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WO2008107509A1 - Procédé de modification de l'architecture de l'inflorescence de plantes - Google Patents

Procédé de modification de l'architecture de l'inflorescence de plantes Download PDF

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Publication number
WO2008107509A1
WO2008107509A1 PCT/ES2008/070043 ES2008070043W WO2008107509A1 WO 2008107509 A1 WO2008107509 A1 WO 2008107509A1 ES 2008070043 W ES2008070043 W ES 2008070043W WO 2008107509 A1 WO2008107509 A1 WO 2008107509A1
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Prior art keywords
plants
gene
plant
transgenic
branches
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Spanish (es)
Inventor
Luís Antonio CAÑAS CLEMENTE
Mónica MEDINA HERRANZ
Edelín Marta ROQUE MESA
Lourdes Castellblanque Soriano
Benito Pineda Chaza
Begoña GARCIA-SOGO
Vicente Moreno Ferrero
José Pío BELTRAN PORTER
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Politecnica de Valencia
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Politecnica de Valencia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the invention relates, in general, to a method for modifying the architecture of the inflorescence of plants, and, in particular, to a method for obtaining transgenic plants that have an architecture of their inflorescence modified with respect to Ia of the corresponding wild plants, based on the use of a construct comprising a cytotoxic gene under the control of a specific anther promoter.
  • the transgenic plants show an architecture of their inflorescence more complex and with greater number of branches than the corresponding wild plants. These branches initiate and develop a greater number of floral meristems than the wild ones.
  • Plant architecture this is its three-dimensional organization, is a characteristic under strict genetic control by what is specific to each plant species (Reinhardt, D. & Kuhlemeier, C. (2002). Plant architecture. EMBO reports 3: 846-851). However, some modification of the basic structural pattern can occur due to environmental conditions such as light, temperature, humidity or nutritional status of plants.
  • the architecture of the aerial part of the plants is defined by the pattern of distribution of the leaves along the stem or phyloxis, the determination of the apical meristems of the stem, and by the branching patterns of the vegetative parts and the parts reproductive, this last factor also called architecture of the inflorescence. Plants produce lateral branches from the meristems that begin in the armpits of the leaves. The branching pattern is conditioned by the filotactic pattern of the plant stem. In many plants, the initiation of axillary meristems is initially suppressed by the apical meristem, a phenomenon called apical dominance.
  • the floral transition affects the architecture of the plant in several ways since there are usually changes in the phyloxis in addition to those that affect the destination and the identity of the meristems.
  • Many plants have an apical meristem of the stem that is undetermined, that is, it remains active throughout the life of the plant by first differentiating leaves and then flowers. This growth pattern is called monopodial and is characteristic of Arabidopsis or Antirrhinum.
  • the pattern of monopodial growth in Arabidopsis is established in different stages: first, type 1 vegetative metamers are formed, formed by very short internodes, a leaf and a yolk, which are organized in the form of a rosette; and in the second place, and after a floral transition, the main stem of the inflorescence is composed at the beginning of type 2 metamers, which have elongated internodes, a caulinar leaf and a bud, and then by type 3 metamers that contain intermediate length internodes. and a floral bud.
  • the axillary meristems are detected in the stem, first in the axillae of the caulinary leaves and later in those of the rosettes, once the floral transition has occurred.
  • axillary meristems form inflorescent lateral stems with a monopodial development pattern (Alvarez et al., (1992). Terminal flower: a gene affecting inflorescence development in Arabidopsis thaliana. Plant Journal 2: 103-116).
  • Terminal flower a gene affecting inflorescence development in Arabidopsis thaliana. Plant Journal 2: 103-116.
  • the formation of axillary meristems in Arabidopsis is controlled, in part, by three R2R3 genes of the Myb family called RAX genes ⁇ REGULATORS OF AXILAR MERISTEMES) that are homologs of the Blind gene of Solanum licopersicon (Müller et al. (2006). homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis. Plant Ce // 18: 586-597).
  • the meristem apical forms a terminal flower when the floral transition occurs, from here, tobacco, for example, initiates several symposia branches that they consist of a leaf or bract, a new sypodial meristem and a terminal flower, while in tomato only two sypodial meristems of which the lower form three leaves before blooming are started while the upper one divides successively forming a terminal flower each time and a new symposium meristem.
  • Petunia only one sympodial branch is initiated, which forms two leaves and a new sympodial meristem (Huber, KA (1980). ).
  • orthologous genes of CLAVATA1 from Arabidopsis have been characterized in rice and corn that contribute to the establishment of the architecture of the inflorescence in said species as well as various genes that affect the vegetative and reproductive branching patterns in Arabidopsis ⁇ LAS), tomato ⁇ LS and BL), rice ⁇ MOC1) or corn ⁇ BIF2) (Wang, Y. & Li, J. (2006). Genes controlling plant architecture. Current Opinion in Biotechnology 17: 1-7).
  • the RAMOSA 3 gene that codes for the trehalose-6-phosphate phosphatase enzyme and which is expressed in discrete domains near axillary meristems has been characterized.
  • the RAMOSA 3 gene could control the branching pattern of corn inflorescences by modifying a sugar signal that would reach axillary meristems (Satoh-Nagasawa et al., (2006).
  • a trehalose metabolic enzyme controls inflorescence architecture in maize. Nature 441 : 227-230).
  • MAX1 encodes a cytochrome P450 family member that acts downstream of MAX3 / 4 to produce a carotenoid-derived branch-inhibiting hormone.
  • GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for Gibberellin Nature 437: 693-698).
  • the ablation of anthers produced by genetic transformation with cytotoxic genes that are expressed only in the anthers, using specific promoters of some staminal tissue has allowed to obtain andro-sterile transgenic plants and transgenic plants with restored androfertility in species of agronomic importance such as Corn, rapeseed or wheat.
  • the genetic ablation is based on the induction of cell death by means of the expression of any enzyme that is capable of destroying the cellular integrity such as proteases, lipases and RNases.
  • RNA from a gene that confers tolerance to a herbicide.
  • the effect of the "antisense” RNA is to eliminate the chemical resistance specifically in pollen, so that the application of the herbicide produces its destruction. This method converts a herbicide into a gametocide.
  • the promoter of the TA29 tobacco gene specific to tapetum, was used to direct the expression of two RNases (Aspergillus oryzae T1 RNAse and Bacillus amyloliquefaciens barnase) in tobacco and Brassica napus.
  • the obtained andro-sterile transgenic anthers lacked tapetum and their pollen sacs did not produce microspores or pollen grains.
  • the floriculture industry strives to achieve new and different varieties of ornamental plants with improved characteristics ranging from resistance to pathogens.
  • diseases to the modification of the architecture of the plant including the floral architecture (altered inflorescences) or modifications in the color and in the number of flowers.
  • the classic improvement techniques are aimed at crossing plants with desirable characteristics to obtain hybrids that incorporate these characteristics, or the use of hormones to alter the phenotype of the plant.
  • recombinant DNA technology has become the alternative strategy to classical improvement to develop plants with an altered phenotype and have certain improved characteristics.
  • the present invention is aimed at solving the need of the ornamental plants sector to have plants with complex inflorescent architectures that add value due to their attractiveness or the improvement of production costs.
  • the invention relates to a process for the production of transgenic plants with an architecture of its modified inflorescence with respect to that presented by the corresponding wild plant which, among other stages, comprises transforming a susceptible plant cell or tissue if transformed with a gene construct comprising a cytotoxic gene under the control of the promoter of the PsENDI gene, or a fragment thereof capable of specifically regulating the gene expression of said cytotoxic gene in anthers.
  • the transgenic plants obtained by the process of the present invention having the additional advantage that they do not produce horizontal dispersion of genes by not generating neither pollen nor seeds, so that its authorization of use at field level would not be a difficulty.
  • the invention relates to a transgenic plant obtainable by the method described in the present invention that has an architecture of its modified inflorescence compared to that of the corresponding wild plant.
  • said transgenic plant has a complex, different architecture, with greater number of branches and branches of greater order, all of them capable of producing flowers, than the wild plant.
  • a process for producing flowers which comprises cultivating a transgenic plant obtained according to the procedure described above under conditions that allow flowering and flower development.
  • Figure 1 shows the schematic representation of the construction pBI-END1 :: barnasa-barstar and the starting construction pBI101-F3.
  • Plasmid PB1101 consists of the constitutive promoter of nopaline synthetase (nos-pro) fused to the nptll gene that confers resistance to kanamycin, the uidA gene that encodes the enzyme D-glucuronidase (GUS) and the polyadenylation signal of the nopaline synthetase gene (nos-ter) at the 3 'ends of both genes.
  • the uidA gene has been replaced by the fragment that contains the barnasa-barstar genes.
  • Figure 2 shows photographs of wild Arabidopsis thaliana plants (Figure 2A) and transgenic A. thaliana plants obtained according to the procedure described in the present invention ( Figure 2B).
  • Figure 2C shows the flowers present in the wild Arabidopsis plants (left flower) and the flowers shown in the transgenic Arabidopsis plants (right flower).
  • two Arabidopsis plants are shown: one transgenic obtained by the method of the invention (right) and another wild (left).
  • the transgenic plants show a greater development than the wild ones, a greater number of branches and a greater number of flowers when compared with the wild ones.
  • FIGS. 2D and 2E show the longitudinal dissection of a wildflower and a transgenic flower, respectively. In the wild flower the presence of normal anthers is observed while in the transgenic anthers are undeveloped.
  • Figure 3 shows transgenic Nicotiana tabacum plants obtained according to the method of the invention and wild N. tabacum plants.
  • Figure 3A shows a wild plant (left) against two transgenic plants according to the method of the invention
  • Figure 3B shows a detail of the branches of a transgenic plant of N.
  • Figure 3C shows the corresponding wild plant.
  • the transgenic plants show a greater development than the wild ones, a greater number of branches and a greater number of flowers when compared with the wild ones.
  • Figure 3D can be seen how the flowers of wild plants fertilize and produce fruits (capsules) stopping their growth (left), while the transgenic ones do not produce fruits and continue to produce flowers that are senescent on the branches without fertilizing.
  • Figures 4A and 4B show an anther of a complete N. tabacum wild plant and a cross section of one of its pollen sacs respectively observed by scanning electron microscopy (SEM). You can see how it contains pollen grains in its inside.
  • Figures 4C and 4D a transgenic anther of N. tabacum is shown showing its collapsed pollen sacs and a cross section of one of them showing that there are no pollen grains inside respectively.
  • Figure 5 shows the nucleotide sequence of the 5 'region of the PsENDL pea gene.
  • the possible regulatory elements within the sequence are represented in different colors depending on the type of regulatory element.
  • Figures 6A and 6B show the number of branches produced in wild plants against transgenic plants (A) and the number of flowers produced in wild plants against transgenic plants (B). In both graphs the number 1 corresponds to wild plants and the number 2 to transgenic plants.
  • Figures 7A and 7B show a representative diagram of the number of branches produced in wild plants (A) and in transgenic plants (B) of A. thaliana.
  • Figures 8A and 8B show a representative diagram of the number of flowers produced in wild plants (A) and in transgenic plants (B) of A. thaliana.
  • the invention relates to a method for obtaining a transgenic plant with an architecture of its modified inflorescence with respect to that presented by the wild plant (wt), which comprises: (a) transforming a plant cell or tissue capable of being transformed with a gene construct comprising:
  • a first nucleic acid sequence comprising the nucleotide sequence of the promoter of the PsENDI gene, or a fragment thereof capable of specifically regulating the gene expression of a second nucleic acid sequence in anthers
  • a second nucleic acid sequence comprising the nucleotide sequence of a cytotoxic gene, or a functional fragment thereof, under the control of said first nucleic acid sequence, to produce a transformed plant cell or tissue
  • stage (b) regenerate said transformed cell or plant tissue in stage (a) to produce a transgenic plant, and (c) select the transgenic plants of stage (b) that exhibit an architecture of its modified inflorescence compared to that presented The corresponding wild plant.
  • a transgenic plant with an architecture of its modified inflorescence with respect to that presented by the wild plant refers to a transgenic plant capable of developing a more complex branching pattern, which produces a greater number of branches and branches of greater order, all of them capable of producing flowers, than the corresponding wild plant (wt) thanks to the incorporation into its genome of the gene construction described in the present invention; in general, said transgenic plant with an architecture of its modified inflorescence has a more complex branching pattern and is the producer of a greater number of branches and Branches of greater order, all of them producing flowers, than the corresponding wild plant.
  • transgenic plant is not only capable of producing a greater number of branches than the corresponding wild plant, but said transgenic plants have an increase in the number of flowers produced and in their half-life with respect to wild plants. While wild plants senesce at three months, transgenic plants have been running for six months. In general, said transgenic plant has a three-dimensional structure that provides a more colorful appearance.
  • a plant of interest is genetically manipulated to contain and stably and consistently express a phenotype that is not normally present in wild plants. Said phenotype consists of a greater number of branches and branches of greater order, all of them capable of producing flowers, and of flowers than the wild plant.
  • said plant is an ornamental plant.
  • Illustrative, non-limiting examples of said plants of interest susceptible to being genetically manipulated according to the invention to obtain transgenic plants with an architecture of their inflorescence modified with respect to that presented by the wild plant include plants belonging to the Aeschynantus genera; Canna; Column; Anemone; Azalea; Begonia; Calceolaria; Camellia; Dianthus; Freessia; Gerbera; Hibiscus; Hypoestes; Kalanchoe; Nicotiana; Pelargonium; Petunia; Pr ⁇ kmula; Rannunculus; Rhipsalidopsis; Pink; Saintpaulia; Sinningia-gloxinia; Streptocarpus; Tigridia; Verbena; and Zinnia.
  • the method of the invention comprises the preparation of a gene construct comprising (i) a first nucleic acid sequence comprising the nucleotide sequence of the promoter of the PsENDI gene, or a fragment thereof capable of specifically regulating the gene expression of a gene. second nucleic acid sequence in anthers, and (ii) a second nucleic acid sequence comprising the nucleotide sequence of a cytotoxic gene, or a functional fragment thereof, under the control of said first nucleic acid sequence.
  • the promoter of the pea PsENDI gene (Pisum sativum L), hereinafter pEND1, is a promoter capable of directing the specific expression in anther in early stages of plant development as described and evidenced in the WO patent application 07/013088. Indeed, the in situ hybridization assays described in said patent application confirmed the specificity of the expression of the PsENDI gene in the tissues of the pea anther, in particular, in the tissues that make up the pollen sacs of the anthers, during the different stages of its development.
  • the pEND1 present in the gene construct comprises the nucleotide sequence shown from nucleotide -2,736 to nucleotide -6 of the nucleotide sequence shown in Figure 5, which constitutes the complete sequence of said promoter.
  • the gene construct used to transform plant cells or tissues comprises a fragment of pEND1 comprising, at least, the nucleotide sequence comprised from nucleotide -366 to nucleotide -6 of the nucleotide sequence shown in Figure. 5.
  • the previously defined pEND1 fragment maintains the regulatory capacity of the specific expression in anther and is capable of directing the specific expression of anther in early stages of plant development.
  • the pEND1 can be obtained by conventional methods from a pea plant (Pisum sativum L.) or from a host organism transformed with a DNA sequence comprising said promoter, as mentioned in WO 01/073088.
  • the fragments of pEND1 that maintain the regulatory capacity of the specific expression in anther can be obtained, based on the information provided, by conventional methods, for example, from the pEND1, making the appropriate deletions.
  • the tests described in Example 1 of WO 01/073088 can be performed.
  • the gene construct used to transform plant cells or tissues comprises, in addition to pEND1 or a functional fragment thereof, that is, capable of regulating the specific expression in anther, a cytotoxic gene, operably linked to said promoter or functional fragment thereof.
  • cytotoxic gene includes any gene that encodes a protein or enzymatic activity that causes cell death in the tissue where it is expressed, for example, a gene that encodes a protein or enzymatic activity that causes the ablation of the anther.
  • Ia diphtheria toxin A (DTA) produced naturally by Corynebacterium diphteriae, Pseudomonas aeruginosa exotoxin A, Aspergillus oryzae ribonuclease T, Bacillus amyloliquefaciens barnase, etc.
  • DTA Ia diphtheria toxin A
  • Pseudomonas aeruginosa exotoxin A Aspergillus oryzae ribonuclease T
  • Bacillus amyloliquefaciens barnase etc.
  • the genes encoding said proteins can be used as cytotoxic genes in the gene construction described herein for the implementation of the present invention.
  • said cytotoxic gene that is expressed in anther because it is under the control of pEND1 is the barnase gene, a Bacillus amyloliquefaciens ribonuclease [Mariani et al., (1990), cited at supra ] that causes the complete ablation of the anther, from very early stages of its development, preventing the formation of pollen therein, thus giving rise to an andro-sterile plant. Additional examples of cytotoxic genes are cited in European patent application EP 412006 as well as in patent application WO 01/073088, the contents of which are incorporated by reference to the present description.
  • the gene construct used to transform plant cells or tissues by the process of the invention can be obtained by conventional methods using widely known techniques [Sambrook, J., et al, 2001. Molecular cloning:... A Laboratory Manual, 3rd ed, Coid Spring Harbor Laboratory Press, NY, VoI. 1-3]. Said gene construct may also contain, operatively linked, regulatory elements of the expression, for example, sequences of termination of transcription, enhancer sequences of transcription and / or translation, etc.
  • the gene construct used to transform plant cells or tissues according to the method of the invention can be inserted into the genome of a plant cell or tissue by any appropriate method to obtain transformed plant cells and tissues. Such methods may involve, for example, the use of liposomes, electroporation, diffusion, particle bombardment, microinjection, gene bullets ("gene gun"), chemical compounds that increase free DNA uptake, for example, coprecipitation with calcium phosphate, viral vectors, etc.
  • Appropriate vectors for plant transformation include those derived from the Ti plasmid of Agrobacter ⁇ um tumefaciens, such as those described in EP 120516.
  • transformation vectors derived from the Ti or Ri plasmids of Agrobacter ⁇ um can be used to insert Ia gene construction in plant cells and tissues.
  • said gene construct is introduced, by means of the vacuum infiltration protocol.
  • the gene construction described herein can be used to transform any cell or plant tissue that can be transformed.
  • said cell or plant tissue belongs to an ornamental type plant.
  • the term "ornamental plant” includes any plant grown for its generally aesthetic value. Between such aesthetic values include visually appealing characters such as colorful, colorful or scented flowers or inflorescences.
  • Illustrative, non-limiting examples of such ornamental plants include plants belonging to the Aeschynantus genera; Canna; Column; Anemone; Azalea; Begonia; Calceolaria; Camellia; Dianthus; Freessia; Gerbera; Hibiscus; Hypoestes; Kalanchoe; Nicotiana; Pelargonium; Petunia; Pr ⁇ kmula; Rannunculus; Rhipsalidopsis; Pink; Saintpaulia; Sinningia-gloxinia; Streptocarpus; Tigridia; Verbena; and Zinnia.
  • transgenic plants with improved ornamental value which have a greater number of branches and branches of greater order, all those branches having the ability to produce flowers, regardless of their number.
  • the gene construct can be incorporated into a vector that includes a prokaryotic replicon, that is, a DNA sequence capable of directing the autonomous replication and maintaining the extrachromosomally recombinant DNA molecule when introduced into a prokaryotic host cell, such as a bacterium. Said replicons are known in the art.
  • said prokaryotic replicon also includes a gene whose expression confers a selective advantage, such as resistance to a drug (drug), to the transformed host cell.
  • bacterial genes that confer resistance to drugs include those that confer resistance to ampicillin, tetracycline, etc.
  • the neomycin phosphotransferase gene has the advantage that it is expressed in both eukaryotic and prokaryotic cells.
  • the vectors that include a prokaryotic replicon also include, in general, restriction sites for the insertion of the gene construct used for the implementation of the process of the invention. These vectors are known (US 6,268,552).
  • the expression vectors capable of expressing a recombinant DNA sequence in plant cells and capable of directing the stable integration into the genome of the host plant cell are vectors derived from the Ti plasmid of A. tumefaciens and several other known expression systems operating in plants (see, for example, WO 87/00551).
  • the method of the invention to obtain transgenic plants with an architecture of its modified inflorescence with respect to that presented by the corresponding wild plant comprises the introduction, in a cell or in a tissue of a plant, of the gene construction previously defined to produce a cell or a transformed plant tissue and generating a transgenic plant with an architecture of its modified inflorescence compared to that presented by the wild plant by regeneration of said cell or transformed plant tissue, wherein said transgenic plant with a modified inflorescence architecture with respect to The one that presents the wild plant produces a greater number of branches and branches of greater order, all of them producing flowers, than the corresponding wild plant as well as a greater number of flowers than the wild plant when grown under conditions that allow flowering and development. from the flowers.
  • the transgenic plant with an architecture of its modified inflorescence thus obtained has a more complex branching pattern and is not only capable of producing greater number of branches than the corresponding wild plant, but also that these branches are capable of producing flowers and the plant It has a half-life superior to that of wild plants.
  • the introduction of said gene construction to transform plant material and generate a transgenic plant can be carried out, as previously mentioned, by any means known in the state of the art, including, but not limited to, the transfer of DNA. mediated by A. tumefaciens, preferably with an unarmed T-DNA vector, electroporation, direct DNA transfer, particle bombardment, etc.
  • the systems and agents for introducing and selecting markers to check the presence of heterologous DNA in cells and / or plant tissues are well known.
  • the genetic markers that allow the selection of heterologous DNA in plant cells are genes that confer antibiotic resistance, for example, kanamycin, hygromycin, gentamicin, etc.
  • the marker allows the selection of successfully transformed plants that grow in a medium containing the corresponding antibiotic because they carry the appropriate resistance gene.
  • the present invention allows flowers to be obtained without having to apply hormones (gibberellins, auxins, cytokinins, etc.) or agrochemicals.
  • hormones gibberellins, auxins, cytokinins, etc.
  • an added advantage of the procedure of Ia The invention is that it allows to obtain not only more showy plants but also to increase the production of cut flowers, with which a reduction in production costs is achieved.
  • the transgenic plants thus obtained have a longer half-life of several months compared to wild plants.
  • the process provided by this invention supposes the additional possibility that the plants thus obtained have dominant androsterility.
  • One of the advantages of the process provided by this invention is that it offers the possibility of having andro-sterile flower-producing plants, with a greater number of branches and branches of greater order, all capable of producing flowers, than the wild plant, with the that the unwanted horizontal transfer of genes is avoided by not producing pollen or seeds, which is especially relevant in their authorization of use at the field level, since the horizontal transfer of genes is one of the major concerns of environmental groups and part of the citizens that today oppose the cultivation of transgenic plants.
  • the availability of andro-sterile plant genotypes may be relevant to avoid pollen contamination in urban areas or decrease the production of pollen allergens.
  • the plant species used in the example of the present invention are shown in Table 1.
  • Samples of the plant tissues used in the present invention for the extraction of nucleic acids were collected directly from the plant, frozen in liquid nitrogen and stored at -8O 0 C until later use. Samples destined for microscopy studies were fixed for further processing.
  • Arabidopsis plants were grown in phytotrons under controlled photoperiod and temperature growth conditions.
  • the temperature was 21 0 C and the illumination came from cold white fluorescent tubes with an intensity of 150 ⁇ E m "2 s " 2 (Sylvania Standard F58W / 133-T8).
  • the plants were grown under conditions of inductive photoperiod, which were 16 hours of light and 8 hours of darkness (long day, DL) and non-inductive photoperiod that were 8 hours of light and 16 hours of darkness (short day, DC).
  • the seeds were sown in alveoli or in pots depending on the subsequent use of the plants generated.
  • the seeds were sown in 6.5 * 6.5 * 5 cm plastic cells for long-day or short-day crops in a mixture of peat: perlite: vermiculite (1: 1: 1). They were placed in trays inside culture chambers and irrigated with immersion with Hoagland solution number 1 supplemented with trace elements. After planting, the trays were covered with plastic to maintain moisture and avoid contamination with other seeds from nearby plants. They were kept in darkness at 4 0 C for 3 days in order to synchronize the germination and after those days they were transferred to cabins.
  • Planting in pots was carried out in plastic pots of 11 cm in diameter, for DL or DC cultures and the same process was performed as for planting in alveoli.
  • the seeds were sown in 6.5 x 6.5 x 5 cm plastic cells in a mixture of peat: perlite: vermiculite (1: 1: 1). They were placed in trays in culture chambers and irrigated by immersion in a Hoagland solution number 1 supplemented with trace elements (Hewitt,
  • the seeds were sterilized by immersion for 3 minutes in a 70% (v / v) ethanol solution and 0.005% Triton X-100. During this time, the seeds were mixed with the previous solution by moving the tube containing them. Subsequently, the solution was removed and 96% ethanol was added with stirring for 1 minute. Immediately afterwards the seeds with the ethanol were placed on sterile filter paper until they dried.
  • the sterilized seeds (approximately 30 mg of seeds) were uniformly distributed in 15 cm diameter Petri dishes containing selection medium with kanamycin [2.2 g / l of MS salts (Murashige culture medium) and Skoog) (Duchefa), 20 g / l sucrose, 0.1 g / l MONTH (morpholinoethane sulfonic acid) pH 5.9, 0.6% agar (Pronadisa), 50 mg / l kanamycin, 300 mg / l timentin ].
  • the boxes with the seeds were stored for three days at 4 0 C in the dark after which they were moved to an in vitro culture cabin. After 7-10 days of planting the transformants that were distinguished by their green and elongated root, they were transplanted with the help of tweezers to plastic cells.
  • Tobacco plants from in vitro culture were individually grown in 13 cm diameter plastic pots containing a previously sterilized peat mix: vermiculite (1: 1), in a greenhouse cabin under controlled conditions and with a temperature of 24 0 C during the day and 18 0 C during the night.
  • Natural light is supplemented with artificial light using 400 W mercury vapor lamps [Phillips HDK / 400 HPI ®, N], to maintain a long day photoperiod.
  • Irrigation consisted of Hoagland solution number 1 provided by an automated drip irrigation system for 2 minutes, 4 times a day
  • the in vitro culture of snuff was performed in booths with constant temperature of 25 0 C under photoperiod conditions long (16 hours light and 8 hours dark) day, with a light intensity of 90 ⁇ E m "2 s" 2 supplied by fluorescent tubes of light type Grolux 36W (Sylvania).
  • Kanamycin-resistant plants (primary transformants, T1) whose cultivation had begun in Petri dishes, were subsequently transplanted into plastic alveoli of 6.5 x 6.5 x 5 cm with a mixture of peat: vermiculite (1: 1) . These crops remained covered with a transparent plastic, in which holes were gradually made in order to avoid excessive water condensation for 9 days. After the acclimatization period, the seedlings were transplanted into individual pots, where they were grown in greenhouse cabins under controlled temperature and photoperiod conditions. 2. Cultivation of microorganisms
  • microorganisms used in the example of the present invention are shown in Table 2.
  • the medium used for the growth of microorganisms was: - LB medium (Luria-Bertani medium): 1% tryptone, 0.5% yeast extract, 1% NaCI, pH 7.0.
  • LB medium Lia-Bertani medium
  • tryptone 0.5% yeast extract
  • NaCI 1% NaCI
  • pH 7.0 pH 7.0
  • the solid medium was solidified by the addition of 1.5% agar (Pronadisa).
  • Cloning was done in different plasmids depending on the origin of the DNA fragments and the required purposes.
  • PCR products polymerase chain reaction
  • pGEM-T Easy Promega
  • Plasmid pBI101 [Vancanneyt, G., et al., (1990). Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. VoI 220 (2): 245-50] was used to obtain transgenic Arabidopsis and tobacco plants through the transformation with Agrobacter ⁇ um tumefaciens. This plasmid contains the nptll gene that offers resistance to kanamycin and the ⁇ -glucuronidase gene that allowed to perform histochemical analysis of the transformed plants. Plasmid pBI101 was used to carry out the expression of the END1 :: barnase transgene in the two plants mentioned. 4. Enzymatic reactions
  • Ligation reactions were performed maintaining a molar ratio between vector and insert of 1: 2 in the case of plasmid pGEM-T Easy (Promega) and 1: 5 in the case of vector pBI101.
  • the final volume of the reactions was 10 or 20 ⁇ l. This volume included the vector / insert volume, 1 unit of phage T4 ligase DNA (Roche Molecular Materials).
  • ligation buffer (5 mM MgC ⁇ 1 mM DTT, 1 mM ATP, 66 mM Tris-HCI pH 7.5). The ligation reactions were carried out at 4 0 C overnight in the case of using the plasmid pGEM-T Easy and at 16 0 C overnight in the case of using pBI101.
  • the reaction was stopped with 2 ul of 0.5M EDTA per 100 ⁇ l total reaction volume, heating 20 min at 7O 0 C.
  • the solution was extracted twice with phenol / chloroform, precipitated with 1/10 v sodium acetate (NaOAc) 3 M pH 5.2, 2.5 v absolute ethanol (EtOH) and 1 ⁇ l of glycogen (Boehringer Mannheim) and the DNA precipitate was resuspended in a suitable volume (about 15 ⁇ l) and quantified by agarose gel electrophoresis.
  • PCR polymerase chain reaction
  • Plasmid DNA amplification reactions were carried out in a total volume of 50 ⁇ l, from 10-50 ng of template DNA, 1 ⁇ M of specific primers of the fragment to be amplified or of the plasmid where it was cloned, dN 4 TPs 200 ⁇ M, 50 mM KCI, MgCb
  • Tris-HCI pH 8.3 1.5 units of Thermus aquaticus polymerase (Taq polymerase, Roche Molecular Biochemicals).
  • the PCR reaction was carried out using the Ribo 1 and Inhi 2 primers (Table 3). These reactions were carried out in a total volume of 20 ⁇ l, from 50-100 ng of genomic DNA, 0.6 ⁇ M of specific primers, dN 4 TPs 200 ⁇ M, 50 mM KCI, 1.5 mM MgCl 2 , 10 mM Tris-HCI pH 8.3, 2.5 units of Thermus aquaticus polymerase (Taq polymerase, Roche Molecular Biochemicals).
  • the reactions were run in a Perkin Elmer 2400 at the following conditions: 1 cycle at 94 0 C 2 minutes; 30 cycles of 94 0 C 30 s; banding temperature (T) in degrees Celsius for 30 s; 72 0 CT ex t minutes, and 1 final cycle at 72 0 C for 10 minutes.
  • T banding temperature
  • the banding temperature was estimated as a function of the temperature Tm (estimated melting temperature) of the primers used (Table 3); the extension time used It depended on the length of the fragment to be amplified; In general, 1 minute was used for each kb of expected product.
  • the italicized sequences correspond to the nucleotides that are not part of the template DNA, and of them, the underlined ones, correspond to the restriction targets introduced to subclone the cDNAs in the corresponding plasmids.
  • the sequencing of cloned DNA fragments was carried out according to the enzyme sequencing protocol developed by Sanger et al., (Sanger, F., Nicklen, S., Coulso, AR 1977. DNA sequencing with chain termination inhibitors. PNAS. USA 74, 5463-5467) automatically in a sequencer "ABI PRISM 377" (Perkin Elmer).
  • the DNA extracted according to the protocol of isolation and purification of plasmid DNA from the QIAGEN-tiplOO system described in section 5.1 was brought to a concentration of 0.2 ⁇ g / ⁇ l, and amplified with Ampli Taq DNA polymerase in the presence of ddNTPs, each marked with a different fluorophore (Perkin Elmer).
  • the primers of the plasmid vector pGEM-T Easy, T7 and SP6 were used.
  • the alkaline lysis method was used as described in Sambrook, J., et al., (1989) [cited at supra], starting from a 3 ml culture grown during a night in liquid medium LB supplemented with the corresponding antibiotic.
  • Medium or large-scale plasmid DNA preparations were made from cultures, grown overnight in 100 ml or 500 ml of liquid medium LB supplemented with antibiotic, according to the method of extraction and purification of plasmid DNA from the systems of Qiagen Plasmid Midi Kit (Qiagen tip-100 columns) and Qiagen Plasmid Maxi Kit (Qiagen tip-500 columns), respectively, following the manufacturer's instructions.
  • the cell pellet was resuspended in 100 ⁇ l of solution I (50 mM glucose; 50 mM Tris-HCI (pH 8.0); 10 mM EDTA) after which 200 Dl of Il solution (NaOH) was added 0.2 N, 1% SDS) and mixed by quickly inverting the tube. Subsequently, solution III (60 ml of 5 M KAc; 11.5 ml of glacial acetic acid; 28.5 ml of water) was added and mixed using the vortex. After 5 minutes on ice, the sample was centrifuged at 12,000 rpm for 5 minutes at 4 0 C.
  • solution I 50 mM glucose; 50 mM Tris-HCI (pH 8.0); 10 mM EDTA
  • 200 Dl of Il solution NaOH
  • solution III 60 ml of 5 M KAc; 11.5 ml of glacial acetic acid; 28.5 ml of water
  • the selection of bacterial recombinants was carried out by sowing the bacterial cells transformed into plates with LB medium supplemented with the antibiotic to which the plasmid under study conferred resistance, and in the event that the plasmid allowed the selection by color, added 40 ⁇ l (25 mg / ml) of IPTG and 25 ⁇ l (20 mg / ml) of X-GaI to the solid culture medium.
  • antibiotics used for the selection of bacterial recombinants and the concentration at which they were used appear in Table 4. Table 4. Antibiotics used and their concentrations
  • Figure 1 the pBI101-F3 construction [described in patent application WO 01/73088] ( Figure 1) was started.
  • This construction contained 2,731 bp of the promoter region of the PsENDI gene isolated from the screening of a genomic pea library. The region comprised from fragment -2,736 to nucleotide -6 of the 5 'region, the first nucleotide of the previously isolated cDNA (clone 162) from a pea flower cDNA library [Gómez, MD, et. al., (2004).
  • the pea END1 promoter drives anther-specific gene expression in different plant species. VoI plant. 219: 967-981].
  • the -2,736A6 fragment of the promoter region of the PsENDI gene was fused to the uidA gene encoding the enzyme ⁇ -glucuronidase (GUS), (Gómez et al., 2004) [cited at supra].
  • GUS ⁇ -glucuronidase
  • This gene was released with the restriction enzymes BamHI and Sacl and the fragment corresponding to plasmid pBI101 plus the promoter of the PsENDI gene was extracted from the agarose gel.
  • the barnasa-barstar fragment previously cloned into the BamHI site of plasmid pBluescript KS (+) (Stratagene), was amplified using the primers Ribo 1 and Inhi 2 [Table 3]. With the first one, the cutting site for the BamHI enzyme of the original clone at the ATG level of barnase is maintained, and with the latter a cutting site for Sacl is created at the level of the stop codon of the barstar gene.
  • the fragment product of the PCR reaction was ligated to the pGEM-T Easy vector (Promega), and subsequently released with the enzymes BamHI and Sacl. This insert was cloned into the site created by these same enzymes in the pBI-END1 construct, thus creating the pBI-END1 :: barnasa-barstar construct ( Figure 1).
  • a tube was inoculated with 10 ml of LB medium, containing 100 ⁇ g / ml of rifampicin and 50 ⁇ g / ml of kanamycin, from a glycerinated with strain C58 pMP90 of A tumefaciens (Koncz and Schell, 1986) [cited at supra] carrier of the constructions of interest. This was incubated place overnight in the dark at 28 0 C with agitation of 200 rpm.
  • the pots were inverted and placed in a lunch box containing 200 ml of the Agrobacterium suspension in the middle of infiltration, so that not only the floral apexes but also the rosette leaves were submerged in the liquid .
  • the assembly was placed in a vacuum hood connected to a pump (EDWARDS RV3 pump, 110-120 / 220-240V, 50-60 Hz, single phase A652-01-903) and subjected to vacuum for 30 minutes in the high position vacuum and flow under "position I" (final total pressure: 3 x 10 ⁇ 2 mbar, 3 Pa). The time began to count when the suspension of Agrobacter ⁇ um began to bubble.
  • the plants were removed from the hood and dried slightly, draining them on a piece of absorbent paper.
  • the plants treated in this way were covered with plastic bags and returned to the cultivation booths where they were allowed to continue growing under the conditions described in section 1.1.
  • During the 2-3 days following the infiltration holes were made in the bags, in order to acclimatize the plants to the usual humidity conditions, until these were definitively eliminated.
  • the plants were grown to obtain mature seeds.
  • the siliques of transformed plants were mature seeds were collected, stored in cellophane bags and incubated in an oven at 37 0 C for at least a week.
  • the primary transformants Ti
  • the seeds from individual Ti plants were sterilized, planted in 15 cm diameter Petri dishes with kanamycin selection medium and grown in in vitro culture cabins. After 7-10 days from planting, the transformants were clearly identifiable by their green color and their developed roots; at that time they were transplanted into alveoli (6.5 X 6.5 x 5 cm) with a peat mixture: vermiculite: perlite (1: 1: 1) and transferred to a phytotron for cultivation under the conditions described in the section 1.1.
  • the phenotype of the population corresponding to the first (Ti) and second generation (T 2 ) of plants transformed with the construction was analyzed pBI-END1 :: barnasa-barstar.
  • the plants were photographed with a Nikon F-601 M camera, coupled to an MZ8 (Leica) magnifying glass.
  • the anthers of the END1 : barnasa plants were fixed and observed by SEM (scanning electron microscopy) and optical microscopy.
  • a segregation analysis of the andro-sterile phenotype was performed to determine the index of the transgene segregation for 4 independent transgenic lines based on the proportion of sterile plants versus fertile plants obtained. For this, the seeds coming from the crossing of independent Ti lines with wild plants were sown in individual alveoli for each one. The phenotype of the resulting plants was observed in terms of morphology of the anthers and fruit formation to quantify the percentage of sterility of the germinated plants.
  • Nicotiana tabacum plants were carried out following the method described by Horsh, R. B., et al., (1984). Inheritance of functional foreign genes in plants. Science VoI 223: 496-498, with the modifications proposed by Fisher and Guiltinan (1995) [Fisher, D. K., Guiltinan, J., (1995). Rapid, efficient production of homocygous transgenic tobaceous plants with Agrobacterium tumefaciens: a seed to seed protocol. Plant Mol. Biol. Rep. VoI. 13: 278-289].
  • a tube was inoculated with 5 ml of LB medium, 10 mM MgSO 4 , 100 ⁇ g / ml rifampin and 50 ⁇ g / ml kanamycin from a glycerinated with the strain LBA4404 of A. tumefaciens carrying the construction of interest. This place overnight incubated in the dark at 28 0 C with agitation of 200 rpm.
  • MSSABCK regeneration and selection medium MSSABCK regeneration and selection medium
  • MSS medium with 0.2 mg / l IAA (indole acetic acid), 2.2 mg / l 6-BAP, 400 mg / l carbenicillin (to inhibit the growth of Agrobacterium) and 130 mg / l kanamycin (to select the growth of cells that would have incorporated T-DNA)].
  • the plates with the explants were incubated in booths in vitro culture at 25 0 C, under conditions of long - day photoperiod (I see 1.2.2.), And every 7-10 days were changed to new boxes with the same medium.
  • the regenerated shoots (one of each explant, to ensure that independent transformation events were selected) that were appearing were cut avoiding the callus and transferred to bottles 6 cm in diameter by 9.5 cm high with rooting medium MSSACK ( solid MSS medium with 0.2 mg / l of IAA, 200 mg / l of carbenicillin and 130 mg / l of kanamycin). From each rooted outbreak, two internodes were isolated, each with a leaf, which was transferred to MSSABCK medium bottles, from which two were regenerated whole plants.
  • the phenotypic analysis of the first generation (Ti) of END1 :: barnasa plants was carried out by analyzing the morphology of the anthers of these plants by means of photographs taken with a Nikon F-601 M camera, coupled to a magnifying glass MZ8 (Leica) and by observing the anthers through SEM and optical microscopy.
  • Vegetable samples were introduced in 4% p-formaldehyde (w / v) in 1XPBS pH 7.0 immediately after collection. Subsequently, they were subjected to two or three vacuum pulses of 3 minutes each, the fixative solution was changed to a fresh one and they were kept overnight at 4 0 C. After the tissue fixation process they were washed with 1XPBS and dehydrated to absolute ethanol by a series of successive 30-minute washes at 4 0 C in increasing ethanol solutions (15%, 30%, 50%, 70%, 85%, 96%, 100%). From this point on, the samples underwent a different process depending on whether they were included in paraffin (Paraplast Plus, Sigma), resin (Historesin, Leica) or used to be analyzed by scanning electron microscopy. 9.2 Critical point and sample analysis
  • the samples stored in 100% ethanol were dried with liquid CO2 in a Polaron E300 critical point dryer, mounted on metal slides with activated carbon adhesive tape on which they were oriented and dissected conveniently. After assembly, the samples were coated with 200 nm gold-palladium particle shading, under an ionized argon atmosphere in a Sputter Coater SCD005 (BALTEC).
  • the images were obtained by means of the Autobeam program of the ISIS platform (Oxford Instruments), with a scanning speed of 200 s per image, in a JEOL JSM-5410 scanning electron microscope operating under the conditions of 10 kV microanalysis and distance of 25 mm work.
  • transgenic flowers do not show anthers (Figure 2C, right flower) and if we remove sepals and petals we can observe hook-shaped structures instead of the pollen sacs of the anther and a very short filament compared to that of the control stamens ( Figure 2E).
  • Transgenic plants continue to develop and produce branches and flowers (Figure 2C right) while control plants enter senescence and their fruits open to release the seeds inside (Figure 2C left). The half-life of these plants increased three months compared to the control.
  • the transgenic plants generated showed, as in the previous case, the same characteristics in terms of development and flowering:
  • the flowers present in the control plants have normal anthers with pollen inside ( Figure 4A and 4B) and the filament of the yarn has its normal length.
  • the transgenic flowers show deformed and necrotic anthers with abundant trichomes covering their collapsed pollen sacs, which do not show pollen inside ( Figure 4C and 4D).
  • Table 5 shows that the percentage of germination shown for the six lines studied (Bi, B 2 , B 3 , B 8 , Bi 4 , B 2 o) ranges between 30-100%, while the germination percentage of Wild seeds is 88.88%.
  • Table 8A Total values, average values and standard deviations of the number of branches and number of flowers for the andro-sterile plants of the transgenic lines. Wt: wild plants, TR: transgenic plants
  • Table 8 B Total values, average values and standard deviations of the number of branches and number of flowers for the wild plants studied
  • the number of branches produced by plants with an andro-sterile phenotype is approximately 8 times higher than in wild plants, since, while in these the average value of branches obtained is 36 branches, in transgenic plants, the average value of branches obtained is 288.
  • a greater number of branch orders is observed (tertiary and quaternary branches are obtained) while in the wild plants only primary and secondary branches are observed ( Figures 7A and 7B).
  • Figures 8A and 8B Regarding the number of flowers (Tables 8A and 8B), in andro-sterile plants it is observed that the number of flowers is approximately 4.5 times higher than in wild plants ( Figures 8A and 8B).
  • transgenic plants In transgenic plants, lateral meristems remain active for longer than in wild plants; thus, while wild plants are able to live for three months, transgenic plants remain alive in the greenhouse for twice as long (six months).

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Abstract

L'invention concerne un procédé consistant à transformer une matière végétale au moyen d'un gène hybride, qui comprend un gène cytotoxique sous le contrôle du promoteur du gène PsEND1 de petit pois, spécifique aux anthères, à régénérer les plantes et à sélectionner les plantes transgéniques dont l'architecture de leur inflorescence est modifiée par rapport à celle de la plante sylvestre correspondante. La plante transgénique ainsi obtenue présente un patron de ramification plus complexe, avec un plus grand nombre de branches et des branches plus importantes, toutes produisant des fleurs. Applications : agriculture, notamment pour la production de plantes ornementales.
PCT/ES2008/070043 2007-03-08 2008-03-07 Procédé de modification de l'architecture de l'inflorescence de plantes Ceased WO2008107509A1 (fr)

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US10472642B2 (en) 2015-02-05 2019-11-12 British American Tobacco (Investments) Limited Method for the reduction of tobacco-specific nitrosamines or their precursors in tobacco plants

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073088A1 (fr) * 2000-03-31 2001-10-04 Consejo Superior De Investigaciones Cientificas Sequence regulatrice de l'expression specifique d'un gene vers l'anthere et son utilisation dans la production de plantes androsteriles et de semences hybrides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073088A1 (fr) * 2000-03-31 2001-10-04 Consejo Superior De Investigaciones Cientificas Sequence regulatrice de l'expression specifique d'un gene vers l'anthere et son utilisation dans la production de plantes androsteriles et de semences hybrides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BELTRAN, J. P .: "La ingeniería genética de las plantas cultivadas, clave para mejorar la nutrición y la salud humanas.", ANALES DE LA REAL ACADEMIA NACIONAL DE FARMACIA., vol. 71, no. 3, 2005, pages 587 - 608, XP003023726, ISSN: 1697-428X *
ROQUE, E. ET AL.: "The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation.", PLANT CELL REPORTS., vol. 26, no. 3, March 2007 (2007-03-01), pages 313 - 325, XP019490249, ISSN: 1432-203X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10472642B2 (en) 2015-02-05 2019-11-12 British American Tobacco (Investments) Limited Method for the reduction of tobacco-specific nitrosamines or their precursors in tobacco plants

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