0305 0.359 HMGR2 56 0.0314 0.0514 0.0381 0.1421 0.0724 0.0224 0.358 HMGR2 44 0.0292 0.0519 0.0320 0.1407 0.0726 0.0276 0.354 HMGR2 38 0.0197 0.0490 0.0397 0.1456 0.0510 0.0375 0.342 HMGR2 30 0.0195 0.0475 0.0371 0.1384 0.0552 0.0375 0.335 SRI 4(control) 0.0276 0.0503 0.0364 0.1528 0.0721 0.0396 0.379 SRI 5(control) 0.0297 0.0517 0.0368 0.1526 0.0784 0.0336 0.383 SR1 6(control) 0.0290 0.0499 0.0346 0.1439 0.0754 0.0317 0.364 SRI 7(control) 0.0272 0.0550 0.0390 0.1481 0.0726 0.0260 0.368 SR1 8(control) 0.0324 0.0547 0.0405 0.1468 0.0744 0.0369 0.386 SR1 10(control) 0.0256 0.0503 0.0419 0.1483 0.0731 0.0354 0.375 SRI 12(control) 0.0251 0.0508 0.0383 0.1531 0.0712 0.0333 0.372 SRI 13(control) 0.0322 0.0501 0.0354 0.1445 0.0762 0.0304 0.369 WO 01/31027 PCT/EPOO/09374 20 Example 3 Assay of HMGR activity in transgenic tobacco seeds 5 Tobacco seeds were collected 18-19 days after anthesis and extracts were prepared by homogenising seeds in 200mM potassium phosphate pH 7.5, 0.35M sorbitol, 10mM EDTA, 5mM MgCl 2 , 5mM glutathione and 4g/l PVPP in a ratio of 1:2 (seeds:buffer w/v). Total homogenate was assayed 10 immediately for HMGR activity according to the method of Chappell et al Plant Physiol (1995) 109: 1337, except TLC analysis was performed as described by Schaller et al (1995) Plant Physiol 109: 762. 15 Seeds from two plants with enhanced levels of sterol (HMGR2 and HMGR36 of table 1) were assayed for HMGR activity along with seeds from two control plants (SR4 and SR5 of table 1). Table 2 shows that the two transgenic seed extracts contain significantly higher activities of HMGR compared to 20 control plants. Thus expression of a 'deregulated' form of an HMGR gene enhances the overall HMGR activity in seed tissue leading to elevated levels of seed sterols. Sample HMGR activity (pmol/hr/mg seed) HMGR2 36 2,520 HMGR2 2 2,480 SR1 4 1,780 SR1 5 1,220 25 Table 2: HMGR activity of trangenic seeds compared to control WO 01/31027 PCT/EPOO/09374 21 Example 4 Transformation of tobacco with another Hevea brasiliensis hmgr 1 cDNA construct 5 Hevea brasiliensis hmg 1 cDNA was placed under control of the double Cauliflower Mosaic Virus 35S (2x35S) promoter and, to terminate transcription, the pea ribulose bisphosphate small subunit terminator (TRBCS) has been placed down stream of the hmg 1 gene. The chimaeric gene 10 was cloned into a pGPTV- KAN [Becker et al Plant Mol Biol (1992) 20: 1195-97] based binary vector, SJ 34. Plasmids CJ151, CJ157, PP5LN and SJ34 are shown in Figures 2 to 5. E. coli strain DH5a (Gibco BRL) was used as the 15 host strain in all cloning procedures. Bacteria were cultivated in LB medium (10 g/l tryptone, 5g/l yeast extract, 5 g/l NaCl) supplemented with the appropriate selection pressure (ampicillin (100 pg/ml) or kanamycin (50 pg/ml) on a rotary shaker (210 rpm) at 37 *C. 20 Plasmid CJ157 was digested with HindIII and NcoI to obtain the CERV promoter fragment. This fragment was inserted in the corresponding sites of plasmid PP5lN resulting in plasmid pNH1. A SalI containing DNA linker was assembled by 25 mixing 4 pmol of oligonucleotides Sall and Sal2 with annealing buffer (10 mM MgCl 2 , 100 mM NaCl, 1 mM dithioerythritol, Tris-HCl pH 7.5) in 100 p.l water. The mixture was heated to 80 0 C in a 5 L water bath and cooled down to room temperature over night. The synthetic linker 30 holding the SalI site was inserted between the EcoRI and XbaI sites of pNH1 yielding pNH2. Oligonucleotides Xmal and Xma2 were also assembled using the above outlined protocol WO 01/31027 PCT/EPOO/09374 22 rendering a DNA-linker containing a XmaI site. The synthetic linker holding the XmaI site was inserted between the HindIII and ClaI site of pNH2 rendering pNH3. Plasmid CJ151 was digested with ClaI and NcoI to obtain a 785 base 5 pair fragment containing the 2x35S promoter. This fragment was inserted into the corresponding sites of pNH3 in place of a CERV promoter fragment (pNH4). The 729 base pair pea ribulose bisphosphate small subunit terminator [TRBCS) was amplified by PCR with primers TRBSC5 and TRBSC3N using 25 10 thermal cycles (30 s. 94 *C, 30 s. 53 *C, 120 s. 72 *C) and a mixture of Thermus aquaticus (Taq) and Pfu DNA polymerase (9:1). The amplification product was purified using the Qiagen PCR product purification kit. This fragment was digested with SacI and EcoRI and inserted into pNH4 in 15 place of the nopaline synthase terminator rendering pNH5. Several pNH5 clones were identified by restriction enzyme digestion analysis using SacI and EcoRI. These clones all exhibited the characteristic DNA fragment pattern, i. e. 631 and 3509 base pair fragments, when separated in an 20 agarose gel. One of the positive clones was sequenced using primers 35S and U19 (Figure 9 A) on an automatic Perkin Elmer 373 sequencer using dyed fluorescent nucleotides according to the supplier's recommendations. The sequencing confirmed that the TRBCS fragment was correctly amplified. 25 Moreover, sequencing also confirmed that the polylinker region, holding sites NcoI, NheI, MunI and SacI, was intact. A cloning scheme covering these steps is shown in Figure 6.

WO 01/31027 PCT/EPOO/09374 23 Table 3. Oligonucleotides used in vector construction (given in 5' to 3' direction) Primer Sequence Sall AAT TCG CTG GTG TCG ACT TTA CTT Sa12 CTA GAA GTA AGG TCG ACA CCA GCG Xmal AGC TTA CTC TTC CCG GGA TTG TTA T Xma2 CGA TAA CAA TCC CGG GAA GAG TA HMGR5 ATA TTT TTC CAT GGA CAC CAC C HMGR3 GGA CCG AAT TCC CAC TAA GAT GC TRBCS5 GGA ATG AGC TCT AAA GAG CTA GAG CTT TCG TTC TRBCS3N GTC AAT GAA TTC GCA AGT CAT AAA ATG U19 TTT CCC AGT CAC GAC GTT GT HMGRisF GGA TCC CAA CTA CCT CAT HMGRisR TCC ACC CAA AGC ACC AG ISHMGR5 CTG TTC CAA TGG CGA CC 35S TCC ACT GAC GTA AGG GAT GAC F72 GCC ATA ATA CTC GAA CTC AG 5 A 1727 base pair gene fragment encoding the Hevea brasiliensis hmg 1 was amplified by PCR from a cDNA clone in order to introduce cloning sites in either end of the gene (accession number X54659, Chye et al., 1991). The hmgl cDNA was amplified by gene specific primers (HMGR5 and 10 HMGR3) using 25 thermal cycles (30 s. 94 0C, 30 s. 53 *C, 120 s. 72 *C) and the proof reading enzyme Pyrococcus furiosus (Pfu) DNA polymerase to enhance the fidelity. The obtained fragment was digested by NcoI and EcoRI and inserted between the NcoI and MunI sites of pNH5 yielding 15 pNH8 (Figure 8). Six pNH8 clones were identified based on WO 01/31027 PCT/EPOO/09374 24 restriction enzyme digestion pattern. These clones displayed 2 fragments of 2378 and 3487 base pairs when digested by NcoI and EcoRI. Two independent positives clones were chosen for sequencing using the primers shown 5 in Figure 9 B. In both clones the hmg 1 genes contained five identical nucleotide substitutions as compared to the published sequence (X54659) (Figure 10). Furthermore, when sequencing the obtained cDNA clone, which had previously been used as the template to amplify the hmg 1 gene, it 10 also contained the same five nucleotide substitutions. The codon changes due to the nucleotide substitutions did not give rise to amino acid substitutions, i. e. all nucleotide substitutions were silent mutations. Hence it was concluded that the most probable explanation for these nucleotide 15 substitutions are sequencing errors when the clone was initially cloned and deposited in the gene bank. This conclusion is supported by the fact that all substitutions are confined to a 225 base pairs region in the central part of the hmg 1 gene. 20 Plasmid pNH8 was digested by HindIII and EcoRI to obtain the 3158 base pair 2x35S-hmgrl-TRBCS cassette which was subsequently inserted into the binary vector pSJ34 rendering pNH16 (Figure 7). The steps of constructing pNH16 25 are schematically drawn in Figure 8. Positive pNH16 clones were selected based on restriction enzyme digestion analysis. Clones exhibiting the correct pattern when digested by HindIII and EcoRI, i. e. 3183 and 30 11106 base pair fragments, were selected. One of the positive clones was sequenced as shown in Figure 9 C. This confirmed that 5' and 3' parts of the hmg 1 gene were WO 01/31027 PCT/EPOO/09374 25 correctly fused to the 2x35S promoter and the TRBSC terminator, respectively. Vectors pNH16 and pSJ34 (vector control) were transformed 5 into Agrobacterium LBA4404 using electroporation according to the method of Wen-Jun and Forde (1989). Transformants were analysed for presence of the gene of interest by PCR. Transformation of tobacco was carried out as described in Example 1. As well as the vector control plants a number of 10 untransformed tobacco plants were generated via tissue culture. Sterol levels were determined in accordance to example 2. 15 Table 4 shows the sterol analysis of mature seeds obtained from tobacco transformed with the Hevea brasiliensis hmg 1 gene fragment under control of the 35S promoter. Seeds from 23 independent transgenic plants (NH16) were analysed along with seeds of 12 independent untransformed plants (SR1) 20 which had been generated via tissue culture. The total sterol content of the SR1 had a mean of 0.337 % dry weight (S.D.0.019). The HMGR seeds contained total sterol levels of up to 0.389 % dry weight which corresponds 25 to increases of up to 15 % compared to the mean of control seeds.

WO 01/31027 PCT/EPOO/09374 26 Table 4: Sterol Analysis of seed from tobacco transformed with 35S - Hevea HMGR (NH16) Total sterols as % of dry wt Sample Choles Campes Stigmas Sitoste Isofuco Cycloar Total terol terol terol rol sterol tenol sterols NH16 18 0.0257 0.0545 0.0375 0.1665 0.0723 0.0327 0.389 NH16 21 0.0272 0.0509 0.0356 0.1681 0.0754 0.0275 0.385 NH16 37 0.0293 0.0536 0.0427 0.1589 0.0714 0.0263 0.382 NH16 31 0.0287 0.0485 0.0317 0.1556 0.0749 0.0350 0.374 NH16 28 0.0307 0.0483 0.0340 0.1553 0.0735 0.0265 0.368 NH16 1 0.0266 0.0500 0.0322 0.1432 0.0727 0.0395 0.364 NH16 47 0.0294 0.0459 0.0374 0.1578 0.0710 0.0221 0.364 NH16 23 0.0245 0.0515 0.0368 0.1517 0.0671 0.0301 0.362 NH16 48 0.0268 0.0476 0.0352 0.1518 0.0660 0.0292 0.357 NH16 46 0.0317 0.0469 0.0410 0.1493 0.0595 0.0245 0.353 NH16 12 0.0215 0.0478 0.0443 0.1594 0.0581 0.0212 0.352 NH16 14 0.0248 0.0474 0.0376 0.1528 0.0661 0.0231 0.352 NH16 22 0.0289 0.0478 0.0347 0.1436- 0.0687 0.0275 0.351 NH16 45 0.0220 0.0474 0.0406 0.1595 0.0598 0.0214 0.351 NH16 32 0.0231 0.0497 0.0363 0.1461 0.0626 0.0285 0.346 NH16 19 0.0221 0.0491 0.0395 0.1407 0.0614 0.0297 0.342 NH16 13 0.0218 0.0502 0.0340 0.1420 0.0636 0.0303 0.342 NH16 42 0.0249 0.0467 0.0347 0.1438 0.0630 0.0257 0.339 NH16 27 0.0257 0.0458 0.0339 0.1445 0.0665 0.0183 0.334 NH16 10 0.0262 0.0415 0.0308 0.1451 0.0650 0.0220 0.331 NH16 44 0.0300 0.0436 0.0413 0.1446 0.0536 0.0171 0.330 NH16 3 0.0221 0.0467 0.0373 0.1459 0.0580 0.0178 0.328 NH16 40 0.0270 0.0450 0.0337 0.1338 0.0633 0.0221 0.325 SRi 18(control) 0.0268 0.0497 0.0325 0.1533 0.0766 0.0330 0.372 SR1 6(control) 0.0314 0.0497 0.0347 0.1416 0.0684 0.0337 0.359 SRI 3(control) 0.0290 0.0466 0.0317 0.1427 0.0725 0.0306 0.353 SRI 17(control) 0.0244 0.0459 0.0305 0.1471 0.0678 0.0346 0.350 SRI 2(control) 0.0267 0.0489 0.0400 0.1391 0.0627 0.0212 0.339 SR1 1(control) 0.0271 0.0449 0.0329 0.1357 0.0654 0.0310 0.337 SRI 9(control) 0.0235 0.0459 0.0312 0.1391 0.0681 0.0292 0.337 SRi 7(control) 0.0243 0.0468 0.0365 0.1334 0.0647 0.0305 0.336 SRI 8(control) 0.0274 0.0427 0.0284 0.1261 0.0627 0.0334 0.321 SRi 5(control) 0.0226 0.0442 0.0413 0.1413 0.0547 0.0125 0.317 SRi 4(control) 0.0220 0.0431 0.0367 0.1357 0.0599 0.0176 0.315 SRi 20(control) 0.0160 0.0427 0.0407 0.1346 0.0495 0.0246 0.308 Example 5 Transformation of tobacco with a truncated Hevea 5 brasiliensis HMG 1 gene A truncated form of Hevea brasiliensis (H.B.K.) Mull. Arg. tHMG1, encoding the enzyme lacking the N-terminal membrane binding domain, was cloned using the primers based on the 10 published sequence Chye et al., 1991. The forward primer WO 01/31027 PCT/EPOO/09374 27 5'-CCTACCTCGGAAGCCATGGTTGCAC-3' incorporates a new start codon (bold) and a Nco I restriction site (underlined) for cloning applications. The reverse primer 5' CATTTTACATTGCTAGCACCAGATTC-3' contains a Nhe I restriction 5 site (underlined) for downstream sub-cloning purposes. The plasmid pNH8 (Figure 8) was used as the template DNA in the PCR (30 cycles) using Pfu polymerase under standard conditions and produced a fragment of the expected size ~1.3 kb. The resulting thmgl gene (Figure 11 a) codes for 10 amino acids 153-575 of the full-length (575) hmgl sequence (Figure 11 b). The PCR product was cloned into the pGEM-T vector (Promega) according to the manufacturers instructions and sequenced to confirm correct sequence. 15 The H. brasiliensis thmgl was inserted into pNH4 (Fig.6) between the Nco I and Nhe I sites of the polylinker, which lie between the CaMV 35S double promoter and nos terminator to give pMH3 (Figure 13). This chimeric gene was isolated by digestion with Xma CI and Sal I, purified and cloned 20 into the corresponding polylinker sites in pSJ34 (Figure 5), this binary construct was named MH3 (Figure 15). MH3 was sequenced to check that the hmgl genes had been inserted correctly and there were no mistakes in the promoter-initiation and terminator sequences. Vectors MH5 25 and pSJ34 (vector control) were then transferred into A. tumefaciens strain LBA4404 by electroporation. Transformation of tobacco was carried out as described in Example 1. 30 Sterol levels in leaf and seeds were determined in accordance to example 2, but with the following modifications. After extraction and transmethylation, WO 01/31027 PCT/EPOO/09374 28 sterols are dissolved in 250-300pl of toluene and silyated by the addition of 125-150pl of 95:5 N,O bis(trimethylsilyl)acetamide:trimethylchlorosilane followed by incubation at 50'C for 10 minutes. GC analysis is 5 carried out using a 25 m x 0.32 mm i.d. (0.25 pm film thickness) 5% BPX5 column (ex SGE) in a Perkin-Elmer Autosystem XL GC. The temperature program is 80-230 at 45 'C/min, 230-280 at 4 'C/min, 280-355 at 20 "C/min, and 5 min. at 355 0 C. The FID temperature is 370 *C, the helium 10 pressure 8 psi, the injection volume 1.0 pL and the split flow 10 mL/min. A GC response factor of 1.0 for each of the sterols with respect to the dihydrocholesterol internal calibrant is assumed. This method afforded improved separation of sterol intermediate compounds. As a result, 15 in addition to the sterol compounds identified in Example 2, A-7-avenasterol, squalene, 24-methylene cycloartanol, 24-methylene lophenol and 24-ethylidene lophenol were also identified by comparison with authentic samples, library spectra and literature data following GC-MS analysis as 20 described in Example 2. Table 5 shows the sterol analysis of leaves from 29 independent transgenic plants (MH5) and five untransformed control plants (SR1). The average total sterol content of 25 the SR1 leaves was 0.180% dry weight (S.D.=0.017), whereas the sterol content of the MH5 leaves ranged from 0.189 1.931% dry weight. The MH5 figures correspond to increases in total sterol content of up to 10.7-fold over the control mean. 30 Table 6 shows the sterol analysis of mature seeds from 27 independent transgenic plants (MH5) and 8 SR1 untransformed WO 01/31027 PCT/EPOO/09374 29 control plants. The average total sterol content of the SR1 seeds was 0.368% dry weight (S.D.=0.039), whereas the total sterol content of the MH5 seeds ranged from 0.352-0.874% dry weight. The MH5 figures correspond to increases of up 5 to 2.4-fold in total sterol and 1.7-fold in 4-desmethylsterol levels over the respective control means. Further analysis of MH5 33 seed was carried out to 10 determine the proportion of free and esterified sterol in the sample. The total lipid fraction is isolated as described in Example 2, but not subjected to the transmethylation process. The lipid residue, which contains dihydrocholesterol as internal standard, is dissolved in 15 40-60 petroleum ether (250 pL) and applied to a glass backed 20 cm x 20 cm x 0.5 mm silica gel thin layer chromatography (TLC) plate. The vial that contained the lipid residue is washed out with a further 250 pL aliquot of petroleum ether, which is also applied to the plate. A 20 10 pL aliquot of a solution consisting of a mixture of B sitosterol (10 mg) and cholesterol oleate (10 mg) dissolved in acetone (1 mL) is spotted to act as a marker. The plate is developed using 60-80 petroleum ether-diethyl ether acetic acid (80:20:2, v/v/v). The sterol fractions are 25 visualised by spraying with a 0.01 % w/v ethanolic solution of rhodamine 6G and viewing the plate under UV light. Approximate Rf values are 0.25 for free sterols and 0.9 for steryl esters. The free sterol band is scraped off the plate and transferred to a vial. The free sterol fraction 30 is isolated by washing the band with three volumes of diethyl ether. The ether washings are combined and filtered. The free sterol fraction, isolated by blowing off WO 01/31027 PCT/EPOO/09374 30 the solvent with nitrogen gas, is silylated and analysed by gas chromatography (GC) as described in Example 2. Amounts of esterified sterol are determined by subtracting amounts of free sterol from total sterol. 5 Table 7 shows the analyses of the free sterol and sterol ester fractions of transgenic MH5 seed samples 6 and 33, alongside that of an SR1 control sample. The additional sterol present in the transgenic samples compared to the 10 control is found primarily in the form of sterol esters. The total sterol content of the SR1 control is 0.388% dry weight, of which 52.4% is in the form of esters. The total sterol contents of MH5 6 and 33 are 0.711% and 0.866% dry weight respectively, of which 68.8% and 74.2% respectively 15 are esterified.

WO 01/31027 PCT/EPOO/09374 31 a) 0H co C4ci im 0 mCD I 'D _' m -I w 000 a) ' ) 0 ) r-~ C-' ' ) wo 100 com OU) m) (0 m r- 0') r- OD r- (Y) w r-4 -1:3 CNj 1 (Y) r- I V (' C'1 Z0 r) (n (n 4 0 C 0 CD- C, C' CDo( C:) 01 ) C)a)I C C ) C) ID C) CD (D CDJ C) CM 4-) 10 (D 1D 0 C D (D 0 C )) C)H D c) C') C; m .H 1 0 C) I 100 0 10 -4 r-oCo )m ) - mcy - ooc) cy) o a)cD ) D a)-i a-) c a)04 10 -4( ) C) 1CD )- CoC' ) CD -0 CC D -C) (D -1 0 4C)i' U) (1)11 1 11 \ m 4-) ( 1) I- i -H4j0 C' ) ( ID r Co ID ID H Co ID 0 1 C; H ID ;1 C;) H ID <D H U)) U) 1'H O HH H . HO H1OO O 4) ) 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00L m m -q r Nr- ( rA 4.) ~0 m m) -o - riv m m( - nt (d' >)- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 p , c) ) ) o L j j 4J J0q ) aDZ)c -11 - 1 w -1~ 0000000000000000000000000)N - ko w n C4 (D (D D CD C). D ID (D OCD HD a)CD C CD - C) C ) C") (.m 1r 00 "a m U 4-) 0m c)c -0 w " w m rjw m m o 4- n) .rqN -0000000000000CDm L I m000000000000 mLO 0 C HH 4)4-) C: C (0D D( 0 0 000000000000000 C C (DCD 0 Da0000D C -P a) OH 0' a) >0 -P ~ ~ I 010 Cl CD ID aH H H ID CD C> Co H) CD 0 ID C ( )0a( Ca) CD r 0- 0 * 0 r H4ci 0 4-)1 r 0 -1 44 d HC ;( 10 o '1 0 - 0( " )3- C \ r-4;C C CD* -ID10 c:>C', CD 00 H 010 m 'H 0'J'") H~m'C'1.-C)m>).

H0(o E4 _ _ _'_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _* _ _ _ ( c WO 01/31027 PCT/EPOO/09374 32 I'l 1--i .- 1 00 CD r- ir- 'n ((N N (N C (-4 -1 .- 1 -4 1-- (N I- w~ (N (-- a) In of Cf) ,- Cf) 0, t- CN;1 00 :) CD .l-4 ~'z 1 Wz w n '.D r-~ r- LO 00 IV -;r "v m m m m~( C') (N C' ,-4 IC) (D' a) a C') L IN I' I I' I I I' IN 0' ) 0 m - m) 00 r- w O c . 1 0) (N In C')C C') .- C' ,-I :I 'a' 'a' In m m' IH m) (N (N C(N CIA (N (N (N H (N (N4 t- (N "T ca, ma C n OD a) 00 r- H- (N4 r- (N C(n C') ~ ~ C 00C)) a (N C) C) (N HD (N ( CD( ID a C) M a) CD) 'a'0 r CD ( DC) ) C) C) CH (D -LO - 0In In 'N OD m In31r CH (D (D CD) 'a CD' 0 C, ( (Ni In c0 m -y (N S 'ai a Hi kD -a'IV 'O 'IV z"N In (N - CDa) ) 0 C) .- ) mC) C) H CD C' a) CD CD C) CD C') H H 000 H 0 H 0 000000000 i mm 000000000 -D<DM D -1 H D < C r- I 0 0(N CD 'a' ) C) HD CD CD In In* In In H; H .4 H CDCDC)C)(DCD (D CD WO 01/31027 PCT/EPOO/09374 33 r- (N N NCDjr- r- 110 'cj r-i mc c coCr- r- inn )m m m o)co 0 'I 00(Y (Y) -a m LO IVJ I- mn~ r- I-Q, r- -1 .- 1 -V iO N4 iO m r- 4 OOOOOoooooOO 00O0 C)0 O'oI 0000000000000000 00000 0 in LO 0 -IT mD mD m m m C) D CY r- (, 0 - _qC (4 0 0 [ CO r- mD 04 CD00 0 C 0 ) (' 0 0 0 0D 0- 0 0~ 0N CD in (D 0n CD 0CD i 0 (D) (0~~ ~ ~ 4- 0D(40- )C)C 0 C) in in0 CD in CD in 0 04 CD (D 0 C) CD in Cn ('4C -. COcnC c I(c) I(' m I~~m m ' -yG 0000000 c Lo u o n ,o 00 oo 0 too oo Tco 0 m 4JG -P4 ~ - - U) CIU) 040m -H0C-D-CMUl(D0. - (D 0 H DCD ' -)r 0 CD - 'D inCO C D o0 M DMC rr-C )) - Dr Dr 00 00 0 00 0 0000r 0000000 ITm 4)) U) J 0 r0l DCi CDi ClC) - DCDCOC'1rHr- r D 0 C ) - CCO a 0 0) CD CD) V 4- 4 0 - 0 0 ,40 0 0 0 0 0 0 0) 0 () 00000000000000*00000000000(::; C 4- r-); 4 0 0 0 00 0 0 0 00 0 0 0 -H U) 'A CD0(DC) CD C0CD CD C D CDC CD D(D D ( (DC) ) C >, 3 0 -A ( C ) 4 r- 'CDD Co in CD -1 3' l Dc o I 'D I'D~C C 0) ) oo o O O O O O O O OOmO a) c0 CD 4 CD 0 CD C CD (D D (D a- CO C- a CD CD (' 4 C) C)Q 0 0 00 - ~ ~ 0 0 4-1 ~~~I~-1 0 00 0 0 0 0 0 0 0 0 0 0 0 444o )C ~0 CD(DC> 0CDi(ln C) ) ( CDC)CDDCD0 C(DCD (D (DCD CC a) 0~ o 04 c-I 0 0 0 0 00 0 0 0 00 0 0 0 (L) 0) 'AHO o) r- 0 C ' 0 * ininiinCD0 CD0'C C C; C; CD inO C rICCDC.

4-) 04 $4 D4 - wD(' m~ N w 4 N ' Ci ri r- i m 0n r' -- n C D ri (4c) r-4 44 m) CU n-~.-) Y R 4MM r- (') N OD - C DiCD 0. .~ C) -inCOCOC--r)m Y)(y o0 0 N - r-0 s- r-q 1 1 - C) i .- 0H lr-1 00 , HD (DCDC)(DC)C0C00C0C V a 1- 00 0 0 0 0 0 0 0 0 0 0 0 0) 44 >,( H 0 U 4 ,,zJ op N- r-o)N m c w , - n m ( rr D4 0 0D (')0 -1 CDC A ' 4r- - ri(nC lCDC D - -i- -in , 0 m . 04 O D C- C)C 0( w1 id '-4 ) C; 0 innii ini ini ini in ini ini ini inni inin ini'ninc E I WO 01/31027 PCT/EPOO/09374 34 0) wO in 0D m~ o r- 110 r- m m l

-

c I m c m 0 m N' L in m~ in a) -j -i m~ u-L i co a in r-I ;3 ' in w m c, in in ,oD N~ q N~ N" Nl CN N N Nl 1 -1 a) :I N l -- 0 ) N~ r- r - in N r N ) in 0 D ) D 0 in CD4 C) CD CD m' m~ r- v' in r-i m~ v r- in -w 0D a) M IV N 0D M r- M 0D r Cl) (Y) N M~ M~ M N Cy) N r (Y) r- Mf a) Ir aO) 00 N r- r- in .q. N a)c' m m N r- -i in -Z r (M(f N -m Mv r~ M M C N r-I in r- a) m~ N~ ,v to w -w N -i 11 O a) ) N a) w~ in w N inr . CD) C) ND C C') CD) CD'D CD C) ND 000C in CD 0 aC ND a) 0CD CD CD in in 'CD ( C') CD ND C') NCN C' C) (D C'0 C) CD) Nl (D a) C0 a) N r- OD w in m' IV m' w w i n in in in in -i .- i m~ 00 a) Om in a) a) qv q, k N r N\ N N N N N m' N -1 0 CD CD ND C) CDi a) CDa) a m' N N m' m' N m' N N .- 1 .-A C) CD 00 CD a ' C) 0 C C) inC in r [- [r- a) w) w a) w N N IV~~~ in IV) -iTWL - r- a) in in in C4 C- -~.4 .H 4 .- 4 , En w cn U)EnU U U WO 01/31027 PCT/EPOO/09374 35 o LO m 14 N m3 %D ) Cn co o C) -4 N OD %0 N 0 )*o o C 0 C C 0 C C LO C) 1 N rr L ' N N C) w~ [~v w C) m N C) a-i N C N N ) r-I W a O a- o) .- oa L0 -- ) 0 C) 0 C LfD CD LD 0 (DO I N N 1O N - LO 04C) C) C) C) 0) C) (D C) C) u U) 020 N O C) m N m 0) w o o N ko o o o o Hj O $4 C) C) C) C) 0) C) C) C) C) 4-J) C) C) C) C) C) C) () L 0 M w r- L r- o0 'H -H N CC) n o) ) -- 'H 0) (D 0 r- 0) 0) o 'H m o 'H 0 O 4 (D C C) 'HA C) 'Hi -H C) ' .) o Q) .114-) C C) C) C) C) C) C) C) C (fU) U *-IT CCC) ' (f ) N w0 w 0 ) 0q M o C) r- r 0 -1H Ln r- m e-1 m CO C\ O 3 o OH 10 ' C' H C' a) N N 0) 0C) C) C) .H C. C. . C) C) 0 (1) C ) C) C) C) C) C) C), C) U) 4-) L- C o-- o r-r 01 c- N C Oq C) -1 kOO N N N N- C) Ln(a 0 C) C) C) C) C) 'H C) 'H C) C)0 C) C) C) C) C) C) C) o ,C o o C) C- m o S>, 0 N 'H C) 10 N N N H fC) C) C) () C) CD C C) C) o - ~ Q) C) C) C)DC C) C) C) C) .. ' N a.a , ~ -HO0 N.H-4 )C C) L N CC C) o CO $4 44 C D C 1 . ) L ' >, 'HDC C) C) CD 10T LO N (N 4 IQ. 0 C) C) C N C N CD N 0 .4- i C) C) C) C) CD C) C) C) C) 41) (1) 01) Z00 (6N .- i 0~ ~ _q N N' rD LO IV LO rs) o 'H 0) o o ' o 'H C' 14 >1 w ' N 10i 'H -1 I-l a) w (0 . 4 -i' CD 'H C') 'H N NP N- 1 43 41 (0 C) C) C) C) C) C) C) C) C) i a ) O. . . . C) C) C) C) H C w C' N - 1 N r 0 4 4-) CO N C' () a) C) F-- 0) -1 CO0 10 N -- I --I ~ N Nq 1) Nl C'n 0 1C) C) C) (D C) ' N C) N 4- 0 C)2C C)> ) C) C ) C) C) C) C) 0 44 1-_ 14 0 0 dP 4-J 'H H -4 ' 44 0 -IO H Q) 0 H ) O H 1) 0 02 rrj 04-p 0 4-) I-4 0 41i 4 0 41) (d 4 14 4) 4 () (1) 4 U) (1) 4-4 U) f 4 -) (1) 4-) 0) W 4-) a) w 3 - -H (A U) 41 U) U) 4. c UC) 41) U' 02 H o 0 ) -43 F4 U) -A 1.4 H C 0 -1 0 - IIDOH OI O 1 H M 0II E4, w 1 - a)y - - W _ p n ' ia - a) -- (0 4JU ( )a)E JU ) l)W a) n - ) )U)a __ 1 42__ ___ 0______-_1_ Z44) 1 WO 01/31027 PCT/EPOO/09374 36 ,,:v LO m ci N CY) D -11Lfl C) 0 (n r-) n ') GO C Ch 0-) LnlC r O (Y) (Y)11 L n 'I Da) M( UL L a ) r-c mk Cr or-m GO;a LO ~ '0c 0)C CNJ- 00 0) (D CNr- r-q 0 Cl tf m C mh 00 C c'Nr- a 4J * 1 0 0 a) a VJ 0) 0 a)s i a -I a)4 0) 04' 04-) 0 4-J * O:) w D ) a)) 0 0 4J -) 4-) > c) H ) H m ) -fli 0 %'D 0C'm 0 CP U) C ) 44 ) WO 01/31027 PCT/EPOO/09374 37 Example 6 Transformation of tobacco with a truncated S.cerevisae HMGR gene Based on the nucleotide sequence of cosmid 8248 from the 5 Saccharomyces cerevisae chromosome XIII sequencing project, primers were designed to clone the tHMG1 gene by polymerase chain reaction. The forward primer 5' GCTTGGATAAGGCCATGGGTCCTTTAG-3' incorporates a new start codon (bold) and a Nco I restriction site (underlined) for 10 cloning purposes. The reverse primer 5' GAATACCAATGAGCTCTGACTAAG-3' contains a Sac I restriction site (underlined) for sub-cloning applications. Prior to PCR the genomic DNA from S. cerevisae, NCYC 957, X2180, a, SUC2, mal, gal2, CUA was digested with Eco RI and the DNA 15 fractionated on a 0.7 % agarose gel. DNA fragments -2.0 kb in size were excised from the gel and purified using the Qiagen QIAquick gel extraction kit, according to the manufacturers protocol. This DNA was used as the template in the subsequent PCR. The PCR (35 cycles) was performed 20 using Taq and Pfu polymerase (3:1) under standard conditions and produced a DNA fragment of the expected size -1.4 kb. The resulting tHMG1 gene (Figure 12a) codes for amino acids 598-1054 of the full length (1054) HMG1 sequence (Figure 12 b). The tHMG1 PCR product was cloned 25 into the pGEM-T vector (Promega) according to the manufacturers instructions and sequenced to confirm the correct sequence. The S. cerevisae tHMG1 was inserted into pNH4 (Figure 6) between the Nco I and Sac I sites of the polylinker to produce pMH4 (Figure 14). This chimaeric 30 gene was isolated by digestion with Xma CI and Sal I, purified and cloned into the corresponding polylinker WO 01/31027 PCT/EPOO/09374 38 sites in pSJ34 (Figure 5), to create the binary plasmid pMH6 (Figure 16). pMH4 was sequenced to check that the HMG1 gene had been inserted correctly and there were no mistakes in the promoter-initiation and terminator sequences. 5 Vectors MH6 and pSJ34 (vector control) were then transferred into A. tumefaciens strain LBA4404 by electroporation. Transformation of tobacco was carried out as described in Example 1. 10 Seeds were analysed in accordance to Example 5. The results showing (see table 8) an increase in total sterol levels of the transgenic plants (MH 6) of up to 16 % compared to the mean of the control plants (SR1, mean 0.373).

WO 01/31027 PCT/EPOO/09374 39 m 1-1 r0A00) D)OCD 0 -C' YrW- - ,o , ai rmO0m2m-wr-- Ln r 0 LI -W (N (n) 0 m - 0nc) (n k COO n O r-i w ) -T " f " f (N (n- w N ;il w 0 00 C1 N liN ' (N C\ 0 ( 14 N(N N " " cq " C(N " "N( -1 -1 ( ( '4 CN(4 0 )0 0 C 0 0 " 0 0 0 0 0 0 0 0k W 0 0 0 0 0 0 0 0 0 0 0 00D 14 rl IV ( - r A 0 4 0 00 00000000 0 0000 0000 000 w n r- m (N C ~- ) 0j) 0m a) oo -iC H p aO ) 0o 02 0 co ri co r- 02 -0 v0 r- C O 10 )H 0 r ( n r O- war- -v.Dm nr-w -w ) (N W0 ( i) LO -A 0T ) r- (NO4 0c) m - (N I) u-) Coo ) [- (Y) c (d) 00 (Y) '. -A 00 00 00 00 00 00 00 00 0 4)4) C; - C CD) C)-1 )(( O U )C C C C 1 O (N1 0O C; 1-nO C - . 4 EQ OCU)C O(~C C NOn~'0 O C onNt--4O( 0- Lo - D m L ik - t - - n ri -A C , r- H H r r- N co m w D D W 00H, Z, m cq ,H 1 - -1 ID M w w~ 02 ~i()( r-C>( "-Jc)r LO'Dr Lf)( N (M .- 4MnMC r4')O)MO r- ) 0i W 0r 0000000000000000 0000r VL)'0r-L)NNr4()00 M()000 ( 4.) -) o -i -i ,z (N4 v r- 00m (Y) r-i a) i) w. (NO m . w . w ' (N r- i) -v (N r- 0) m W 0) 4 -) r -0 >f 41 0 C C CO a '0 * (N O C O C O ( C') 0 n . C ') C; '. 0 0 ) LX) >1 0 m m(Y()(Y) (N (YC) Y) ( NNN (n (Y)N(" N N H(N N ("C) (N (N N .4-J) ) 0).0 D C, C;( 0 0 0 000000 00000000000000; C C* 0 04 00 0 0 0 0 0 0 0 0 0 0 0 0 0)i 0 C0). Oo OO O OO O OO OO O OO O >1 ~ ~ ~ ~ ~ ~ ~ ~ U v U) nL L nm rL ( rn p ' :: n -w) m (mN ,w' v n (Nm a.) r>0 Q W 0 oN -4 -1 0 ~ ~ ~ U 00 C.a DC)CC CO . 0 (N C) r-I 002 Ci).0)0 (Nm CD 0 C C 0DCDCD C: C.0CD .0 C) <D C; m; mm C; (N 0N (N mm (N( m ( ( ( N ( (N ' N ( 00 00 0 00 0 00 00 0 00 0 4) HO 4i 0 H 0-J 44 (p()L -mL -L n vm rm( wo r(NODOm '-0c0(n0 - o)02iN) (N(NN a0 H- .0 mr) wr- m.--0,ow "-'T n(NNv .- 40)(n4(nt--COl)-mU N(oLo 0 , H$4 () C4 C)C DC DC C DC )C D DC DC D(DC D0( D( * 0 (d ) 4.) CD CD0 CDl CDCD0 0'.D0 CD'l'0. CD.0. '0..0 C). C) CD . . CD C, (D ) 00)( C CD1i ED C D C C D ;C ;C C ;C C ;C C ;("C WO 01/31027 PCT/EPOO/09374 40 Example 7 Transformation of tobacco with truncated Hevea brasiliensis HMGR1 cDNA linked to a seed-specific promoter The H. brasiliensis tHMGR1 was also cloned into the 5 polylinker region of pNH12 in the Nco I and Nhe I restriction sites, which lie between the ACP (acyl-carrier protein) promoter and the nos terminator to give construct pMH1l. The chimeric gene was cloned into the binary vector pSJ34 after digestion and purification with XmaC I and EcoR 10 I and named pMH15. The binary vector pMH15 was sequenced to check that the hmgrl gene had been inserted correctly and there were no mistakes in the promoter-initiation and terminator sequences. The binary plasmid was used to transform the A. tumefaciens strain LBA4404 by 15 electroporation. Tobacco was transformed with this plasmid in accordance to example 1. Example 8 Transformation of Brassica napus (oil seed rape) 20 with truncated Hevea brasiliensis gene of example 5 Electrocompetent Agrobacterium tumefaciens cells (strain LBA4404) were defrosted on ice and 5ng of vector plasmid MH5 (as above) added. Cells plus plasmid were then placed 25 into a pre-chilled electroporation cuvette and electroporated in a Bio Rad Gene Pulser at a capacitance of 25 and at 600 ohms. Immediately after electroporation 950pF of 2X TY broth was added, the cells mixed gently and placed in a sterile vial. The cells were shaken at 28 C for 2 30 hours and 2 5pl aliquots plated on solid Lennox media containing rifampicin 50ig/ml and kanamycin 50pg/ml and WO 01/31027 PCT/EPOO/09374 41 incubated at 28'c for 3 days. Single colonies were used to inoculate 10ptl of water (for PCR confirmation) and 500pl of Lennox media containing rifampicin 50pg/ml and kanamycin 50pig/ml. 5 Seeds of B.napus cv.Westar were surface sterilised in 1% sodium hypochlorite for 20 mins. The seeds were washed in sterile distilled water 3 times and plated at a density of 10 seeds per plate on MSMO with 3% sucrose pH 5.8. Seeds 10 were germinated at 24 0 C in a 16 h light / 8 h dark photoperiod. After 3-4 days, the cotyledons, including 2mm of petiole, were excised. Care was taken to remove the apical meristem and to keep the cotyledon out of the medium. The excised cotyledons were placed on MS medium, 3% 15 sucrose and 0.7% agar with 20 pM 6-benzylaminopurine (BAP). Petioles with attached cotyledons were embedded in this medium to a depth of approximately 2mm at 10 per plate. For transformation, individual excised cotyledons were taken from the plates and the cut surface of their petiole 20 immersed into the agrobacterium suspension for a few seconds. They were then returned to the MS plates and co cultivated with the agrobacterium for 72 h. After co cultivation, the cotyledons were transferred to regeneration medium (MS medium with 20pM BAP, 3% sucrose, 25 0.7% agar, pH 5.8 with 400mg/l augmentin and 15 mg/l kanamycin sulphate). The petioles were, as before, embedded to a depth of 2mm at a density of 10 explants per plate, and again the cotyledon was kept out of the medium. After 2 or 3 weeks, shoots had appeared, some of which bleached by 30 the fourth week, the remaining green shoots were sub cultured onto shoot elongation medium (regeneration medium WO 01/31027 PCT/EPOO/09374 42 minus BAP). After 1 or 2 weeks, when apical dominance had been established, the shoots were transferred to rooting medium [MS medium, 3% sucrose, 2 mg/l indole butyric acid (IBA), 0.7% agar and 400mg/l augmentin (no kanamycin)]. As 5 soon as a small root mass was obtained, the plantlets were transferred to potting mix supplemented with fertiliser granules. The plants were grown in a misting chamber (average humidity 75%) for 2- 3 weeks at 24*C, 16h light / 8h dark photoperiod. After 3 weeks the plants were 10 transferred to the glasshouse and allowed to flower and set seed.

Claims (27)

1. The use of a gene expressing a non-feed back inhibited HMG-reductase to increase the level of 4-desmethyl sterols in the seeds of plants.

2. The use according to claim 1, wherein the level of 4 desmethylsterols is increased in the seeds by at least 10%.

3. The use according to claim 1, wherein the seeds are oilseeds.

4. The use according to claim 3, wherein the oilseeds are from tobacco, canola, sunflower, rape or soy.

5. The use according to claim 1, wherein the non feedback inhibited HMG-reductase is expressed by a truncated non plant HMG gene.

6. The use according to claim 5, wherein the HMG reductase expressed by the truncated HMG gene lacks the membrane binding domain.

7. The use according to claim 1, wherein the non-feedback inhibited HMG-reductase is expressed by a truncated plant HMG gene.

8. The use according to claim 1, wherein the HMG reductase can be derived from Asteraceae.

9. The use according to claim 8, wherein the HMGR gene can de derived from Hevea brasiliensis or the HMGR gene is WO 01/31027 PCT/EPOO/09374 44 a truncated version of a gene which can be derived from Hevea brasiliensis.

10. Use according to claim 9, wherein the HMGR gene is the hmg 1 gene derived from Hevea brasiliensis or a truncated version of said gene.

11. Use of a heterologous gene expressing a truncated non feed back inhibited HMG-reductase to increase the level of sterols in plants.

12. Use according to claim 11 wherein the heterologous gene is derived from Hevea brasiliensis.

13. Method of obtaining seeds by (a) transforming a plant by: 1. transforming a plant cell with a recombinant DNA construct comprising a DNA segment encoding a polypeptide with non feedback inhibited HMGR activity and a promoter for driving the expression of said polypeptide in said plant cell to form a transformed plant cell. 2. regenerating the transformed plant cell into the transgenic plant. 3. selecting transgenic plants that have enhanced levels of 4-desmethylsterols in the seeds compared to wild type strains of the same plant (b) cultivating the transformed plant for one or more generations; (c) harvesting seed from the plant grown under(b). WO 01/31027 PCT/EPOO/09374 45

14. Method of obtaining seeds by (a) transforming a plant by: 1. transforming a plant cell with a recombinant DNA construct comprising a heterologous plant DNA segment encoding a truncated polypeptide with HMGR activity and a promoter for driving the expression of said polypeptide in said plant cell to form a transformed plant cell. 2. regenerating the transformed plant cell into the transgenic plant. 3. selecting transgenic plants that have enhanced levels of sterols compared to wild type strains of the same plant (b) cultivating the transformed plant for one or more generations; (c) harvesting the plant grown under(b).

15. Plant obtainable by a method according to claim 14.

16. Plant tissue obtained from a plant according to claim 15.

17. Plant tissue according to claim 16, selected from the group of leaves, fruit and seeds.

18. Plant having incorporated in its genome a heterologous gene encoding a truncated polypeptide HMGR activity.

19. Plant according to claim 18 wherein the heterologous gene is derived from Asteraceae. WO 01/31027 PCT/EPOO/09374 46

20. Plant according to claim 19 wherein the heterologous gene is derived from Hevea brasiliensis.

21. Plant according to claim 18-20 wherein the truncated polypeptide lacks the membrane binding domain.

22. Plant according to one or more of claims 18-21 selected from vegetables, oilseeds or fruit-trees.

23. Plant tissue having enhanced levels of sterols and produced by a plant according to one or more of claims 18 21.

24. Plant tissue according to claim 22 selected from the group of leaves, fruits or seeds.

25. Seeds having enhanced level of 4-desmethyl sterols and produced by a plant having non-feedback inhibited HMGR activity.

26. Method of obtaining oil comprising 4-desmethyl sterols by extracting oilseeds in accordance to claim 10 or 11.

27. Food product comprising an oil obtained in accordance to claim 12.