CA2324944A1 - 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

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

WO 9915234Q PCT / F'R99 / 00806 SEEDS AND PLANTS OF WHICH YOU ARE ABLE
GERMINATIVE IS DESTROYED BY TOTAL OR PARTIAL ABSENCE
AT LEAST ONE ESSENTIAL ENERGY RESERVE
GERMINATION.
The present invention relates to a method to destroy the germinative power of a seed of plant by the introduction, into said seed, of a nucleic acid sequence encoding an enzyme capable of rapidly metabolize an energy reserve essential for the germination of said seed.
The invention is based on the fact that it can happen when cultivating in full field, that a significant quantity of seeds newly formed falls to the ground during the cultivation and / or harvest. These seeds scattered involuntarily will be able to germinate and grow multiply as soon as the climatic conditions there will lend. Control of the non-multiplication of plants resulting from this dissemination require monitoring binding cultivated plots, consisting of example in 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 specifically aims to destroy the germinative power of seeds left behind field in order to prevent their multiplication without resorting when using a toxic substance.
This goal is achieved thanks to the presence in the seeds of enzymes capable of rapidly burning their energy reserve before the initiation of the germination. By quickly, we mean that the reserves energy are burned within a period of between about 4 weeks and about 12 weeks. This modification gives the seeds of the invention a very clear disadvantage which limits considerably, see prohibited, their involuntary multiplication. Indeed, when the climatic conditions adapted to the germination will occur, seeds modified according to the invention will no longer have the necessary energy to their germination and will therefore be unable to multiply.
If, on the other hand, the semen is stored, from harvesting, under controlled conditions and regular, the enzyme activity introduced will be inhibited and the seed will retain its germinative power and can be multiplied.
The energy reserves essential to the germination of a seed are, most often, in the form of lipid or starch. Conditions normal, suitable for allowing germination, are characterized by humidity, oxygenation and sufficient temperature, depending on the species concerned. Through example, a sufficient temperature for rapeseed and tobacco is around 4 ° C to 10 ° C.
The invention therefore relates to seeds of plants whose germinative power is destroyed by the total or partial absence of at least one reservation energy essential for germination, in which the destruction of germinative power is imparted by an enzyme present in the seed to a level allowing rapid consumption of said energy reserve. By this is meant that the enzyme is expressed in the plant with an expression level of at least minus 0.3 ~ compared to the total proteins expressed in the plant.
The invention therefore relates to everything especially transgenic seeds and plants whose germination power is destroyed by the absence total or partial of at least one energy reserve essential for germination.
A first embodiment of a seed S of the invention is a transgenic seed which contains at least one heterologous gene encoding an enzyme which confers the destruction of the germinative power of said seed by rapid consumption of at least one reserve energy essential for germination.
More particularly, the invention is relates to a transgenic seed which contains at hands a heterologous gene encoding an enzyme responsible for the consumption of an energy reserve essential for germination.
Such transgenic seed contains in its cells at least one chimeric gene comprising successively.
- a promoter, for example the promoter constitutive double 35S or more particularly a specific seed promoter, - possibly a sequence coding for a signal peptide, - a gene coding for a chosen enzyme in the group of lipases, proteases, amylases, whose action on the seed metabolism is identical to that of germinating enzymes, - a termination sequence.
The promoter is advantageously chosen among.
- The double 35S promoter of the cauliflower mosaic, or other promoters of viral origin generally recognized as such by The skilled person.
- The pCRU promoter of the radish cruciferin allowing expression of proteins or polypeptides only in seeds VlrO 99/52344 4 PCT / FR99 / 00806 or in the seeds (Depigny-This et al., ~ Plant Mol.
Biol., 20, 467-479, 1992).
- The promoters pGEAl and pGEA6 corresponding to the 5 ′ non-coding region of the genes of the seed reserve protein, GEAI and GEA6, of Arabidopsis thaliana allowing expression specific in seeds (Gaubier et al., Mol. Gen.
Genet., 238, 409-418, 1993).
The sequence encoding a signal peptide is advantageously chosen from.
- The nucleotide sequence of 69 nucleotides encoding the 23-acid prepeptide amino acids of sporamine A in sweet potatoes, signal peptide allowing the entry of polypeptides recombinants in the cell secretion system processed plants, mainly in the reticulum endoplasmic.
- The nuc sequence: Leotidic of 42 nucleotides encoding the N-terminal propetide of vacuolar addressing of 14 amino acids of the sporamine A in sweet potatoes, allowing the accumulation of recombinant polypeptides in the vacuoles of transformed plant cells.
- The sequence of 111 nucleotides coding for the 37 amino acid prepropetide of sporamine A
consisting of the N-terminal part towards part C-terminal of the 23 amino acids of the prepeptide mentioned above followed by the 14 amino acids of propetide mentioned above, this prepropetide allowing the entry of recombinant polypeptides in the secretion system and their accumulation in cell vacuoles transformed plants.
The three sequences above are described in 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 encoding the enzyme can be for example that of the gastric l.
dog described in the PCT patent application published under WO number 96/33277 or that of pancreatic lipase human (Lowe et al., J. Biol. Biochem., 264, 20042-20048, 1989). Lipase activity in seeds of plants according to the invention results, when it is not not stopped or slowed down by storage conditions, a decrease 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 those of the enzymes in the 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 a enzyme responsible for consuming a reserve energy essential for germination, placed under control of a promoter chosen from those allowing overexpression and activation of said gene before initiation 'of the germination process, as a more or less early specific seed promoter.
The invention also relates to a plant transgenic or part of this plant, such as cells, characterized in that it contains.
- at least one heterologous gene coding for an enzyme that confers the destruction of power germinating the seeds of said plant by rapid consumption of at least one energy reserve essential for germination, as described previously, or - at least one endogenous gene coding for a enzyme responsible for consuming a reserve energy essential for germination, placed under WO 99/52344 6 PCT / P'R99 / 00806 control of a promoter chosen from those allowing overexpression and activation of said gene before the initiation of the germination process. These promoters may be those already described above.
Seeds and plants or parts of plants according to the invention may further contain a or several heterologous genes of interest, such as genes capable of giving the plant character agronomic or resistance to a predator or herbicide or a gene encoding a protein of interest pharmaceutical. These heterologous genes of interest can be introduced into seeds at the same time or before chimeric genes used to destroy power germinative of said seeds in accordance with the invention.
The seeds of the invention are then remarkable in what they do to limit the spread not voluntary seed carrying these genes of interest.
Seeds and plants according to the invention can be dicotyledonous seeds and plants or monocots, like those selected in the group made up of solanaceae (tomatoes, tobacco), cruciferous (rapeseed), cereals (corn, wheat).
The method according to the invention presents advantages over techniques coupling a cytoplasmic male sterility and a gene of interest. In effect, the destruction of the germinative power of the seed prohibits the multiplication of seeds from male and female gametes. While sterility male does not prevent obtaining seeds from female gametes. In addition, obtaining seeds homozygous for the heterologous gene is easy then that it is complex in the case of a technique male sterility, because it is necessary to have a fertility restoration technique.

WO 99/52344 ~ PCT / FR99 / 00806 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 plant transgenic whose germination is controlled. More particularly the invention relates to the use of a gene encoding an enzyme responsible for consumption of an energy reserve essential for germination to prepare a transgenic plant whose germinative power is destroyed. We prefer everything particularly use according to the invention, a gene which code for an enzyme chosen from the lipase group, proteases, amylases, whose action on the seed metabolism is identical to that of germination and / or digestion enzymes.
Other features and benefits of the invention will appear in the description which follows relating to the preparation of transgenic seeds of the invention of tobacco and rapeseed expressing a lipase heterologous, and in which reference will be made to the drawing in annex where figure 1 represents the conservation of transgenic rapeseed seeds expressing a lipase under a specific seed promoter.
1) Preparation of tobacco seeds expressing the dog li ~ ase under the control of a promoter constitutive.
The promoter used is the double promoter 35S. Upstream of the gene encoding dog lipase is placed a sequence coding for a signal peptide secretion.
Tobacco plants used for transformation experiences (Nicotiana tabacum var.
Xanthi NC and PBD6) are cultured in vitro on the medium of Murashige and Skoog (1962) added to vitamins from Gamborg et al. (1968, Sigma reference WO 99/52344 g PCT / F'R99 / 00806 M0404) sucrose 20g / 1 and agar (Merk-) 8g / 1 The pH of the medium is adjusted to 5.8 with a solution of potash before autoclaving at 120 ° C for 20 min. The tobacco seedlings are transplanted by cuttings from the s knots every 30 days on this multiplication medium MS20.
All in vitro cultures are performed in an air-conditioned enclosure, under the conditions defined above below.
- light intensity of 30uE. m-2.S-1;
4 p.m. photoperiod;
- thermoperiod of 26 ° C during the day, 24 ° C
night.
The processing 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 pBIOC25 is carried out for 48 hours at 28 ° C under agitation, in LB medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) supplemented with adequate antibiotics (rifampicin and tetracycline). The preculture is then diluted to 50th in the same medium and cultivated under the same conditions. After one overnight, 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 to the 10th.
Explants of approximately lcm2 are cut out from the leaves of the seedlings described above.
They are then brought into contact with the suspension bacterial for an hour and then dried quickly on filter paper and placed on a coculture medium (Solid MS30).

After 2 days, the explants are transferred in Petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200 mg / 1), bacteriostatic, augmentin (400 mg / 1) and hormones necessary for the induction of buds (BAP, 1mg / 1 and ANA, 0.1 mg / 1). A transplanting of the explants is carried out on the same medium after 2 weeks of culture.
After 2 more weeks, buds are transplanted into petri dishes on the middle of development composed of MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted in half. Rooting takes around 20 days after which the seedlings can be cloned by internode cuttings or taken out in the greenhouse.
Using the same process, it is possible to obtain tobacco seeds expressing lipase human pancreatic under the control of a promoter constitutive.

2) Pret ~ aration of rapeseed crraines expressing dog linase under the control of a promoter seed shattered.
The promoter used is the promoter of the cruciferin. Upstream of the gene coding for lipase is placed a sequence coding for a signal peptide secretion.
Spring rapeseed (Brassica) napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a Domestos solution at 15 ~.
After 4 rinses with sterile water, the seeds are put to germinate, 20 seeds per 7 cm pot diameter and 10 cm high, on mineral medium Murashige and Skoog (Sigma M 5519) with 30g / 1 of sucrose and solidified with 5g / l agargel.
pots are placed in a culture chamber at 26 ° C with WO 99/52344] 0 PCT / FR99 / 00806 a photoperiod of 16h / 8h and under united intensity luminous of the order of 80 uE m-2 s-1.
After 5 days of germination, the cotyledons are taken sterile by cutting each petiole about 1mm above the cotyledon node.
At the same time a preculture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pGAZE
in which was inserted the sequence coding for a PPS (or PS) addressing signal under control of pCRU promoter (or pd35S), is carried out in Erlenmeyer flask 50 ml, for 36 h at 28 ° C in 10 ml of medium 2YT bacterial (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) supplemented with antibiotics useful for strain selection used.
This preculture is used to seed 1 ~ a new bacterial culture carried out in the same conditions. After 14 h the culture is centrifuged 15 min at 3000g and the bacteria are taken up in a equivalent volume of liquid germination medium.
This suspension is distributed in the petri dishes 5 cm in diameter at a rate of 5m1 / box.
The severed end of the petiole is immersed for a few seconds in the solution of agrobacteria thus prepared, then the petiole is sunk a few millimeters into the middle of regeneration. This medium has the same basic composition the germination medium with an additional 4 mg / 1 of benzyl-amino-purine (BAP), phytohormone promoting new bud formation. Ten explants (cotyledon with petiole) are cultured in a 9 cm Petri dish in diameter (Greiner reference 664102).
After 2 days of coculture in the same ~ environmental conditions such as germination, explants are transplanted. in phytatray boxes (Sigma, reference P1552) containing the previous medium supplemented with a selective agent. 45 mg / l ~ sulfate kanamycin (Sigma, reference K4000) and a bacteriostatic. mixture of 1/6 (by weight) of salt potassium clavulanic acid and 5/6 sodium salt amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.
Twice in a row, at 3 weeks interval, the explants are sterile transplanted onto new medium under the same conditions.
The green buds that appeared at the end of second or third transplanting are separated from the explant and cultivated individually in transparent pots 5cm in diameter and 10cm high containing a medium identical to the previous one but lacking from BAP. After 3 weeks of cultivation the bud stem transformed is cut and the bud is transplanted in a jar of fresh medium. After three to four weeks the roots are developed enough to allow acclimatization in phytotron (temperature 21 ° C, photoperiod 16h / 8h and 84 ~ relative humidity), the seedlings are repotted in 12 cm pots diameter filled with the same soil enriched with fertilizer delay (Osmote, at a rate of 4 g / 1 of potting soil) then transported in a greenhouse (class S2) regulated at 18 ° C, with two daily waterings of 2 minutes with water.
As soon as flowers appear, these are bagged (Crispac, reference SM 570y 300 mm * 700 mm) of so as to prevent cross-fertilization.
When the pods arrived at when ripe, they are harvested, dried and then beaten.
The seeds obtained are used to measure the activity biochemical. Selection of transgenic offspring is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on genotypes). The operating conditions are identical to those described above except that germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots the first three weeks are acclimatized in phytotron before being spent in the greenhouse.
Using the same process, it is possible to obtain rapeseed expressing lipase human pancreatic under the control of a promoter specific seed.

3) Measures of the effect of the activity of the lipase on the power of aermination in seeds examples 1 and 2.
Lipase activity, when not not slowed down or stopped by conditions of storage, cold at 4 ° C and reduced humidity, causes a decrease in triglyceride content without modification of the fatty acid composition.
Lipase activity is determined using a pH-STAT by the Gargouri titrimetric method and al. (Gastroenterology, 91, 919-925, 1986) in which the substrate used is tributyrin. The emulsion of tributyrin (4 ml for 30 ml of emulsion) is produced using vortex in the presence of bile salts (1.04 g / 1) bovine albumin (0.1 g / 1) and NaCl (9 g / 1). The dosage consists of neutralizing the released butyric acid under the action of lipase with a sodium hydroxide solution a set pH of 5.5 to 37 ° C. One lipase unit is the amount of enzyme that causes release of a micromole of fatty acid in 1 minute at 37 ° C and under optimal pH conditions (5.5). The purified dog gastric lipase has activity specific of 570 units / mg of protein. The activity lipase can be measured on the total ground material, on the pellet or on the centrifugation supernatant. The lipase activity measurements confirm expression, in the seeds of transformed plants, lipase encoded by the heterologous gene.

WO 99/5234

4 [3 PCT / FR99 / 00806 If we follow the evolution of the power of germination of these seeds with lipase activity at over time, a rapid degradation of the ability of these seeds to germinate until disappearance after 16 weeks. However, if these seeds are kept under conditions which do not allow expression of lipase activity (4 ° C), their power germination is not degraded (Figure 1). What confirms the effect of lipase activity on potency germplasm and the value of its use according to the invention.
4) Field trials.
To highlight the limitation of the dissemination generated by the invention, it suffices to to cultivate in the same plots of crops transgenic plants according to the invention, for example rapeseed expressing dog gastric lipase under control of the radish cruciferin promoter, and the non-transgenic lines, each on spaces spotted, and harvest the plants in the same conditions. Part of the seeds produced will fall into the plot during cultivation or when harvest, and the following year, we note the interest of seeds and plants according to the invention by comparing the number of regrowths on each plot.
A field trial of rapeseed transgenic homozygous for the transgene. cDNA
coding for gastric lipase under promoter constitutive has been realized. This field of rapeseed transgenic was surrounded by borders of rapeseed not transgenic. This test made it possible to confirm in real conditions the potential for re germination of transgenic seeds expressing the cDNA encoding the gastric lipase.

WO 99/52344 ~ 4 PCT / FR99 / 00806 Observation of the number of regrows on each plot.
Each of these plots has been harvested independently with the same combine harvester. The rapeseed production yield has been low nearly equivalent in the two plots. I1 is therefore reasonable to think that the amount of rapeseed fallen to the ground during harvest is equivalent for each plot.
Two years after cultivation, a sample system of rapeseed regrowth was carried out in each of these plots and gave the results reported in the table below.
Border Culture Border central left of right of colzas no colzas colzas no Trans ni es Trans ni es Trans ni es Surface of sample 1155 m2 924 m2 924 m2 redheads Number of regrowth 474 44 297 observe Number of regrowth / m2 0.41 0.05 0.32 So on average there were 7, 8 times less regrowth per square meter on the plot cultivated in transgenic rapeseed only on plots cultivated in non-transgenic rapeseed.
C ~ n ~; rmar; nn r ~ P la narurP transaéniaue des regrowth.
Taking into account seed projections outside boundaries of each plot that may occur during harvesting rapeseed or during tillage during the following two years, it was verified that the regrowth found on a plot came from good ; this by checking whether or not they included the lipase gene. Part of the regrowth was therefore WO 99/52344 l 5 PCT / FR99 / 00806 sampled and transplanted in the greenhouse. 45 for the border right, 44 (all) for central crop, 52 for the left border. These plants were brought to maturity and carried out in self-fertilization. Samples sheets were made to determine the presence or absence of cDNA encoding lipase in these plants. After grinding in liquid nitrogen the sample, the extraction buffer (0.2 M Tris-HC1;
0.25mM Na-EDTA; 0.25M NaCl; SDS 0.5 ~; pH8.0) is added. Then, the reaction medium is subjected to the vortex for 5 sec. followed by phenol extraction chloroform-isoamyl alcohol (25/24/1 v / v / v). The sentence aqueous is then precipitated by adding isopropanol at -80 ° C for 15 min. After centrifugation at 4000 g for 15 min. the base is washed in 70 ~ ethanol, then recentrifuged and dried. The pellet is taken up in 100 μl of H2O. The reaction PCR amplification of DNA is carried out in a GeneAmp PCR System 9700 thermal cycler in the presence of 0.36 μl of each of the oligodeoxynucleotide primers

5 'TTCTACCCACACCACTTC 3' and 5 'ACATGCATGTCTGTCAGG 3' to 50 pmoles / 1z1, 0.5 μl dNTP lOmM, 3 μl glycerol 98 ~, 1.8 μl 25mM MgCl2, 0.36 μl BSA 10 mg / ml, 3 μl of DNA buffer Promega x10 polymerase and 0.36 μl of Taq DNA polymerase Promega at 5 U / μl in a reaction medium of 30 μl final. The DNA is denatured for 5 min at 94 ° C., subjected to cycles each consisting of 30 denaturing at 94 ° C, 1 min. hybridization at 50 ° C, and 1 min.
elongation at 72 ° C, then elongation is continued 30 for 5 min.
The results obtained show that.
- cDNA coding for gastric lipase is not detected in any border sample right and left.
35 - only 64 ~ (27/42, not 44 because 2 plants were lost during transplanting in the greenhouse) WO 99/52344 ~ 6 PCT / FR99 / 00806 regrowth from the central plot: cultivated in transgenic rapeseed, have cDNA coding for lipase.
No regrowth was found.
transgenic plants in the initial part grown in non-transgenic rapeseed (or at a non-transgenic rate detectable due to sampling. about 11 ~
regrowths have been analyzed). It does not mean that there was no passage of transgenic seeds in the other plots, but that if there were (which is very likely) they have not been able to close again.
These analyzes show that the potential for re-germination in the field of transgenic seeds was overvalued by 36 ~. The number of regrowths transgenic in the area originally cultivated in foreign rapeseed is 0.3 plants / m2. The potential of re-germination of transgenic seeds is therefore in this test 22 times less important than that of non-transgenic rapeseed.
Verification of the expression of the transaene coding for Qastricrue linase Finally it was checked that the transgene expressed well in test positive seeds PCR. For this, the lipase activity was dosed for each of the 27 seed lots harvested from the 27 PCR + plants, using the pH Stat technique. All lots express lipase. The average lipase activity is 74 UTC4 / g of grain (extreme values. 40; 104 UTC4 / g grain, see attached results) versus initially 125 UTC4 / g grain harvested. a decrease of about 41 ~
lipase activity. Only seeds whose level of expression of the transgene to decreased (for a few reasons whatever. due to conditions physiological, extinction phenomena, ...) are partially able to close again.

WO 99/52344 (7 PCT / FR99 / 00806 This full-scale test therefore shows clearly that the seeds of transgenic rapeseed expressing gastric lipase possess a very distinct selective disadvantage, making their dissemination very highly unlikely due to their loss of power germinative.
- the grain regrowth potential transgenic is about 12 times less important than for non-transgenic seeds - the transgenic seeds which have germinated as part of this essay express half the Recombinant lipase as the original seeds. I1 therefore seems that when the seeds express lipase at least up to 120 UTC4 / g, they have no re-germination potential in open field conditions.
Furthermore, it was found that the gastric lipase has no significant effect on the fatty acid composition, fat content transgenic seeds being significantly diminished. Rapeseed seeds (T2 and T3) expressing the lipase have a fat content of around 22 to 24 ~ instead of 40 ~ on average, a drop about 40 ~.

Claims (13)

-18- 1) Plant seed whose power germinative is destroyed by the total or partial absence at least one energy reserve essential to the germination, in which the destruction of power germinative is conferred by an enzyme present in the semen at a level allowing the rapid consumption of said energy reserve. 2) Transgenic seed whose power to germination is destroyed by the total absence or partial of at least one energy reserve essential for germination. 3) Transgenic seed according to claim 2, characterized in that it contains at at least one heterologous gene encoding an enzyme responsible for the consumption of an energy reserve essential for germination. 4) Transgenic seed according to one of the claims 2 or 3, characterized in that it contains at least one heterologous gene encoding a enzyme that confers the destruction of germinative power of said semen by rapid consumption of at least one energy reserve essential for germination. 5) Seed according to any of the claims 2 to 4, characterized in that it contains at least one chimeric gene comprising successively.
- a promoter, a sequence coding for a signal peptide, - a gene coding for a chosen enzyme in the group of lipases, proteases, amylases, whose action on semen metabolism 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 gene for radish cruciferin allowing the expression of proteins or polypeptides only in seeds or in seeds.
- pGEA1 and pGEA6 promoters corresponding to the 5' non-coding region of the genes of the seed reserve protein, GEA1 and GEA6, of Arahidopsis thaliana allowing an expression specific in 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 encoding the 23-acid prepeptide sporamine A amino acids in sweet potato.
- The nucleotide sequence of 42 nucleotides encoding the N-terminal propeptide of vacuolar addressing of 14 amino acids of the sporamin A in sweet potato.
- The 111-nucleotide sequence coding for the 37 amino acid prepropetide of sporamine A
consisting of the N-terminal part towards the C- part terminal of the 23 amino acids of the prepeptide of the sporamin A in sweet potato followed by the 14 acids vacuolar addressing N-terminal propeptide amines sporamin A in sweet potato. 8) Seed according to any of the claims 3 to 7, characterized in that the gene heterologous 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 consumption of an energy reserve essential to germination, placed under the control of a promoter selected from those allowing overexpression and the activation of said gene before the initiation of the process of sprouting. 10) Transgenic plant or part thereof plant, characterized in that it contains:
- at least one heterologous gene coding for an enzyme that confers power destruction germination of the seeds of the said plant by rapid consumption of at least one energy reserve essential for germination, or - at least one endogenous gene coding for a enzyme responsible for the consumption of a reserve energy essential for germination, placed under the control of a promoter chosen from among those allowing the overexpression and activation of said gene before the initiation of the germination process. 11/ Transgenic seed according to one any of claims 2 to 9 or plant transgenic according to claim 10, characterized in that it also contains one or more genes heterologs of interest, such as genes capable of confer on the plant an agronomic character or a resistance to a predator or a herbicide or a gene coding for a protein of pharmaceutical interest. 12) Use of a gene encoding a enzyme responsible for the consumption of a reserve energy essential for germination to prepare a transgenic plant whose germination power is destroy. 13) Use according to claim 12, characterized in that the gene codes for an enzyme selected from the group of lipases, proteases, amyloidosis, whose action on semen metabolism is identical to that of the germination enzymes and/or digestion.