EP1069818A1

EP1069818A1 - Seeds and plants whereof the germinating capacity is destroyed by total or partial lack of at least one energy reserve indispensable to germination

Info

Publication number: EP1069818A1
Application number: EP99911897A
Authority: EP
Inventors: Philippe Bournat
Original assignee: Groupe Limagrain Holding SA
Current assignee: Groupe Limagrain Holding SA
Priority date: 1998-04-09
Filing date: 1999-04-07
Publication date: 2001-01-24
Also published as: AU3042099A; AU758821B2; FR2777155B1; CA2324944A1; FR2777155A1; WO1999052344A1

Abstract

The invention concerns seeds and plants whereof the germinating capacity is destroyed by total or partial lack of at least one energy reserve indispensable to germination, wherein the destruction of germinating capacity is ensured by an enzyme present in the seed to an extent enabling said energy reserve to be quickly consumed.

Description

SEEDS AND PLANTS OF WHICH THE GERMINATING POWER IS DESTROYED BY THE TOTAL OR PARTIAL ABSENCE OF AT LEAST ONE ENERGY RESERVE ESSENTIAL TO GERMINATION.

The present invention relates to a method 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 essential for the germination of said seed.

The invention is based on the fact that it can happen when cultivating in the open field that a non-negligible quantity of newly formed seeds falls on the ground during cultivation and / or harvesting. These seeds, involuntarily disseminated, will be able to germinate and multiply as soon as the climatic conditions are suitable. Controlling the non-multiplication of plants resulting from this dissemination requires binding monitoring of cultivated plots, consisting for example of a treatment with herbicides, a crop rotation, sometimes for several years for certain plants such as rapeseed, whose seeds can survive a decade.

The present invention aims precisely to destroy the germinative power of seeds remaining in the field in order to prevent their multiplication without resorting to the use of a toxic substance.

This goal is achieved thanks to the presence in the seeds of enzymes capable of rapidly burning their energy reserve before germination is initiated. By rapidly, it is meant that the energy reserves are burned within a period of between approximately 4 weeks and approximately 12 weeks. This modification gives the seeds of the invention a very clear disadvantage which considerably limits, or even prohibits, their involuntary multiplication. In fact, when the climatic conditions suitable for germination occur, the seeds modified according to the invention will no longer have the energy necessary for their germination and will therefore be unable to multiply.

If, on the other hand, the semen is stored, from harvest, under controlled and regular conditions, the enzymatic activity introduced will be inhibited and the semen will retain its germinative power and may be multiplied.

The energy reserves essential for the germination of a seed are, most often, in the form of lipid or starch. Normal conditions, suitable for allowing germination, are characterized by sufficient humidity, oxygenation and temperature, depending on the species concerned. For example, a sufficient temperature for rapeseed and tobacco is around 4 ° C to 10 ° C.

The subject of the invention is therefore plant seeds whose germinative power is destroyed by the total or partial absence of at least one energy reserve essential for germination, in which the destruction of the germinative power is imparted by an enzyme present in the seed at a level allowing the rapid consumption of said energy reserve. By this is meant that the enzyme is expressed in the plant with an expression rate of at least 0.3% relative to the total of the proteins expressed in the plant.

therefore the invention particularly relates to transgenic seeds and plants whose germination power is destroyed by the total or partial absence of at least one energy reserve essential for germination.

A first embodiment 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 essential 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 essential for germination. Such a transgenic seed contains in its cells at least one chimeric gene successively comprising:

a promoter, for example the double 35S constitutive promoter or more particularly a specific seed promoter,

- optionally a sequence coding for a signal peptide,

- a gene coding for an enzyme chosen from the group of lipases, proteases, amylases, the action of which on the metabolism of the seed is identical to that of the enzymes of germination,

- a termination sequence.

The promoter is advantageously chosen from:

- The double promoter 35S of the cauliflower mosaic virus, or other promoters of viral origin generally recognized as such by one skilled in the art. - The pCRU promoter of the radish cruciferin gene allowing the expression of proteins or polypeptides only in seeds or in the seeds (Depigny-This et al., ^: Plant Mol. Biol., 20, 467-479, 1992).

The pGEAl and pGEAβ promoters corresponding to the 5 'non-coding region of the genes of the seed reserve protein, GEAI and GEA6, of Arabidopsis thaliana allowing specific expression in the seeds (Gaubier et al., Mol. Gen. Genêt. , 238, 409-418, 1993).

The sequence coding for a signal peptide is advantageously chosen from:

The nucleotide sequence of 69 nucleotides coding for the prepeptide of 23 amino acids of sporamine A in sweet potatoes, this signal peptide allowing the entry of the recombinant polypeptides into the secretion system of transformed plant cells, mainly in the endoplasmic reticulum.

The nucleotide sequence of 42 nucleotides coding for the N-terminal propetide for vacuolar addressing of 14 amino acids of sporamine A in sweet potato, allowing the accumulation of recombinant polypeptides in the vacuoles of transformed plant cells. - The sequence of 111 nucleotides coding for the prepropetide of 37 amino acids of sporamine A consisting of the N-terminal part towards the C-terminal part of the 23 amino acids of the aforementioned prepeptide followed by the 14 amino acids of the aforementioned propetide, this prepropetide allowing the entry of recombinant polypeptides into the secretion system and their accumulation in the vacuoles of 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). The heterologous sequence coding for the enzyme can be, for example, that of dog gastric lipase described in the PCT patent application published under the number WO 96/33277 or that of human pancreatic lipase (Lowe et al., J. Biol Biochem., 264, 20042-2008, 1989). The activity of lipase in the seeds of plants according to the invention leads, when it is not stopped or slowed down by the storage conditions, a reduction in the triglyceride content without modification of the fatty acid composition. Indeed, 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 embodiment 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 essential for germination, placed under the control of a promoter chosen from those allowing 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 relates 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 essential for germination, as described above, or - at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination, placed under the control of a promoter chosen from those allowing the overexpression and activation of said gene before the initiation of the germination process. These promoters can be those already described above. The seeds and plants or parts of plants according to the invention may also contain one or more heterologous genes of interest, such as genes capable of conferring on the plant an agronomic character or a resistance to a predator or to a herbicide or a gene encoding a protein of pharmaceutical interest. These heterologous genes of interest can be introduced into seeds at the same time or before the chimeric genes making it possible to destroy 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 according to the invention can be dicotyledonous or monocotyledonous seeds and plants, such as those selected from the group consisting of nightshades (tomatoes, tobacco), crucifers (rapeseed), cereals (corn, wheat).

The method according to the invention has advantages over techniques coupling male cytoplasmic sterility and a gene of interest. Indeed, the destruction of the germinative power of the seed prohibits the multiplication of seeds from male and female gametes. While male sterility does not prevent the obtaining of seeds from female gametes. In addition, obtaining seeds homozygous for the heterologous gene is easy whereas it is complex in the case of a male sterility technique, since it is necessary to have a technique of restoring fertility. The invention therefore also relates to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination to prepare a transgenic plant whose germination is controlled. More particularly, the invention relates to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination to prepare a transgenic plant whose germinative power is destroyed. It is particularly preferred to use, according to the invention, a gene which codes for an enzyme chosen from the group of lipases, proteases, amylases, the action of which on the metabolism of semen is identical to that of the enzymes of germination and / or digestion.

Other characteristics and advantages of the invention will appear in the following description relating to the preparation of transgenic seeds of the invention of tobacco and rapeseed expressing a heterologous lipase, and in which reference will be made to the drawing in the appendix. FIG. 1 represents the conservation of transgenic rapeseed seeds expressing a lipase under a specific seed promoter.

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

The promoter used is the double 35S promoter. Upstream of the gene coding for dog lipase is placed a sequence coding for a secretory signal peptide.

Tobacco plants used for processing experiments {Nicotiana tabacum var. Xanthi NC and PBD6) are cultured in vitro on the basic medium of Murashige and Skoog (1962) supplemented with vitamins from Gamborg et al. (1968, Sigma reference M0404) sucrose at 20 g / l and agar (Merk) at 8 g / 1. The pH of the medium is adjusted to 5.8 with a potassium hydroxide solution before autoclaving at 120 ° C for 20 min. Tobacco seedlings are transplanted by cutting internodes every 30 days on this MS20 multiplication medium.

All in vitro cultures are carried out in an air-conditioned enclosure, under the conditions defined below: - light intensity of 30 μE. πT ^ .S-- ¹ ; 4 p.m. photoperiod;

- thermoperiod of 26 ° C during the day, 24 ° C at night.

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

A preculture of Agrobacterium tumefaciens strain LBA4404 containing the plasmids pBIOC29 or pBIOC26 or pBI0C25 is carried out for 48 h at 28 ° C with stirring, in LB medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press , 1989) supplemented with appropriate antibiotics (rifampicin and tetracycline). The preculture is then diluted to 50th in the same medium and cultivated under the same conditions. After one night, the culture is centrifuged (10 min., 3000 g), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / 1 sucrose) and this suspension is diluted with 10.

Explants of about 1 cm 2 are cut from the leaves of the seedlings described above. They are then brought into contact with the bacterial suspension for one hour, then quickly dried on filter paper and placed on a coculture medium (solid MS30). After 2 days, the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200 mg / 1), a bacteriostatic, augmentin (400 mg / 1) and the necessary hormones. to the induction of buds (BAP, 1 mg / 1 and ANA, 0.1 mg / 1). The explants are subcultured on the same medium after 2 weeks of culture.

After 2 additional weeks, the buds are subcultured in petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted in half. Rooting takes about 20 days, at the end of which the seedlings can be cloned by cutting internodes or taken out in the greenhouse. According to the same method, it is possible to obtain tobacco seeds expressing human pancreatic lipase under the control of a constitutive promoter.

2) Preparation of rapeseed seeds expressing dog lipase under the control of a specific seed promoter.

The promoter used is the cruciferin promoter. Upstream of the gene coding for lipase is placed a sequence coding for a secretory signal peptide.

The spring rapeseeds (Brassica napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% Domestos solution. After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per pot of 7 cm in diameter and 10 cm high, on mineral medium of Murashige and Skoog (Sigma M 5519) with 30g / 1 of sucrose and solidified with agargel at 5 g / 1. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 16h / 8h and under a light intensity of the order of 80 μE m ^~ 2 s ^~ ^.

After 5 days of germination, the cotyledons are removed sterile by cutting each petiole about 1mm above the cotyledon node.

In parallel, a preculture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pGAZE into which the sequence coding for a PPS (or PS) addressing signal has been inserted under the control of the pCRU promoter (or pd35S), is carried out in Erlenmeyer flask of 50 ml, for 36 h at 28 ° C in 10 ml of 2YT bacterial medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) supplemented with antibiotics useful for the selection of the strain used .

This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 g and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box.

The severed end of the petiole is immersed for a few seconds in the solution of agrobacteria thus prepared, then the petiole is pressed a few millimeters into the regeneration medium. This medium has the same basic composition as the germination medium with an additional 4 mg / l of benzylamino-purine (BAP), a phytohormone promoting the new formation of buds. Ten explants (cotyledon with petiole) are cultured per Petri dish 9 cm in diameter (Greiner reference 664102).

After 2 days of coculture under the same environmental conditions as germination, the explants are subcultured. In phytatray boxes (Sigma, reference P1552) containing the preceding medium supplemented with a selective agent: 45 mg / 1. kanamycin sulfate (Sigma, reference R4000) and a bacteriostatic: mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 sodium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.

Twice in succession, 3 weeks apart, the explants are transplanted sterile onto new medium under the same conditions. The green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5cm in diameter and 10cm high containing a medium identical to the previous one but devoid of BAP. After 3 weeks of culture, the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium. After three to four weeks the roots are sufficiently developed to allow acclimatization in phytotron (temperature 21 ° C, photoperiod 16h / 8h and 84% relative humidity), the seedlings are repotted in pots 12 cm in diameter filled with the same potting soil enriched with late fertilizer (Osmote, at the rate of 4 g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at 18 ° C, with two daily waterings of 2 minutes.

As soon as flowers appear, these are bagged (Crispac, reference SM 570y 300 mm * 700 mm) so as to prevent cross-fertilization.

When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for assaying biochemical activity. The selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes). The operating conditions are identical to those described above except that the germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse. According to the same process, it is possible to obtain rapeseed expressing human pancreatic lipase under the control of a specific seed promoter.

3) Measurements of the Effect of the Activity of Lipase on the Germination Power in the Seeds of Examples 1 and 2.

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

The lipase activity is determined using a pH-STAT by the titrimetric method of Gargouri et al. (Gastroenterology, 91, 919-925, 1986) in which the substrate used is tributyrin. The tributyrin emulsion (4 ml for 30 ml of emulsion) is produced using a vortex in the presence of bile salts (1.04 g / 1) of bovine albumin (0.1 g / 1) and NaCl (9 g / 1). The assay consists in neutralizing the butyric acid released under the action of the lipase with a sodium hydroxide solution at a set pH of 5.5 to 37 ° C. One lipase unit corresponds to the quantity of enzyme which causes the release of a micromole of fatty acid in 1 minute at 37 ° C and under optimal pH conditions (5.5). Purified dog gastric lipase has a specific activity of 570 units / mg of protein. The lipase activity can be measured on the total ground material, on the pellet or on the centrifugation supernatant. Measurements of lipase activity confirm the expression, in the seeds of transformed plants, of the lipase encoded by the heterologous gene. If we follow the evolution of the germination power of these seeds to lipase activity over time, we observe a rapid deterioration in the capacity of these seeds to germinate until disappearance after 16 weeks. On the other hand, if these seeds are stored under conditions which do not allow the expression of lipase activity (4 ° C), their germination power is not degraded (Figure 1). This confirms the effect of lipase activity on transgenic germination power and the advantage of its use according to the invention.

4) Field trials.

To demonstrate the limitation of the spread generated by the invention, it suffices to carry out in the same plots cultures of transgenic plants according to the invention, for example rapeseed expressing dog gastric lipase under the control of the cruciferin promoter radish, and non-transgenic lines, each on identified spaces, and harvest the plants under the same conditions. A part of the seeds produced will fall into the plot during the cultivation or during the harvest, and the following year, the value of the seeds and plants according to the invention is noted by comparing the number of regrowths on each of the plots.

A field trial of transgenic colza homozygous for the transgene: cDNA coding for gastric lipase under constitutive promoter was carried out. This field of transgenic rapeseed was surrounded by borders of non-transgenic rapeseed. This test made it possible to confirm in real conditions the potential for re-germination of transgenic seeds expressing the cDNA coding for gastric lipase. Observation of the number of regrows on each plot.

Each of these plots was harvested independently with the same combine harvester. The production yield of rapeseed was roughly equivalent in the two plots. It is therefore reasonable to think that the quantity of rapeseed grains that fell on the ground during the harvest is equivalent for each plot.

Two years after cultivation, a systematic sampling of rapeseed regrowth was carried out in each of these plots and gave the results reported in the table below:

Culture border Left border of central right of colzas No colzas colzas No

Transgenic Transgenic Transgenic

Collection area 1,155 m2 924 m2 924 m2 of regrowth

Number of regrowths 474 44 297 observed

Number of regrowth / m2 0.41 0.05 0.32

On average, there was therefore 7.8 times less regrowth per square meter on the plot cultivated in transgenic rapeseed than on the plot cultivated in non-transgenic rapeseed.

Confirmation of the transgenic nature of regrowth.

Taking into account the projections of seeds outside the limits of each plot that may occur during the colza harvest or during tillage for the following two years, it was verified that the regrowth found on a plot came from it; this by checking whether or not they contain the lipase gene. Part of the regrowth was therefore sampled and transplanted in the greenhouse: 45 per. ^' the right border, 44 (all) for the central crop, 52 for the left border. These plants were brought to maturity and carried out in self-fertilization. Leaf samples were taken to determine the presence or absence of cDNA coding for lipase in these plants. After grinding in liquid nitrogen of the sample, the extraction buffer (0.2 M Tris-HCl; 0.25 mM Na-EDTA; 0.25 M NaCl; 0.5% SDS; pH 8.0). Then, the reaction medium is subjected to the vortex for 5 sec. followed by extraction with phenol- chloroform-isoamyl alcohol (25/24/1 v / v / v). The aqueous phase is then precipitated by adding isopropanol at -80 ° C for 15 min. After centrifugation at 4000 g for 15 min. the pellet is washed in 70% ethanol, then recentrifuged and dried. The pellet is taken up in 100 μl of H20. The PCR amplification reaction of the DNA is carried out in a GeneAmp PCR System 9700 thermocycler in the presence of 0.36 μl of each of the oligodeoxynucleotide primers 5 'TTCTACCCACACCACTTC 3' and 5 'ACATGCATGTCTGTCAGG 3' at 50 pmol / μl, 0, 5 μl lNm dNTP, 3 μl 98% glycerol, 1.8 μl MgC12 25mM, 0.36 μl BSA 10 mg / ml, 3 μl of Promega xlO DNA polymerase buffer and 0.36 μl of Taome DNA polymerase 5 U / μl in a reaction medium of 30 μl final. The DNA is denatured for 5 min at 94 ° C, subjected to 35 cycles each consisting of 30 denaturation at 94 ° C, 1 min. hybridization at 50 ° C, and 1 min. elongation at 72 ° C., then the elongation is continued for 5 min.

The results obtained show that:

- the cDNA coding for gastric lipase is not detected in any sample of the right and left borders. - only 64% (27/42, and not 44 because 2 plants were lost during transplanting in the greenhouse) of regrowth from the central plot cultivated in transgenic rapeseed, have the cDNA coding for lipase.

No regrowth of transgenic plants was therefore found in the part initially cultivated in non-transgenic rapeseed (or at a rate that was not detectable due to sampling: around 11% of the regrowth was analyzed). This does not mean that transgenic seeds have not been passed into the other plots, but that if there have been (which is very likely) they have not been able to germinate.

These analyzes show that the potential for re-germination in the open field of transgenic seeds was overstated by 36%. The number of transgenic regrowths in the area initially cultivated with foreign rapeseed is 0.3 plants / m2. The re-germination potential of transgenic seeds is therefore 12 times less in the context of this trial than that of non-transgenic rapeseed.

Verification of the expression of the transgene coding for gastric lipase

Finally, it was checked that the transgene expressed well in seeds positive for the PCR test. For this, the lipase activity was assayed for each of the 27 batches of seeds harvested from the 27 PCR + plants, by the pH Stat technique. All lots express lipase. The average lipase activity is 74 UTC4 / g of grain (extreme values: 40; 104 UTC4 / g of grain, see results attached) against initially 125 UTC4 / g of harvested grain: a drop of about 41% of l lipase activity. Only seeds whose level of transgene expression has dropped (for a few reasons: due to physiological conditions, extinction phenomena, etc.) are partially able to germinate. This full-scale trial therefore clearly shows that the seeds of transgenic colza expressing gastric lipase have a very marked selective disadvantage, making their dissemination very highly improbable due to their loss of germinative power:

- the regrowth potential of the transgenic seeds is approximately 12 times less important than for non-transgenic seeds - the transgenic seeds which germinated within the framework of this test express half the recombinant lipase than the initial seeds. It therefore seems that when the seeds express lipase at least up to 120 UTC4 / g, they have no potential for re-germination in open field conditions.

Furthermore, it has been found that gastric lipase has no significant effect on the fatty acid composition, the fat content of the transgenic seeds being significantly reduced. Rapeseed lipase-expressing rapeseed (T2 and T3) has a fat content of the order of 22 to 24% instead of 40% on average, a drop of around 40%.

Claims

1) Plant seed whose germinative power is destroyed by the total or partial absence of at least one energy reserve essential for germination, in which the destruction of the germinative power is conferred by an enzyme present in the seed at a level allowing the rapid consumption of said energy reserve.

2) Transgenic seed whose germination power is destroyed by the total or partial absence of at least one energy reserve essential for germination.

3) transgenic seed according to claim 2, characterized in that it contains at least one heterologous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination.

4) transgenic seed according to one of claims 2 or 3, characterized in that it 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 essential for germination.

5) Seed according to any one of claims 2 to 4, characterized in that it contains at least one chimeric gene successively comprising:

- a promoter, a sequence coding for a signal peptide, - a gene coding for an enzyme chosen from the group of lipases, proteases, amylases, the action of which on the metabolism of semen is identical to that of the enzymes of germination and / or digestion, and

- a termination sequence.

6) Seed according to claim 5, characterized in that the promoter is chosen from:

The pCRU promoter of the radish cruciferin gene allowing the expression of proteins or polypeptides only in seeds or in seeds.

The promoters pGEAl and pGEA6 corresponding to the 5 ′ non-coding region of the genes of the seed reserve protein, GEAI and GEA6, of Arajidopsis thaliana allowing specific expression in the seeds.

7) Seed according to one of claims 5 or 6, characterized in that the sequence coding for a signal peptide is chosen from: - The nucleotide sequence of 69 nucleotides coding for the prepeptide of 23 amino acids of sporamine A in potatoes sweet.

The nucleotide sequence of 42 nucleotides coding for the N-terminal propeptide for vacuolar addressing of 14 amino acids of sporamine A in sweet potatoes.

- The sequence of 111 nucleotides coding for the prepropetide of 37 amino acids of sporamine A consisting of the N-terminal part towards the C-terminal part of the 23 amino acids of the prepeptide of sporamine A in sweet potato followed by the 14 acids amino acids of the vacuolar targeting propeptide of sporamine A in sweet potatoes.

8) Seed according to any one of claims 3 to 7, characterized in that the heterologous gene codes for dog gastric lipase. 9) Seed according to one of claims 2 or 3, characterized in that it contains at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination, placed under the control of a promoter chosen from those allowing the overexpression and activation of said gene before the initiation of the germination process.

10) Transgenic plant or part of this plant, 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 essential for germination, or

- at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination, placed under the control of a promoter chosen from those allowing the overexpression and activation of said gene before the initiation of the process germination.

11 / transgenic seed according to any one of claims 2 to 9 or transgenic plant according to claim 10, characterized in that it also contains one or more heterologous genes of interest, such as genes capable of conferring on the plant a agronomic character or resistance to a predator or to a herbicide or a gene coding for a protein of pharmaceutical interest.

12) Use of a gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination to prepare a transgenic plant whose germinative power is destroyed.

13) Use according to claim 12, characterized in that the gene codes for an enzyme chosen from the group of lipases, proteases, amylases, whose action on the metabolism of semen is identical to that of the enzymes of germination and / or digestion.