CA2224736A1 - Inhibition of cell respiration and production of male sterile plants - Google Patents

Abstract

A method of inhibiting gene expression in a target plant tissue which comprises stably transforming a plant cell of a type from which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells of the said target tissue resulting in death of the cells, the said disrupter gene is selected from the group consisting of the T-urfl3 gene, genes encoding an .alpha.- or .beta.-tubulin, short sense co-suppression of two essential maize cell cycle genes, cdc25 and replication origin activator (ROA) and a short sense construct to the adenine nucleotide translocator (ANT) of the inner mitochondrial membrane.

Description

INHIBITION OF CELL RESPIRATION AND PRODUCTION OF MALE STERILE PLANTS
The present invention relates to a method of producing male sterile plants by use of a gene, which is expressible in plants and inhibits an essential cell function. hence disrupting full - e~cpression of a selected plant characteristic.
Our International Patent Application No. WO 90/08831 describes and claims the disruption of respiration using a variety of disrupter genes which we refer to also as "pollen-inactivating genes".
The ability of such inactivating genes to function in this way varies and, therefore, there is a need for further improved gene sequences so that appropriate selection for specific u applications may be made.
An object of this invention is to provide genes for use in inhibiting gene expression.
According to the present invention there is provided a method of inhibiting geneexpression in a target plant tissue comprising stably transforming a plant cell of a type from which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when e~cpressed, of inhibiting respiration in the cells of the said target tissue resulting in death of the cells characterised in that the said disrupter gene is selected from the group consisting of the T-urfl 3 gene, genes encoding an a - or ,~-tubulin, short sense co-suppression of two essenti~l maize cell cycle genes. cdc25 and 2u replication origin activator (ROA) and a short sense construct to the adenine nucleotide translocator (ANT) of the inner mitochondrial membrane.
Down regulation of gene activity due to short sense co-suppression is described in our International Patent Application No.WO 90/08299.
The a- or ~-tubulin genes act as disrupters by de-stabilizing microtubule arrays in plant cells. hence inhibiting essential microtubule function in the said target tissue resulting in death of the cells.
The use of short sense co-suppression of two essential maize cell cycle genes, cdc25 and replication origin activator (ROA) disrupts cell division and hence provide a ~rowth defect in the targeted organ or tissue.
Preferably the promoter is an anther- and/or tapetum-specific promoter or a pollen-specific promoter, so that on e~pression of the said disrupter protein therein the re_enerated plant is in male sterile. More preferably the said anther and/or tapetum-specific promoter was isolated using the cDNA sequences shown in Figure 1 or 2 or 3 of the accompanying drawings and using the techniques described in our International Patent Application No. WO 90/08826.
Plasmids cont~ininv the DNA sequences shown in Figures 1, 2 and 3 have been deposited under the terms of the Budapest Treaty, details being as follows:
Plasmid pMS10 in an ~scherichia coli strain RR1 host, cont~ininv the gene sequence shown in Figure I herewith, and deposited with the National Collection of Industrial &
Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40090.
Plasmid pMS 14 in an ~scherichia coli strain DH5a host, cont~ining the gene sequence shown in Figure 2 herewith, and deposited with the National Collection of Industrial & Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40099.
Plasmid pMS 18 in an ~scherichia coli strain RR1 host, Cont~inin~ the gene sequence shown in Figure 3 herewith, and deposited with the National Collection of Industrial &
Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40100.
The isolation and characterisation of these gene sequences of this invention aredescribed in full in WO 93/01294.
Other promoters may also be used, for example a promoter such as the tapetum specific MFS14 promoter.
The present invention also provides a plant having stably incorporated in its genome 2u by transformation a gene construct carrying a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting an essenti~l cell function such as respiration, microtubule arrays or cell division in the cells of the said target tissue resulting in death of the cells.
The invention also provides a plant, particularly a monocotyledonous plant, and more particularly a corn plant, having stably incorporated within its genome a gene construct carrying a tissue-specific promoter which operates in the cells of the said target tissue and a disrupter gene encoding a protein which is capable of inhibiting an essenti~l cell function such as respiration or microtubules in the said cells of the said target tissue resulting in death of the 3() cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct of the adenine nucleotide translocator, genes encoding an a- or ,~-tubulin and short sense down-regulation of the essential cell cycle genes, cdc25 and ROA.

These gene constructs may be used as a means of inhibiting cell growth in a range of organisms from simple unicells to complex multicellular org~nicmC such as plants and animals.
Bv the use of tissue- or cell-specific promoters, particular cells or tissue may be targeted and - destroyed within complex org~nicmC. One particular application intended for this invention is in the destruction of cells essential for male flower development, leading to male sterility.
The invention therefore provides a method of preventing or inhibiting growth anddevelopment of plant cells based on gene constructs which inhibit an essenti~l cell function such as respiration or microtubules. The technique has wide application in a number of crops where inhibition of particular cells or tissue is required.
Of particular interest is the inhibition of male fertility in maize for the production of Fl hybrids in si~l. The concept of inhibition of mitochondrial function as a mecll~nicm for male sterility arises from some previous research on T-type cytoplasmic male sterilitv in maize (cms-T) which has shown an association between the male sterile phenotype and mitochondrial dysfuction. Although a direct causal relationship has yet to be established 15 between mitochondrial dysfunction and cms-T, an increasing body of evidence suggests that fully functional mitochondria, particularly in the tapetal cells, are essential. This is particularly critical during microsporogenesis since the metabolic dem~n(ls placed on the tapetal cells results in a 40-fold increase in mitochondrial number.
Thus we provide a number of negative mutations which act upon mitochondria to 2() inhibit functional respiration. When specifically expressed in maize anther tissue these mutations will result in a male sterile phenotype.
We also use ~ yres~ion of a- or ~-tubulin genes to disrupt cell function. Duringnormal cell life, expression of tubulin genes is closely regulated by their endogenous promoters and closely matches the requirements of cells for these proteins which are 2~ polymerised and assembled into microtubules during growth and development of the plant. By expressing tubulin genes in an unre_ulated fashion using non-tubulin promoters in a particular tissue or stage of development~ the equilibrium between free tubulin monomers and those polmerised in microtubules is disrupted resulting in instability of the microtubule complex and cellular dysfuntion. When expressed in the tapetum or other cells of the anther this latter 3() effect will cause the plants to be sterile.

We also propose the use of short sense down-regulation of essenti~l cell cvcle genes, eg cdc25 and ROA. When expressed in the tapetum or other cells of the anther this latter effect will cause the plants to be sterile.
The method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed. Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agluinfe~;Lion usingAgrobac~erium one~aci~ns or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few.
o Reference may be made to the literature for full details of the known methods.
The development and testing of these gene constructs as disrupters of mitochondrial function in the unicellular organism, yeast, will now be described. A mech~ni~m by which these gene constructs may be used to inhibit plant cell growth and di~rel~liation in transformed plants will also be described. The object of these procedures is to use yeast as a I j model system for the identification and optimisation of gene constructs for ~:Apl essing proteins which disrupt mitochondrial function. Plant cells will then be transformed with the selected constructs and whole plants regenerated therefrom.
The accompanying drawings are as follows:
Figure I shows the DNA sequence of an anther-specific cDNA, carried by plasmid 2~ pMS 10, Figure 2 shows the DNA sequence of a tapetum-specific cDNA, carried by plasmid pMS I 4;
Figure 3 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMS18;
Figure 4 shows the sequence of the T-urfl3 gene (SEQ ID NO I ) with the primers Turf-l (SEQ ID NO 2) and Turf-2R (SEQ ID NO 3) underlined;
Figure 5 shows DNA encoding the 59 amino acid region from the ATP-2 gene of Ni~olinia plumbaginifolia ((SEQ ID NOS 4 and 5) with primers PREB-IB (SEQ ID NO 6) and PREB-R (SEQ ID NO 7) shown;
3() Figure 6 shows the cleavage site of the pre-,~ sequence;
Figure 7 is a map of vector pCaMVIIN, Figure 8 is a map of vector RMS 17;

Figure 9 is a map of vector pIE109;
Figure 10 shows the MFS14 promoter sequence (SEQ ID NO 8) with the following features:
position 2198 transcription start CCT"A"CAA (concensus CTC"A"TCA) position 2167 ATCCATT (possible TATA box motif) position 2141 CCAT (possible CAAT box motif) position 2233 cdna start CAC"A"CAG
position 2295 translation start GCAACAATGGCG (concensus TAAACAATGGCT), Figure 11 is a map of vector RMS I l;
Figure 12 is a map of vector pMANT3 Figure 13 illustrates the contruction of vectors for maize cell line transformation: and Figure 14 is a plot showing numbers of transformants produced in the various different experiments.
The invention will now be illustrated by the following Examples.
Example I
Construction of the maize transformation vector. RMS 17.
We used the PCR to amplify the T-urfl3 gene from cms-T maize (line RW33:TMS) and the mitochondrial targeting sequence, pre-~, from Nic-7~ianu plumbuYinolia DNA
samples from plant material were prepared using the method described by Edwards e~ al 2() (Nucliec Acids Research 1991, 19, 1349).
The complete T-urfl3 gene was amplified in the PCR using primers turf-l (5'ATCGGATCCATGATCACTACTTTCTTAAACCTTCCT-3', SEQ ID NO 2) and turf-2R (5'TAGTCTAGATCACGGTACTTGTACGCTATCGGT-3', SEQ ID NO 3) designed from sequence information provided by Dewey et al ( 1986, Cell, 44, 429-449). The PCR
2~ conditions were 35 cycles of denaturing at 94~C for 0.8 min, annealing at 65~C for I min and extension at 72~C for 2.5 mins. To aid subsequent cloning the PCR primers were designed such that they introduce unique BamHI and XbaI restriction sites at the 5' and 3' ends of the gene respectively. The position of these primers relative to the T-urfl 3 gene sequence is shown in Figure 4.
3() Similarly the 59 amino region from the ATP2 gene of ~lico~iana plumbaginifolia which encodes the functional pre-~ mitrochondrial targeting sequence was amplified in the PCR using the primers PREB-IB
(5'ATCGGTACCGCCATGGCTTCTCGGAGGCTTCTCGCCT-3', SEQ ID NO 6) and PREB-R (5'ATCGGATCCCGCTGCGGAGGTAGCGTA-3', SEQ ID NO 7) designed using 3~ sequence information provided from Boutry et al (1987, Nature, 328, 341). The PCR

CA 02224736 l997-l2-l6 conditions were as described above except that the annealing temperature was reduced to 60~C. To aid subsequent cloning PBEB-IB and PREB-2R were designed such that theyintroduce unique ~nl and BamHI restriction sites at the 5' and 3' ends of the amplified fragment respectively. The position of these primers relative to the ATP2 gene is shown in Fi_ure 5.
Following amplification the pre-B PCR fragment was digested with ~nI and BamHI
to generate cohesive ends and cloned into the corresponding sites of the vector pUC 18 to give plasmid pPB I . The TURF- 13 PCR product was then digested with BamHI and XZ~aI
and cloned into the corresponding sites in pPB 1 to give plasmid pPB2. In pPB2 the pre-~
lo sequence is fused in frame with the T-urfl3 gene so that following e~y,ession in a plant cell the full product will be transported to mitochondria. Cleavage of the pre-~ sequence at the predicted site between residues 55-56 will release the T-urfl3 protein which includes at its NH~-terminus an additional 4 residues from the pre-,B sequence (Figure 6).
The pre-,B/T-urfl 3 gene fusion in pBB2 removed by digestion with the enzymes K~n I
ls and Sal I, blunted-ended and cloned into the plasmid pCAMVIlN (Figure 7) which was digested with BamHI and blunt-ended to give pPB3. This cloning step places the pre-~/T-urfl 3 gene fusion under transcriptional control of the CAMV 35S promoter. The Ad~lI intron is present in this construct to boost ~,uression levels in corn cells (Mascarenhas ct al., 1990.
Plant Mol. Biol., 15, 913-920) and the nos 3' sequence provides a polyA addition site. To produce the final vector, RMS17 (Figure 8) the PAT selection c~csette from plE109 (Figure 9) which allows in vitro selection of transformed corn cells on bialaphos was introduced as an EcoRI fragment into the unique l~coRl site of pPB3.
Example 2 Transformation of BMS corn cells with RMS 17.
The objective ofthis experiment was to show that expression ofthe pre,B/TU~-13 gene construct in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation. The RMS 17 vector (Figure 8) was introduced into cultured BMS cells using a silcon carbide fibre-mediated transformation technique as follows:
Preparation of silicon carbide whiskers Dry whiskers were alwavs handled in a fume cabinet, to prevent inhalation and possible lung damage. These whiskers may be carcinogenic as they have similar properties to asbestos. The Silar SC-9 whiskers were provided by the Advanced Composite Material Corporation Greer, South Carolina,USA. The sterile whisker suspensions were prepared in advance as follows. Approximately 50mg of whiskers were deposited into a pre-weighed 1.5 ml Eppendorf tube, which was capped and reweighed to determine the weight of thewhiskers. The cap of the tube was perforated with a syrin e needle and covered with a double layer of aluminium foil. The tube was autoclaved (121~C, 15psi, for 20 minutes) and dried. Fresh whisker suspensions were prepared for each experiment, as it had been reported that the level of DNA transformation when using fresh suspensions was higher than that of older suspensions. A 5% (weight/volume) whisker suspension was prepared using sterile u deionised water. This was vortexed for a few seconds to suspend the whiskers immediately before use.
DNA transformation into cells All procedures were carried out in a laminar air flow cabinet under aseptic conditions. The DNA was transformed into the cells using the following approach. Specific modifications to this method are indicated in the text.
Cell and whisker suspensions were pipetted using cut down Gilson pipette tips. 100~
of fresh BMS medium (see appendix 1 ) was measured into a sterile Eppendorf tube. To this was added 40ul ofthe 5% (w/v) whisker suspension and 25~1 (Im~!ml) ofthe plasmid DNA, which was vortexed at top speed for 60 seconds using a desktop vortex unit (Vortex Genie 2 2u Scientific industries, Inc). Immediately after this period of vortexing, 500~11 of the cell suspension was added ie 250,~1 of packed cells. The Eppendorf tube was then capped and vortexed at top speed for 60 seconds in an upright position. The same procedure was used to transform the other cell lines.
Three controls were included in this experiment. Two positive control vectors were 25 pPG3 which contains the PAT selection cassette alone and RMS 15, which is identical to RMS 17 except that the T-urfl 3 gene is replaced by the mitochondrial uncoupling protein gene UCP, which jhas no effect on cultured BMS cells. The pre-,~ targeting sequence is present in both constructs. A negative control, which should completely prevent establishment of transgenic calli, was provided by RMS 13. which is identical to RMS 17 3u except that the pre~/T-urfl3 gene fusion is replaced by the cytotoxic ribonuclease gene, barnase.
The mean numbers of transgenic calli established in this e,~pe~ ent are shown inTable 1.

TABLE I
No. of transgenic calli per replicate Vector Mean 1 2 3 pPG3 ~ 44 33 39 These data show that relative to the two positive controls, pPG3 and RMS 15, expression of the preB/T-urfl 3 gene fusion results in a signficant decrease (p~5% or better) in the establishment of transgenic calli. This suggests that targeting T-urfl 3 protein to mitochondria has a deleterious effect on these cells, presumably due impartment of mitochondrial function.
Expression of the cytotoxic ribonuclease, barnase, completely abolishes the establishment of transformed calli.
o Example 3 Construction of the maize transformation vector RMS 11 RMS11 is a transformation vector in which e~p-ession ofthe pre-,B/T-urfl3 gene fusion is controlled by the maize tapteum promoter, MFS14. The sequence ofthe MFS14 promoter and untranslated leader region from position -2198 to +97 is shown in Figure 10. In this way, expression of the T-urfl 3 protein is limited to the cells producing pollen and not throu_hout the whole plant.
To construct RMS 11, the K~n I - SalI fragment from pPB2 cont~ining the pre-~/T-urfl 3 gene fusion was blunt-ended and ligated into the blunt ended BamHI site of plasmid pSC9 to yield pPB4. In pPB4 the pre-,~/T-urfl3 gene fusion is now positioned between the -152 to +97 MFS14 promoter fragment and the nos 3' polyadenylation sequence. Thiscomplete cassette was removed from pPB4 by digestion with Sacl and EcoR~ and cloned into the corresponding sites of pSC7 to give plasmid pB5. pSC7 contains the - 153 to -5800 region of the MFS 14 promoter so that the introduction of the SacI - EcoRI fragment from pPB4 recreates the full 5.8 kb MFS14 promoter.
2j RMS I I (Figure 11) was completed by introduction of the PAT in vitro selection cassette from p IE 109 into the unique EcoF'I site of pPB5.

g Example 4 Transformation of maize cells with RMS I I bv particle bombardment to provide stably transformed~ male sterile plants The maize transformation vector, RMS 1 1 was used to transform regenerable maize5 cell cultures by particle bombardment.
Culture Material Friable embryoyenic Type II callus was initiated from immature zygotic embryos e,xcised from either greenhouse or filed grown A188 plants 10-12 days after pollination with pollen from the inbred B73. The medium used for callus initiateion and maintenance was u based onN6 medillm as modified by Armstrong and Green. Specifically, the medium contained 6mM L-proline, 2% (w/v) sucrose, 2 mg/l 2,4-dichlorophenoxy- acetic acid (2,4-D) and 3% (w/v) Gelrite (Trade Mark, Caroline Biological Supply) at pH 6Ø Callus was grown for 4-4 weeks prior to suspension culture initiation. Suspension cultures were initiated in a MS-based liquid medium cont~ininv 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l 1~ I-naphthalPn~cetic acid (NAA), 6mM proline, 200 mg/l casein hydrolysate (Difco Laboratories), 35 (w/v sucrose and 5% (v/v) coconut water (Difco Laboratories) at pH 6Ø
Cell suspensions were m~int~ined in theis me~iurn in 125 ml Erlenmeyer flasks at 28~C in the dark on a gyrating shaker at 125 rpm. Suspension were subcultured every 3.5 days by addition of 3ml packed volume of cells and 10 ml culture medium to 20 ml of fresh culture 2u medium. The suspension cultures were typically 6 months to one year old at the time of bombardment. Suspension cultures recovered from cryopreservation were used in some transformations .
Microprojectile Bolllbaldlllelll Cell suspensions were sieved through a 1.0 mm and then a 0.5mm screen. A packed 25 volume of 0.2 ml of the cells which passed through the sieves was then suspended in 5 ml of suspension medium and evenly distributed on to a Whatman No.4 filter paper disc l,ia vacuum filtration using a 4.7 cm microanalysis holder. Precipitation of supercoiled plasmid DNA on to tungsten particles and bombardment using the DuPont PDS-1000 Biolistics (Trade Mark) apparatus were essenti~ly as described by the manufacturers. Target plates were bombarded 3() once.

Transformant selection and plant re~eneration Following bombardment, each filter disc (with cells) was tlansr~lled to N6 basedmedium cont~ining 100 mg/l myo-inositol, 2 mg/l 2,4-D, 3% (w/v) sucrose, and 0.3% (w/v) Gelrite at pH 6Ø For selection using the NPTII gene, this medium was supplemented with 5 200 mgtl kanamvcin sulphate. The filter discs were transferred to fresh medium cont~ining the selection agent after seven and again after 14 days. The suspension was divided into two equal aliquots and each was evenly plated over 20 ml of solidified media cont~ining the selective agent and 3% (w/v) Gelrite in 100 x 20 mm Petri dishes. After 2-5 weeks, rapidly growing, putatively transformed calli were removed and transferred to the surface of fresh 1() selection me~ m Plants were renerated by transferring tissue to MS based medium cont~ining I g/l myo-inositol, 1 mg/l NAA, 6% (w/v) sucrose, and 3% (w/v) Gelrite at pH
6Ø After 2-3 weeks. the tissue was transferred to MS media cont~ininu 0.25 mg/l NAA, and 3% (w/v) sucrose and placed in the light, where embryo ge~nh,dlion occurred. Plants were then grown in half-strength MS based mer~ m cont~ in~ 500 mg/l myo-inositol, 3% (w/v) sucrose and 0.3% Gelrite at pH 6.0 for approximately 1-2 weeks priro to transfer to the greenhouse.
After transfer to the glasshouse, plants within each independently transformed clone were tested for the presence ofthe MFS14/pre-B/T-urfl3 gene construct using the PCR.
DNA was extracted from small leaf samples using the technique described by Edwards e~ al 2() ( 1991, Nucleic Acids Research, 19, 1349) and used in the PCR with the primers, 14-SA (5'-AGACGCTGAGCTCAAGGACGTGA-3' SEQ ID NO 9)and turf-2R (see Example I for the sequence of this primer).
At flowering the plants within each independently transformed clone were assessed in the glasshouse using a visual scale developed for CMS lines and described in Table 2. Plants scoring 4 and below are functionally sterile. The accu~ ted sterility scores for each of the independent PCR positive clones is shown in Table 3 and compared to a maize line which was generated by bombardment with RMS 1 1 but which is PCR negative for the MFS 14/pre-B/T-urfl3 gene construct.
Sterile plants were backcrossed with pollen from fertile, non-transgenic BE70 plants.
the progeny seeds arising from one of these crosses (clone YK23, plant 5 x BE70) were planted in the glasshouse and allowed to flower. The presence of the MFS 1 4/pre-B/T-urfl 3 gene construct as assessed by PCR and PAT test (the latter determines whether the selectable marker used inthe transformation process is present) and the fertility scores of the plants are shown in Table 4. Plants which were PCR negative were rogued from the glasshouse prior to flowering. Plants I and 2 had an inconclusive PCR test and were kept until flowering. Plant 12 was kept as a control. As can be seen in Table 4, 6 ofthe pro_eny plants were sterile and s this sterilitv correlated with the presence of the transgene as assessed either by PCR or PAT
testin_ This is consistent the the presence of a sin_le transgenic locus imparting sterility.

Anther Classification (After L.M. Josephson) Class 0 No anthers exerted Class I Less than half of the anthers exerted and all were small, dry and hard with no pollen shed.
Class 2 Most of anthers exerted but all were small. dary and hard with no pollen shed.
Class 3 Partially fertile anthers exerted with some pollen shed, proportion of anthers exerted was highly variable.
Class 4 Slightly abnormal anthers with approximately 75 to 100 percent exertion.
Class S Normal anthers and fully fertile.

1~ * Class 4 through 5 tassels were considered fertile.

CLONE NO. OF PLANTS FERTILITY SCORE P.C.R.
WK23 2 0 +
3 3 +
WK23 2 0 +
YK23 3 0 +
4 +

PLA PCR TEST PAT TEST FERTILITY SCORE
NT
YK23/51 1 +/ O
YK23/5/2 +/- + o YK23/516 + +
YK23/5/7 + + O

YK23/5/9 + + O

YK23/5/1 1 + + O
Y~C23/5/12 - - 5 Example 5 Construction of the maize tran~rn,dlion vector pRMS-23 We have tested whether e~ression of a short-sense construct from the maize adenine nucleotide translocator (ANT) gene will give rise to a defect in the growth of maize cells. A
fragment of the maize ANT was isolated using the PCR and primers designed from the sequence ofthe maize gene published by Bathgate et al (1989, Eur. J. Biochem., 83, 303-310).
o The fragment of theANT gene was amplified in the PCR using primers MANT- 1(5'-ATGCCCGGGCTTGCAATGTCTGTTAGCGGTGGCATCA-3', SEQ ID NO 10) and MANT-2RB (5-ATGCCCGGGCGATGGGGTAAGATGCAAGACCA-3', SEQ ID NO 11).
The PCR conditions were 35 cycles of denaturing at 94~C for 0.8 min, ~nn~linv at 65~C for 1 min and extension at 72~C for 2.5 mins. To aid subsequent cloning the PCR primers were designed such that they introduce unique SmaI restriction sites at the 5' and 3' ends of the gene. The sequence of the maize ANT gene was published in Eur. J. Biochem. ( 1989) 183, 303-310. The MANT-1 primer sequence appears at the bevinninv ofthe coding sequence of the gene and the MANT-2R primer sequence is near the end of the gene.

Following PCR which produced a DNA fragment of the predicted size of 1050 bp, the DNA was digested withSmaI and subcloned into theS~nal site of pUC18 to give pMANTI.
Subsequently the nos 3' polyadenylation signal sequence was introduced 3' to the ANT gene as a SacI- l~coRI fragment into the corresponding sites in pMANTI to yield pMANT2. A
/~indIII - Ba~nHI fragment carrying the CaMV 35s promoter and ADH lintron from pCaMVI~N (Figure S) was introduced into the corresponding sites of pMANT2 to yield pMANT 3. pRMS-23 (Figure 12) was completed by introduction ofthe PAT in vitro selection cassette from pIE 109 (Figure 7) into the unique ~coR~ site of pMANT3 .
1~ Example 6 Transformation of BMS corn cells with pRMS-23 The objective of this experiment was to show that expression of pRMS-23 in cultured BMS corn cells results in a reduction in cell viability as measured by the establichm~nt of transgenic calli following transformation in two separate experiments. The vector DNAs 1~ were introduced into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.
Following transformation with pRMS23 the mean numbers of transgenic calli established were determined relative a positive control pPG3 wihich contains the in vi~ro selection cassette alone (Table 5). These data show that expression of a short sense adeneine 2() nucleotide translocator gene results in a significant decrease in the establishment of transgenic calli.

No of transgenic calli per replicate 2 3 Mean Experiment I
pPG3 16 20 22 19 pRMS-23 3 4 3 3 Experiment 2 pPG3 49 48 40 46 pRMS-23 10 15 23 16 Example 7 Construction of the maize tran~r~l ,llation vectors. pTBR and pTBS
We tested whether un-regul~ted expression of a-tubulin genes will give rise to adefect in the growth of maize cells. Two constructs cont~ining the coding sequence from a-tubulin cDNAs isolated from two biotypes of Eleusine indica were prepared. pTBR (Figure 13) contains the a-tubulin cDNA from a dinitroaniline resistant biotype of Eleusine indica cloned as a blunt-ended h'inf I fragment into the blunt-ended BamHI site of pCaMVIlN
(Figure 7). pTBS (Figure 13) contains the a-tubulin cDNA from a dinitroaniline sensitive biotype cloned exactly as described for pTBR.
IU Example 8 Transformation of BMS corn cells with pTBR and pTBS.
The objective of this experiment was to show that e.~l es~ion of pTBR and pTBS in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment oftransgenic calli following Lldllsrollllation. The vector DNAs were introduced 15 into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.
Following transformation with pTBR and pTBS the mean numbers of transgenic calliestablished were determined relative a positive control pPG3 wihich contains the in ~,itro selection cassette alone (Figure 14). These data show that non-regul~ted e~,ression of an a-20 tubulin gene from either biotype of Eleusine indica results in a significant decrease in theestablishment of transgenic calli.

CA 02224736 l997-l2-l6 1~
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(A) ORGANISM: T-urfl3 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
I () ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCTCCCT .TGATCAAGG TTTGGTATTT 60 ~5 ATGGATGATT CCTATTTGGC TCAACTCTCC GAGTTAGCCA ACCACAATAG AGTGGAAGCG 180 (2) INFORMATION FOR SEQ ID NO: 2:
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(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 6~
(ii) MOLECULE TYPE: DNA
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(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Dl TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
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(A) LENGTH: 269 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: ATP-2 gene of Nicotinia plumbaginifolia (ix) FEATURE:
4() (A) NAME/KEY: CDS
(B) LOCATION:l..267 (D) OTHER INFORMATION:~codon_start= l (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gln Ser Ala Gln l 5 l0 15 Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu 60 Asn Arg Ala Val Gln Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gln Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr 5 Asp Glu Phe Thr Gly Ala Gly Ser Ile (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gln Ser Ala Gln 20 l 5 l0 15 Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu Asn Arg Ala Val Gln Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gln Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr Asp Glu Phe Thr Gly Ala Gly Ser Ile (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~5 (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: PREB-IB primer ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

- -ivi) ORIGINAL SOURCE:
(A) ORGANISM: PREB-2R primer ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATCGvALCCC GCTGCGGAGG TAGCGTA 27 l() !2) INFORMATION FOR SEQ ID NO: 9:
i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2285 base pairs (B) TYPE: nucleic acld (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 2() (vi) ORIGINAL SOURCE:
(A) ORGANISM: MFSl4 Promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

3~
CCAGAAAACT TAGAAAACTC TGGTATCCTT GCCCCTTGTG GATATGGGAC AATGTCAAAC l80 I() 5~

~5 ATACACGTTA ATTTTCTAAT TGGTTCTTCT AACCCTCTAA TCTAATGCTT CATTTGATTA 900 ACTTGAAGCG AAGATAGAAG AGATGTGACG TCGGTATCGC ATGTTTGACA ACTTTCTGGT l020 6() (5 AGATATCGGT GTGATCTTTA GAACCGCCAT TTGATGGCCT GAGTTTTAGT AGATCTAGAC 1200 -2~ ACTGATTCTA CTCATGCGCG AACCATTCGT GCGCCACCGC TGCCCGTCCC GCGATCGCTC 1800 It) (2) INFORMATI..~ FOR SEQ ID NO: 9:
(i) SEQ YNCE CHARACTERISTICS:
(A LENGTH: 23 base pairs (E) TYPE: nucleic acid '; (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: 14-SA Primer (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
6~
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~o (i ) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: MANT-i Primer (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: l0:
1() (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base palrs (B) TYPE: nucleic acld (C) STRANDEDNESS: single (D) TOPOLOGY: linear 2() ! ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: MANT-2RB Primer (xij SEQUENCE DESCRIPTION: SEQ ID NO: ll:
3() ATGCCCGGGC GATGGGGTAA GATGCAAGAC CA 32

Claims (9)

1. A method of inhibiting gene expression in a target plant tissue which comprises stably transforming a plant cell of a type from which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells of the said target tissue resulting in death of the cells, the said disrupter gene is selected from the group consisting of the T-urf13 gene, genes encoding an .alpha.- or .beta.
-tubulin, short sense co-suppression of two essential maize cell cycle genes, cdc25 and replication origin activator (ROA) and a short sense construct to the adenine nucleotide translocator (ANT) of the inner mitochondrial membrane.

2. A plant having stably incorporated in its genome by transformation a gene construct carrying a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting an essential cell function such as respiration, microtubule arrays or cell division in the cells of the said target tissue resulting in death of the cells wherein the disrupter gene is selected from the group consisting of the T-urf13 gene, genes encoding an .alpha.- or .beta.-tubulin, short sense co-suppression of two essential maize cell cycle genes, cdc25 and replication origin activator (ROA) and a short sense construct to the adenine nucleotide translocatos (ANT) of the inner mitochondrial membrane.

3. A plant having stably incorporated within its genome a gene construct carrying a tissue-specific promoter which operates in the cells of the said target tissue and a disrupter gene encoding a protein which is capable of inhibiting an essential cell function such as respiration or microtubules in the said cells of the said target tissue resulting in death of the cells characterised in that the said disrupter gene is selected from the T-urf13 gene, a short sense construct of the adenine nucleotide translocator, genes encoding an .alpha.- or .beta.-tubulin and short sense down-regulation of the essential cell cycle genes, cdc25 and ROA.

4. A plant as claimed in claim 2 or claim 3 which is a monocotyledonous plant.

5. A plant as claimed in claim 4 which is a corn plant.

6. A method as claimed in claim 1 or a plant as claimed in any one of claims 2 to 5, wherein the promoter is an anther- and/or tapetum-specific promoter.

7. A method or a plant as claimed in claim 6 wherein the promoter may be isolated using the cPNA sequences of any of Figures 1 to 3.

8. A method or a plant as claimed in claim 6, wherein the promoter is the promoter from the MFS14 gene (SEQ ID NO 8)

9. A male sterile corn plant having stably incorporated within its genome a gene construct carrying a tapetum-specific promoter which operates in the cells of tapetum and a disrupter gene encoding a protein which is capable of inhibiting essentialcell function such as respiration or microrubles in the cells of the tapetum resulting in death of the cells characterised in the the said disrupter gene is selected from the T-urfl3 gene, a short sense construct of the adenine nucleotide translocator, genes encoding an .alpha.- or .beta.-tubulin and short sense down-regulation of the essential cell cycle genes, cdc25 and ROA.