AU758821B2 - Seeds and plants whereof the germinating capacity is destroyed by total or partial lack of at least one energy reserve indispensable to germination - Google Patents

Description

SEEDS AND PLANTS WHOSE GERMINATIVE POWER IS DESTROYED BY THE TOTAL OR PARTIAL ABSENCE OF AT LEAST ONE ENERGY RESERVE THAT IS INDISPENSABLE FOR GERMINATION The present invention concerns a process for destroying the germinative power of a plant seed by the introduction into said seed of a nucleic acid sequence coding for an enzyme capable of rapidly metabolizing an energy reserve that is indispensable for the germination of said seed.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that S any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood oooo that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other o country.

The invention is based on the fact that when a crop is grown on an open field basis, it is possible that a non-negligible quantity of newly formed seeds can fall to the ground during the culture and/or harvest phase. These involuntarily disseminated seeds are capable of germinating and multiplying as soon as the weather conditions allow. The control of the multiplication of the plants resulting from this dissemination requires strict monitoring of the cultivated parcels, consisting, for example, of treatment with herbicides or crop rotations, sometimes for several years for certain plants such as rape, the seeds of which can survive for ten years.

\Vnelbfiies\Jome$\cintae\Keep\speci\30420.99.doc 16/01/03 The goal of the present invention is to destroy the germinative power of the seeds remaining in the field so as to prevent their multiplication without employing any toxic substances.

This goal is attained by means of the presence in the seeds of enzymes that are capable of rapidly burning up their energy reserve before germination can be initiated. Rapidly is understood here to mean that the energy reserves are burned up within a period of between circa 4 weeks and circa 12 weeks. This modification confers on the seeds of the invention a very clear disadvantage, which considerably limits, or even prevents, their involuntary multiplication. In *o oooo** \\melbiles'home\cintae\Keep\speci30420-99.doc 16/01/03 fact, when the weather conditions that are suitable for germination appear the seeds modified according to the invention no longer have the energy required for their germination and are thus incapable of multiplying.

If, in contrast, the seed is stored after harvest under controlled and regular conditions the introduced enzymatic activity is inhibited and the seed preserves its germinative power and can multiply.

The energy reserves that are indispensable for the germination of a seed are, most frequently, in lipid or starch form. The normal conditions which are capable of allowing germination are characterized by a certain sufficient humidity, oxygenation and temperature which varies with the species. For o*oo example, a sufficient temperature for rape and tobacco is on the order of 4 to 100C.

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Thus the invention provides transgenic plant seeds whose germinative power is destroyed by the total or partial absence of at least one energy reserve that is indispensable for germination, in which the destruction of the germinative o* power is conferred by an enzyme encoded by the transgene, which is present in the seed at a level allowing the rapid consumption of said energy reserve. This is understood to mean that the enzyme is expressed in the plant with a level of expression of at least 0.3% in relation to the total proteins expressed in the plant.

The invention therefore pertains more specifically to transgenic seeds and plants whose germinative power is destroyed by the total or partial absence of at least one energy reserve that is indispensable for germination.

\\melbfiles\home$\cintae\Keepspeci\30420-99.doc 16/01/03 '1 'I WO 99/52344 3 PCT/FR99/00806 A first mode of implementation of a seed of the invention is a transgenic seed which contains at least one heterologous gene coding for an enzyme which confers the destruction of the germinative power of said seed by rapid consumption of at least one energy reserve that is indispensable for germination.

More particularly, the invention relates to a transgenic seed which contains at least one heterologous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination.

Such a transgenic seed contains in its cells at least one chimera gene comprising successively: a promoter, for example the double constitutive promoter 35S or more particularly a specific seed promoter, possibly a sequence coding for a signal peptide, a gene coding for an enzyme selected from the group of the lipases, proteases or amylases, whose action on the metabolism of the seed is identical to that of the germination enzymes, a terminator sequence.

The promoter is advantageously selected from among: The double promoter 35S of the cauliflower mosaic virus, or other promoters of viral origin generally recognized as such by the expert in the field.

The promoter pCRU of the radish cruciferin gene enabling expression of proteins or polypeptides solely in seeds (Depigny-This et al., Plant Mol. Biol., 467-479, 1992).

I, WO 99/52344 4 PCT/FR99/00806 The promoters pGEA1 and pGEA6 corresponding to the noncoding region of the reserve protein genes of seeds, GEA1 and GEA6, of Arabidopsis thaliana, enabling a specific expression in the seeds (Gaubier et al., Mol. Gen. Genet., 238, 409-418, 1993).

The sequence coding for a signal peptide is advantageously selected from among: The nucleotide sequence of 69 nucleotides coding for the prepeptide of 23 amino acids of sporamine A in the sweet potato; this signal peptide enables the entry of the recombinant polypeptides into the secretion system of the transformed plant cells, principally into the endoplasmic reticulum.

The nucleotide sequence of 42 nucleotides coding for the N-terminal propeptide of vacuolar addressing of 14 amino acids of sporamine A in the sweet potato enabling the accumulation of recombinant polypeptides in the vacuoles of the transformed plant cells.

The sequence of 111 nucleotides coding for the prepropeptide of 37 amino acids of sporamine A constituted by the N-terminal part near the C-terminal part of the 23 amino acids of the aforementioned prepeptide followed by the 14 amino acids of the aforementioned propeptide; this prepropeptide enables the entry of recombinant polypeptides into the secretion system and their accumulation in the vacuoles of the transformed plant cells.

The three sequences above are described in the articles by Murakami et al.

(Plant Mol. Biol., 7, 343-355, 1986) and Matsuoka and Nakamura (Pro. Natl.

Acad. Sci. USA, 88, 834-838, 1991).

WO 99/52344 PCT/FR99/00806 The heterologous sequence coding for the enzyme can be, for example, that of canine gastric lipase described in the PCT patent application published as Number WO 96/33277 or that of human pancreatic lipase (Lowe et al., J. Biol.

Biochem., 264, 20042-20048, 1989). When it is not stopped or slowed down by the storage conditions, the activity of lipase in the plant seeds according to the invention results in a decrease in the triglyceride level with modification of the fatty acid composition. In fact, the action of lipase on the composition of the seed must be identical to that of the enzymes of germination and/or digestion.

A second mode of implementation of a seed according to the invention is a transgenic seed which contains at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination, which gene is placed under the control of a promoter selected from among those that enable the overexpression and activation of said gene before the initiation of the germination process, such as a more or less early specific seed promoter.

The invention also pertains to a transgenic plant or part of this plant, such as cells, characterized in that it contains: at least one heterologous gene coding for an enzyme which confers the destruction of the germinative power of the seeds of said plant by rapid consumption of at least one energy reserve that is indispensable for germination, as described above, or at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination, which gene is placed under the control of a promoter selected from among those enabling the overexpression and activation of said gene before the initiation of WO 99/52344 PCT/FR99/00806 the germination process. These promoters can be those that were already described above.

The seeds and plants or plant parts according to the invention can also contain one or more heterologous genes of interest, such as genes capable of conferring on the plant an agricultural character or resistance to a predator or to an herbicide or a gene coding for a protein of pharmaceutical interest. These heterologous genes of interest can be introduced into the seeds at the same time or prior to the chimera genes enabling destruction of the germinative power of said seeds in accordance with the invention. The seeds of the invention are then remarkable in that they make it possible to limit the involuntary dissemination of seeds carrying these genes of interest.

The seeds and plants of the invention can be dicotyledon or monocotyledon seeds and plants, such as those selected from the group constituted by the Solanaceae family (tomato, tobacco), the crucifers (rape) or the cereals (corn, wheat).

The process according to the invention exhibits advantages over the techniques that combine cytoplasmic male sterility with a gene of interest. In fact, the destruction of the germinative power of the seed prevents the multiplication of the seeds stemming from the male and female gametes whereas male sterility does not prevent the occurrence of seeds stemming from female gametes. In addition, obtaining seeds homozygotous for the heterologous gene is easy whereas it is complex in the case of a male sterility technique because it is necessary to have available a technique for the restoration of the fertility.

Thus the invention also relates to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination for the preparation of a transgenic plant whose germination is controlled. More particularly, the invention pertains to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination for the preparation of a transgenic plant whose germinative power is destroyed. It is preferred particularly to use according to the invention a gene that codes for an enzyme selected from among the group of lipases, proteases and amylases, whose action on the metabolism of the seed is identical to that of the enzymes of germination and/or digestion.

For the purposes of this specification it will be clearly understood that the .:word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Other characteristics and advantages of the invention will become apparent from the description below in relation to the preparation of transgenic tobacco o5 and rape seeds of the invention expressing a heterologous lipase; reference will be made in the description to the attached drawing in which figure 1 represents the conservation of transgenic rape seeds expressing a lipase under a specific seed promoter.

1) Preparation of tobacco seeds expressing canine lipase under the control of a constitutive promoter.

The promoter employed is the double promoter 35S. Upstream of the gene coding for canine lipase is placed a sequence coding for a signal peptide of secretion.

H:\cintae\Keep\speci\30420-99.doc 16/01/03 The tobacco plants employed for the transformation experiments (Nicotiana tabacum var. Xanthi NC and PBD6) were cultured in vitro on the base medium of Murashige and Skoog (1962) with the addition of the vitamins of Gamborg et al.

(1968, Sigma reference M0404), sucrose at 20 g/l and agar (Merck) at 8 g/l. The pH of the medium was adjusted to 5.8 with a potash solution prior to

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H:\cintae\Keep\speci\30420-99.doc 16/01/03 WO 99/52344 PCT/FR99/00806 autoclaving at 120 0 C for 20 minutes. The tobacco sprouts were planted by cuttings of the internodes every 30 days on this multiplication medium All of the in vitro cultures were carried out in a climate-controlled enclosure under the following conditions: luminous intensity of 30 pE m 2 photoperiod of 16 h; thermoperiod of 26 0 C during the day, 24 0 C at night.

The transformation technique employed was derived from that of Horsch et al. (Science, 227, 1229-1231, 1985).

A preculture of Agrobacterium tumefaciens strain LBA4404 containing plasmids pBIOC29 or pBIOC26 or pBIOV25 was implemented for 48 h at 28 0 C under agitation in LB medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989) with the addition of suitable antibiotics (rifampicin and tetracycline). The preculture was then diluted to the 5 0 th in the same medium and cultured under the same conditions.

After one night, the culture was centrifuged (10 min., 3000 the bacteria were taken up in an equivalent volume of liquid MS30 medium (30 g/1 sucrose) and this suspension was diluted to the 10 t h Explants of circa 1 cm 2 were cut from the leaves of the sprouts described above. They were then brought into contact with the bacterial suspension for one hour, then dried rapidly on filter paper and placed in a coculture medium (solid WO 99/52344 PCT/FR99/00806 After 2 days, the explants were transferred to Petri dishes on the regeneration medium MS30, containing a selective agent, kanamycin (200 mg/1), a bacteriostatic agent, Augmentin (400 mg/1) and the hormones required for the induction of buds (BAP, 1 mg/1 and ANA, 0.1 mg/1). Planting of the explants was performed on the same medium after 2 weeks of culture.

After 2 additional weeks, the buds were planted in Petri dishes on the development medium composed of medium MS20 with the addition of kanamycin and Augmentin. After 15 days, the buds were planted by half.

Rooting took circa 20 days at the end of which the sprouts could be cloned by cutting of internodes or moved to the greenhouse.

Using the same process, it was possible to obtain tobacco seeds expressing human pancreatic lipase under the control of a constitutive promoter.

2) Preparation of rape seeds expressing canine lipase under the control of a specific seed promoter.

The promoter employed was the promoter of cruciferin. Upstream of the gene coding for the lipase was placed a sequence coding for a signal peptide of secretion.

The spring rape seeds (Brassica napus cv WESTAR or Limagrain lines) were disinfected for 40 minutes in a 15% solution of Domestos. After 4 rinsings with sterile water, the seeds were allowed to germinate with 20 seeds per pot with a diameter of 7 cm and a height of 10 cm, on the mineral medium of Murashige and Skoog (Sigma M 5519) with 30 g/l of sucrose and solidified with agar gel at g/1. These pots were placed in a culture chamber at 26 0 C with a photoperiod of 16 h/8 h and under a luminous intensity on the order of 80 pE m 2 s'.

WO 99/52344 PCT/FR99/00806 After 5 days of germination, the cotyledons were collected under sterile conditions by cutting each petiole circa 1 mm above the cotyleodonal node.

At the same time, a preculture was prepared of Agrobacterium tumefaciens strain LBA4404 containing the plasmid pGAZE in which was inserted the sequence coding for an addressing signal PPS (or PS) under the control of the promoter pCRU (or pd35S) in a 50-ml Erlenmeyer flask for 36 h at 28 0 C in 10 ml of bacterial medium 2YT (Sambrook et al., Molecular Cloning Laboratory Manual, 2n d Edition, Cold Spring Harbor Laboratory Press, 1989) supplemented with the antibiotics that are useful for the selection of the strain employed.

This preculture was used to seed at 1% a new bacterial culture prepared under the same conditions. At the end of 14 h the culture was centrifuged for minutes at 3000 g and the bacteria were taken up in an equivalent volume of liquid germination medium. This suspension was distributed in Petri dishes with a diameter of 5 cm at a dose of 5 ml/jar.

The sectioned end of the petiole was immersed for several seconds in the agrobacterial solution prepared in this manner; the petiole was then pushed in several millimeters in the regeneration medium. This medium had the same base composition as the germination medium with the addition of 4 mg/1 of benzylaminopurine (BAP), a phytohormone that promotes the neoformation of buds. Ten explants (cotyledon with petiole) were cultured per Petri dish with a diameter of 9 cm (Greiner reference 664102).

After 2 days of coculture under the same environmental conditions as the germinations, the explants were planted in Phytatray boxes (Sigma, reference P1552) containing the preceding medium with the addition of a selective agent, mg/1 of kanamycin sulfate (Sigma, reference K4000), and a bacteriostatic agent, mixture (by weight) of 1/6 potassium salt of clavulanic acid and 5/6 amoxicillin sodium (injectable Augmentin) at the dose of 600 mg/1.

WO 99/52344 PCT/FR99/00806 Twice in succession at an interval of 3 weeks the explants were planted in a sterile manner on new medium under the same conditions.

The green buds that appeared at the end of the second or third planting were separated from the explant and cultured individually in transparent pots with a diameter of 5 cm and a height of 10 cm, and containing a medium identical to the preceding medium but without BAP. After 3 weeks of culture, the stem of the transformed bud was sectioned and the bud was planted in a pot of fresh medium. At the end of three to four weeks, the roots were sufficiently developed to allow acclimatization in the phytotron (temperature 21'C, photoperiod 16 h/8 h and 84% relative humidity), the shoots were repotted in pots with a diameter of 12 cm filled with the same soil enriched with timedrelease fertilizer (Osmote, at the dose of 4 g/l of soil) then transferred to the greenhouse (class S2) regulated to 18 0 C with two daily waterings with 2 minutes of water.

As soon as the flowers appeared, they were bagged (Crispac, reference SM 7 0y 300 mm x 700 mm) so as to prevent cross fertilization.

When the siliques reached maturity, they were collected, dried and then threshed. The resultant seeds were used for the quantitative determination of the biochemical activity. The selection of the transgenic descendence was implemented by germination on a medium containing kanamycin sulfate at the dose of 100 to 150 mg/l (depending on the genotype). The operating conditions were identical to those described above except that the germinations were implemented in glass tubes with a single seed per tube. Only the sprouts that developed secondary roots during the first three weeks were acclimatized in the phytotron before being transferred to the greenhouse.

Using the same procedure, it is possible to obtain rape seeds that express human pancreatic lipase under the control of a specific seed promoter.

WO 99/52344 PCT/FR99/00806 3) Measurements of the effect of the activity of the lipase on the germinative power in the seeds of examples 1 and 2.

The activity of the lipase, when it is not slowed down or stopped by the storage conditions, cold of 4°C and reduced hygrometry, leads to a decrease in the triglyceride content without modification of the fatty acid composition.

The lipase activity is determined by means of a pH-STAT by the titrimetric method of Gargouri et al. (Gastroenterology, 91, 919-925, 1986) in which the substrate employed is tributyrin. The tributyrin emulsion (4 ml per 30 ml of emulsion) is implemented in the vortex in the presence of bile salts (1.04 g/1), bovine albumin (0.1 g/l) and NaCl (9 The quantitative determination comprises neutralizing the butyric acid released under the action of the lipase by a soda solution with a set pH of 5.5 at 37 0 C. One lipase unit corresponds to the amount of enzyme which induces the release of one micromole of fatty acid in 1 minute at 37C under optimal pH conditions Purified canine gastric lipase has a specific activity of 570 units/mg of protein. The lipase activity can be measured on the total ground product, on the residue or on the centrifugation supernatant. The measurements of the lipase activity confirm the expression in the seeds of transformed plants of the lipase coded by the heterologous gene.

WO 99/52344 PCT/FR99/00806 If we follow the evolution of the germinative power over time of these seeds with lipase activity, we will observe a rapid degradation of the capacity of these seeds to germinate up to its disappearance after 16 weeks. In contrast, if these seeds are stored under conditions that do not allow expression of the lipase activity (4 0 their germinative power does not degrade (Figure This confirms the effect of the lipase activity on the transgenic germinative power and the value of its use according to the invention.

4) Field trials In order to demonstrate the limitation of the dissemination stemming from the invention, it is sufficient to implement in the same parcels crops of transgenic plants according to the invention, for example rape expressing canine gastric lipase under control of the radish cruciferin promoter, and nontransgenic seed lines, with each group of seeds being planted on plotted spaces, and to harvest the plants under the same conditions. A portion of the seeds produced will fall to the ground during the culture or harvest activities, and the following year it is possible to determine the value of the seeds and plants according to the invention by comparing the amount of regrowth in each of the parcels.

An open field trial of transgenic rape homozygotous for the transgene cDNA coding for gastric lipase under a constitutive promoter was performed. This field of transgenic rape was surrounded by borders of nontransgenic rape. This trial made it possible to confirm under real conditions the regerminative potential of transgenic seeds expressing cDNA coding for gastric lipase.

WO 99/52344 PCT/FR99/00806 Observation of the number of new sprouts on each parcel Each of these parcels was harvested independently with the same combine harvester. The rapeseed production yield was approximately equivalent in the two parcels. It is therefore reasonable to assume that the quantity of rapeseed that fell to the ground during the harvest was equivalent for the parcels.

Two years after the culture, a systematic collection of the new rape sprouts was performed in each of these parcels and yielded the results presented in the table below: Left border of Central crop of Right border of nontransgenic transgenic rape nontransgenic rape rape Surface area of the regrowth 1155 m 2 924 m 2 924 m 2 collection field Number of new sprouts observed 474 44 297 Number of new sprouts/m 2 0.41 0.05 0.32 Thus, on average there was 7.8 times less regrowth per square meter on the parcel on which transgenic rape had been grown compared with the parcels on which nontransgenic rape had been grown.

Confirmation of the transgenic nature of the regrowth Taking into account that seeds can be thrown outside of the limits of each parcel during the harvesting of the rapeseed or when working the soil during the following two years, it had to be confirmed that the new sprouts found on a parcel indeed originated from that parcel; this was done by verifying whether or not the new sprouts had the lipase gene. A portion of the new sprouts were therefore collected and planted in the greenhouse: 45 for the right border, 44 (the totality) for the central planting and 52 for the left border. These plants were WO 99/52344 PCT/FR99/00806 brought to maturity and allowed to self-fertilize. Samples were collected from the leaves to determine the presence or absence of the cDNA coding for lipase in these plants. After grinding the sample in liquid nitrogen, the extraction buffer (Tris-HCl 0.2 M, Na-EDTA 0.25 mM, NaCl 0.25 M, SDS pH 8.0) was added.

The reaction medium was then subjected to the vortex for 5 seconds followed by an extraction with phenol-chloroform-isoamyl alcohol (25/24/1 The aqueous phase was then precipitated by adding isopropanol at -80 0 C for minutes. After centrifugation at 4000 g for 15 minutes, the residue was washed in 70% ethanol, then centrifuged again and dried. The residue was taken up in 100 pl of H 2 0. The PCR amplification of the DNA was performed in a GeneAmp PCR System 9700 thermocycler in the presence of 0.36 pl of each of the oligodesoxynucleotide primers 5' TTCTACCCACACCACTTC 3' and ACATGCATGTCTGTCAGG 3' at 50 pmole/pl, 0.5 pl dNTP 10 mM, 3 p1 98% glycerol, 1.8 pl MgCl 2 25 mM, 0.36 pl BSA 10 mg/ml, 3 pl of Promega polymerase DNA buffer x 10 and 0.36 pl of Promega polymerase DNA Taq at 5 U/ul in a final reaction medium of 30 pl. The DNA was denatured for 5 minutes at 94°C, subjected to 35 cycles each constituted by 30 seconds of denaturation at 94 0 C, 1 minute of hybridization at 50 0 C and 1 minute of elongation at 72 0 C; the elongation was then continued for 5 minutes.

The results obtained showed that: the cDNA coding for gastric lipase was not detected in any of the samples from the right and left borders.

only 64% (27/42; not 44 because 2 plants wee lost during the greenhouse planting process) of the new sprouts from the central parcel planted in transgenic rape had the cDNA coding for the lipase.

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WO 99/52344 16 PCT/FR99/00806 Thus, no new shoots from transgenic plants were found in the part that had initially been planted in nontransgenic rape (or at a level that was not detectable because of the sampling rate: circa 11% of the regrowth was analyzed). This does not mean that there was no passage of transgenic seeds into the other parcels, but that if it took place (which is quite probable), they were not capable of germinating.

These analyses show that the open field regerminative potential of the transgenic seeds was overestimated by 36%. The number of new transgenic shoots in the zone that had initially been planted in transgenic rape was 0.3 plants/m'. Thus, within the framework of this trial, the regerminative potential of the transgenic seeds was 12 times lower than that of the nontransgenic seeds.

Verification of the expression of the transgene coding for gastric lipase Finally, it was verified that the transgene was expressed in the seeds that were positive in the PCR test. In order to do this, the lipase activity was quantitatively determined for each of the 27 lots of seeds collected from the 27 PCR+ plants by the pH Stat technique. All of the lots expressed lipase. The average lipase activity was 74 UTC4/g of seed (extreme values: 40-104 UTC4/g of seed, cf. attached results) versus initially 125 UTC4/g of seed collected, a drop in the lipase activity of circa 41%. Only the seeds whose level of expression of the transgene dropped (for whatever reason: because of physiological conditions, extinction phenomena, etc.) are partially capable of germinating.

1.

WO 99/52344 17 PCT/FR99/00806 This real scale trial therefore clearly demonstrates that the seeds of transgenic rape expressing gastric lipase have a very clear selective disadvantage, making their dissemination very strongly improbable due to the fact of their loss of germinative power: the regrowth potential of the transgenic seeds was circa 12 times smaller than that of the nontransgenic seeds.

the transgenic seeds that germinated in the framework of this trial expressed two times less recombinant lipase than the initial seeds. It would therefore appear that when the seeds express lipase at least at the level of 120 UTC4/g, they do not have germinative potential under open field conditions.

In addition, it was found that the gastric lipase did not have a significant effect on the fatty acid composition, with the fat content of the transgenic seeds being significantly decreased. The seeds (T2 and T3) of rape expressing lipase have a fat content on the order of 22 to 24% compared to 40% on average, a drop of circa

Claims (19)

1. A transgenic plant seed whose germinative power is destroyed by the total or partial absence of at least one energy reserve that is indispensable for germination, in which the destruction of the germinative power is conferred by an enzyme encoded by the transgene, which is present in the seed at a level enabling the rapid consumption of said energy reserve.

2. A transgenic plant seed whose germinative power is destroyed by the total or partial absence of at least one energy reserve that is indispensable for germination.

3. A transgenic plant seed according to claim 2, which comprises at least one heterologous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination.

4. A transgenic plant seed according to claim 2 or claim 3, which comprises at least one heterologous gene coding for an enzyme that confers the destruction of the germinative power of said seed by rapid consumption of at least one energy reserve that is indispensable for germination.

A transgenic plant seed according to any one of claims 2 to 4, which comprises at least one chimeric gene comprising successively: a promoter, a sequence coding for a signal peptide, a gene coding for an enzyme selected from the group consisting of lipases, proteases and amylases, whose action on the metabolism of the seed is identical to that of the enzymes of germination and/or digestion, and a terminator sequence. \\melbfiles\home$\cintae\Keep\speci3O42o99.doc 16/01/03

6. A transgenic plant seed according to claim 5, in which the promoter is selected from the group consisting of: a) The promoter pCRU of the radish cruciferin gene enabling expression of proteins or polypeptides solely in seeds, and b) The promoters pGEA1 and pGEA6 corresponding to the noncoding region 5' of the reserve protein genes of seeds, GEA1 and GEA6, of Arabidopsis thaliana, enabling a specific expression in the seeds.

7. Seed according to claim 5 or claim 6, in which the sequence coding for a signal peptide is selected from the group consisting of: a) The nucleotide sequence of 69 nucleotides coding for the prepeptide of 23 amino acids of sporamine A in the sweet potato, b) The nucleotide sequence of 42 nucleotides coding for the N-terminal propeptide of vacuolar addressing of 14 amino acids of sporamine A in the sweet potato, and c) The sequence of 111 nucleotides coding for the prepropeptide of 37 amino acids of sporamine A constituted by the N-terminal part near the C- terminal part of the 23 amino acids of the prepeptide of sporamine A in the sweet potato followed by the 14 amino acids of the N-terminal propeptide of vacuolar addressing of sporamine A in the sweet potato.

8. A transgenic plant seed according to any one of claims 3 to 7, in which the heterologous gene codes for canine gastric lipase.

9. A transgenic plant seed according to claim 2 or claim 3, which comprises at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination, placed under the control of a promoter selected from among those enabling the \\melbfiles1ome$\cintae\Keep\speci\O42-99.doc 16/01/03 overexpression and activation of said gene before the initiation of the germination process. A transgenic plant or part thereof, which comprises: a) at least one heterologous gene coding for an enzyme that confers the destruction of the germinative power of the seeds of said plant by rapid consumption of at least one energy reserve that is indispensable for germination, or b) at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve that is indispensable for germination, placed under the control of a promoter selected from among those enabling the S: overexpression or activation of said gene before the initiation of the germination process.

S:

11. A transgenic plant seed according to any one of claims 2 to 9, or a transgenic plant according to claim 10, which also comprises one or more heterologous genes of interest, such as genes capable of conferring on the plant an agricultural character or a resistance to a predator or to an herbicide or a gene coding for a protein of pharmaceutical interest.

12. Use of a gene coding for an enzyme which is responsible for the consumption of an energy reserve that is indispensable for germination for producing a transgenic plant whose germinative power is destroyed.

13. Use according to claim 12, in which the gene codes for an enzyme selected from among the group of the lipases, proteases and amylases, the action of which is identical to that of the enzymes of germination and/or digestion. \\melb-files\Iome$\cintae\Keep\speci\30420-99.doc 16/01/03

14. A method of producing a transgenic plant whose germinative capacity is destroyed, comprising the step of transforming seeds of the plant with a gene encoding an enzyme which is responsible for consumption of an energy reserve which is indispensable for germination.

A method according to claim 14, in which the gene codes for an enzyme selected from among the group of the lipases, proteases and amylases, the action of which is identical to that of the enzymes of germination and/or digestion.

16. A transgenic plant seed according to claim 1 or claim 2, substantially as herein described with reference to the Examples. S:

17. A transgenic plant or part thereof according to claim 10, substantially as herein described with reference to the Examples. oo

18. A use according to claim 12, substantially as herein described with reference to the Examples.

19. A method according to claim 14, substantially as herein described with reference to the Examples. SDated this 16th day of January 2003 GROUPE LIMAGRAIN HOLDING By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\cintae\Keep\speci 3O42-99.doc 16/01/03 "I' WO 99/52344 22 PCT/FR99/00806 Key to Figure 1: Top of graph: Conservation of transgenic rape seeds expressing a lipase under a specific seed promoter. Bottom of graph: Duration of conservation. sem weeks At right of graph: ambient 4°C silica gel