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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
• • C—CHEMISTRY; METALLURGY
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
  • C12N15/8289—Male sterility

Definitions

• the present invention relates to a promoter sequence which is specific for pollen, to constructs and transgenic plant cells and plants comprising the promoter as well as to methods of transforming pollen and controlling fertility in plants using this promoter.
• cross-breeding represents the traditional approach.
• Various techniques for producing male sterility are known and have been proposed in the art. One method involves removal of the anthers or tassels of the female parent plant, either manually or mechanically. This plant may then only be fertilised by pollen from the male parent and therefore its progeny will be hybrid.
• WO 90/08830 describes the induction of male sterility in a plant by a cascade of gene sequences which express a protein which disrupts pollen biosynthesis.
• WO 93/18171 describes the use of a GST promoter to inducibly express chalcone synthase (chs) and restore fertility to a male sterile plant made sterile by "knocking out" the endogenous chs genes.
• a component of a system to genetically engineer male sterility is the availability of a promoter which specifically drives expression either in pollen or in tissue on which pollen production or development depends.
• genes which are specifically or highly expressed in pollen and germinating pollen encode proteins that are likely to play a role in cell wall metabolism, for example, those having homology to genes encoding enzymes involved in pectin degradation; polygalacturonases (SM Brown et al.., Plant Cell (1990) 2: 263-274, SJ Tebbutt et al.., Plant Mol. Biol.
• genes highly expresed in pollen include those that encode cytoskeletal proteins (I. Lopez et al.., Proc. Natl, Acad Sci USA 93: 7415-7420 (1996), HJ Rogers et al.., Plant J. 4: 875-882 (1993) and CJ Staiger et ⁇ /.., Plant J. 4: 631-641 (1993)), putative ascorbate oxidases, a Kunitz protein inhibitor and many others whose function cannot be inferred by homology to known genes. The temporal expression of such genes has been studied and many are found to be expressed late in microsporogenesis reaching a maximum in mature microsporocytes.
• the applicants have isolated a further promoter which is specifically expressed only in pollen tissue.
• the promoter is derived from a 'late' pollen expressed gene isolated from maize, ZmC5.
• a recombinant nucleic acid sequence which comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen.
• fragment includes one or more regions of the basic sequence which retain promoter activity. Where the fragments comprise one or more regions, they may be joined together directly or they may be spaced apart by additional bases.
• variant with reference to the present invention means any substitution of, variation of, modification of, replacement of, deletion of or the addition of one or more nucleotides from or to the nucleic acid sequence providing the resultant sequence exhibits pollen promoter expression.
• the term also includes sequence that can substantially hybridise to the nucleic acid sequence.
• ZmC5 gene refers to the gene of maize which encodes a 563 amino acid sequence as described herein.
• a cDNA sequence encoding this sequence is defined in EMBL Y13285
• the promoter sequence of the present invention is comprised within the clone deposited National Collection of Industrial and Marine Bacteria as NCIMB 40915 on 26 Jan 1998. This is a Sal I fragment derived as described hereinafter.
• the promoter region lies within a region which consists of approximately 2kb of sequence upstream of the transcription start site of the ZmC5 gene of maize as shown in Figure 1 hereinafter. - 4 -
• a recombinant nucleic acid sequence which comprises a promoter sequence comprising at least part of the DNA sequence as shown in Figure 5 or at least part of a sequence encoding a promoter which has substantially similar activity to the promoter encoded by Figure 5 or a variant or fragment thereof.
• substantially similar activity includes DNA sequences which are complementary to and hybridise to the DNA of the present invention and which code for a promoter which acts in pollen.
• hybridisation occurs at, or between, low and high stringency conditions.
• low stringency conditions can be defined as 3 x SCC at ambient temperature of between about 60°C to about 65°C
• high stringency conditions as 0.1 x SSC at about 65°C.
• SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate.
• 3 x SSC is three times as strong as SSC and so on.
• the pollen specific promoter of the present invention may be used to engineer male sterility by driving genes capable of interfering with pollen production or viability, or to express genes of interest specifically in pollen grains.
• the promoter sequence may form part of an expression cassette in combination with genes whose expression in pollen, and particularly in late pollen production, may be desirable. These include genes which have an impact on pollen or pollen production. Such genes may be those involved in the control of male-fertility, genes which encode insecticidal toxins (which would then be targeted to insect species which feed on pollen), or genes which would enhance or modify the nutritional value of the pollen.
• the promoter sequence could be used to drive expression of a selectable marker for use in pollen transformation. Examples of suitable selectable marker genes include antibiotic resistance genes such as kanamycin resistance gene, hygromycin resistance gene and the PAT resistance gene so as to enable stable transformants to be identified depending on the species e.g. corn, rice, wheat.
• expression cassette which is synonymous with terms such as "DNA construct", “hybrid” and “conjugate” - includes an effect gene directly or indirectly attached to the regulator promoter, such as to form a cassette.
• An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence intermediate the promoter and the target gene.
• the DNA sequences may furthermore be on different vectors and are therefore not necessarily located on the same vector.
• fused in relation to the present invention which includes direct or indirect attachment.
• constructs also include plasmids and phage which are suitable for transforming a cell of interest.
• expression cassettes of the present invention comprise a promoter sequence as described above which is arranged to control expression of a gene which is deleterious to pollen development, such as genes encoding barnase, adenine nucleotide translocator, mutant tubulins, T-urf (as claimed in WO 97/041 16) or trehalose phosphate phosphatase (TPP).
• a gene which is deleterious to pollen development such as genes encoding barnase, adenine nucleotide translocator, mutant tubulins, T-urf (as claimed in WO 97/041 16) or trehalose phosphate phosphatase (TPP).
• a gene which is deleterious to pollen development such as genes encoding barnase, adenine nucleotide translocator, mutant tubulins, T-urf (as claimed in WO 97/041 16) or trehalose phosphate phosphatase (
• Ribozymes are RNA molecules capable of catalysing endonucleolytic cleavage reactions. They can catalyse reactions in trans and can be targeted to different sequences. They are therefore potential alternatives to antisense as a means of modulating gene expression. (Hasselhof and Gerlach (1988) Nature Vol 334: 585-591) or Wegener et al. (1994) Mol Gen Genet 245: (465-470) have demonstrated the generation of a trans -dominant mutation by expression of a ribozyme gene in plants. If required, the pollen specific promoter of the present invention may be used to control expression of the ribozymes such that they are specifically expressed in pollen.
• Baulcombe (1997) describes a method of gene silencing in transgenic plants via the use of replicable viral RNA vectors (AmpliconsTM) which may also be useful as a means of knocking out expression of endogenous genes.
• This method has the advantage that it produces a dominant mutation i.e. is scorable in the heterozygous state and knocks out all copies of a targeted gene and may also knock out isoforms.
• This is a clear advantage in wheat which is hexaploid. Fertility could then be restored by using .an inducible promoter to drive the expression of a functional copy of the knocked out gene.
• the pollen specific promoter as the elements of the AmpliconTM vector, expression of the gene would then take place specifically in the pollen.
• the use of cytotoxic or disrupter genes as means of disrupting pollen production requires the expression of restorer genes to regain fertility.
• the construct further - 6 -
• a cassette comprising a nucleotide sequence which is able to overcome the effect of said deleterious gene, such as a restorer gene such as barstar in the case of barnase or TPS in the case of TPP, or a sequence which encodes a construct which is sense or antisense to a deleterious gene.
• a deleterious gene such as a restorer gene such as barstar in the case of barnase or TPS in the case of TPP, or a sequence which encodes a construct which is sense or antisense to a deleterious gene.
• An alternative means of controlling expression of deleterious genes is to use operator sequences. Operator sequences such as lac, tet, 434 etc. may be inserted into promoter regions as described in WO 90/08830. Repressor molecules can then bind to these operator sequences and prevent transcription of the downstream gene, for example a gene deleterious to pollen development (Wilde et al. (1992) EMBO J. 1 1, 1251).
• expression system means that the system defined above can be expressed in an appropriate organism, tissue, cell or medium.
• the system may comprise one or more expression cassettes and may also comprise additional components that ensure to increase expression of the target gene by use of the regulator promoter.
• an expression system comprising
• Elements (a) and (b) and (c) and (d) above may be provided by one or two individual vectors, but preferably are contained in the same vector to ensure co-segregation. These can be used to transform or co-transform plant cells so as to allow the appropriate interaction between the elements to take place.
• the second promoter sequence and the second gene provide for chemical
• switching on and off of the first gene.
• application of the chemical inducer to pollen or to a plant will have the effect of switching on the second gene which thereby counteracts the effect of the first gene.
• the absence of the chemical inducer will have a similar effect where the second promoter sequence is active only in the absence of the chemical inducer.
• Elements (c) and (d) are suitably in the form of an expression cassette comprising a nucleotide sequence which is able to overcome the effect of said deleterious gene, such as a restorer gene such as barstar in the case of barnase or TPS in the case of TPP, or a sequence which encodes a construct which is sense or antisense to a deleterious gene, or a gene encoding a repressor molecule in the case of operator sequences being used operatively interconnected with an inducible promoter.
• a deleterious gene such as a restorer gene such as barstar in the case of barnase or TPS in the case of TPP
• a sequence which encodes a construct which is sense or antisense to a deleterious gene or a gene encoding a repressor molecule in the case of operator sequences being used operatively interconnected with an inducible promoter.
• the expression system of the present invention may further comprise a selectable marker, such as herbicide resistance genes or antibiotic resistance genes so as to allow stable transformants to be identified depending on the species eg corn, rice, wheat.
• a selectable marker such as herbicide resistance genes or antibiotic resistance genes so as to allow stable transformants to be identified depending on the species eg corn, rice, wheat.
• the presence of a herbicide resistance gene also allows selection of male sterile progeny in a segregating population.
• Transformation of a plant with such an expression system will result in the production of male sterile plants and methods of producing such a plant form a fourth aspect of the present invention.
• Expression systems in accordance with this embodiment of the present invention wherein the gene is deleterious to viable pollen production, are useful in the production of hybrids but are especially useful when the male sterile line can be made homozygous.
• "late" promoters such as the ZmC5 promoter described above, because the gene products are expressed late in pollen development, primary transformants and heterozygotes produce pollen which segregates 1 : 1 for sterility i.e. 50% of the pollen is fertile and so self pollination leading to non-hybrid seed may occur.
• the inducible promoter In order to obtain a homozygous sterile plant, the inducible promoter must be switched on to drive expression of the restorer gene using the appropriate chemical such as ethanol in the case of the AlcA/R switch or safener for the GST switch. This then inactivates the deleterious gene, so allowing self pollination to occur in accordance with the model below.
• male sterile parents can be identified and selected for hybrid production.
• this homozygous sterile line may be used in the production of F 1 hybrids by cross pollination by an unmodified inbred male parent line. See below.
• FI seed is hybrid and heterozygous for sterility, meaning that 50% of the pollen from each plant is fertile.
• a crop species such as com this is ample viable pollen to ensure complete pollination across a field due to the sheer volume of pollen produced by each tassel.
• reduced pollen viability is not a factor to be taken into account.
• These methods may allow for reversal of the sterility, for example in hybrid production, by activation using an inducible promoter.
• a method of controlling the fertility of a plant which comprises transforming said plant with an expression system as described above, and when fertility is to be restored, activating the inducible promoter.
• Suitable inducible promoters include those which are controlled by the application of an external chemical stimulus, such a herbicide safener.
• inducible promoters include, for example, a two component system such as the alcA/alcR gene switch promoter system described in our published International Publication No. WO 93/21334, the ecdysone switch system as described in our International Publication No. WO 96/37609 or the GST promoter as described in published International Patent Application Nos. WO 90/08826 and
• switch promoters Such promoter systems are herein referred to as "switch promoters".
• the switch chemicals used in conjunction with the switch promoters are agriculturally acceptable chemicals making this system particularly useful in the method of the present invention.
• pollen specific promoter of the present invention is used to obtain male sterility, full restoration of fertility may not be achievable by this method as pollen is haploid. This means that only 50% of pollen produced following activation of the restorer gene is fertile.
• the use of the promoter of the present invention particularly useful in some very particular applications.
• transformation of pollen is required.
• the use of the pollen specific promoter be may highly desirable.
• An example of such an application is known as MAGE LITER (male germ line transformation) and is described by Stoger et al. (Plant Cell Reports 14 (1995) 273-278).
• pollen is transformed by micro projectile bombardment.
• a pollen specific promoter is used to drive, for example a selectable marker gene.
• the fact that the promoter is pollen specific confers several advantages. First of all, the marker is expressed only in the pollen, not the rest of the plant and so the remaining plant tissue does not contain unwanted marker.
• pollen transformation is important are that pollen can be made to undergo sporophytic development i.e. will give rise to haploid and doubled haploid plants. This means that homozygosity is achieved in one step.
• the transformed pollen can be used to pollinate wildtype plants thus giving seed carrying the introduced transgene, again a faster process than the traditional transformation route.
• the expression systems of the present invention can be introduced into a plant or plant cell via any of the available methods including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of plant cells and protoplasts, microprojectile bombardment, bacterial bombardment, particularly the "fibre” or “whisker” method, and pollen tube transformation, depending upon the particular plant species being transformed.
• the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.
• the method of the present invention would be useful in the production of a wide range of hybrid plants, such as wheat, rice, com, cotton, sunflower, sugar beet, and lettuce, oil seed rape and tomato.
• Plant cells which contain a plant gene expression system as described above, together with plants comprising these cells form further aspects of the invention.
• a replicable viral DNA vector which comprises a recombinant nucleic acid as defined above. - 12 -
• Figure 1 is a diagram showing the alignment of the ZmC5 cDNA with a 2.4kb fragment from the 5 'region of its corresponding gene.
• the transcriptional start point is indicated (*), and the putative translational start is underlined.
• Sa Sal I
• S Sma I
• Sp Sph I
• H Hind III
• X Xho l
• ? Pst I;
• FIG. 2 shows a Southern blot of maize (inbred line A188) genomic DNA. Each lane contains 15 ⁇ g of genomic DNA digested in lane 1 with Eco RI, in lane 2 with Hind Til, and in lane 3 with Bam HI. The Southern blot was hybridised with radiolabelled ZmC5 cDNA probe.
• Figure 3 shows ethidium bromide stained gels showing the 18S RNAs of various maize tissue total RNAs and the northern blots of the s.ame gels probed with the ZmC5 cDNA probe.
• the gel was loaded with lO ⁇ g of total RNA from various maize tissues.
• the gel was loaded with lO ⁇ g of total RNA isolated from shoot, a developmental staged series of spikelets, pollen and germinating pollen.
• Hind 111 site (for ease of cloning) and the 3' terminus of the ZmC5 promoter region, respectively.
• the uidA translation start is underlined.
• Bud stages are as follows: Bud-1 corresponds to buds of 5-8mm, (microspores at meiosis/tetrad stage), Bud-2: buds of 10-12mm (uninucleate microspores), Bud-3: 13- 15mm,
• Figure 5 shows the DNA sequence encoding the ZmC5 promoter sequence in maize.
• the underlined A is the putative transcriptional start point and the bold and underlined ATG is the translational start point.
• Figure 6 shows an expression cassette comprising C5-barnase/barstar-nos.
• a maize (inbred line B73) genomic library (8x10 6 plaques) was screened using a PCR fragment corresponding to the 5' 270bp of cDNA clone ZmC5c labelled by random priming (Ausubel et al.., supra).
• One positive clone ZmC5g was plaque purified.
• Comparison of the sequence of a 2.5kb Sail fragment of ZmC5g subcloned into pUC19 with ZmC5c (and deposited as NCIMB 40915) showed that the two sequences overlapped and that they were - 14 -
• a transcriptional start point was mapped using two oligonucleotide primers complementary to nucleotides 1 to 21 (5'- ACCTAGGAGAGCCTTTGCCAT-3') and 56 to 82 (5'-AGCGGGTGACGGTGGCGACCACACCGA-3') of the coding sequence (data not shown).
• the products unequivocally locate the transciptional start point on the A nucleotide at only 15 bases upstream of the putative ATG (Fig 1).
• Other pollen-specific genes have been found to have long 5 '-untranslated sequences (SJ Tebbutt et al.., Plant Mol. Biol. (1994) 25: 283-299). Thus this region of ZmC5g appears to be unusually short.
• the calculated free energy for this short 5' UTL is of 0.9kJ/mol(M. Zuker, Meths Enzymol.
• the predicted amino acid sequence of ZmC5 (563 amino acids) was compared to the EMBL and GenBank databases, and revealed a high degree of homology to both plant (between 30.9% and 41.4%) and microbial PMEs (between 18.6% and 20.8%).
• An alignment of amino acid sequences showed conservation across both plant and microbial sequences restricted primarily to the C-terminal end of the protein which includes four regions likely to be the catalytic domains or active sites of the enzyme (D. Alb-ani et al.., Plant Mol Biol (1991) 16:501 -513),0 Marcovic et al.., Protein Sci 1 : 1288-1292 (1992)).
• In vitro mutagenesis of the A In vitro mutagenesis of the A.
• niger PME B Duwe et al.., Biotechnol. Letts 18: 621-626 (1996)) indicated that a histidine residue, which is conserved in ZmC5, within the region I may be located at the active site of the enzyme, and in A. niger is required for enzyme activity. However this histidine is replaced by other amino acid residues in several PMEs of both plant and fungal origin, suggesting that is it not essential in all PMEs.
• ZmC5 shows a closer relationship to the P. inflata 'late' pollen expressed PPE gene (JH Mu et al.., Plant Mol Biol 25: 539-544 (1994)), than to B. napus 'early' pollen expressed Bpl9 gene (D. Albani et al . supra.)
• a maize inbred line A 188, genomic Southern blot containing 15 ⁇ g of DNA digested with either BamHl, EcoRI, or Hind III was probed with radiolabelled full-length ZmC5 cDNA insert.
• Two strong hybridising bands in each lane of the blot in Figure 2 suggests the presence of at least two similar genes in the maize genome. Several further bands with show a much weaker signal suggests that this gene family may also comprise several less related members.
• FIG. 3(A) A northern blot containing 1 O ⁇ g total RNA from eight maize tissues was probed with the cDNA ZmC5c to determine the expression programme of the gene.
• a transcript of approximately 2.0kb was detected only in pollen and germinating pollen ( Figure 3(A)) , indicating that, within the limits of detection of this technique, expression of this gene appears to be restricted to these two tissues. No signal was detectable in leaf, root, shoot, cob, endosperm or embryo. The expression programme during spikelet development was also determined.
• Figure 3(B) shows a Northern blot containing total RNA from 0.25, 0.5 and 1.0cm spikelets, mature pollen and germinating pollen. The ethidium bromide stained gels demonstrate the equal loadings of RNA in the lanes of each gel. - 16 -
• Spikelets were staged by staining the anthers with acto-carmine, and the anthers were found to contain cells at the following stages: pre-meitotic sporpogenous cells (0.25cm), mid-prophase I (0.5 cm), maturing pollen grains (1.0 cm). Some overlap between consecutive stages is however inevitable due to the variation in the developmental stage between the two florets within the same spikelet.
• This Northern analysis shows that ZmC5 expression is restricted to mature dehisced pollen and germinating pollen with no detectable expression in any other maize tissues including spikelets containing cells in earlier stages of microsporogenesis.
• Transciptional fusions were made between the 5' region of ZmC5g and the reporter gene ⁇ -glucuronidase (Figure 4A), and used to transform tobacco by Agrobacterium transformation.
• the construct was made as follows:- the two Sph I sites, one within the 2.5kb Sail fragment which contains 2kb of 5'sequence relative to the ATG on one within the polylinker, were used to remove the 3' end from position -61 to +403 ( Figure 1).
• Transformants were selected on kanamycin and primary transgenic plants were regenerated, two of which, positive for expression of the transgene, were taken to the T2 generation. Pollen grains from dehisced anthers of the transgenic plants were harvested and stained for GUS activity as described by J.A. Jefferson (Plant Mol Biol Rep (1987) 5: 387- 405). Two plants were positive showing approximately 50% blue staining pollen ( Figure 4B). No blue colouration was detected in non-transgenic controls. To investigate the number of integration sites, plants from two transgenic lines were selfed, and progeny were scored for resistance to kanamycin. Of the progeny assessed from transgenic plant GC5-2, - 17 -
• transgenic plant GC5-7 gave a mean ratio of kanamycin resistant to kanamycin sensitive of 3.8: 1 indicative of a single integration site (expected ratio for one integration site is 3: 1, for two, 15: 1 and for three 63:1).
• Extracts were made from a range of tissues including five stages of developing anthers, and analysed fluorimetrically fir GUS expression (Jefferson, supra.)
• Figure 4(C) shows GUS activities from two transgenic plants. Only very low levels of expression are detectable in tissues other than developing and mature dehisced anthers. In tobacco the stage of bud development can be correlated with bud length (Tebutt et al.., supra.) but this is dependent on the growth conditions.
• Bud-1 corresponds to microspores at mitosis/tetrad stage
• Bud-2 uninucleate microspores
• Bud-3 microspore mitosis Bud-4
• early to mid-stage binucleate gametophyte Bud-5 mid- to late-stage binucleate gamteophyte.
• Microspore stages as assessed by DAPI staining indicate that the timing of expression of the ZmC5 promoter in tobacco agrees well with its expression in maize based on the Northern data ( Figure3; Figure 4C).
• Figure 3 Figure 4C
• a plant transformation vector comprising the Ale A promoter driving expression of GUS .and a 35S CaMV promoter driving the expression of AlcR has been introduced into tobacco and tomato plants.
• GUS expression may be studied in all tissues before and after induction with ethanol as a root drench.
• GUS staining of tomato anthers and pollen shows clear expression of GUS after induction. The same result is expected from pollen from other species. - 18 -
• the resulting plasmid was named pSK- C5-BB ⁇ ( Figure 6).
• the entire cassette is then removed as an EcoRl- Notl fragment to a binary plant transformation vector pVB6.
• the construct is then introduced into Agrobacterium Tumefaciens by the freeze-thaw method. Standard techniques are used to introduce the D ⁇ A into tobacco.

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  • 1999-01-22 KR KR1020007009173A patent/KR20010041129A/ko not_active Application Discontinuation
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  • 1999-01-22 HU HU0100787A patent/HUP0100787A3/hu not_active Application Discontinuation
  • 1999-01-22 IL IL13797199A patent/IL137971A0/xx unknown
  • 1999-01-22 AU AU22876/99A patent/AU751438B2/en not_active Ceased
  • 1999-01-22 JP JP2000532527A patent/JP2002504335A/ja active Pending
  • 1999-02-17 DZ DZ990026A patent/DZ2725A1/xx active

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