WO1993018168A2 - Transgenic wheat - Google Patents
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- WO1993018168A2 WO1993018168A2 PCT/EP1993/000485 EP9300485W WO9318168A2 WO 1993018168 A2 WO1993018168 A2 WO 1993018168A2 EP 9300485 W EP9300485 W EP 9300485W WO 9318168 A2 WO9318168 A2 WO 9318168A2
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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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
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- the present invention provides a method for producing transgenic wheat plants wh ch comprises introducing foreign DNA via the pollen tubes, thereby obtaining a transgenic embryo which can develop into a mature transgenic plant.
- the present invention also provides transgenic wheat plants.
- One such method involves the preparation of protoplasts from plant tissue, the transformation thereof in vitro and subsequently the regeneration of the transformed protoplasts into transgenic whole plants.
- the plasmids were introduced into exposed pollen tubes by first removing the tips of pollinated stigmas in which the pollen tubes were growing to expose these tubes and then adding thereto a solution containing the plasmids.
- the plasmid DNA apparently migrated down the growing pollen tubes together with the male nuclei and so entered the ovules during fertilization.
- about 1% of the seeds which were obtained were resistant to kanamycin as demonstrated by their ability to germinate and grow on media containing kanamycin and by the ability of such germinating plants to grow into mature wheat plants in soil always soaked with water containing 100 mg/l kanamycin.
- a still further object of the present invention is to provide the appropriate recombinant DNA molecules for the purposes of producing the aforesaid transgenic wheat plants. All other objects of the invention will be revealed in the following description and claims.
- step (d) protecting the treated plants of step (c) from additional pollination by any nearby plants, growing said plants and collecting the seeds developed in said florets;
- the stigmas are truncated after pollination and the droplet of an aqueous DNA solution is applied onto the truncated stigmas.
- the droplet of an aqueous DNA solution is applied onto intact stigmas following a time period after pollination.
- the aforesaid truncation is carried out by truncating the stigma tips, about 1 ⁇ 2-21 ⁇ 2, more preferably about 1-2 hours after pollination.
- cultivars such as cultivars ATIR (CNO "S”/PJ62/4/GLL/3/TOB//JAR "S”/CRESPO, Hazera Seed Co., Israel), BAGULA “S” (CM 59123, CIMMYT, Mexico), CIANO 79 (CM31678, CIMMYT, Mexico), Genaro 81 (VEERY#3"S", CIMMYT, Mexico), KAUZ “S” (CM67458, CIMMYT, Mexico), KEA"S” (CM21335, CIMMYT, Mexico), MILAN (23IBWSN37, CIMMYT, Mexico), OPATA M 85 (CM40038, CIMMYT, Mexico), Papago M86 (CM40038, CIMMYT, Mexico), Papago M86 (CM40038
- the interval between pollination and truncation of the stigma tips should be chosen in accordance with the wheat genotype used and the environmental conditions.
- this interval may be in the range of 1-11 ⁇ 2 hours.
- the type of wheat cultivar and the environmental conditions favour a slower attachment of the pollen and subsequent pollen tube development, the interval may be in the range of 11 ⁇ 2-2 hours.
- the above described timing between pollination and DNA application should also be adhered to when performing the intact stigma embodiment of the invention, contingent, as noted above, with the wheat genotype used, with the environmental conditions etc.
- the droplet of DNA solution applied in step (b) of the method is suitably maintained on the pollinated stigma (step (c)) for a time period sufficient to ensure that the DNA vector transported by the growing pollen tube reaches the ovule.
- a suitable time period was found to be at least 12 hours, e.g. for about 12-14 hours.
- Also provided by the invention is a recombinant DNA vector for use in the above method.
- the DNA vector of the invention comprises a gene which can induce in the plant the expression of a trait, the transfer of which into the plant is desired.
- a gene may for example be one expressing a nitrogen fixation structural protein or one expressing a protein that confers herbicide resistance to the plant.
- the vector may also suitably comprise a reporter gene, e.g. the uidA gene encoding E. coli ⁇ -glucuronidase, which serves as an indicator of successful transformation: cells expressing this gene when provided with the substrate (X-Gluc) for the expressed enzyme are capable of converting this susbstrate into a detectable product.
- the DNA vector preferably comprises also a plant selectable marker gene which may, for example, be one encoding an antibiotic resistance, e.g. kanamycin resistance.
- a plant selectable marker gene which may, for example, be one encoding an antibiotic resistance, e.g. kanamycin resistance.
- the APH(3')II ( NPTII) gene encodes the enzyme aminoglycoside phosphotransferase and thus confers kanamycin resistance to the transformed plants.
- Another bacterial selectable marker gene e.g. Amp R may also be included in the vector for the purpose of propagating the DNA vector in bacteria in order to obtain large quantities thereof needed for the preparation of said DNA solution.
- Non-limiting examples of such DNA vectors used in the experiments conducted within the framework of the present invention, are the plasmids pPCV702nifD and ⁇ PCV702GUS, both of which comprise the afore-mentioned APH(3')II gene and, in pPCV702nifD, the Klebsiella pneumoniae nifD coding region linked to a CaMV 35S promoter and a NOS terminator and, in pPCV702GUS, the coding region of ⁇ -glucuronidase (GUS) also linked to a CaMV 35S promoter and a NOS terminator.
- pPCV702nifD the Klebsiella pneumoniae nifD coding region linked to a CaMV 35S promoter and a NOS terminator
- GUS ⁇ -glucuronidase
- the invention also provides transgenic wheat plants of agronomic, commercial crop varieties.
- transgenic wheat plants of several commercial varieties have been obtained for the first time.
- Transgenic wheat plants obtained in accordance with the invention were grown through 5 generations without a loss of the foreign gene with which the original plants were transformed.
- Fig. 1 is a schematic representation of the plasmid pPCV702nifD
- Fig. 2 is a schematic representation of the plasmid pPCV702GUS
- Fig. 3a shows results of Southern blot hybridization of a NPTII gene probe to DNA samples, digested with BamHI and HindIII restriction endonucleases, from wild type wheat varieties; plants transformed according to the procedure of the present invention and from the plasmid pPCV702nifD, described in Example 4;
- Fig. 3b is a schematic restriction map illustrating the pertinent restriction endonuclease sites of pPCV702nifD integrated into the wheat genome in the transformant designated TAU89107-1, as determined from
- Fig. 4a shows results of Southern blot-hybridization of a NPT II probe to DNA samples, digested with PstI restriction endonuclease, from wild type wheat varieties and plants transformed with pPCV702nifD according to the present invention, as described in Example 4;
- Fig. 4b is a schematic restriction map illustrating the pertinent restriction endonuclease sites for pPCV702nifD integrated into the wheat genome as determined from the Southern blot results (Figs. 3 a, 4a and Fig. 5), as described in Example 4;
- Fig. 5 shows results of Southern blot-hybridization of a NPT II fragment and the rest of the 702nifD vector with DNA samples digested with Bam HI and Pst I from a wild-type wheat plant and second generation
- Fig. 6 shows the results of Southern blot hybridization of a NPTII fragment with BamHI and HindIII digested DNA sample of the second generation (F 2 ) of transformed wheat plants, obtained by selfing of the transformant TAU89107-1, as described in Examples 4 and 5;
- Fig. 7 shows the results of Southern blot hybridization of a NPIII fragment with BamHI and HindIII digested DNA samples of the third generation (F 3 ) of transformed wheat plants obtained by selfing of plants from the TAU89107 F 2 families, as described in Examples 4 and 6;
- Fig. 8a shows the results of an agarose gel electrophoresis of DNA samples obtained by PCR amplification of the APH(3')II gene present in the transformed wheat plants of the Shafir cultivar.
- DNA samples were extracted from F 2 , F 3 and F 4 plants originating from the transformant TAU89107-1 and resulting from self pollination of each generation of plants, as described in Example 7;
- Fig. 8b shows the results of an agarose gel electrophoresis of DNA samples obtained by PCR amplification of the APH(3')II gene and its gene
- Fig. 9 shows the results of Southern blot hybridization of a gene 4 terminator fragment with DNA samples of a wild type Shafir wheat plant and the F 4 generation progenies of the transformant TAU89107-1.
- DNA samples were digested with BamHI and HindIII. All four generations resulted from self-pollination, as described in Examples 4 and 8;
- Fig. 10a shows the F 5 progeny CC44 (TAU89107-1-J18-9-1B-4A) ⁇ of the TAU89107-1 transformant which produced a normal shoot upon growing on kanamycin containing medium as compared to the wild type Shafir plant which turned white under the same growth conditions, as described in Example 9;
- Fig. 10b demonstrates gel separation analysis of the PCR amplification products of the NPTII tail (51bp) and its gene 4 terminator using DNA templates extracted from CC44 transformant or from plants of the wheat cultivar Shafir, as described in Example 9;
- Fig. 11 shows the results of Southern-blot hybridizations of the first generation (F 1 ) of wheat plants of the commercial cultivars Pavon 76 and Seri 82 transformed with pPCV702GUS and probed with the same plasmid, the DNA samples having been digested with EcoRI and HindIII, as described in Example 10;
- Fig. 12 shows the results of an agarose gel electrophoresis of DNA samples obtained from PCR amplification of the APH(3')II and GUS genes from DNA extracts from F 1 transformants of Seri 82 cultivar, as described in Example 10;
- Fig. 13 shows a representation of the electrophoresis on an agarose gel of the 0.6 kb PCR product obtained from F 1 generation transformants, transformed with the plasmid pPAT-NPTII 100.1-28 carrying the Streptomyces hygroscopicus bar gene, as described in Example 12.
- plasmids Two plasmids, pPCV702nifD and pPCV702GUS, were constructed as illustrated in Figs. 1 and 2, respectively, both derived from the vector pPCV702 (Koncz and Schell, 1986; and Koncz et al., 1989).
- the APH(3')II or NPT II gene encoding aminoglycoside (kanamycin) phosphotransferase, linked to the NOS (nopaline synthase) promoter and to the TL-DNA ipt gene (gene 4) polyadenylation signal, or PolyA, (terminator), served as a selective marker. This gene, when expressed in plants, confers kanamycin resistance.
- the plasmid pPCV7702nifD contains the coding region of Klebsiella pneumoniae nifD, situated between the CaMV 35S promoter and NOS terminator.
- the plasmid pPCV702GUS includes the coding region of the ⁇ -glucuronidase (GUS) gene, a reporter gene, linked to the CaMV 35S promoter and to the NOS terminator.
- GUS ⁇ -glucuronidase
- the glassine paper bag was cut across the top and a male pollinator spike (of same or different cultivar) with its spikelets cut in half, to allow mature stamens to protrude outward from the glumes, was inserted into the bag.
- the pollinator spike was left inside the bag for 20-30 min. to allow its stamens to burst and release their pollen.
- the pollinator spike was swirled around several times to allow pollen to reach all florets of the spike. Thereafter, the pollinator spike was removed and the glassine paper bag sealed with a clip.
- the aforesaid pollination procedure provides a synchronized initiation of pollen tube formation within the stigmas.
- the aforesaid period between pollination and clipping of the pollinated stigma tips varies according to environmental conditions and the strain of wheat plants used, such that a period of about 1-11 ⁇ 2 hrs is optimal for situations favouring relatively rapid pollination attachment and subsequent development of pollen tubes within the stigmas and into the styles, a period of 11 ⁇ 2-2 hrs being optimal in situations where the aforesaid pollen attachment and pollen tube development is slower.
- Seeds obtained by the transformation procedure of Example 2 using the pPCV702nifD vector were immersed in hypochlorite for 5 minutes, washed in distilled water and then transferred to water-agar petri dishes with 150 ⁇ g/ml kanamycin and placed in a growth chamber at 25°C. Ten days later, all seeds were transferred to MS medium and thereafter scored for the appearance of bleaching of the shoot and the number and length of roots recorded. In some experiments the seedlings were kept in the growth chamber for 4 days on a kanamycin containing medium and thereafter transferred to MS growth medium for a further 4 days and scored as noted above.
- APTII activity i.e. expression of the NPTII gene carried by the vectors used to transform the original parent plants
- seedlings of the F 3 generation were germinated on water-agar containing 150 ⁇ g/ml kanamycin.
- These green seedlings were transferred to the greenhouse and their leaf DNA was analyzed by Southern blotting and PCR amplification of specific sequences (See Examples 4 to 11 below).
- TAU89107-1 The above kanamycin selection procedure has been performed in all consecutive generations of the transformed plant line termed TAU89107-1, up to and including the F 5 (fifth) generation, this further indicating that the above transformation is stable.
- the histochemical assay described by Jefferson et al. (1987) was used to localize the appearance of the blue color of the X-Gluc cleavage product.
- TAU89104-3 and TAU89107-1 which were transformed by plasmid pPCV702nifD. All the extracted DNAs were digested with the restriction endonucleases BamHI and HindIII and then separated on an agarose gel and transferred onto a nylon filter, by the above-noted standard procedures.
- the schematic restriction map shown in Fig. 3b illustrates the possible mode of insertion of a part of the plasmid pPCV702nifD (Fig. 1) into the wheat plant genome, "genomic" denoting this genomic DNA.
- This map was constructed on the basis of the hybridization results shown in Fig. 3a and Fig. 4a and also on the basis of the known restriction enzyme sites of the plasmid pPCV702nifD (Fig. 1), and the absence of nifD sequence in the DNA of the transformant TAU89107-1.
- Fig. 4a total DNA was extracted from the same wild type cultivars and transformant wheat plants as described for Fig. 3a, but these DNA samples were digested with the restriction endonuclease PstI only, which cuts each of the two plasmid vectors in the NPTII gene and in the amp R gene.
- the digested DNA samples were separated on an agarose gel and were subsequently transfened to a nylon filter and hybridized as before with the NPTII ApaI/HindIII DNA fragment probe.
- the results of the hybridization reveal that transformant TAU89107-1 has two PstI DNA fragments (approx. 6.9 and 0.8 kb) which are capable of hybridizing said probe.
- the schematic restriction map shown on Fig. 4b) is the same as that of Fig. 3b) and was constructed on the basis of the hybridization results shown in Figs. 3a) and 4a) and on the basis of the known restriction sites of pPCV702nifD (Fig. 1).
- total DNA was extracted from the wild type wheat cultivar Shafir and from nine F 2 transformants of TAU89107-1 F 1 plants (1, 2, 3, 4, 5, 6, 7, 8 and 9), (see Figs. 3a and 4a). These DNA samples were digested with the restriction endonucleases PstI and BamHI, were separated on an agarose gel and transferred to a nylon filter as before. The DNA samples on the nylon filter were hybridized with the probe NPTII Apal/HindHI DNA fragment + pPCV702nifD vector as noted above (Figs. 3a, 4a) under the aforesaid hybridization conditions.
- Lanes 1 and 2 represent the F 2 transformant TAU89107-1-J18; lane 4 the F 2 TAU89107-1-K10 transformant derived from selfing of F 1 plants of TAU89-107-1; and lanes 3 and 5 represent the controls being DNA from the plasmid ⁇ PCV702nifD digested with EcoRI.
- the Southern blot procedures were as described above in Example 4. The probes used in these analyses were the following: the HindIII-ApaI NPTII fragment or the PCR synthesized gene 4 terminator (described in Example 8), both of which resulted in identical hybridization patterns.
- Fig. 8(a) there is shown a representation of the electrophoresis on an agarose gel, under standard conditions, of the DNA fragments obtained after the PCR procedure, namely, the results of the PCR amplification of the inserted NPTII coding sequence in F 2 , F 3 and F 4 generation plants of sub-families of the transformant Shafir TAU89107- 1Km R (for the PCR procedures see also Example 11 below).
- the DNA templates used in the PCR procedure were as follows:
- Lane No. 1 control sample, no DNA template in reaction
- Lane No. 2 control sample, non-transformed, wild-type Shafir DNA
- Lane No. 3 transformant sample, F 2 generation, Shafir TAU89107-1- B19 DNA
- Lane No. 4 transformant sample, F 2 generation, Shafir TAU89107-1- J20 DNA
- Lane No. 5 transformant sample, F 3 generation, Shafir TAU89107-1- J18-X DNA;
- Lane No. 6 transformant sample, F 3 generation, Shafir TAU89107-1- J18-9 DNA
- Lane No. 7 transformant sample, F 4 generation, Shafir TAU89107-1- J18-9-1B DNA;
- Lane No. 8 transformant sample, F 4 generation, Shafir TAU89107-1- J18-9-1K DNA
- Lane No. 9 size markers for DNA fragments from ⁇ X 174 bacteriophage DNA cleaved with HaeIII restriction endonuclease.
- Figure 8(b) shows the results of the PCR amplification of the APH(3')II gene and its adjacent gene 4 terminator using total DNA extracted from the F 4 progenies of transformant TAU89107-1Km R obtained by selfing.
- the primers used in the PCR reactions were as follows:
- Primer No. 1 corresponds to nucleotide 21 until the 47th nucleotide of the NPTII coding region (26 nucleotides) as follows:
- Primer No. 2 corresponds to nucleotide 744 until nucleotide 775 of the NPTII coding region (31 nucleotides) as follows:
- Primer No. 3 is a reverse primer and represents the 3' end of gene 4 terminator as follows:
- the DNA templates and the primers used in the PCR amplification were as follows:
- Lane No. 1 size markers being bacteriophage ⁇ DNA digested with
- Lane No. 2 control template non-transformed wild-type Shafir DNA with primers 1 and 3;
- Lane No. 3 transformant sample, F 4 generation, Shafir TAU89107-1- H29-21-1A2 with primers 1 and 3 (about 1.15 kb);
- Lane No. 6 transformant sample, F 4 generation, Shafir TAU89107-1- J18-X-1B with primers 1 and 3;
- Lane No. 7 transformant sample, F 4 generation Shafir TAU89107-1- J18-X-1B with primers 2 and 3;
- Lane No. 8 Bacteriophage ⁇ x174 DNA digested with HaeIII.
- the linkage between the NPTII coding region and the adjacent gene 4 terminator was maintained throughout 4 generations of selfing of the TAU89107-1Km R family.
- the stability of the NPTII sequence in different plant organs was evaluated in various F 4 transformants of the TAU89107-1.
- Lane No. 1 control template, non-transformed wild-type Shafir DNA.
- Lane No. 3 transformant sample, F 4 generation Shafir TAU89107-1- H29-21-1A2C (a central tiller form another plant originated from the same F 3 spike as in Lane No. 2).
- Lane No. 4 transformant sample, F 4 generation Shafir TAU89107-1- J18-9-1BC (a central tiller from which DNA was extracted).
- Lane No. 5 transformant sample
- F 4 generation Shafir TAU89107-1- J18-9-1BR (R refers to the roots of the plant from which
- Lane No. 6 transformant sample, F 4 generation Shafir TAU89107-1- J18-X-1BC (a central tiller from which DNA was extracted).
- Lane No. 7 transformant sample, F 4 generation Shafir TAU89107-1- J18-X-1BR (roots of the plant from which DNA was extracted).
- Resistance to kanamycin was expressed in seedlings of F 5 plants germinated on 150 ⁇ g/ml kanamycin.
- a representative F 5 plant is shown in Fig. 10a together with kanamycin-sensitive seedling of the wild-type Shafir.
- the F 5 plant (CC44) is a progeny of the TAU89107-1-J18-9 F 3 family.
- the mature F 5 kanamycin-resistant Shafir transformant plant resembles phenotypically (plant height, leaf and spike shape) the non-transformed wild-type Shafir.
- This representative F 5 plant was examined for resistance to kanamycin at the seedling stage in F 1 , F 3 and the F 5 generations, whereas seed multiplication via selfing without kanamycin was performed in the F 2 and F 4 generations.
- Lane No. 1 bacteriophage ⁇ x174 DNA digested with Haelll.
- Lane No. 2 control template, non-transformed wild-type Shafir DNA with primers 2 and 3.
- Lane No. 3 transformant template extracted from kanamycin-resistant F 5
- Lane No. 2 represents the F 1 wheat transformant Serf 82 TAU90-56-13
- Lane No. 3 represents the F 1 transformant Pavon 76 TAU90-84-7.
- the above observed results are evidence of the presence of the introduced 2.9 kb GUS fragment in F 1 transformants of the high-yielding commercial wheat cultivars Pavon 76 and Seri 82.
- Lane No. 3 control sample, GUS primers and Seri 82 wild type DNA as template added (500 ng DNA);
- Lane No. 4 control sample, NPTII primers and wild type Seri 82 DNA as template added (500 ng DNA);
- Lane No. 5 transformant sample, GUS primers and TAU9055-9 DNA as template added (50 ng DNA);
- Lane No. 6 transformant sample, NPTII primers and TAU9055-9 DNA as template added (50 ng DNA);
- Lane No. 7 transformant sample, GUS primers and TAU9054-10 DNA as template added (50 ng DNA);
- Lane No. 8 transformant sample, GUS primers and TAU9054-11 DNA as template added (50 ng DNA) (no synthesis observed);
- Lane No. 9 transformant sample, NPTII primers and TAU9056-13 DNA as template added (200 ng DNA);
- Lane No. 10 size markers for DNA fragments from ⁇ x174 bacteriophage
- Example 2 The above wheat strains were transformed using the procedures described in Example 2 above, the foreign DNA being administered to the plants being that detailed in Example 1 above. Following the transformation procedure, the treated plants were tested to determine the success and frequency of transformation using the bioassay methods described in Example 3 above and by a PCR assay carried out as follows:
- the NPTII fragment was synthesized by PCR using total transformant DNA as template and the following primers:
- Primer No. 1 (5' region of NPTII coding sequence):
- Primer No. 2 (a reverse primer of the 3' region of NPTII coding sequence):
- GUS fragment was synthesized by PCR using total transformant DNA as template and the following primers:
- Primer No. 4 (a reverse primer of the 3' region of GUS):
- Km R Transformant is capable of expressing the NPTII gene, i.e. is kanamycin resistant, as observed from green seedlings capable of growing on water agar containing 150 ⁇ g/ml kanamycin
- NPTII PCR Presence of NPTII fragment detected directly in the transformant by use of PCR methods (see PCR procedure hereinbelow).
- GUS + Transformant exhibits GUS activity in the roots, as observed in a bioassay using X-Gluc as substrate (see Example 3 above).
- GUS PCR Presence of GUS fragment detected directly in the transformant by use of PCR methods (see PCR procedure hereinbelow).
- the high yielding, semi-dwarf, spring bread wheat cultivars Seri 82 (VEERY #5"S", CIMMYT, Mexico) and Shafir (SON64A/TZPP//NAI60/3/FA, Hazera Seed Co., Israel) were transformed with the plasmid pPAT-NPTII 100.1-28 carrying the Streptomyces hygroscopicus bar gene (DeBlock et al., 1987, White et al., 1990). This gene encodes an acetyl CoA transferase capable of inactivating the herbicides bialaphos and glufosinate by acetylation.
- the gene was linked to the CaMV 35S promoter and PolyA (terminator) sequences to serve as a marker gene in plants.
- the plasmid pPAT-NPTII 100.1-28 is a pUC-derivative (Topfer et al., unpublished, personal communication) which also carries the NPTII gene with the first intron of the maize Shrunken gene (shl) (Wen et al., 1985) inserted downstream to the NPTII translational start site (Christoph, Maas and J. Schell, personal communication).
- the bar sequence was detected by PCR amplification in transformants of the F 1 generation as shown in Figure 13, which is a representation of the electrophoresis on an agarose gel, under standard conditions of the 0.6 kb PCR product obtained, when specific bar primers were used.
- the first one corresponds to the nucleotide -8 until 19th nucleotide of the bar coding region (White et al., 1990) as follows:
- the translation start site is underlined.
- Asterisks denote nucleotide substitutions to form the NdeI and BamHI sites and additional two deoxycytidines that were added to the 5' end.
- the second primer is a reverse primer complementary to the sequence of nucleotide 533 to nucleotide 548 of the coding region, two stop signals and 16 nucleotides downstream according to White et al., (1990) and additional two deoxycytidines as follows: 5'-CCGGATCCCCCGGGTCATCAGATCTCGGTGACGGGC (37 mer)
- Lane No. 1 transformant sample, F 1 generation, TAU92P4SHF9-2, reamplified fragment after gel purification of the 0.6 kb fragment synthesized by PCR;
- Lane No. 2 transformant sample, F 1 generation TAU92P4SER14-1
- Lane No. 3 transformant sample, F 1 generation TAU92P4SER14-1, reamplified fragment after gel purification of the 0.6 kb fragment obtained in the first PCR synthesis, (demonstrated in Lane 2);
- Lane No. 4 control sample, non-transformed, wild type Shafir DNA
- Lane No. 5 control sample, non-transformed, wild type Seri 82 DNA
- Lane No. 6 control sample, no template added in PCR reaction
- Lane No. 7 control sample, plasmid pPAT-NPTII 101.1-28 as a template (30 ng);
- Lane No. 8 size markers for DNA fragments from ⁇ x174 bacteriophage
- transgenic wheat plants in particular, those of commercial agronomic varieties which have stably integrated the foreign DNA and can transmit this DNA to the next generation.
- T 2 -DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Gen. Vol. 204, 386-396.
- sucrose synthase gene on chromosome 9 of Zea mays L. EMBO J. 4, 1373-1380.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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AU36303/93A AU3630393A (en) | 1992-03-03 | 1993-03-03 | Transgenic wheat |
JP5515316A JPH07507445A (en) | 1992-03-03 | 1993-03-03 | transformed wheat |
EP93905293A EP0631628A1 (en) | 1992-03-03 | 1993-03-03 | Transgenic wheat |
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IL101119A IL101119A0 (en) | 1992-03-03 | 1992-03-03 | Transgenic wheat |
IL101119 | 1992-03-03 |
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WO1993018168A2 true WO1993018168A2 (en) | 1993-09-16 |
WO1993018168A3 WO1993018168A3 (en) | 1993-10-28 |
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JP (1) | JPH07507445A (en) |
AU (1) | AU3630393A (en) |
CA (1) | CA2117652A1 (en) |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994013822A2 (en) * | 1992-12-16 | 1994-06-23 | Ciba-Geigy Ag | Methods for stable transformation of wheat |
WO2005123926A1 (en) | 2004-06-18 | 2005-12-29 | Thomas Schmulling | Method for modifying plant morphology, biochemistry and physiology comprising expression of cytokinin oxydase in the seeds |
US7026528B2 (en) | 1996-06-21 | 2006-04-11 | Monsanto Technology Llc | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
EP2045327A2 (en) | 2005-03-08 | 2009-04-08 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2159289A2 (en) | 2005-06-23 | 2010-03-03 | BASF Plant Science GmbH | Improved methods for the production of stably transformed plants |
EP2163635A1 (en) | 2004-08-02 | 2010-03-17 | BASF Plant Science GmbH | Method for isolation of transcription termination sequences |
US9187761B2 (en) | 2006-09-25 | 2015-11-17 | Thomas Schmulling | Transcriptional repressors of cytokinin signaling and their use |
EP2925870A4 (en) * | 2012-12-03 | 2016-07-13 | Adi Zaltsman | Plant self nitrogen fixation by mimicking prokaryotic pathways |
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- 1993-03-03 EP EP93905293A patent/EP0631628A1/en not_active Withdrawn
- 1993-03-03 JP JP5515316A patent/JPH07507445A/en active Pending
- 1993-03-03 AU AU36303/93A patent/AU3630393A/en not_active Abandoned
- 1993-03-03 HU HU9402528A patent/HUT68066A/en unknown
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Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5610042A (en) * | 1991-10-07 | 1997-03-11 | Ciba-Geigy Corporation | Methods for stable transformation of wheat |
US5955362A (en) * | 1991-10-07 | 1999-09-21 | Novartis Finance Corporation | Methods for stable transformation of wheat |
WO1994013822A2 (en) * | 1992-12-16 | 1994-06-23 | Ciba-Geigy Ag | Methods for stable transformation of wheat |
WO1994013822A3 (en) * | 1992-12-16 | 1994-09-15 | Ciba Geigy Ag | Methods for stable transformation of wheat |
US7026528B2 (en) | 1996-06-21 | 2006-04-11 | Monsanto Technology Llc | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
WO2005123926A1 (en) | 2004-06-18 | 2005-12-29 | Thomas Schmulling | Method for modifying plant morphology, biochemistry and physiology comprising expression of cytokinin oxydase in the seeds |
EP2163635A1 (en) | 2004-08-02 | 2010-03-17 | BASF Plant Science GmbH | Method for isolation of transcription termination sequences |
EP2166103A1 (en) | 2004-08-02 | 2010-03-24 | BASF Plant Science GmbH | Method for isolation of transcription termination sequences |
EP2166104A1 (en) | 2004-08-02 | 2010-03-24 | BASF Plant Science GmbH | Method for isolation of transcription termination sequences |
EP2045327A2 (en) | 2005-03-08 | 2009-04-08 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2166102A2 (en) | 2005-03-08 | 2010-03-24 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2166101A2 (en) | 2005-03-08 | 2010-03-24 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2166100A2 (en) | 2005-03-08 | 2010-03-24 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2166099A2 (en) | 2005-03-08 | 2010-03-24 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2169058A2 (en) | 2005-03-08 | 2010-03-31 | BASF Plant Science GmbH | Expression enhancing intron sequences |
EP2159289A2 (en) | 2005-06-23 | 2010-03-03 | BASF Plant Science GmbH | Improved methods for the production of stably transformed plants |
US9187761B2 (en) | 2006-09-25 | 2015-11-17 | Thomas Schmulling | Transcriptional repressors of cytokinin signaling and their use |
EP2925870A4 (en) * | 2012-12-03 | 2016-07-13 | Adi Zaltsman | Plant self nitrogen fixation by mimicking prokaryotic pathways |
Also Published As
Publication number | Publication date |
---|---|
AU3630393A (en) | 1993-10-05 |
JPH07507445A (en) | 1995-08-24 |
HU9402528D0 (en) | 1994-12-28 |
HUT68066A (en) | 1995-05-29 |
EP0631628A1 (en) | 1995-01-04 |
CA2117652A1 (en) | 1993-09-16 |
IL101119A0 (en) | 1992-11-15 |
WO1993018168A3 (en) | 1993-10-28 |
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