EP0853674A1

EP0853674A1 - Inhibition of cell respiration and production of male sterile plants

Info

Publication number: EP0853674A1
Application number: EP96924071A
Authority: EP
Inventors: Andrew James Greenland; Simon William Jonathan 24 Pound Lane Bright; Paul Richard Drayton; Philip John Bell
Original assignee: Zeneca Ltd
Current assignee: Syngenta Ltd
Priority date: 1995-07-24
Filing date: 1996-07-11
Publication date: 1998-07-22
Also published as: BR9609535A; JPH11509417A; WO1997004116A1; TR199801884T2; CN1197481A; RU2168545C2; NO980314D0; TR199800112T1; GB9515161D0; AU6465296A; CA2224736A1; HUP9802858A3; HUP9802858A2; PL324656A1; BG102274A; NO980314L; TR199801883T2; MX9800575A; AU705759B2; NZ312750A

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 α- or β-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 expression 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 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 gene expression 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 ofthe target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells ofthe said target tissue resulting in death ofthe cells characterised in that the said disrupter gene is selected from the group consisting ofthe T-urfl3 gene, genes encoding an α - or β-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) ofthe 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 α- or β-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 ofthe 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 growth 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 expression ofthe said disrupter protein therein the regenerated 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 ofthe accompanying drawings and using the techniques described in our International Patent Application No. WO 90/08826.

Plasmids containing the DNA sequences shown in Figures 1, 2 and 3 have been deposited under the terms ofthe Budapest Treaty, details being as follows:

Plasmid pMSlO in an Escherichia coli strain RR1 host, containing the gene sequence shown in Figure 1 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 Escherichia coli strain DH5α host, containing 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 Escherichia coli strain RR1 host, containing 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 are described in full in WO 93/01294.

Other promoters may also be used, for example a promoter such as the tapetum specific MFS 14 promoter.

The present invention also provides 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 ofthe 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 ofthe said target tissue resulting in death ofthe 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 ofthe 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 ofthe said target tissue resulting in death ofthe cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct ofthe adenine nucleotide translocator, genes encoding an α- or β-tubulin and short sense down-regulation ofthe 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 organisms such as plants and animals. By the use of tissue- or cell-specific promoters, particular cells or tissue may be targeted and destroyed within complex organisms. 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 and development of plant cells based on gene constructs which inhibit an essential 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 situ. The concept of inhibition of mitochondrial function as a mechanism for male sterility arises from some previous research on T-type cytoplasmic male sterility 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 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 demands 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 inhibit functional respiration. When specifically expressed in maize anther tissue these mutations will result in a male sterile phenotype.

We also use expression of - or β-tubulin genes to disrupt cell function. During normal 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 polymerised and assembled into microtubules during growth and development ofthe plant. By expressing tubulin genes in an unregulated 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 ofthe microtubule complex and cellular dysfuntion. When expressed in the tapetum or other cells ofthe anther this latter effect will cause the plants to be sterile. We also propose the use of short sense down-regulation of essential cell cycle genes, eg cdc25 and ROA. When expressed in the tapetum or other cells ofthe anther this latter effect will cause the plants to be sterile.

The method employed for transformation ofthe 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 agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few. Reference may be made to the literature for full details ofthe 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 mechanism by which these gene constructs may be used to inhibit plant cell growth and differentiation in transformed plants will also be described. The object of these procedures is to use yeast as a model system for the identification and optimisation of gene constructs for expressing 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 1 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMSlO;

Figure 2 shows the DNA sequence of a tapetum-specific cDNA, carried by plasmid pMS 14;

Figure 3 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMS 18; Figure 4 shows the sequence ofthe T-urfl3 gene (SEQ ID NO 1) with the primers

Turf-1 (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 Nicotinia phimbaginifolia ((SEQ ID NOS 4 and 5) with primers PREB-IB (SEQ ID NO 6) and PREB-R (SEQ ID NO 7) shown; Figure 6 shows the cleavage site ofthe pre-β 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 MFS 14 promoter sequence (SEQ ID NO 8) with the following features: position 2198 transcription start CCT"A"CAA (concensus CTC'A' CA) position 2167 ATCC ATT (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 1 1 is a map of vector RMS 11; 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 1

Construction ofthe 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-β, from Nicotiana plitmbapinolia. DNA samples from plant material were prepared using the method described by Edwards et al (Nucliec Acids Research 1991, 19, 1349).

The complete T-urfl3 gene was amplified in the PCR using primers turf-1

(5^*ATCGGATCCATGATCACTACTTTCTTAAACCTTCCT-3' , SEQ ID NO 2) and turf-

2R (5 AGTCTAGATCACGGTACTTGTACGCTATCGGT-3', SEQ ID NO 3) designed from sequence information provided by Dewey et al (1986. Cell, 44, 429-449). The PCR conditions were 35 cycles of denaturing at 94°C for 0.8 min, annealing 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 BamHI z.nά Xbal restriction sites at the 5' and 3' ends ofthe gene respectively. The position of these primers relative to the T-urfl3 gene sequence is shown in Figure 4. Similarly the 59 amino region from the ATP2 gene of Nicotiana plumbagi ifolia which encodes the functional pre-β mitrochondrial targeting sequence was amplified in the

PCR using the primers PREB-IB

(5ΑTCGGTACCGCCATGGCTTCTCGGAGGCTTCTCGCCT-3', SEQ ID NO 6) and

PREB-R (5ΑTCGGATCCCGCTGCGGAGGTAGCGTA-3', SEQ ID NO 7) designed using sequence information provided from Boutry et al (1987, Nature, 328. 341). The PCR 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 they introduce unique Kpnl and BamHI restriction sites at the 5' and 3' ends ofthe amplified fragment respectively. The position of these primers relative to the ATP2 gene is shown in Figure 5.

Following amplification the pre-B PCR fragment was digested with Kpnl and BamHI to generate cohesive ends and cloned into the corresponding sites ofthe vector pUC18 to give plasmid pPB 1. The TURF- 13 PCR product was then digested with BamHI and Xbal and cloned into the corresponding sites in pPB 1 to give plasmid pPB2. In pPB2 the pre-β sequence is fused in frame with the T-urfl3 gene so that following expression in a plant cell the full product will be transported to mitochondria. Cleavage ofthe pre-β sequence at the predicted site between residues 55-56 will release the T-urfl3 protein which includes at its NH₂-terminus an additional 4 residues from the pre-β sequence (Figure 6).

The pre-β/T-urfl3 gene fusion in pBB2 removed by digestion with the enzymes Kpn I and Sal I, blunted-ended and cloned into the plasmid pCAMVIiN (Figure 7) which was digested with BamHI and blunt-ended to give pPB3. This cloning step places the pre-β/T- urfl3 gene fusion under transcriptional control ofthe CAMV 35S promoter. The Adhl intron is present in this construct to boost expression levels in corn cells (Mascarenhas et al., 1990. Plant Mol. Biol., 15, 913-920) and the nos 3' sequence provides a polyA addition site. To produce the final vector, RMS 17 (Figure 8) the PAT selection cassette from p IE 109 (Figure 9) which allows in vitro selection of transformed corn cells on bialaphos was introduced as an EcoRI fragment into the unique EcoRI site of pPB3. Example 2 Transformation of BMS corn cells with RMS 17. The objective of this experiment was to show that expression ofthe preβ/TURF-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 always 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 ofthe whiskers. The cap ofthe tube was perforated with a syringe 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 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. lOOμl of fresh BMS medium (see appendix 1) was measured into a sterile Eppendorf tube. To this was added 40μl ofthe 5% (w/v) whisker suspension and 25μl (lmg/ml) ofthe plasmid DNA, which was vortexed at top speed for 60 seconds using a desktop vortex unit (Vortex Genie 2 Scientific industries, Inc). Immediately after this period of vortexing, 500μl ofthe cell suspension was added ie 250μl 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 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-β 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 except that the preβ/T-urfl3 gene fusion is replaced by the cytotoxic ribonuclease gene, barnase.

The mean numbers of transgenic calli established in this experiment are shown in Table 1. TABLE 1

No. of transgenic calli per replicate

Vector Mean 1 2 3 pPG3 - 44 33 39

RMS13 0 0 0 0

RMS15 40 30 43 38

RMS17 12 13 23 16

These data show that relative to the two positive controls, pPG3 and RMS 15, expression of the preB/T-urfl3 gene fusion results in a signficant decrease (p<5% or better) in the establishment of transgenic calli. This suggests that targeting T-urfl3 protein to mitochondria has a deleterious effect on these cells, presumably due impartment of mitochondrial function. Expression ofthe cytotoxic ribonuclease, barnase, completely abolishes the establishment of transformed calli. Example 3

Construction ofthe maize transformation vector, RMS11 RMS1 1 is a transformation vector in which expression ofthe pre-β/T-urf!3 gene fusion is controlled by the maize tapteum promoter, MFS 14. The sequence ofthe MFS 14 promoter and untranslated leader region from position -2198 to +97 is shown in Figure 10. In this way, expression ofthe T-urfl3 protein is limited to the cells producing pollen and not throughout the whole plant.

To construct RMS1 1, the Kpn I - Sail fragment from pPB2 containing the pre-β/T- urfl3 gene fusion was blunt-ended and ligated into the blunt ended BamHI site of plasmid pSC9 to yield pPB4. In pPB4 the pre-β/T-urf!3 gene fusion is now positioned between the - 152 to +97 MFS 14 promoter fragment and the nos 3' polyadenylation sequence. This complete cassette was removed from pPB4 by digestion with Sacl and EcoRI and cloned into the corresponding sites of pSC7 to give plasmid pB5. pSC7 contains the -153 to -5800 region ofthe MFS 14 promoter so that the introduction ofthe Sacl - EcoRI fragment from pPB4 recreates the full 5.8 kb MFS 14 promoter. RMS 1 1 (Figure 11) was completed by introduction ofthe PAT in vitro selection cassette from plΕ109 into the unique EcoRI site of pPB5. Example 4

Transformation of maize cells with RMS1 1 by particle bombardment to provide stably transformed, male sterile plants.

The maize transformation vector, RMS 1 1 was used to transform regenerable maize cell cultures by particle bombardment. Culture Material

Friable embryogenic Type II callus was initiated from immature zygotic embryos excised 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 based onN6 medium 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 6.0. Callus was grown for 4-4 weeks prior to suspension culture initiation. Suspension cultures were initiated in a MS-based liquid medium containing 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l 1-naphthaleneacetic 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 6.0. Cell suspensions were maintained in theis medium 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 3 ml packed volume of cells and 10 ml culture medium to 20 ml of fresh culture 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 Bombardment

Cell suspensions were sieved through a 1.0 mm and then a 0.5mm screen. A packed volume of 0.2 ml ofthe 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 via 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 essentialy as described by the manufacturers. Target plates were bombarded once Transformant selection and plant regeneration

Following bombardment, each filter disc (with cells) was transferred to N6 based medium containing 100 mg/l myo-inositol, 2 mg l 2,4-D, 3% (w/v) sucrose, and 0.3% (w/v) Gelrite at pH 6.0. For selection using the NPTII gene, this medium was supplemented with

5 200 mg/l kanamycin sulphate. The filter discs were transferred to fresh medium containing 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 containing 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 lϋ selection medium. Plants were renerated by transferring tissue to MS based medium containing 1 g/1 myo-inositol, 1 mg/l NAA, 6% (w/v) sucrose, and 3% (w/v) Gelrite at pH 6.0. After 2-3 weeks, the tissue was transferred to MS media containing 0.25 mg/l NAA, and 3% (w/v) sucrose and placed in the light, where embryo germination occurred. Plants were then grown in half-strength MS based medium containing 500 mg/l myo-inositol, 3% (w/v)

15 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 et al

20 (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 1 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

25 scoring 4 and below are functionally sterile. The accumulated sterility scores for each ofthe independent PCR positive clones is shown in Table 3 and compared to a maize line which was generated by bombardment with RMS1 1 but which is PCR negative for the MFS14/pre-B/T- urf!3 gene construct.

Sterile plants were backcrossed with pollen from fertile, non-transgenic BE70 plants.

30 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 ofthe MFS14/pre-B/T-urfl3 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 ofthe plants are shown in Table 4. Plants which were PCR negative were rogued from the glasshouse prior to flowering. Plants 1 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 progeny plants were sterile and this sterility correlated with the presence ofthe transgene as assessed either by PCR or PAT testing. This is consistent the the presence of a single transgenic locus imparting sterility.

TABLE 2 Anther Classification (After L.M. Josephson)

Class 0 No anthers exerted

Class 1 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 5 Normal anthers and fully fertile.

Class 4 through 5 tassels were considered fertile.

TABLE 3

CLONE NO. OF PLANTS FERTILITY SCORE P C R.

WK 23 2 0 +

3 3 +

WK 23 2 0 +

YK 23 3 0 +

1 4 +

YE 23 4 5 -

_. TABLE 4

PLA PCR TEST PAT TEST FERTILITY SCORE NT

YK23/5/1 +/- - 0

YK23/5/2 +/- + 0

YK23/5/3 - ROGUED

YK23/5/4 - ROGUED

YK23/5/5 NO GERMINATION

YK23/5/6 + + 1

YK23/5/7 + + 0

YK23/5/8 - ROGUED

YK23/5/9 + + 0

YK23/5/10 - ROGUED

YK23/5/1 1 + + 0

YK23/5/12 - 5

Example 5

Construction ofthe maize transformation vector pRMS-23 We have tested whether expression 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 ofthe 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). The fragment of the ANT 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, annealing 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 Smal restriction sites at the 5' and 3' ends ofthe gene. The sequence ofthe maize ANT gene was published in Eur. J. Biochem. (1989) 183, 303-310. The MANT-1 primer sequence appears at the beginning ofthe coding sequence of the gene and the MANT-2R primer sequence is near the end ofthe gene. Following PCR which produced a DNA fragment ofthe predicted size of 1050 bp, the DNA was digested with Smal and subcloned into the Smal site of pUC18 to give pMANTl. Subsequently the nos 3' polyadenylation signal sequence was introduced 3' to the ANT gene as a Sacl- EcoRI fragment into the corresponding sites in pMANTl to yield pMANT2. A Hindlll - Ba?nHl fragment carrying the CaMV 35s promoter and ADH lintron from pCaMVCN (Figure 5) 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 pIE109 (Figure 7) into the unique EcoRI site of pMANT3. 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 establishment of transgenic calli following transformation in two separate experiments. The vector DNAs 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 vitro selection cassette alone (Table 5). These data show that expression of a short sense adeneine nucleotide translocator gene results in a significant decrease in the establishment of transgenic calli.

TABLE 5

No of transgenic calli per replicate

1 2 3 Mean

Experiment 1 pPG3 16 20 22 19 pRMS-23 ->

J 4 3

Experiment 2 pPG3 49 48 40 46 pRMS-23 10 15 23 16 Example 7

Construction ofthe maize transformation vectors. pTBR and pTBS

We tested whether un-regulated expression of α-tubulin genes will give rise to a defect in the growth of maize cells. Two constructs containing the coding sequence from α- tubulin cDNAs isolated from two biotypes of Eleusine indica were prepared. pTBR (Figure 13) contains the α-tubulin cDNA from a dinitroaniline resistant biotype of Eleusine indica cloned as a blunt-ended Hinϊl fragment into the blunt-ended BamHI site of pCaMVCN (Figure 7). pTBS (Figure 13) contains the α-tubulin cDNA from a dinitroaniline sensitive biotype cloned exactly as described for pTBR. Example 8

Transformation of BMS corn cells with pTBR and pTBS.

The objective of this experiment was to show that expression of pTBR and pTBS in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation. The vector DNAs were introduced 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 calli established were determined relative a positive control pPG3 wihich contains the in vitro selection cassette alone (Figure 14). These data show that non-regulated expression of an α- tubulin gene from either biotype of Eleusine indica results in a significant decrease in the establishment of transgenic calli.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: ZENECA LIMITED

(B) STREET: 15 Stanhope Gate

(C) CITY: London (E) COUNTRY: UK

(F) POSTAL CODE (ZIP) : W1Y 6LN

(ii) TITLE OF INVENTION: Production of Male Sterile Plants (iii) NUMBER OF SEQUENCES: 11

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 357 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE: (A) ORGANISM: T-urfl3 gene

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCTCCCT TTGATCAAGG TTTGGTATTT 60

TTCGGTTCTA TTTTTATTTT TTTTTTGTGC ATATTATTGA TAAAGGGATA TCTCCGTAAA 120 ATGGATGATT CCTATTTGGC TCAACTCTCC GAGTTAGCCA ACCACAATAG AGTGGAAGCG 180

GCAAAAGCGG GCCACGTGGC CCTGCATGAG CTATCCTTCT CGTGGTTGAG GGGGGTTCAA 240

ATTAGGGTGA GGACCTTACC TATACAACGG AATGAAGGAG GGGGTCGAAG CAACGACCAA 300

TCCACTCTCT CTAAGCCTAA GTATTCCTCA ATGACCGATA GCGTACAAGT ACCGTGA 357

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Turf-1 primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCT 36

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Turf-2R primer

ixi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TAGTCTAGAT CACGGTACTT GTACGCTATC GGT 33

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(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: (A) NAME/KEY: CDS

(B) LOCATION: 1..267

(D) OTHER INFORMATION: /codon_start= 1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ATG GCT TCT CGG AGG CTT CTC GCC TCT CTC CTC CGT CAA TCG GCT CAA 48

Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gin Ser Ala Gin 1 5 10 15

CGT GGC GGC GGT CTA ATT TCC CGA TCG TCA GGA AAC TCC ATC CCT AAA 96

Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lvs 20 25 30 TCC GCT TCA CGC GCC TCT TCA CGC GCA TCC CCT AAG GGA TTC CTC TTA 144 Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu 35 40 45

AAC CGC GCC GTA CAG TAC GCT ACC TCC GCA GCG GCA CCG GCA TCT CAG 192 Asn Arg Ala Val Gin Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gin 50 55 60

CCA TCA ACA CCA CCA AAG TCC GCC AGT GAA CCG TCC GGA AAA ATT ACC 240 Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr 65 70 75 80

GAT GAG TTC ACC GGC GCT GGT TCG ATC GG 269

5 Asp Glu Phe Thr Gly Ala Gly Ser Ile

85

(2) INFORMATION FOR SEQ ID NO: 5: 0

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 89 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear 5

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:

Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gin Ser Ala Gin 0 1 5 10 15

Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys 20 25 30 5 Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu 35 40 45

Asn Arg Ala Val Gin Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gin 50 55 60 0

Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr 65 70 75 80

Asp Glu Phe Thr Gly Ala Gly Ser Ile 5 85

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: 0 (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

50

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATCGGTACCG CCATGGCTTC TCGGAGGCTT CTCGCCT 37

->->

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs 60 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

65 ivi) ORIGINAL SOURCE:

(A) ORGANISM: PREB-2R primer

ixi) SEQUENCE DESCRIPTION: SEQ ID NO: ATCGGATCCC GCTGCGGAGG TAGCGTA (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2285 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: MFS14 Promoter

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

AAAAGCGTAC CAGTAAGGGA TAAAGAAAAT AAACAAACAC GAAATGCTTC CCCATCGGCC 60

AATTCGCCTA GGTGTGCTAG GAACTGGCCT ATATGTTCGT GTGTGCTTCT CCTATTTTCA 120

CCAGAAAACT TAGAAAACTC TGGTATCCTT GCCCCTTGTG GATATGGGAC AATGTCAAAC 180

CGGTGATCAT ATGGCTTCTG ATATGATTGC CCCACTCTAT CCACACCAAC TCCGAGTCTA 240 TCTCAAAATA GTTTAGCCAT CTCTTCCCTA ATTTTCTACA TTGCACTCGG TGGCAGACCA 300

CCGGACCCTA GGCTGTGGGG TTCATTCGGT CGGGCATTGT TATGCCGACC TTCTTGCCAT 360

GACCGATTGA TAATGTTGAT CGGCCTGTGA TCATATGGCG TGTTGTGGGT TAAATATGTA 420

GGGGGCAGAA CATACTGCCG TTGTGGTATG TAATAATTTG GTGCATAGTG TGCGACAGTA 480

GGTTCTGTGT ATGTGTATCC GATATGTCCG GTGGTACATC TGAACTGGCC GGTTGTGTTA 540 GCTATTATTG GGGCGCCACG CGTAGCCCTG GTGCGGCCCG GACTATCCGG CAGAGAAAGC 600

CGACGGTCTG TGTAAGGGCC GAACTATCCA GACAAAAGCT CGGACGGTCC GACCGTGTAG 660

AGGGCCGTCG ATCTGCCAAG CAAGGACGAT GGTGATGGTA TTTGCCCTGG ATATGAGTTC 720

ATCAACATAC CATATAATGG ATGGGGCTGC AAATCCCCAT TTGTCGCCGA TGTATTAGAC 780

ATAAATATCA TGTTACTAGT TTCATATGAT GGAAAACTAG GAGCAACAGA CTTCTCCAAC 840 ATACACGTTA ATTTTCTAAT TGGTTCTTCT AACCCTCTAA TCTAATGCTT CATTTGATTA 900

TGCAAATGGT CTACATACTG TTTAATAGAT TGGATGTCGT CGGGTTTACT TACGTTAGGG 960

ACTTGAAGCG AAGATAGAAG AGATGTGACG TCGGTATCGC ATGTTTGACA ACTTTCTGGT 1020

GACGATCCAC CATGTATTGT GACAAGAATT TCTCCTTCGT TTGACACATG TAGTCCTCGT 1080

ATTGTTGTTG CTCATCGGTC GTCGGACTCT TAATAGCCGG CTTTAGGATA TTGTCCGGGG 1140 AGATATCGGT GTGATCTTTA GAACCGCCAT TTGATGGCCT GAGTTTTAGT AGATCTAGAC 1200 ACATTTCCCC AACGGAGTCG CCAAAAAGTG TGTTGGCGCC GATCCAGGCG CGAAACACTG 1260

GAGATGGACC GTTTGGCGGT GTTCTCCGGG TGAGGACGGT CCGCGACCTG GGTCCAGCAG 1320

CGACTCTCCT CTACGTGTGT CCGGACGGTC CGTCGTCTGG GGCTCGGACG GTCGCGATGG 1380

CGCAGAGGGT CTTCTTCTTC GCAGCCGACC TAGATCTCGC CTCCCGGGAG GGACCGTCGG 1440 GGAGGAGAGA TTGTAGGGTG TGTCTTGGCG TCGACAGGCC ACACAATACG CCTCTAGTCG 1500

ACGTAGAGCC GAAGAGAGGT GAAGGATTGA GGTAGAAGGA GGCTAAACTT GGGCTAAACT 1560

AGAACTACTG CTAATGCATA AGGTAAAAAC GAGAAGTGGA CTTCATTTGA TCGATTGTGG 1620

AAGTAATCTG ACTGTAGCCC TTTATCTATA TAAAGGGGAG GTATGGACCC GTTACAAGCC 1680

GTTTTCCGAG CTAATCTCAC GGTTTTAGTT AATAAATCCT GCGAGAAACT CGGAACTCTA 1740 ACTGATTCTA CTCATGCGCG AACCATTCGT GCGCCACCGC TGCCCGTCCC GCGATCGCTC 1800

AGTTAACCCT GTGTTGTGCG CTGTGATTTG GTGGCATATA AAACCACATT TGCAATAAAA 1860

ATTTGTAGGG ATTTAACATA CCAAGTGCTG CGAAAGGAAT CGTTTTCGGA GGACCCAAAA 1920

TTAAAGAGGC AGATGCTAGA GCTCGTCCAG CTCAGCGCTG AGCACCTGTG TTGTCTTCCT 1980

CGTCCACGCC GGCGGAGATG AACGGCAACA AAGGCGGAAA GGCCGAGACG CTGAGCTCAA 2040 GGACGTGACA CCGCGCGTAC CTCGCGTTCA GTTGGCTCAC ACAACAGCAG CTCGCTCGCC 2100

CCAAGCTCCC GCGTCCTGAT CCGTAGGTGA GCCATGCAAA GGTCGCCGCG CGCCCTGATC 2160

CATTGCACCC TTCAAAGCTC GAACCTACAA ATAGCGTGCA CCAGGCATCC TGGCCACACC 2220

CACACAGCAA GCCAGCAGAG CAGAAAGCAG CCGCAGCCCC AGCCCCCACA AAGACGAAGG 2280

CAACA 2285 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: 14-SA Primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

AGACGCTGAG CTCAAGGACG TGA 23

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: MANT-1 Primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATGCCCGGGC TTGCAATGTC TGTTAGCGGT GGCATCA 37

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: MANT-2RB Primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGCCCGGGC GATGGGGTAA GATGCAAGAC CA 32

Claims

CLAIM

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 ofthe target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells ofthe said target tissue resulting in death ofthe cells, the said disrupter gene is selected from the group consisting ofthe T-urfl3 gene, genes encoding an α- or β-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) ofthe 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 ofthe 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 ofthe said target tissue resulting in death ofthe cells.

3. A plant having stably incorporated within its genome a gene construct carrying a tissue-specific promoter which operates in the cells ofthe 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 ofthe said target tissue resulting in death ofthe cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct ofthe adenine nucleotide translocator, genes encoding an α- or β-tubulin and short sense down-regulation ofthe 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 cDNA 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 MFS 14 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 an essential cell function such as respiration or microtubules in the cells ofthe tapetum resulting in death ofthe cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct ofthe adenine nucleotide translocator, genes encoding an α- or β-tubulin and short sense down-regulation ofthe essential cell cycle genes, cdc25 and ROA.