WO2000056904A1 - A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape - Google Patents

A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape Download PDF

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Publication number
WO2000056904A1
WO2000056904A1 PCT/BR1999/000018 BR9900018W WO0056904A1 WO 2000056904 A1 WO2000056904 A1 WO 2000056904A1 BR 9900018 W BR9900018 W BR 9900018W WO 0056904 A1 WO0056904 A1 WO 0056904A1
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cells
meristematic
cotton
cocoa
banana
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PCT/BR1999/000018
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French (fr)
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Elíbio Leopoldo RECH FILHO
Francisco José LIMA ARAGAO
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EMBRAPA-Empresa Brasileira De Pesquisa Agropecuária
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Priority to MXPA00011341A priority Critical patent/MXPA00011341A/en
Priority to AU35897/99A priority patent/AU3589799A/en
Priority to PCT/BR1999/000018 priority patent/WO2000056904A1/en
Priority to CN99808805A priority patent/CN1309715A/en
Priority to EP99917693A priority patent/EP1080213A1/en
Publication of WO2000056904A1 publication Critical patent/WO2000056904A1/en
Priority to HK02100891.8A priority patent/HK1039354A1/en

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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the present invention refers to the use of biolistic for introducing exogenous genes associated with a molecule capable of translocate and select transgenic meristematic cells of cotton, coffee, cocoa, banana or grape plants and obtaining transgenic plants by regenerating the transformed tissue.
  • the low obtaintion frequency of the genetically transformed tissue the low capacity of regenerating a fertile plant from said transformed tissue, together with the use of transformation methods, the efficiency of which depends upon the genotype, have rendered it difficult to obtain transgenic plants of most cultivated plants.
  • biolistic process was proposed by Sanford with a view to introduce genetic material into the nuclear genome of higher plants. Since then its universality of application has been appraised, and it has proved to be an effective and simple process for the intro- duction and expression of genes into bacteria, protozoa, fungi, algae, insects, vegetable and animal tissue, as well as isolated organells as chloroplast and mitochondria, according to the results observed by Sanford J C, Smith F D & Russel J.A. (1993) Optimizing the biolistic process for different biological application. Methods in Enzymology :217:413-510 . In the specialized literature there are several other examples of the use of biolistic for the obtain- tion of transgenic organisms such as, for instance, US Patents 5,565,346, US 5,489,520 and WO 96/04392, among others.
  • microprojectiles accelerated at high speed are used for carrying and introducing nucleic acids and other substances into cells and tissues in vivo (Rech E.L. & Aragao F.J.L. (1997).
  • the biolistic process - In: Brasileiro A.C.M. & Carneiro V.T.C. (Ed) - Manual of genetic transformation of plants: EMBRAPA/Cenargen.
  • This process has also been called as method of bombardment with microprojectiles, "gene gun” method, particle- acceleration method, among others.
  • Different systems have been developed and constructed which are capable of accelerating microparticles (made of tungsten or gold), coated with nucleic acids sequences, at speeds higher than 1500 km/h "1 .
  • shock wave can be generated by a chemical explosion (dry gunpowder), a discharge of helium gas under high pres- sure, by vaporization of a drop of water through a electric discharge at high voltage and low capacitance or at low voltage and high capacitance.
  • a plant infection by Agrobacterium is initiated by the penetration thereof into the vegetal tissue through a lesion caused in the plant.
  • the bacteria are attracted towards the vegetal cell by positive chimotactism in connection to phenolic compounds, sugars and aminoacids (signal-molecules) that exude the damaged tissue in response to the wound.
  • the signal-molecules also activates the genes of the virulence region located in a plasmid referred to as Ti (Tumor inducing), and present in all virulence strains of Agrobacterium.
  • T-region another Ti plasmid region from the bacteria to the vegetal cell genome
  • T-DNA transferred DNA
  • the synthesized vegetal hormones modify the hormonal balance thus causing a not controllable multiplication of the transformed cells as well as of the pe- ripheral cells.
  • Such random proliferation of the cells leads to the formation of a tumor.
  • the opines are simple molecules resulting form condensation of an aminoacid with carbohydrate.
  • the opines are excreted from the cells and cataboiized specifically by the infecting bacteria present in the tumor intercellular spaces.
  • Several opines types have been identified (nopaline, octopine, agropine and others), and only the tumor inducing strain is capable of catabolizing its own opine.
  • T-DNA genes integrated into the vegetal cell genome these genes do not participate of transference and integration processes, for which only the vir region and the T-DNA ends (constituted by repetitive direct sequences with 25 base pairs) are essential. Therefore, all genes present in the T-DNA may be deleted by a double recombination process, whereby producing de- sarmed strains but not interfering in the transformation process. Those strains maintain the function virulence region, the original T-DNA genes being substituted by marking and interesting genes flanked by the repetitive sequences essential for the transference process (Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M & Schell JS. (1983) Ti plasmid vector for introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO Journal. 2:2143-2150). For this reason, the strains containing a modified T-DNA do not induce tumors and maintain the transformation capability.
  • the infected tissues should usually be submitted to a step of production of callus which should present the ability of further forming new embryos or meristems.
  • This method is very inefficient and was disclosed for Arabidopsis thaliana (Feldmann KA & Marks MD. (1987) "Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Molecular & General Genetics. 208:1-9).
  • Another publication disclose a similar methodology for soybean (Chee P, Fober KA & Sligntom JL.
  • Agrobacterium system as a tool for the process of obtaining transgenic plants depends upon two factors: firstly, there must be a positive pathogen-host relationship. Some dicotyledons and most of the monocotyledons and gimnosperms are not susceptible or present very low susceptibility towards Agrobacterium (Christou P. (1993) Phylosophy and practice of variety-independent gene transfer into recalcitrant crops. In Vitro Cellular & Developmental Biolog 29P:119-124). Secondly, it is necessary to develop tissue culture systems in order to obtain explants that have organogenic capability and are able to be transformed.
  • transgenic plants from the apical region of embrionic axis there are two essential requirements, namely: 1) introduction of exogenous genes with high frequencies into the cells of the apical regions, and integration of exogenous genes into the vegetable genome, and 2) regeneration and production of fertile transgenic plants from the resulting transformed cells.
  • the objective of the present invention to provide a process with high production frequency for the obtaintion of transgenic higher plants and which enables the selection of the transformed cells containing a gene exhibiting resistance to a molecule that translocate through the plant tissues, concentrating in the meristematic region.
  • the present invention refers to a process for selecting transgenic meristematic cells in cotton, coffee, cocoa, banana or grape plants which comprises the steps of:
  • step (b) inducing the multiple shooting of the cells in the meristematic region modified in step (a) by cultivating said meristems, tissues or organs containing meristems in a medium containing a multiple shooting inducer; and c) selecting the transgenic meristematic cells as obtained in step (b) by further cultivation of said meristems, tissues or organs containing meristems in a medium containing a molecule which translocate and concentrates in the apical meristematic; or
  • step (d) selecting the transgenic meristematic cells of the apical region as obtained in step (b) by applying a molecule which translocate and concentrates in the apical meristematic region of embryos to the multiple shoots of the apical region.
  • transgenic meristematic cells comprising a gene that shows resistance to a molecule capa- ble of translocating through the tissues and concentrating in the meristematic region of embryonic axis or tissues containing meristems.
  • This selection is related to the steps of multiple shooting and ulterior production of fertile transgenic plants at a high frequency in the range of 1 % to 20%.
  • This value represents a magnitude of about 2 to 100 times as high as the frequencies obtained by the processes known at present, which are of the order of 0.02% - 0.5%.
  • the process of the present invention enables the obtaintion of transgenic plants in a period of time shorter than those described in the prior art.
  • the embryonic axis of the apical meristematic cells or tissues or organs containing meristems in cotton, coffee, cocoa, banana or grape plants to be transformed are prepared in laboratories in a conventional way for the bombardment (biolistic) process and/or inoculation with Agrobacterium.
  • the genes to be used for the bombardment will depend upon the specific objective of each process in question, that is to say, they will be chosen in accordance with the new characteristic which one desires to impart to the transformed plant. For instance, in the case where the objective of the process is to obtain plants resistant to herbicides, genes which impart such a resistance to herbicides would be utilized.
  • the embryonic axes or tissues containing meristems are then contacted with a culture medium containing a multiple shoot inducer and a selective agent, and should be maintained in this medium for a period of time sufficient to guarantee the desired selection and induction, preferably during a period ranging from 1 to 30 days.
  • cytokinins namely 6-benzylaminopurin (BAP) are used as a multiple shooting-inducing agent and the herbicides such as imazapyr (commercialized by American Cyanamid) or basta (commercialized by AgrEvo) are used as the selective agent.
  • a molecule which concentrates in the apical meristematic region such as the above-cited herbicides, for instance, is carried through the vascular system of the embryonic axes, then concentrating in the meristematic region acting as an selective agent. In this way, it is possible to carry out the selection of the cells without deleterious effects to the embryonic axes.
  • Mature cotton seeds were disinfected in 70%-ethanol for 1 minute and in 4.0%- calcium hypochlorite for 7 minutes.
  • the disinfected seeds were washed with sterile distilled water and incubated for 16-18 hours in sterile distilled water at room temperature. The water was removed and the seeds were further incubated in a dark environment for 16-18 hours at room temperature.
  • cocoa coffee and grape the seeds were disinfected in 70%-ethanol for 1 minute and in 1.0%-sodium hypochlorite for 20-30 minutes.
  • the disin- fected seeds were washed with sterile distilled water and incubated for 16-18 hours in sterile distilled water at room temperature.
  • the cotton, cocoa and grape seeds were opened for removal of the embryonic axes .
  • the primary leaves were cut so as to expose the region of the apical meristem.
  • the embryonic axes were removed and cultivated during 1 week in WPM me- dium. After this time, the primary leaves were removed in order to expose the meristematic region.
  • the embryonic axes were placed in the culture plates containing the bombardment medium (10-15 axes/plate), said bombardment medium (herein after called BM) consisting of a medium of Murashige and Skoog (1962), supplemented with 3% of sucrose, 0.7% phytagel, pH 5.7.
  • BM said bombardment medium
  • the axes were arranged in a circle, equidistant by 6 - 12 mm from the center of the plate and with a region of the apical meristem directed upwards.
  • the material to be transformed had been positioned on the plate containing the bombardment medium BM, it was bombarded with the gene of interest.
  • various vectors containing genes which create resistance to the herbicide imazapyr or basta were used.
  • Selected banana tissues were disinfected in 70%-ethanol for 1 minute and in 4.0%-9.0% sodium hypochlorite for 20-30 minutes. The disinfected tissues were washed with sterile distilled water.
  • the tissues were placed in the culture plates containing the bombardment medium (10-15 tissues/plate), said bombardment medium (herein after called BM) consisting of a medium of Murashige and Skoog (1962), supplemented with 3% of sucrose, 0.7% phytagel, pH 5.7.
  • BM bombardment medium
  • the tissues were arranged in a circle, equidistant by 6 - 12 mm from the center of the plate and with a region of the apical meristem directed upwards.
  • the material to be transformed had been positioned on the plate containing the bombardment medium BM, it was bombarded with the gene of interest.
  • various vectors containing genes which create resistance to the herbicide imazapyr or basta were used.
  • microparticles responsible for carrying the exogenous DNA into the cells were sterilized and washed.
  • 60 mg of microparticles of tungsten M10 (Sylvania) or gold (Aldrich, 32,658-5) were weighed, transferred to a microcentrifuge tube, to which 1.0 ml of 70% ethanol was added.
  • the mixture was vigorously stirred and kept under stirring for 15 minutes at the lowest speed of the stirrer. 15,000g was centrifuged for 5 minutes and the supernatant was removed and discarded with the help of a micropipette of 1 ,000 ⁇ l.
  • 1 ml of sterile distilled water was added and mixed vigorously in a stirrer and centrifuged as in the preceding step. The supernatant was discarded, and the washing operation was repeated two more times.
  • the supernatant was discarded, and the microparticles were again suspended in 1 ml of 50% glycerol (v/v). Equal parts of glycerol and distilled wa- ter were mixed, the mixture was autoclaved and kept at room temperature.
  • the exogenous DNA was precipitated onto the microparticles and, for this purpose, an aliquot part of 50 ⁇ l of the microparticle suspension (60mg/ml) was transferred to a microcentrifuge tube. From 5 to 8 ml of DNA (1 mg/ ⁇ l) was added. The mixture was rapidly homogenized (3 - 5 seconds) by stirring the outer part of the tube with help of the fingers. 50 ⁇ l of CaCI 2 2.5 M was added, rapidly homogenized and 20 ⁇ l of spermidine 0.1 M (Sigma S-0266) was added, which is an extremely hygroscopic and oxidizable reactant.
  • the resulting mixture was incubated at room temperature under slow stirring for 10 minutes, centrifuged for 10 sec, and the supernatant was carefully removed. 150 ⁇ l of absolute ethanol was added, and then the outer part of the tube was again stirred with the help of the fingers. The resulting mixture was centrifuged at 15,000 g for 10 seconds, and the supernatant was removed. The preceding step was repeated, adding 24 ⁇ l of absolute ethanol, vigorously homogenized and sonicated for 1 - 2 seconds.
  • the discs containing the microparticles covered with DNA were immediately stocked on a plate containing drying material (silica gel) and placed in a desiccator.
  • the bombardment of the apical meristematic region of the embryonic axis of higher plants was carried out with a microparticle accelerator which utilizes high pressure of helium gas, as described in Aragao FJL, Barros LMG, Brasileiro ACM, Ribeiro SG, Smith FD , Sanford JC, Faria JC, Rech EL : Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theoretical Applied Genetics 93:142-150 (1996).
  • Example 1 Obtaintion of Transgenic Cotton Plants (Gossypium spp.), through the selection with the herbicides imazapyr or ammonium glyphosinate Immediately after the bombardment of the embryonic axes with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing multiple shooting-inducing and selecting medium (ISM) (MS medium supplemented with 22.2 ⁇ l BAP, 3% glucose or sucrose, 500-1000 nM of Imazapyr, 0.8% of agar, pH 5.7).
  • ISM multiple shooting-inducing and selecting medium
  • the bombarded embryonic axes remained immersed in the ISM for 2-3 weeks and were transferred to plates with elongation medium (EM) (SM medium, 3% sucrose, 0.8% of agar, pH 5.7) and kept in a growth chamber at a temperature of 27° C with 16 hours photoperiod (50 umols m ⁇ 2 s "1 ).
  • EM elongation medium
  • the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1 % of sucrose, 0.8% of agar, 0.1 % carbon black, pH 5.7), with photoperiod of 16 hours (50 ⁇ mols m "2 s "1 ) at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days.
  • the elastic band was removed and after 6-7 days the plastic bag was also removed.
  • the plantlets were transferred to vessels containing soil for the production of seeds.
  • Example 2 Obtaintion of Transgenic Coffee Plants (Cofea. spp), with the herbicides imazapyr or ammonium glyphosinate
  • the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing multiple shooting inducing medium (IM) (MS medium supplemented with 11.1 to 44.4 ⁇ M BAP, 3% sucrose, 0.6% of agar, pH 5.7).
  • BM bombardment medium
  • IM multiple shooting inducing medium
  • the embryonic axes were, then, transferred to plates with culture medium containing herbicide (CMH) (SM medium, 3% sucrose, 100-500 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in a growth chamber at a temperature of 27° C with 16 hours photoperiod (50 ⁇ mols m-2s-1) until the induction of multiple shoots.
  • CMH herbicide
  • the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1%) or imazapyr (0.2%), were applied directly to the multiple shoots.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1 % of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 ⁇ mols m " 2 s "1 ) at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels con- taining an autoclaved soil: vermiculite (1 :1) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days.
  • the elastic band was removed and after 6-7 days the plastic bag was also removed.
  • the plantlets were transferred to vessels containing soil for the production of seeds.
  • Example 3 Obtaintion of Transgenic Cocoa Plants (Theobroma cacao .), through selection with the herbicides imazapyr or ammonium glyphosinate
  • the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing induction medium IM (MS medium supplemented with 11.1 to 44.4 ⁇ M BAP, 3% of sucrose, 0.6% of agar, pH 5.7).
  • BM bombardment medium
  • IM MS medium supplemented with 11.1 to 44.4 ⁇ M BAP, 3% of sucrose, 0.6% of agar, pH 5.7.
  • the embryonic axes were transferred to plates with culture medium comprising the herbicide (MCH) (MS medium, 3% of sucrose, 500- 1000 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in growth chamber at a temperature of 27° C with 16 hours photoperiod (50 ⁇ mols m "2 s "1 ) until the induction of the multiple shootings.
  • MCH herbicide
  • the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium
  • MS1S MS, 1% of sucrose, 0.8% of agar, pH 5.7
  • photoperiod 50 ⁇ mols m " 2 s ⁇ 1 ) at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days.
  • the elastic band was removed and after 6-7 days the plastic bag was also removed
  • the plantlets were transferred to vessels containing soil for the production of seeds.
  • Example 4 Obtaintion of Transgenic Banana Plants (Musa spp), through selection with the herbicides imazapyr or ammonium glyphosinate
  • the tissues containing the meristems were cultivated in the same culture medium (MB) for 7 days at a temperature of 27° C with 16 hours photoperiod (50 ⁇ mols m "2 s "1 ) for induction of the multiple shoots.
  • MB culture medium
  • the embryonic axes which germinated were transferred to a "Magenta"-type box containing the culture medium MSBH (MS medium supplemented with 10-20 ⁇ M BAP, 3% of sucrose, 50-300 nM of IMAZAPYR, 0.8% of agar, pH 5.7), for 7 days at a temperature of 27° C with 16 hours photoperiod (50 umols m "2 s "1 ) to reduce the total number of multiple shoots.
  • MSBH MS medium supplemented with 10-20 ⁇ M BAP, 3% of sucrose, 50-300 nM of IMAZAPYR, 0.8% of agar, pH 5.7
  • the embryonic axes were again transferred to the "Magenta"-type culture box containing the culture medium MS3S (MS supplemented with 5-10 ⁇ M BAP, 3% of sucrose, 0.8% of agar, pH 5.7) at a temperature of 27° C with 16 hours photoperiod (50 ⁇ mols m "2 s "1 ) to enable the elongation of the multiple shoots.
  • MS3S MS supplemented with 5-10 ⁇ M BAP, 3% of sucrose, 0.8% of agar, pH 5.7
  • photoperiod 50 ⁇ mols m "2 s "1 .
  • the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium MS1S (MS, 1 % of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 ⁇ mols rr ⁇ 2 s "1 ) at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1 ) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed.
  • the plantlets were transferred to vessels containing soil for the production of seeds.
  • Example 5 Obtaintion of Transgenic Grape Plants (Vitis Vinifera L,), trough se- lection with the herbicides imazapyr or ammonium glyphosinate
  • the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing induction medium IM (MS medium supplemented with 22.2 to 44.4 ⁇ M BAP, 3% of sucrose, 0.6% of agar, pH 5.7).
  • BM bombardment medium
  • IM MS medium supplemented with 22.2 to 44.4 ⁇ M BAP, 3% of sucrose, 0.6% of agar, pH 5.7.
  • the embryonic axes were transferred to plates with culture medium comprising the herbicide (MCH) (MS medium, 3% of sucrose, 100-600 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in growth chamber at a temperature of 27° C with 16 hours photoperiod (50 ⁇ mols m "2 s "1 ) until the induction of the multiple shootings.
  • MCH herbicide
  • the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1% of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 ⁇ mols m " V) at 27° C to enable the plantlets to grow and take root.
  • MS1 S MS, 1% of sucrose, 0.8% of agar, pH 5.7
  • photoperiod 16 hours at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days.
  • the elastic band was removed and after 6-7 days the plastic bag was also removed
  • the plantlets were transferred to vessels containing soil for the production of seeds.
  • the shoots that reached 2 - 4 cm length were transferred to a culture medium
  • MS1 S MS, 1% of sucrose, 0.8% of agar, pH 5.7
  • photoperiod 50 ⁇ mols m ⁇ 2 s "1 ) at 27° C to enable the plantlets to grow and take root.
  • a section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
  • the shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
  • the plantlets were covered with a plastic bag closed with an elastic band for 7 days.
  • the elastic band was removed and after 6-7 days the plastic bag was also removed.
  • the plantlets were transferred to vessels containing soil for the production of seeds.

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Abstract

The present invention refers to a process for selecting transgenic meristematic cells in cotton, coffee, cocoa, banana or grape plants and the consequent production of transgenic plants. The process comprises the steps of introducing exogenous genes into cells of the apical meristem of embryonic axes or tissues or organs containing meristems of cotton, coffee, cocoa, banana or grape plants; induction of multiple shooting of the cells in the apical meristematic region modified in the preceding step by cultivating their embryonic axes or tissues containing meristems in a medium comprising a multiple shooting inducer; and selecting the transgenic meristematic cells of the apical region, transformed by further cultivation of said embryonic axes or tissues containing meristems in a medium containing a selecting agent or by applying a selective agent to the multiple shootings originated from the cells in the meristematic region.

Description

A PROCESS FOR THE SELECTION OF TRANSGENIC CELLS IN THE MERISTEMATIC REGION OF COTTON, COFFEE, COCOA, BANANA OR GRAPE
Field of the invention The present invention refers to the use of biolistic for introducing exogenous genes associated with a molecule capable of translocate and select transgenic meristematic cells of cotton, coffee, cocoa, banana or grape plants and obtaining transgenic plants by regenerating the transformed tissue.
Background of the invention The use of genetic engineering techniques for introducing genes which are responsible for agronomic characteristics of interest may facilitate the development of new varieties of cultivated plants. The obtaintion of a transgenic plant requires methods of introducing the exogenous DNA into the vegetable tissue and regenerating the whole plant from such genetically transformed tissue. Depending upon the species to be transformed, various types of tissue have been used for the introduction of an exogenous DNA, the meristematic tissue been preferably employed in various transformation processes, primarily due to the ease regeneration of a plant from this type of tissue. Various processes have been proposed for introducing exogenous genes into apical meristematic cells of superior plants, among which the following can be pointed out: a) the biolistic system and b) the _Agrobacterium system. The introduction and integration of exogenous DNA into cells of superior plants have been demonstrated by various scientists and described in different publications such as (McCabe D., Martinelli B.J. (1993) Transformation of elite cotton cultivars via particle bombardment of meristems. Bio/Technology 11 :596-598; Keller G., Spatoa L, McCabe D., Martinelli B., Swain W., John M.E. (1997) Transgenic cotton resistant to herbicide bialaphos. Transgenic Research 6:385-392).
However, the low obtaintion frequency of the genetically transformed tissue, the low capacity of regenerating a fertile plant from said transformed tissue, together with the use of transformation methods, the efficiency of which depends upon the genotype, have rendered it difficult to obtain transgenic plants of most cultivated plants.
With the development of the biolistic process for the direct introduction of genes into vegetable cells at the end of the '80 (Sanford J.C. Klein T.M., Wolf E.D. & Allen N. (1987). Delivery of substances into cell tissues using a particle bombardment process; Journal of Particle Science and Technology, 5:27-37), a great number of transgenic plants of several species have been obtained, including those species which proved to be recalcitrant to the transformation by the using other methods. This is due to the fact that it has become possible to introduce and express exogenous genes in any kind of vegetable tissue. Thus, any type of tissue having a potential ability to regenerate a whole fertile plant is suitable for transformation.
The biolistic process was proposed by Sanford with a view to introduce genetic material into the nuclear genome of higher plants. Since then its universality of application has been appraised, and it has proved to be an effective and simple process for the intro- duction and expression of genes into bacteria, protozoa, fungi, algae, insects, vegetable and animal tissue, as well as isolated organells as chloroplast and mitochondria, according to the results observed by Sanford J C, Smith F D & Russel J.A. (1993) Optimizing the biolistic process for different biological application. Methods in Enzymology :217:413-510 . In the specialized literature there are several other examples of the use of biolistic for the obtain- tion of transgenic organisms such as, for instance, US Patents 5,565,346, US 5,489,520 and WO 96/04392, among others.
In biolistic, microprojectiles accelerated at high speed are used for carrying and introducing nucleic acids and other substances into cells and tissues in vivo (Rech E.L. & Aragao F.J.L. (1997). The biolistic process - In: Brasileiro A.C.M. & Carneiro V.T.C. (Ed) - Manual of genetic transformation of plants: EMBRAPA/Cenargen. This process has also been called as method of bombardment with microprojectiles, "gene gun" method, particle- acceleration method, among others. Different systems have been developed and constructed which are capable of accelerating microparticles (made of tungsten or gold), coated with nucleic acids sequences, at speeds higher than 1500 km/h"1. All these systems are based on the generation of a shock wave with enough energy for displacing a carrying membrane containing the microparticles coated with DNA. The shock wave can be generated by a chemical explosion (dry gunpowder), a discharge of helium gas under high pres- sure, by vaporization of a drop of water through a electric discharge at high voltage and low capacitance or at low voltage and high capacitance.
Those systems which use helium gas under high pressure and electric discharge have shown a wide spectrum of utilization. The accelerated particles penetrate the cellular wall and membrane in a non-lethal way, locating themselves randomly in the cellular organ- ells. Then the DNA is dissociated from the microparticles by the action of the cellular liquid, and the process of integrating the exogenous DNA in the genome of the organism to be modified takes place (Yamashita T. lada, A. & Morikawa H. (1991) - Evidence that more than 90% of b-glucuronidase-expressing cells after particle bombardment directly receive the for- eign gene in their nucleus; Plant Physiol. 97:829-831). In spite of the efficiency and universality of utilization of the biolistic process, it depends upon the optimization of various physical and biological parameters, which is fundamental to the effective introduction of heterolo- gous genes into a vegetable tissue.
In 1985, a transference of exogenous genes to a plant was described for the first time, using a genetically modified Agrobacterium tumefaciens strain (Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG & Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229-1231). Since then, a great number of species have been transformed through Agrobacterium system. The obtaintion of transgenic plants through the introduction of genes mediated by Agrobacterium is based on the capabil- ity of such bacteria for transferring specific sequences from their DNA to the vegetal genome. A plant infection by Agrobacterium is initiated by the penetration thereof into the vegetal tissue through a lesion caused in the plant. The bacteria are attracted towards the vegetal cell by positive chimotactism in connection to phenolic compounds, sugars and aminoacids (signal-molecules) that exude the damaged tissue in response to the wound. In addition to attract the bacteria, the signal-molecules also activates the genes of the virulence region located in a plasmid referred to as Ti (Tumor inducing), and present in all virulence strains of Agrobacterium. After being activated, the virulence region genes induce the transference of another Ti plasmid region (called T-region) from the bacteria to the vegetal cell genome (Jouanin L, Brasileiro ACM, Lepe JC, Pilate G & Corn D. (1993) Genetic transfor- mation: a short review of methods and their applications, results and perspectives for forest trees. Annales des Sciences Forestieres. 50:325-336.; Tinland B. (1996) The integration of T-DNA into plant genomes. Trends in Plant Sciences. 1 :178-184). Once integrated in vegetal genome, the T-region is referred to as T-DNA (transferred DNA) where it basically induces the synthesis of vegetal hormones and opines. The synthesized vegetal hormones (auxines and cytokinins) modify the hormonal balance thus causing a not controllable multiplication of the transformed cells as well as of the pe- ripheral cells. Such random proliferation of the cells leads to the formation of a tumor. The opines, are simple molecules resulting form condensation of an aminoacid with carbohydrate. The opines are excreted from the cells and cataboiized specifically by the infecting bacteria present in the tumor intercellular spaces. Several opines types have been identified (nopaline, octopine, agropine and others), and only the tumor inducing strain is capable of catabolizing its own opine.
Although the formation of the tumor is a result of the expression of T-DNA genes integrated into the vegetal cell genome, these genes do not participate of transference and integration processes, for which only the vir region and the T-DNA ends (constituted by repetitive direct sequences with 25 base pairs) are essential. Therefore, all genes present in the T-DNA may be deleted by a double recombination process, whereby producing de- sarmed strains but not interfering in the transformation process. Those strains maintain the function virulence region, the original T-DNA genes being substituted by marking and interesting genes flanked by the repetitive sequences essential for the transference process (Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M & Schell JS. (1983) Ti plasmid vector for introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO Journal. 2:2143-2150). For this reason, the strains containing a modified T-DNA do not induce tumors and maintain the transformation capability.
The transference of genes into vegetable cells through Agrobacterium tumefaci- ens is still one of the most used methods (De Block M. (1993) The cell biology of plant transformation: current state, problems, prospects and the implication for the plant breeding. Euphytica. 71 :1-14.). This system is simple and efficient and not very exprensive in several cases. Generally, the resulting plants show a low number of copies of exogenous genes, and the DNA delimited by the two T-DNA ends is exclusively transferred (De Block M. (1993) The cell biology of plant transformation: current state, problems, prospects and the implication for the plant breeding. Euphytica. 71 :1-14).
The infected tissues should usually be submitted to a step of production of callus which should present the ability of further forming new embryos or meristems. However, it is possible directly to infect embryos taken from mature seeds. This method is very inefficient and was disclosed for Arabidopsis thaliana (Feldmann KA & Marks MD. (1987) "Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Molecular & General Genetics. 208:1-9). Another publication disclose a similar methodology for soybean (Chee P, Fober KA & Sligntom JL. (1989) Transformation of soybean (Glycine max (L.) Merril) by infecting germinating seeds with Agrobacterium timefaciens. Plant Physiology. 91 :1212-1218), but it does not present any evidence of a stable transformation.
The use of the Agrobacterium system as a tool for the process of obtaining transgenic plants depends upon two factors: firstly, there must be a positive pathogen-host relationship. Some dicotyledons and most of the monocotyledons and gimnosperms are not susceptible or present very low susceptibility towards Agrobacterium (Christou P. (1993) Phylosophy and practice of variety-independent gene transfer into recalcitrant crops. In Vitro Cellular & Developmental Biolog 29P:119-124). Secondly, it is necessary to develop tissue culture systems in order to obtain explants that have organogenic capability and are able to be transformed.
Recently a biolistic method in combination with the Agrobacterium system (agrobiolistic) has been used for obtaining transgenic plants. Microparticles made of tungsten are accelerated at very high speed towards the vegetal tissue. As a result, microw- ounds are produced that allow the infection caused by the agrobacteria. This method has turned it possible to obtain tobacco transgenic plants. (Bidney DL. Scelonge CJ, Martich J, Burrus M, Sims L & Huffman G. (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Molecular Biology. 18:301-313), sun-flower (Malone-Schoneberg J, Scelonge CJ, Martich J, Burrus M & Bidney DL. (1994) Stable Plant Science 103:199-207), banana (May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzem CJ (1995) Generation of transgenic banana (Musa acumi- nata) plants via /Agroόacter/um-mediated transformation. Bio/technology 13:486-492) and grape (Scorza R, Cordts JM, Ramming DN, Emers RL. (1995) Transformation of grape (Vitris vinifera L.) sygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Reports 14:589-592).
For the obtaintion of transgenic plants from the apical region of embrionic axis , there are two essential requirements, namely: 1) introduction of exogenous genes with high frequencies into the cells of the apical regions, and integration of exogenous genes into the vegetable genome, and 2) regeneration and production of fertile transgenic plants from the resulting transformed cells.
With the development of the transformation systems it was possible to modify meristems cells. However, the development and further production of fertile transgenic plants require the regeneration and production of the plant from the transformed cells.
During the last few decades several attempts have been made to obtain the regeneration of fertile plants of commercially important plants. For instance, some methodologies of multiple shooting of apical and lateral meristems of embryos in different plants such as soybean, bean, eucalyptus, cocoa, pineapple, have been developed.
Other regeneration systems developed for certain plants such as soybean, bean, cotton and peanuts involve the induction of somatic embryogenesis from cultivated mature and immature embryos. The systems already known for the obtaintion of transgenic plants based on the transformation of meristematic cells present the disadvantages of impossibility of selecting the transformed cells, low production frequencies of transgenic plants and high frequency of chimeras (plants with an organ or groups of some transgenic cells and other non-transgenic cells).
It is, therefore, the objective of the present invention to provide a process with high production frequency for the obtaintion of transgenic higher plants and which enables the selection of the transformed cells containing a gene exhibiting resistance to a molecule that translocate through the plant tissues, concentrating in the meristematic region.
Summary of the invention
The present invention refers to a process for selecting transgenic meristematic cells in cotton, coffee, cocoa, banana or grape plants which comprises the steps of:
a) introducing exogenous genes into cells of meristem of embryonic axis, tissues or organs containing meristems of cotton, coffee, cocoa, banana or grape plants;
b) inducing the multiple shooting of the cells in the meristematic region modified in step (a) by cultivating said meristems, tissues or organs containing meristems in a medium containing a multiple shooting inducer; and c) selecting the transgenic meristematic cells as obtained in step (b) by further cultivation of said meristems, tissues or organs containing meristems in a medium containing a molecule which translocate and concentrates in the apical meristematic; or
d) selecting the transgenic meristematic cells of the apical region as obtained in step (b) by applying a molecule which translocate and concentrates in the apical meristematic region of embryos to the multiple shoots of the apical region.
Detailed description of the invention
It has now been surprisingly found that it is possible to effect the selection of transgenic meristematic cells comprising a gene that shows resistance to a molecule capa- ble of translocating through the tissues and concentrating in the meristematic region of embryonic axis or tissues containing meristems. This selection is related to the steps of multiple shooting and ulterior production of fertile transgenic plants at a high frequency in the range of 1 % to 20%. This value represents a magnitude of about 2 to 100 times as high as the frequencies obtained by the processes known at present, which are of the order of 0.02% - 0.5%. In addition, the process of the present invention enables the obtaintion of transgenic plants in a period of time shorter than those described in the prior art.
The process as claimed now is suitable for transformation, regeneration and selection of different higher plants.
According to the present invention, the embryonic axis of the apical meristematic cells or tissues or organs containing meristems in cotton, coffee, cocoa, banana or grape plants to be transformed are prepared in laboratories in a conventional way for the bombardment (biolistic) process and/or inoculation with Agrobacterium. Of course, the genes to be used for the bombardment will depend upon the specific objective of each process in question, that is to say, they will be chosen in accordance with the new characteristic which one desires to impart to the transformed plant. For instance, in the case where the objective of the process is to obtain plants resistant to herbicides, genes which impart such a resistance to herbicides would be utilized.
After the bombardment, the embryonic axes or tissues containing meristems are then contacted with a culture medium containing a multiple shoot inducer and a selective agent, and should be maintained in this medium for a period of time sufficient to guarantee the desired selection and induction, preferably during a period ranging from 1 to 30 days. In a preferred embodiment of the invention, cytokinins, namely 6-benzylaminopurin (BAP) are used as a multiple shooting-inducing agent and the herbicides such as imazapyr (commercialized by American Cyanamid) or basta (commercialized by AgrEvo) are used as the selective agent. An additional advantage of the present invention is that the now claimed process enables the multiple shooting to be completed in a relatively short period of time, thus avoiding the occurrence of genetic variations that are common to other known processes.
During the step for selecting the transformed cells, a molecule which concentrates in the apical meristematic region, such as the above-cited herbicides, for instance, is carried through the vascular system of the embryonic axes, then concentrating in the meristematic region acting as an selective agent. In this way, it is possible to carry out the selection of the cells without deleterious effects to the embryonic axes.
The invention can be better understood with the help of the examples given below, which are merely illustrative, and the parameters and conditions described should not be regarded as being limiting of the invention.
Examples
Preparation of the embryonic axes of cotton, coffee, cocoa and grape for bombardment (biolistic )
Mature cotton seeds were disinfected in 70%-ethanol for 1 minute and in 4.0%- calcium hypochlorite for 7 minutes. The disinfected seeds were washed with sterile distilled water and incubated for 16-18 hours in sterile distilled water at room temperature. The water was removed and the seeds were further incubated in a dark environment for 16-18 hours at room temperature. In the case of cocoa, coffee and grape the seeds were disinfected in 70%-ethanol for 1 minute and in 1.0%-sodium hypochlorite for 20-30 minutes. The disin- fected seeds were washed with sterile distilled water and incubated for 16-18 hours in sterile distilled water at room temperature.
The cotton, cocoa and grape seeds were opened for removal of the embryonic axes . The primary leaves were cut so as to expose the region of the apical meristem. In the case of coffe, the embryonic axes were removed and cultivated during 1 week in WPM me- dium. After this time, the primary leaves were removed in order to expose the meristematic region. Then the embryonic axes were placed in the culture plates containing the bombardment medium (10-15 axes/plate), said bombardment medium (herein after called BM) consisting of a medium of Murashige and Skoog (1962), supplemented with 3% of sucrose, 0.7% phytagel, pH 5.7. The axes were arranged in a circle, equidistant by 6 - 12 mm from the center of the plate and with a region of the apical meristem directed upwards.
Once the material to be transformed had been positioned on the plate containing the bombardment medium BM, it was bombarded with the gene of interest. In this case, various vectors containing genes which create resistance to the herbicide imazapyr or basta were used.
Preparation of tissues containing apical meristems for bombardment
Selected banana tissues were disinfected in 70%-ethanol for 1 minute and in 4.0%-9.0% sodium hypochlorite for 20-30 minutes. The disinfected tissues were washed with sterile distilled water.
Then the tissues were placed in the culture plates containing the bombardment medium (10-15 tissues/plate), said bombardment medium (herein after called BM) consisting of a medium of Murashige and Skoog (1962), supplemented with 3% of sucrose, 0.7% phytagel, pH 5.7. The tissues were arranged in a circle, equidistant by 6 - 12 mm from the center of the plate and with a region of the apical meristem directed upwards.
Once the material to be transformed had been positioned on the plate containing the bombardment medium BM, it was bombarded with the gene of interest. In this case, various vectors containing genes which create resistance to the herbicide imazapyr or basta were used.
Preparation of the Microparticles and Meristems Bombardment
The microparticles responsible for carrying the exogenous DNA into the cells were sterilized and washed. 60 mg of microparticles of tungsten M10 (Sylvania) or gold (Aldrich, 32,658-5) were weighed, transferred to a microcentrifuge tube, to which 1.0 ml of 70% ethanol was added. The mixture was vigorously stirred and kept under stirring for 15 minutes at the lowest speed of the stirrer. 15,000g was centrifuged for 5 minutes and the supernatant was removed and discarded with the help of a micropipette of 1 ,000 μl. 1 ml of sterile distilled water was added and mixed vigorously in a stirrer and centrifuged as in the preceding step. The supernatant was discarded, and the washing operation was repeated two more times.
After the last washing, the supernatant was discarded, and the microparticles were again suspended in 1 ml of 50% glycerol (v/v). Equal parts of glycerol and distilled wa- ter were mixed, the mixture was autoclaved and kept at room temperature.
Then the exogenous DNA was precipitated onto the microparticles and, for this purpose, an aliquot part of 50 μl of the microparticle suspension (60mg/ml) was transferred to a microcentrifuge tube. From 5 to 8 ml of DNA (1 mg/μl) was added. The mixture was rapidly homogenized (3 - 5 seconds) by stirring the outer part of the tube with help of the fingers. 50 μl of CaCI2 2.5 M was added, rapidly homogenized and 20 μl of spermidine 0.1 M (Sigma S-0266) was added, which is an extremely hygroscopic and oxidizable reactant.
The resulting mixture was incubated at room temperature under slow stirring for 10 minutes, centrifuged for 10 sec, and the supernatant was carefully removed. 150 μl of absolute ethanol was added, and then the outer part of the tube was again stirred with the help of the fingers. The resulting mixture was centrifuged at 15,000 g for 10 seconds, and the supernatant was removed. The preceding step was repeated, adding 24 μl of absolute ethanol, vigorously homogenized and sonicated for 1 - 2 seconds.
Then samples of 3.2 μl of the solution was distributed in the central region of each carrying membrane previously positioned on a membrane support. Each precipitation was sufficient for preparing 6 carrying membranes containing microparticles covered with the
DNA of interest. The discs containing the microparticles covered with DNA were immediately stocked on a plate containing drying material (silica gel) and placed in a desiccator.
The bombardment of the apical meristematic region of the embryonic axis of higher plants was carried out with a microparticle accelerator which utilizes high pressure of helium gas, as described in Aragao FJL, Barros LMG, Brasileiro ACM, Ribeiro SG, Smith FD , Sanford JC, Faria JC, Rech EL : Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theoretical Applied Genetics 93:142-150 (1996).
Example 1 : Obtaintion of Transgenic Cotton Plants (Gossypium spp.), through the selection with the herbicides imazapyr or ammonium glyphosinate Immediately after the bombardment of the embryonic axes with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing multiple shooting-inducing and selecting medium (ISM) (MS medium supplemented with 22.2 μl BAP, 3% glucose or sucrose, 500-1000 nM of Imazapyr, 0.8% of agar, pH 5.7). The bombarded embryonic axes remained immersed in the ISM for 2-3 weeks and were transferred to plates with elongation medium (EM) (SM medium, 3% sucrose, 0.8% of agar, pH 5.7) and kept in a growth chamber at a temperature of 27° C with 16 hours photoperiod (50 umols m~ 2s"1). Alternately, the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
The shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1 % of sucrose, 0.8% of agar, 0.1 % carbon black, pH 5.7), with photoperiod of 16 hours (50 μmols m"2s"1) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene. The shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed. The plantlets were transferred to vessels containing soil for the production of seeds.
Example 2: Obtaintion of Transgenic Coffee Plants (Cofea. spp), with the herbicides imazapyr or ammonium glyphosinate
immediately after the bombardment of the embryonic axes with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing multiple shooting inducing medium (IM) (MS medium supplemented with 11.1 to 44.4 μM BAP, 3% sucrose, 0.6% of agar, pH 5.7). The embryonic axes were, then, transferred to plates with culture medium containing herbicide (CMH) (SM medium, 3% sucrose, 100-500 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in a growth chamber at a temperature of 27° C with 16 hours photoperiod (50 μmols m-2s-1) until the induction of multiple shoots. Alternately, the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1%) or imazapyr (0.2%), were applied directly to the multiple shoots.
The shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1 % of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 μmols m" 2s"1) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene. The shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels con- taining an autoclaved soil: vermiculite (1 :1) mixture.
The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed. The plantlets were transferred to vessels containing soil for the production of seeds.
Example 3: Obtaintion of Transgenic Cocoa Plants (Theobroma cacao .), through selection with the herbicides imazapyr or ammonium glyphosinate
Immediately after the bombardment of the axes of the embryonic axes with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing induction medium IM (MS medium supplemented with 11.1 to 44.4 μM BAP, 3% of sucrose, 0.6% of agar, pH 5.7). Thereafter, the embryonic axes were transferred to plates with culture medium comprising the herbicide (MCH) (MS medium, 3% of sucrose, 500- 1000 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in growth chamber at a temperature of 27° C with 16 hours photoperiod (50 μmols m"2s"1) until the induction of the multiple shootings. Alternately, the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
The shoots that reached 2 - 4 cm length were transferred to a culture medium
MS1S (MS, 1% of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 μmols m" 2s~1) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
The shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed The plantlets were transferred to vessels containing soil for the production of seeds.
Example 4: Obtaintion of Transgenic Banana Plants (Musa spp), through selection with the herbicides imazapyr or ammonium glyphosinate
Immediately after the bombardment of the tissues containing meristems with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the tissues containing the meristems were cultivated in the same culture medium (MB) for 7 days at a temperature of 27° C with 16 hours photoperiod (50 μmols m"2s"1) for induction of the multiple shoots. After this period, the embryonic axes which germinated were transferred to a "Magenta"-type box containing the culture medium MSBH (MS medium supplemented with 10-20 μM BAP, 3% of sucrose, 50-300 nM of IMAZAPYR, 0.8% of agar, pH 5.7), for 7 days at a temperature of 27° C with 16 hours photoperiod (50 umols m"2s"1) to reduce the total number of multiple shoots. Then the embryonic axes were again transferred to the "Magenta"-type culture box containing the culture medium MS3S (MS supplemented with 5-10 μM BAP, 3% of sucrose, 0.8% of agar, pH 5.7) at a temperature of 27° C with 16 hours photoperiod (50 μmols m"2s"1) to enable the elongation of the multiple shoots. After two weeks the axes of embryos began to emit shoots. Alternately, the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
The shoots that reached 2 - 4 cm length were transferred to a culture medium MS1S (MS, 1 % of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 μmols rrϊ 2s"1) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene. The shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1 ) mixture. The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed. The plantlets were transferred to vessels containing soil for the production of seeds.
Example 5: Obtaintion of Transgenic Grape Plants (Vitis Vinifera L,), trough se- lection with the herbicides imazapyr or ammonium glyphosinate
Immediately after the bombardment of the embryonic axes with the microparticles covered with an exogenous DNA which imparts resistance to imazapyr, the embryonic axes were transferred from the bombardment medium (BM) to culture plates containing induction medium IM (MS medium supplemented with 22.2 to 44.4 μM BAP, 3% of sucrose, 0.6% of agar, pH 5.7). Thereafter, the embryonic axes were transferred to plates with culture medium comprising the herbicide (MCH) (MS medium, 3% of sucrose, 100-600 nM of IMAZAPYR, 0.7% of agar, pH 5.7) and kept in growth chamber at a temperature of 27° C with 16 hours photoperiod (50 μmols m"2s"1) until the induction of the multiple shootings. Alternately, the embryonic axes were cultivated in culture medium without the selective agent, and the selection was perfomed after the development of the induced multiple shoots. So, the herbicide ammonium glyphosinate (0.5 to 1 %) or imazapyr (0.2%), were applied directly to the multiple shoots.
The shoots that reached 2 - 4 cm length were transferred to a culture medium MS1 S (MS, 1% of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 μmols m" V) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene. The shoots expressing the exogenous DNA were then individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed The plantlets were transferred to vessels containing soil for the production of seeds.
The shoots that reached 2 - 4 cm length were transferred to a culture medium
MS1 S (MS, 1% of sucrose, 0.8% of agar, pH 5.7), with photoperiod of 16 hours (50 μmols m~ 2s"1) at 27° C to enable the plantlets to grow and take root. A section of 1 mm was removed from the base of the stem and leaf for analysis of the expression of the exogenous gene.
The shoots expressing the exogenous DNA were individually registered and transferred to a new culture flask. Once the plantlets had taken root, they were transferred to vessels containing an autoclaved soil: vermiculite (1 :1) mixture.
The plantlets were covered with a plastic bag closed with an elastic band for 7 days. The elastic band was removed and after 6-7 days the plastic bag was also removed. The plantlets were transferred to vessels containing soil for the production of seeds.

Claims

1- Process for selecting transgenic meristematic cells of cotton, coffee, cocoa, banana or grape plants which comprises the steps of:
a) introducing exogenous genes into cells of apical meristem of embryonic axes or tissues containing meristems of cotton, coffee, cocoa, banana or grape plants;
b) inducing the multiple shooting of the cells in the meristematic region modified in step (a) by cultivating said embryonic axes or tissues containing meristems in a medium containing a multiple shooting inducer; and
c) selecting the meristematic transgenic cells as obtained in step (b) by further cultivation of said meristems in a medium containing a molecule which translocate and concentrates in the apical meristematic of embryonic axes of cotton, coffee, cocoa, banana or grape plants, or
d) selecting the meristematic cells of the apical region as obtained in step (b) by applying to the multiple shoots of the apical region a molecule which translocate and concentrates in the apical meristematic region of embryonic axes of cotton, coffee, cocoa, banana or grape plants.
2- A process according to claim 1 , wherein the multiple shooting-inducing agent is a cytokinin.
i- A process according to claim 2, wherein the cytokinin is 6-benzylaminopurine.
4- A process according to claim 1 , wherein the selection agent of step (c) is a molecule which concentrates in the apical meristematic region of said embryos or tissues containing meristems.
5- A process according to any one of the preceding claims, wherein the cotton, coffee, cocoa, banana or grape plant is selected from the group comprising different culti- vars.
PCT/BR1999/000018 1999-03-19 1999-03-19 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape WO2000056904A1 (en)

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MXPA00011341A MXPA00011341A (en) 1999-03-19 1999-03-19 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape.
AU35897/99A AU3589799A (en) 1999-03-19 1999-03-19 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape
PCT/BR1999/000018 WO2000056904A1 (en) 1999-03-19 1999-03-19 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape
CN99808805A CN1309715A (en) 1999-03-19 1999-03-19 Process for selection of transgenic cells in meristematic region of cotton, coffee, cocoa, banana or grape
EP99917693A EP1080213A1 (en) 1999-03-19 1999-03-19 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape
HK02100891.8A HK1039354A1 (en) 1999-03-19 2002-02-05 A process for the selection of transgenic cells in the meristematic region of cotton, coffee, cocoa, banana or grape

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