WO1990013654A1

WO1990013654A1 - Male sterility in plants

Info

Publication number: WO1990013654A1
Application number: PCT/US1990/002404
Authority: WO
Inventors: Laurence K. Grill; Thomas H. Turpen; Robert L. Erwin
Original assignee: Biosource Genetics Corporation
Priority date: 1989-05-05
Filing date: 1990-05-04
Publication date: 1990-11-15
Also published as: AU5664090A

Abstract

The present invention relates to the induction of male sterility in plants. Male sterility is induced by using viral vectors which are biologically contained, self-replicating and capable of the non-nuclear chromosomal transformation of a plant and which contain a nucleotide sequence capable of inducing male sterility. The invention further relates to viruses containing the viral vectors which are transmissible, i.e. infective. Plant are infected by the viruses of the invention in order to induce male sterility.

Description

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TITLE OF THE INVENTION MALE STERILITY IN PLANTS

BACKGROUND OF THE INVENTION

The present invention relates to the induction of male sterility in plants. Male sterility is induced by using viral vectors which are non-infective (also referred to herein as biologically contained) but which are self-replicating and capable of the non-nuclear chromosomal transformation of a plant and which contain nucleotide sequence capable of inducing male sterility. The invention further relates to viruses containing the viral vectors which are transmissible. A plant is infected by the viruses of the invention in order to induce male sterility. Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication. In fact, a complete viral particle, or virion, may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope such as in the case of alpha viruses. The coat protects the virus from the environment and serves as a vehicle for transmission from one host.cell to another. Unlike cells, viruses do not grow in size and then divide, because they contain within their coats few or none of the biosynthetic enzymes and other machinery required for their replication. Rather, viruses multiply in cells by synthesis of their separate components, followed by assembly. Thus the viral nucleic acid, after shedding its coat, comes into contact with the appropriate cell machinery where it specifies the synthesis of proteins required for viral reproduction. The viral nucleic acid is then itself replicated through the use of both viral and cellular enzymes. The components of the viral coat are formed and the nucleic acid and coat components are finally assembled. With some viruses, replication is initiated by enzymes present in virions. Viruses are subdivided into three main classes — animal viruses, plant viruses and bacterial viruses. Within each class, each virus is able to infect only certain species of cells. With animal anc. bacterial viruses, the host range is determined by the specificity of attachment to the cells which depends on properties of both the virion's coat and specific receptors on the cell surface. These limitations disappear when transfection occurs, i.e., when infection is carried out by the naked viral nucleic acid, whose entry does not depend on virus-specific receptors.

A given virus may contain either DNA or RNA, which may be either single- or double-stranded. The portion of nucleic acid in a virion varies from about 1% to about 50%. The amount of genetic information per virion varies from about 3 to 300 kb per strand. Ths diversity of virus-specific proteins varies accordingly. Examples of double-stranded DNA containing viruses include, but are not limited to, Hepatitis B virus, papovaviruses such as polyoma and papilloma, adenovirus, poxviruses such as vaccinia, caulimoviruses such as Cauliflower mosaic virus (CaMV) , Pseudomonas phage PMS2, Herpesvirus, Bacillus subtilis phage SP8, and the T bacteriophages. Representative viruses which are single-stranded DNA are the parvoviruses and the bacteriophages φX174, fl and M13. Reoviruses, cytoplas- mic polyhedrosis virus of silkworm, rice dwarf virus and wound tumor virus are examples of double-stranded RNA viruses. Single-stranded RNA viruses include tobacco mosaic virus (TMV) , turnip yellow mosaic virus (TYMV) , picornaviruses, myxoviruses, paramyxoviruses and rhabdo- viruses. The RNA in single-stranded RNA viruses may be either a plus (+) or a minus (-) strand. For general information concerning viruses see Grierson, D. et al., Plant Molecular Biology. Blackie, London, pp. 126-146 (1984); Dulbecco, R. et al., Virology. Harper & Row, Philadelphia (1980); White, A. et al., Principles of Biochemistry. 6th Ed., McGraw-Hill, New York, pp. 882-900 (1978) .

One means for classifying plant viruses is based on the genome organization. Although many plant viruses have RNA genomes, the organization of genetic information differs between groups. The genome of most monopartite plant RNA viruses is a single-stranded molecule of (+)- sense. There are at least 11 major groups of viruses with this type of genome. An example of this type of virus is TMV. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two distinct (+)-sense single-stranded RNA molecules that are encapsidated in separate particles. Both RNAs are required for infectivity. Cowpea mosaic virus (CPMW) is an example of a bipartite plant virus. The third major type, containing at least six major types of plant viruses, has three (+)-sense single-stranded RNA molecules, i.e., is tripartite. Each strand is separately encapsidated and all three are required for infectivity. An example of a tripartite plant virus is alfalfa mosaic virus (AMV) . Many plant viruses also have smaller sub-genomic mRNAs that are synthesized to amplify a specific gene product. One group of plant viruses which have a single-stranded DNA genome are the geminiviruses, such as cassava latent virus and maize streak virus.

Techniques have been developed which are utilized to transform many species of organisms. Hosts which are capable of being transformed by these techniques include bacteria, yeast, fungus, animal cells and plant cells or tissue. Transformation is accomplished by using a vector which is self-replicating and which is compatible with the desired host. The vectors are generally based on either a plasmid or a virus. Foreign DNA is inserted into the vector, which is then used to transform the appropriate host. The transformed host is then identified by selection or screening. For further information concerning the transformation of these hosts, see Maniatis, T. et al., Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor (1982) ; DNA Cloning, Ed. Glover, D.M. , IRL Press, Oxford (1985); Grierson, D. et al., supra; and Methods in Enzymology, volumes 68, 100, 101, 118 and 152-155 (1979, 1983, 1983, 1986 and 1987) .

Viruses that have been shown to be useful for the transformation of appropriate hosts include bacterio¬ phages, animal viruses such as adenovirus ype 2 and vaccinia virus and plant viruses such as CaMV and brome mosaic virus (BMV) . An example of the use of a bacteriophage vector is shown in U.S. Patent 4,508,826. U.S. Patent 4,593,002 shows the use of adenovirus type 2 as well as a bacteriophage for the transformation of the appropriate host. The use of a vaccinia virus is shown in U.S. Patent 4,603,112. Transformation of plants using plant viruses is described in EP A 67,553 (TMV) , EP A 194,809 (BMV) and Brisson, N. et al., Methods in Enzymology 118. 659 (1986) (CaMV) .

When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.

The production of hybrid seed is important for many commercial crops. Because of hybrid vigor (heterosis) , maximum yields as well as uniformity are achieved. In order to produce hybrid seed on a commercial level, cytoplasmic and genetic male sterility of the female parent are used for many plant species.

Pollen control in hybrid seed production field is an extremely critical factor. Pollen control is essential to ensure hybridization by . enforced cross- pollination between the female and male parents of the intended cross. Various methods of pollen control in seed fields have been utilized or investigated in recent years, aimed primarily at reducing the cost or easing the difficulty in this critical period while still maintaining the desired genetic purity of the hybrid. Some of the methods include 1) emasculation, 2) cytoplasmic sterility, 3) genie male sterility, and 4) chemical pollen control. The emasculation or detasselling methods are very time consuming, laborious and expensive. Currently, the most widely used method of pollen control in corn involves detasseling or the physical removal of the tassel from the plant, either as a manual operation or in combination with mechanical devices. One way to overcome these costs is to obtain a method of producing male sterile plants.

Much effort has been spent to develop crop plants which are male sterile. The term "male sterile" generally designates a plant where the male inflorescences on the mature plant produce no viable pollen but the plants still has complete female reproductive capability. The use of male sterile plants in a hybrid production system avoids the need for the emasculation or detasselling since the only pollen available for the designated female parent plants, (which are male sterile) is the pollen produced by the designated male parent plants.

Male sterility as a method of pollen control in the production field has progressed significantly in the last thirty years. There are presently three types of sterility either in use or being extensively investigated. The three methods are: 1) cytoplasmic; 2) genie; and 3) chemical male sterility. Cytoplasmic male sterility (CMS) causes pollen to abort leaving the anthers devoid of pollen. Female fertility usually is not effected. Cytoplasmic male sterility is inherited through the female cytoplasm, rather than through the chromosomes, probably through the nucleic acids of the plastids, mitochondria, or virus-like entities. Genie male sterility is another approach to producing hybrids. Chromosomal-genic systems exist in some crops. Certain kinds of altered chromosomes (for instance, those of small deletions) normally are not inherited through the pollen (they abort the pollen grains that carry them) but they are inherited through the egg. Ordinarily the male sterile phenotype can not be propagated in pure form because of male sterility. Homozygous recessive ms/ms plants are generated each generation by segregation in an F-2 or backcross as 1/4 or 1/2 of the progeny. Successful seed production requires the right version of each inbred be used depending on whether a commercial single cross or seed parent single cross desired. In order to multiply the inbred seed stock extra care is required, thus even after the conversion of inbreds (which is complicated, time consuming and expensive) , additional expenses in the form of foundation seed production inventory and quality control are required.

Chemical male sterility has been a topic of many articles concerning the possibilities of development of chemical pollen control agents for crop plants. Chemical induction of male sterility has been demonstrated in cotton, in curcurbits, in sunflower, in wheat, in corn and in several other crop plants. An effective male gametocide would be of tremendous value. This would eliminate the need to convert female parental lines to cytoplasmic or genie male sterile. Foundation seed operations would not be involved in sterile, maintainer and restorer seed increases. Determining the proper dosage, time, and method of application of the male sterility chemical are very important. The timeliness of treatment application is generally very critical since the action of most chemicals studied to date inhibits the early stages of meiosis (pollen formation) similar to the case of cytoplasmic male sterility. The problem of pollen from some female plants in the field would still be present due to some plants not being treated with enough chemical or the plant not being at the proper stage when treated.

SUMMARY OF THE INVENTION

The present invention relates to the induction of male sterility in plants. The male sterility is induced through the use of viral vectors which are biologically contained, self-replicating and capable of the non- nuclear chromosomal transformation of a plant and which contain nucleotide sequences capable of inducing male sterility. The invention further relates to viruses containing the viral vectors which are transmissible, i.e. infective. Plants are infected by the viruses of the invention to induce male sterility in the plants. DETAILED DESCRIPTION OF THE INVENTION

The present invention includes (a) viral vectors which are non-infective but which are self-replicating and capable of the non-nuclear chromosomal transformation of a plant and which contain a nucleotide sequence capable of inducing male sterility in plants, (b) viruses containing the viral vectors which are infective, (c) production cells which are capable of producing the viruses or parts thereof, and (d) a plant infected by the viruses of the invention.

In order to provide a clear and consistent under¬ standing of the specification and the claims, including the scope given to such terms, the following definitions are provided: Adjacent: A position in a nucleotide sequence immediately 5¹ or 3* to a defined sequence.

Anti-sense Mechanism: A type of gene regulation based on controlling the rate of translation of mRNA to protein due to the presence in a cell of an RNA molecule complementary to at least a portion of the mRNA being translated.

Biologically Contained: The viral nucleic acid is not capable of naturally infecting a host since it is not capable of expressing a biologically functional coat protein.

Biologically Functional: The capability of performing an expected biological function in a cell or organism. For example, the biological function of a viral coat protein is the encapsidation of the viral nucleic acid. A non-biologically functional coat protein is not capable of encapsidating viral nucleic acid. A nucleotide sequence which lacks a biologically functional protein coding sequence will produce either no protein or will produce a protein which will not perform an expected function. If the entire coding sequence for the protein is removed, then no protein will be produced. If a significant portion of the coding sequence for the protein is removed, then any protein that is produced will not function as the entire protein would function. If the coding sequence for the protein is mutated such as by a point mutation, the protein might not function as a normal protein would function. For example, a nucleotide sequence which lacks a biologically functional coat protein coding sequence is a nucleotide sequence which does not code for a coat protein capable of encapsidating viral nucleic acid. This term is intended to include a complete deletion of the coat protein sequence. Cell Culture: A proliferating mass of cells which may be in an undifferentiated or differentiated state.

Chimeric Sequence or Gene: A nucleotide sequence derived from at least two heterologous parts. The sequence may comprise DNA or RNA. Coding Seguence: A deoxyribonucleotide sequence which when transcribed and translated results in the formation of a cellular polypeptide, or a ribonucleotide sequence which when translated results in the formation of a cellular polypeptide. Compatible: The capability of operating with other components of a system. A vector which is compatible with a host is one which is capable of replicating in that host. A coat protein which is compatible with a viral nucleotide sequence is one which is capable of encapsidating the viral sequence.

Gene: A discrete chromosomal region which is responsible for a discrete cellular product.

Host: A cell, tissue or organism capable of replicating a viral vector and which is capable of being infected by a virus containing the viral vector. This term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms, such as bacteria, yeast, fungus, animal cells and plant tissue.

Infection: The ability of a virus to transfer its nucleic acid to a host wherein the viral nucleic acid is replicated, viral proteins are synthesized and new viral particles assembled. The terms transmissible and infective are used interchangeably herein. The term non-infective as used herein means non-infective by natural, biological means. Male Sterility: As used herein, male sterility is intended to cover any mechanisms which renders pollen incapable of fertilizing an egg. Such sterility could be caused by lack of viable pollen formation, timing of pollen formation, self-incompatibility, etc. Phenotypic Trait: An observable property resulting from the expression of a gene.

Plant: This term generally refers to a whole plant but is also intended to refer to a plant cell, plant organ or plant tissue as the context dictates. Plant Cell: The structural and physiological unit of plants, consisting of a protoplast and the cell wall. Plant Organ: A distinct and visibly differentiated part of a plant such as root, stem, leaf or embryo.

Plant Tissue: Any tissue of a plant in plant or in culture. This term is intended to include a whole plant, plant cell, plant organ, protoplast, cell culture or any group of plant cells organized into a structural and functional unit.

Production Cell: A cell, tissue or organism capable of replicating a vector or a viral vector, but which is not necessarily a host to the virus. This term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms, such as bacteria, yeast, fungus, animal cells and plant tissue. Pro oter: The 5*-flanking, non-coding sequence adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.

Protoplast: An isolated plant cell without cell walls, having the potency for regeneration into cell culture or a whole plant.

Substantial Sequence Homologv: Denotes nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting the function of the gene products or an RNA coded for by such sequence.

Transcription: The production of an RNA molecule by RNA polymerase as a complementary copy of a DNA sequence.

Vector: A self-replicating DNA molecule which transfers a DNA segment between cells.

Viral Vector: A vector comprising a nucleic acid sequence of a virus which has been modified so that a non-biologically functional coat protein is produced. This may be accomplished by removing at least a part of the coding sequence or by mutating the coding sequence. If the virus codes for one or more virus transmissibility factors, then the nucleic acid sequence of the virus is also modified to make these non- biologically functional.

Virus: An infectious agent which is composed of a nucleic acid encapsidated in a protein. A virus may be a mono-, di-, tri- or multi-partite virus as described above.

The present invention provides for the infection of a plant by a virus which has been modified so that the virus is transmissible but the viral nucleic acid is not infective. Naturally occurring mutant viruses may also have these same properties of being transmissible, but having viral nucleic acid which is not infective. The non-infectivity of the viral nucleic acid is accomplished by modifying the nucleic acid so that biologically non-functional viral coat protein (capsid protein) and any other viral transmissibility factors are produced as described herein.

The present invention has a number of advantages, one of which is that the transformation and regeneration of target organisms is not necessary. Another advantage is that it is not necessary to develop vectors which integrate a desired nucleotide sequence in the genome of the target organism. Existing organisms can be altered with a new nucleotide sequence without the need of going through a germ cell. The present invention also gives the option of applying the nucleotide sequence to the desired plant.

The chimeric genes or sequences and vectors of the present invention are constructed using techniques well known in the art. Suitable techniques have been described in Maniatis, T. et al., Molecular Cloning. Cold Spring Harbor Laboratory, New York (1982) ; Methods in Enzymology. Vols. 68, 100, 101, 118 and 152-155, Academic Press, New York (1979, 1983, 1983, 1986 and 1987); and DNA Cloning. Vols. I, II, III, Glover, D.M. , Ed., IRL Press, Oxford (1985 and 1987). Medium compositions have been described in Miller, J.H. , Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York (1972) , as well as the references previously identified. DNA manipulations and enzyme treatments are carried out in accordance with the manufacturers- recommended procedures.

An important feature of the present invention is the preparation of nucleotide sequences which are capable of replication in a compatible plant but which in themselves are incapable of infecting the plant. The nucleotide sequence has substantial sequence homology to a viral nucleotide sequence. The viral nucleotide sequence is a plant viral nucleotide sequence. A partial listing of suitable viruses has been described above. The nucleotide sequence may be an RNA, DNA, cDNA or chemically synthesized RNA or DNA. The first step in achieving any of the features of the invention is to modify the nucleotide sequences coding for the capsid protein and any transmissibility factors within the viral nucleotide sequence by known conventional techniques such that non-biologically functional proteins are produced by the modified virus. Therefore, any virus for which the capsid protein nucleotide sequence and any transmissibility factor nucleotide sequences have been identified may be suitable for use in the present invention. Other viruses may be used after the nucleic acid has been sequenced.

Some of the viruses which meet this requirement,. and therefore are suitable, include the viruses from the tobacco mosaic virus group such as Tobacco Mosaic virus (TMV) , Cowpea Mosaic virus (CMV) , Alfalfa Mosaic virus

(AmV) , Cucumber Green Mottle Mosaic virus watermelon strain (CGMMV-W) and Oat Mosaic virus (OMV) and viruses from the brome mosaic virus group such as Brome Mosaic virus (BMV) , broad bean mottle virus and cowpea chlorotic mottle virus. Additional suitable viruses include Rice Necrosis virus (RNV) , geminiviruses such as tomato golden mosaic virus (TGMV) , cassava latent virus and maize streak virus. Each of these groups of suitable viruses is characterized below.

TOBACCO MOSAIC VIRUS GROUP

Tobacco Mosaic virus (TMV) is a type member of the Tobamoviruses. The TMV virion is a tubular filament, and comprises coat protein subunits arranged in a single right-handed helix with the single-stranded RNA intercalated between the turns of the helix. TMV infects tobacco as well as other plants. TMV is transmitted mechanically and may remain infective for a year or more in soil or dried leaf tissue. The TMV virions may be inactivated by subjection to an environment with a pH less than 3 or greater than 8, or by formaldehyde or iodine. Preparations of TMV may be obtained from plant tissues by (NH ₂S0₄ precipitation followed by differential centrifugation. The TMV single-stranded RNA genome is about 6400 nucleotides long and is capped at the 5^• end but is not poly-adenylated. The genomic RNA can serve as mRNA for a protein of molecular weight about 130,000 (130K) and another produced by read-through of molecular weight about 180,000 (180K) . However, it cannot function as a messenger for the synthesis of coat protein. Other genes are expressed during infection by the formation of monocistronic, 3'-coterminal subgenomic mRNAs, including one (LMC) encoding the 17.5K coat protein and another (I₂) encoding a 3OK protein. The 3OK protein has been detected in infected protoplasts fVirology 132, 71 (1984)) , and it is involved in the cell-to-cell transport of the virus in an infected plant (Deom, CM. et al., Science 237. 389 (1987)). The functions of the two large proteins are unknown.

Several double-stranded RNA molecules, including double-stranded RNAs corresponding to the genomic, I₂ and LMC RNAs, have been detected in plant tissues infected with TMV. These RNA molecules are presumably intermediates in genome replication and/or mRNA synthesis - processes which appear to occur by different mechanisms.

TMV assembly apparently occurs in the plant cell cytoplasm, although it has been suggested that some TMV assembly may occur in chloroplasts since transcripts of ctDNA have been detected in purified TMV virions. Initiation of TMV assembly occurs by interaction between ring-shaped aggregates ("discs") of coat protein (each disc consisting of two layers of 17 sub-units) and a unique internal nucleation site in the RNA; a hairpin region about 900 nucleotides from the 3 ' end in the common strain of TMV. Any RNA, including subgenomic RNAs, containing this site may be packaged into virions. The discs apparently assume a helical form on interaction with the RNA, and assembly (elongation) then proceeds in both directions (but much more rapidly in the 3'- to 5¹ direction from the nucleation site).

Another member of the Tobamoviruses, the Cucumber green mottle mosaic virus watermelon strain (CGMMV-W) is related to the cucumber virus. Noru, Y_, et al., Virology 45. 577 (1971). The coat protein of- CGMMV-W interacts with the RNA of both TMV and CGMMV to assemble viral particles in vitro. Kurisu et al.. Virology 70, 214 (1976) .

Several strains of the tobamovirus group are divided into two subgroups on the basis of the location of the assembly of origin. Fukuda, M. et al., Proc. Natl. Acad. Sci. USA 78, 4231 (1981) . Subgroup I, which includes the vulgare, OM, and tomato strain, has an origin of assembly at about 800-1000 nucleotides from the 3' end of the RNA genome, and outside of the coat protein cistron. Lebeurier, G. et al., Proc. Natl. Acad. Sci. USA 74. 1913 (1977); and Fukuda, M. et al., Virology 101. 493 (1980) . Subgroup II, which includes CGMMV-W and cornpea strain (Cc) , has an origin of assembly about 300-500 nucleotides from the 3' end of the RNA genome, and within the coat-protein cistron. Fukuda, M. et. al., supra. The coat protein cistron of CGMMV-W is located at nucleotides 176-661 from the 3'end. The 3' noncoding region is 175 nucleotides long. The origin of assembly is positioned within the coat protein cistron. Meshi, T. et al., Virology 127, 52 (1983) .

BROME MOSAIC VIRUS GROUP

Brome mosaic virus (BMV) is a member of a group of tripartite single-stranded RNA-containing plant viruses commonly referred to as the bromoviruses. Each member of the bromoviruses infects a narrow range of plants. Mechanical transmission of bromoviruses occurs readily, and some members are transmitted by beetles. In addition to BMV, other bromoviruses include broad bean mottle virus and cowpea chlorotic mottle virus.

Typically, a bromovirus virion is icosahedral with a diameter of about 26 mm. , and contains a single species of coat protein. The bromovirus genome has three molecules of linear, positive-sense, single- stranded RNA, and the coat protein mRNA is also encapsidated. The RNAs each have a capped 5¹ end and a tRNA-like structure (which accepts tyrosine) at the 3^» end. Virus assembly occurs in the cytoplasm. The complete nucleotide sequence of BMV has been identified and characterized as described by Alquist et al. , J. Mol. Biol. 153. 23 (1981) .

RICE NECROSIS VIRUS

Rice Necrosis virus (RNV) is a member of the Potato Virus Y Group or Potyviruses. The Rice Necrosis virion is a flexuous filament comprising one type of coat protein (molecular weight about 32,000 to about 36,000) and one molecule of linear positive-sense single- standard RNA. The Rice Necrosis virus is transmitted by Polvmyxa graminis (a eukaryotic intracellular parasite found in plants, algae and fungi) . RNV is capable of infecting most monocot species including, but not limited to barley and corn.

GEMINIVIRUSES

Geminiviruses are a group of small, single-stranded DNA-containing plant viruses with virions of unique morphology. Each virion consists of a pair of isometric particles (incomplete icosahedra) , composed of a single type of protein (molecular weight about 2.7-3.4 x 10⁴) . Each geminivirus virion contains one molecule of circular, positive-sense, single-stranded DNA. In some geminiviruses (i.e., cassava latent virus and bean golden mosaic virus) , the genome appears to be bipartite, containing two single-stranded DNA molecules which are of similar size, but differ as to nucleotide sequence. However, other geminiviruses (i.e., the leaf- hopper transmitted viruses such as Chloris striate mosaic virus) , have only one type of single-stranded DNA. Geminivirus replication occurs in the plant cell nucleus where large aggregates of virus particles accumulate. The geminivirus, tomato golden mosaic virus (TGMV) is capable of infecting a wide variety of both dicotyledonous and monocotyledonous plants including tobacco, tomato, bean, soya bean, sugar beet, cassava, cotton, maize, oats and wheat. The nucleotide sequence of any suitable virus can be derived from a viral nucleic acid by modifying the coat protein coding sequence. The modification may be the removal of a coding sequence for at least a part of the viral coat protein. Alternatively, the nucleotide sequence can be synthesized such that it lacks at least a part of the viral coat protein coding sequence. A sufficient amount of the coding sequence is removed such that any coat protein which may be produced by the virus will be incapable of encapsidating the viral nucleic acid. In addition, the coat protein coding sequence may be modified by mutation such that the coat protein which is produced is incapable of encapsidating the viral nucleic acid. In each instance, as non-biologically functional protein is produced. In some instances, part of the gene may be necessary for good promoter activity. If so, then the entire gene should not be deleted. In order to be easily transmissible to other plants, the nucleotide sequence must be encapsidated in a compatible coat protein as described further below. The viral nucleic acid may further be modified to alter the coding sequence for any viral transmissibility factors. The alteration of the coding sequences for these factors will ensure that the nucleotide sequence cannot be transmitted by other vectors, e.g. by insects.

The nucleotide sequence is prepared by cloning the viral nucleic acid in an appropriate production cell. If the viral nucleic acid is DNA, it can be cloned directly into a suitable vector using conventional techniques. One technique is to attach an origin of replication compatible with the production cell to the viral DNA. If the viral nucleic acid is RNA, a full- length DNA copy of the viral genome is first prepared by well known procedures. For example, the viral RNA is transcribed into DNA using reverse transcriptase to produce subgenomic DNA pieces, and a double-stranded DNA made using DNA polymerases. The DNA is then cloned into appropriate vectors and cloned into a production cell. The DNA pieces are mapped and combined in proper sequence to produce a full-length DNA copy of the viral RNA genome. The coding sequences for the viral coat protein and any viral transmissibility factors are identified and altered. The resulting nucleotide sequence is self-replicating but incapable of infecting host itself. Any manner of separating the coding sequences for the viral coat protein and the coding sequences for the viral transmissibility factors from the remainder of the viral nucleotide sequence or of altering the nucleotide sequences of these proteins to produce non-biologically functional proteins is suitable for the present invention.

A second feature of the present invention is a chimeric nucleotide sequence which comprises a first nucleotide sequence and a second nucleotide sequence. The first sequence is capable of self-replication, is not capable of transmission and has substantial sequence homology to a viral nucleotide sequence, as described above. The second sequence is capable of being trans¬ cribed in plant tissue. The second sequence is preferably placed adjacent a viral promoter, although a fusion protein may be produced which also has biological activity. Any viral promoter can be utilized, but it is preferred to use a promoter of the viral coat protein gene, at least a part of the coding sequence of which has been deleted. In those instances where the coat protein coding sequence is altered but not deleted, a viral promoter can be attached to the second sequence by conventional techniques or the second sequence can be inserted into or adjacent the coat protein coding sequence such that a fusion protein is produced. The second sequence which is transcribed may be transcribed as an RNA which is capable of inducing male sterility by an anti-sense mechanism. Alternatively, the second sequence in the chimeric nucleotide sequence may be transcribed and translated in plant tissue to produce a protein which induces male sterility. The second nucleotide sequence may also code for the expression of more than one phenotypic trait. The chimeric nucleotide sequence is constructed using conventional techniques such that the second nucleotide sequence is in proper orientation to the viral promoter. The second nucleotide sequence can be inserted into the first nucleotide sequence prepared above such that it is adjacent a viral promoter. Since the location of the promoter of the viral coat protein gene is known in this sequence as a result of the deletion of the gene, the second nucleotide sequence can be placed adjacent this promoter. Alternatively, an appropriate viral promoter can first be attached to the second nucleotide sequence and this construct can then be inserted either into the first nucleotide sequence or adjacent thereto. In addition, the second nucleotide sequence can be inserted into and adjacent an altered coat protein coding sequence.

A double-stranded DNA of the chimeric nucleotide sequence or of a complementary copy of the chimeric nucleotide sequence is cloned into a production cell. If the viral nucleic acid is an RNA molecule, the chimeric nucleotide sequence is first attached to a promoter which is compatible with the production cell. The chimeric nucleotide sequence can then be cloned into any suitable vector which is compatible with the production cell. In this manner, only RNA copies of the chimeric nucleotide sequence are produced in the production cell. For example, if the production cell is ___ _______ the lac promoter can be utilized. If the production cell is a plant cell, the CaMV promoter can be used. The production cell will be a eukaryotic cell such as yeast, plant or animal, if viral RNA must be capped for biological activity. The chimeric nucleotide sequence can then be cloned into any suitable vector which is compatible with the production cell. Alternatively, the chimeric nucleotide sequence is inserted in a vector adjacent a promoter which is compatible with the production cell. If the viral nucleic acid is a DNA molecule, it can be cloned directly into a production cell by attaching it to an origin of replication which is compatible with the production cell. In this manner, DNA copies of the chimeric nucleotide sequence are produced in the production cell. A promoter is a DNA sequence that directs RNA polymers to bind to DNA and to initiate RNA synthesis. There are strong promoters and weak promoters. Among the strong promoters are lacuv5, trp, tac, trp-lacuv5, λp_x, ompF, and bla. A useful promoter for expressing foreign genes in E___ coli is both strong and regulated. The λp_x promoter of bacteriophage λ is a strong, well- regulated promoter. Hedgpeth, J.M. et al. Mol. Gen. Genet. 163, 197 (1978) ; Bernard, H. M. et al. Gene 5_., 59 (1979); Remaut, E.P. et al., Gene 15, 81 (1981). A gene encoding a temperature-sensitive λ repressor such as λclts 857 may be included in the cloning vector. Bernard et al., supra. At low temperature (31°C) , the p_L promoter is maintained in a repressed state by the cl- gene product. Raising the temperature destroys the activity of the repressor. The _λ promoter then directs the synthesis of large quantities of mRNA. In this way, _____ coli production cells may grow to the desired concentration before producing the products encoded within the vectors. Similarly, a temperature-sensitive promoter may be activated at the desired time by adjusting the temperature of the culture.

It may be advantageous to assemble a plasmid that can conditionally attain very high copy numbers. For example, the pAS2 plasmid containing a lac or tac promoter will achieve very high copy numbers at 42°C. The lac repressor, present in the pAS2 plasmid is then inactivated by isopropyl-_/9-D-thiogalactoside to allow synthesis of mRNA.

A further alternative when creating the chimeric nucleotide sequence, is to prepare more than one nucleotide sequence (i.e., prepare the nucleotide sequences necessary for a multipartite viral vector construct) . In this case, each nucleotide sequence would require its own origin of assembly. Each nucleotide sequence could be chimeric (i.e., each nucleotide sequence has a foreign coding sequence inserted therein) , or only one of the nucleotide sequences may be chimeric.

If a multipartite virus were found to have the coding sequence for its coat protein on one strand of nucleic acid, and the coding sequence for a transmissibility factor on a different strand, then two chimeric nucleotide strands would be created in accordance with the invention. One foreign coding sequence could be inserted in place of the coat protein gene (or inserted next to the altered coat protein gene) on one strand of nucleic acid, and another foreign coding sequence could be inserted in plc.ce of the transmissibility factor gene (or inserted next to the altered transmissibility factor gene) on the other strand of nucleic acid.

Alternatively, the insertion of a foreign coding sequence into the nucleotide sequence of a monopartite virus may result in the creation of two nucleotide sequences (i.e., the nucleic acid necessary for the creation of a bipartite viral vector) . This would be an advantageous situation when it is desirable to keep the replication and translation of the foreign coding sequence separate from the replication and translation of some of the coding sequences of th original nucleotide sequence. Each nucleotide sequence would have to have its own origin of assembly.

A third feature of the present invention is a virus or viral particle. The virus comprises a chimeric nucleotide sequence as described above which has been encapsidated. The resulting product is then capable of infecting an appropriate host, but the chimeric nucleo- tide sequence is incapable of further infection since it lacks the mechanism to produce additional virus or viral particles. However, the chimeric nucleotide sequence is capable of replicating in the host. The chimeric nucleotide sequence is transcribed and/or translated within the host to produce the desired product.

Most viruses are encapsidated by either an icosahedral capsid, a spherical capsid or a rod-shaped capsid. Plant viruses such as the tobacco mosaic virus are commonly encapsidated by a rod-shaped capsid.

The icosahedral capsids and the spherical capsids are more geometrically constrained than a rod-shaped capsid, and therefore are more limited as to the amount of nucleic acid which may be encapsidated. In contrast, a rod-shaped capsid is expandable so that it can encapsidate any amount of nucleic acid as explained in European Patent Application 0 278 667.

In one embodiment of the present invention, the chimeric nucleotide sequence is encapsidated by a heterologous capsid. Most commonly, this embodiment will make use of a rod-shaped capsid because of its ability to encapsidate a longer chimeric nucleotide sequence than the more geometrically constrained icosahedral capsid or spherical capsid. The use of a rod-shaped capsid permits the incorporation of a larger foreign protein coding sequence to form the chimeric nucleotide sequence. Such a rod-shaped capsid is most advantageous when more than one foreign coding sequence is present in the chimeric nucleotide sequence. Another feature of the invention is a vector containing the chimeric nucleotide sequence as described above. The chimeric nucleotide sequence is adjacent a nucleotide sequence selected from the group consisting of a production cell promoter or an origin of replication compatible with the production cell. The vector is utilized to transform a production cell which will then produce the chimeric nucleotide sequence in quantity. The production cell may be any cell which is compatible with the vector. The production cell may be prokaryotic or eukaryotic. However, if the viral RNA (chimeric nucleotide sequence) must be capped in order to be active, then the production cell must be capable of capping the viral RNA, such as a eukaryotic production cell. The production cell may contain only the vector which contains the chimeric nucleotide sequence. In this instance, the production cell will only produce the chimeric nucleotide sequence which must then be encapsidated in vitro to produce an infective virus. The encapsidation is accomplished by adding a compatible viral coat protein to the chimeric nucleotide sequence in accordance with conventional techniques. In this scenario, the viral coat protein would be produced in a second production cell. A second vector comprising a coding sequence for a compatible viral coat protein adjacent a promoter compatible with the production cell is utilized to transform the second production cell. The second production cell then produces the viral coat protein in quantity.

As explained above, the viral coat protein produced by the second production cell may be homologous or heterologous to the viral nucleotide sequence. The viral coat protein must be compatible with the chimeric nucleotide sequence and of sufficient size to completely encapsidate the chimeric nucleotide sequence.

Alternatively, the first production cell further contains a second vector which comprises a coding sequence for a compatible viral coat protein adjacent a promoter compatible with the production cell. Again, this coding sequence for the viral coat protein may be homologous or heterologous to the viral nucleotide sequence. In this instance, the production cell then produces the coat protein and the chimeric nucleotide sequence. The chimeric nucleotide sequence is then encapsidated by the viral coat protein to produce a transmissible virus. The second vector is prepared by conventional techniques, by inserting the coding sequence for the viral coat protein which was deleted in the preparation of the chimeric nucleotide sequence, or a homologous or heterologous viral coat protein coding sequence into an appropriate vector under the control of a promoter compatible with the production cell. There are numerous alternatives whereby different vectors are utilized to transform separate production cells or the same production cell. With most viruses, it is unknown whether transmissibility factors alone are essential for the infectivity of a virion or whether the transmissibility factors simply serve as an adjunct to the capsid, and therefore enhance virion i'.-fectivity, but do not prevent infection if not present on the virion. For purposes of this invention (i.e., to insure non-infectiousness of the viral vector) , it has been assumed that the transmissibility factors alone could render the viral vector infectious, and therefore the vector comprising the chimeric nucleotide sequence will contain neither a nucleotide sequence for the capsid protein nor a nucleotide sequence for any transmissibility factor.

However, this vector comprising the chimeric nucleotide- sequence must contain a promoter and an origin of replication, and preferably contain- an origin of assembly. As stated above, this vector is utilized to transform a production cell. The origin of replication must be compatible with the production cell.

The other necessary vector or vectors may be varied primarily dependent on the virus of interest. If the viral nucleotide sequence for the capsid protein is adjacent the nucleotide sequence for the transmissibility factors, and the sequences share the same promoter (as in the case with alphaviruses) , a second vector comprises the sequence for the capsid protein, the sequence for the transmissibility factors and a promoter. This second vector may optionally contain the origin of assembly if the origin of assembly has not been incorporated in the first vector. When this vector is utilized to transform a production cell, the origin of assembly must be compatible with that production cell. Alternatively, the second vector may comprise the nucleotide sequence for the capsid protein and a promoter, and a third vector may comprise the nucleotide sequence for the transmissibility factors and a promoter. Either the second or third vector could further comprise the origin of assembly which would have to be compatible with the production cell to be transformed by the vector.

With any of the alternative embodiments, each vector may be utilized to transform its own production cell, or all of the vectors may be utilized in one production cell. All of the promoters associated with each vector must also be compatible with the production cell in which the vector is utilized.

As previously discussed, a prokaryotic production cell can be transformed to contain a vector containing the coat protein coding sequence or any other coding sequence necessary for infectivity of the virus. Alternatively, the prokaryotic production cell or a eukaryotic production cell is transformed such that the coding sequences for the coat protein and transmissibility factors (if necessary) are stably incorporated into the genome of the cell. Several conventional techniques can be utilized to accomplish this embodiment. For example, appropriate coding sequences can be inserted into plant cells by transformation with Agrobacterium. such as described by Schell, J. et al., Bio/Technology 1 , 175 (1983); Fillatti, J. et al., Bio/Technology 5, 726 (1987); Everett, N.P. et al., Bio/Technology 5_, 1201 (1987); Pua, E-C. , Bio/Technology 5. 815 (1987); Hinchee, M.A. , et al., Bio/Technology 6., 915 (1988) and Meth. Enzvmol, Vol. 118, supra. Agrobacterium have also been utilized for introducing viruses into plants, both dicots and monocots by a process termed Agro-infection. This technique has been described by Grimsley, N. , et al., Proc. Natl. Acad. Sci. USA 83, 3282 (1986); Elmer, J.S. et al., Plant Mol Biol 10, 225 (1988); Grimsley, N. et al., Nature 325, 177 (1987); and Hayes, R.J. et al., Nature 334. 180 (1988) .

Alternatively, the appropriate coding sequence can be inserted into eukaryotic cells by direct gene transfer including electroporation, calcium chloride or polyethylene glycol mediated transformation, liposome fusion microinjection or microprojectile bombardment. These techniques have been described by Fromm, M.E., Meth. Enzvmol 153. 307 (1987); Shillito, R.D. et al., Meth. Enzy ol 153, 283 (1987); Dehayes, A., et al., EMBO J 4, 2731 (1985); Negrutin, R. et al., Plant Mol Biol 8_., 363 (1987); Reich, T.J. et al., Bio/Technology 4., 1001 (1986); Klein, T.M. et al., Bio/Technology _5, 559 (1988); McCabe, D.E. et al., Bio/Technology 6 , 923 (1988) .

A production cell which has been transformed will contain the coat protein and/or transmissibility factors coding sequence(s) either in a stable vector or stably incorporated in the genome. The vector containing the chimeric nucleotide sequence is then introduced into the production cell as previously discussed.

Once the chimeric nucleotide sequence is replicated and the capsid protein and transmissibility factors have been produced by the appropriate production cells, the intact virions may be assembled in vitro. The replicated chimeric nucleotide sequence is encapsidated by the capsid protein in accordance with known conventional techniques. The transmissibility factors may be added to the virion assembly separately when a third vector is used to cause the production of transmissibility factors separate from the capsid protein. Alternatively, if a single second vector was used to cause the production of both capsid protein and transmissibility factors (as is usually the case with alphaviruses) , then the in vitro virion assembly is complete upon encapsidation of the chimeric nucleotide sequence.

A further feature of the present invention is a plant which has been infected by the virus. After introduction into a plant, the plant contains the chimeric nucleotide sequence which is capable of self- replication but which is not capable of producing additional transmissible viruses or viral particles. The plant can be infected with the virus by conventional techniques. Suitable techniques include, but are not limited to, leaf abrasion, abrasion in solution, high velocity water spray and other injury of a host as well as imbibing host seeds with water containing the chimeric nucleotide sequence alone or the encapsidated virus. Although the chimeric nucleotide sequence is not capable of producing infective agents, it is capable of self-replicating and spreading throughout the plant.

An alternative method for introducing a chimeric nucleotide sequence into a plant is a technique known as agroinfection or Agrobacterium-mediated transformation (sometimes called Agroinfection) as described by Grimsley, N. et al.. Nature. 325. 177 (1987). This technique makes use of a common feature of Agrobacterium which colonizes plants by transferring a portion of their DNA (the T-DNA) into a host cell, where it becomes integrated into nuclear DNA. The T-DNA is defined by border sequences which are 25 base pairs long, and any DNA between these border sequences is transferred to the plant cells as well. The insertion of a chimeric viral nucleotide sequence between the T-DNA border sequences results in transfer of the chimeric sequence to the plant cells, where the chimeric sequence is replicated, and then spreads systemically through the plant. Agroinfection has been accomplished with potato spindle tuber viroid (PSTV) (Gardner, R.C. et al., Plant Mol. Biol. 6 , 221 (1986)), cauliflower mosaic virus (CaMV)

(Grimsley, N. et al., Proc. Natl. Acad. Sci. U.S.A. 83,

3282 (1986)), maize streak virus (Grimsley, N. et al.,

Nature 325, 177 (1987) and Lazarowitz, S.G., Nucl. Acids

Res. 16, 229 (1988)), digitaria streak virus (Donson, J. et al., Virogology 162. 248 (1988)), wheat dwarf virus (Hayes, R.J. et al., J. Gen. Virol. 69, 891 (1988) ) and tomato golden mosaic virus (TGMV) (Elmer, J.S. et al., Plant Mol. Biol. 10, 225 (1988) and Gardiner, W.E. et al., EMBO J. 2_. 899 (1988)). Therefore, agroinfection of a susceptible plant could be accomplished with a virion containing a chimeric nucleotide sequence based on the nucleotide sequence of any of the above viruses.

Several viral products are useful to insure the production and spread of viral nucleic acids. One factor is a replicase which is involved in the replication of the viral nucleic acid. A second factor which may be present is termed herein a transport protein. This protein(s) is(are) involved in the movement of the viral nucleic acid from infected cells to adjacent cells and thus, the spread of the viral nucleic acid throughout the production or host cell, tissue or organism. In order to insure suitable replication and spread of the viral vector, an additional embodiment of the present inventi n includes the stable transformation of the plant or of the production cell, tissue or organism with a coding sequence for viral replicase, a viral transport protein or both. The transformation, including stable integration into the genome of the production cell or host, is accomplished as described above concerning the coat protein gene.

In an additional embodiment of the invention, the host range of a virus is enlarged so that a viral construct, i.e., chimeric nucleic acid, containing different foreign genes may be used in many different plants. The host range of the virus is enlarged by transforming the plant (or production cell since it will also work in this context) to contain parts of viral material useful in enabling infection of the host by the viral construct. For plants, the plants are transformed to contain the viral replicase gene or transport protein gene. Alternatively, the virus can be produced to contain coding sequences for transport proteins capable of functioning in the host. In addition, it is possible to transform protoplasts of the host plant to contain the viral nucleic acid or parts thereof such that all cells of the regenerated plants will be capable of infection. This transformation is carried out by any of the techniques previously described. A still further feature of the invention is a process for the induction of male sterility in plant. Male sterility can be induced by several mechanisms including, but not limited to, an anti.-sense RNA mechanism, a ribozyme mechanism, or a protein mechanism which may induce male sterility or self-incompatibility or interfere with normal gametophytic development. The second nucleotide sequence of the chimeric nucleotide sequence comprises the transcribable sequence which leads to the induction of male sterility. This process involves the infection of the appropriate plant with a virus, such as those described above, and the growth of the infected plant to produce the desired male sterility. The growth of the infected plant is in accordance with conventional techniques.

Male sterility can be induced in plants by many mechanisms including, but not limited to (a) absence of pollen formation, (b) formation of infertile and/or non¬ functional pollen, (c) self-incompatibility, (d) inhibition of self-compatibility, (e) perturbation of mitochondrial function(s) , (f) alteration of the production of hormone or other biomoolecule to interfere with normal gametophytic development, or (g) inhibiting a developmental gene necessary for normal male gametophytic tissue. These mechanisms may be accomplished by using anti-sense RNA, ribozymes, genes or protein products. The viruses of the present apply contain one or more nucleotide sequences (referred to as second nucleotide sequence in the chimeric nucleotide sequence) which function to induce male sterility. The viruses may contain a nucleotide sequence, a single gene or a series of genes to accomplish this function.

Male sterility traits could be formed by isolating a nuclear-encoded male sterility gene. Many of these genes are known to be single genes. For example Tanksley et al., Hort Science 23. 387 (1988) placed ms- 10 in CIS with a rare allele of the tightly linked enzyme-coding gene Prx-2. The Prx-2 allele is codominant,. allowing selection for heterozygous plants carrying the recessive ms-10 allele in backcross populations and eliminating the need for progeny testing during transfer of the gene into parents for hybrid production. A male-sterile anthocyaninless plant (ms- 10 aa/ms-lOaa) was crossed to a heterozygous, fertile plant in which a rare peroxidase allele was in cis with the recessive male-sterile allele (ms-10 Prx-2 V+Prx- 2⁺) . Male sterile plants were selected from the progeny (ms-10 Prx-2 '/ms-lOaa) . Once the male-sterile gene has been transferred into a prospective parental line, sterile plants can be selected at the seedling stage either from backcross or F₂ seed lots. In pearl millet recessive male sterile genes were found in Vg 272 and IP 482, Rao, et al. , Journ. of Heredity 74:34 (1983). Male sterility in pearl millet line Vg 272 and in IP 482 is essentially controlled by a single recessive gene. Male sterility in Vg 272 is due to a recessive gene, ms, which has no effect on meiosis in pollen mother cells, but acts after separation of microspores from tetrads but before the onset of the first mitotic division. Dewey, et al., Cell 44:439-449 (1986) isolated and characterized a 3547 bp fragment from male sterile (cms- T) maize mitochondria, designated TURF 243. TURF 243 contains two long open reading frames that could encode polypeptides of 12,961 Mr and 24,675 Mr. TURF 243 transcripts appeared to be uniquely altered in cms-T plants restored to fertility by the nuclear restorer genes Rfl and Rf2. A fragment of maize mtDNA from T cytoplasm was characterized by nucleotide sequence analysis. To obtain isolation of nucleic acids, mitochondrial RNA (mtRNA) , and mtDNA were prepared from 6 to 7 day old dark grown seedlings of Zea Mays L. as previously described by Pring and Levings (1978) and Schuster et al. (1983).

Another way male sterile traits could be formed is by isolating a male sterility gene from a virus. There are several viruses or virus like particles that induce male sterility in plants. Recent work suggests that viroid-like agents in male sterile beets may occur. (Pearson, O.N. Hort Science 16:482, 1981) . Cytoplasmic male sterility may be conditioned by a discrete particle such as a plasmid or an inclusion. Viruses are not seed transmitted with the regularity of the eytosterile systems. Viroids can be transmitted through the pollen. Transfer of a factor of some kind across a graft union has been demonstrated in petunia, beet, sunflower, and alfalfa. There is no direct effect on the fertility of the scion, but selfs or crosses by a malntainer on the grafted scion produced male sterile plants n the next generation. Cms beets grown at 36°C for 6 weeks, then at 25^βC, produced fertile plants from new shoots possibly due to elimination of "cytoplasmic spherical bodies", but progenies from the plants revereted to sterile after 3 generations at normal growing conditions. Cytoplasmic male sterility in the broad bean plant (Vicia fabal) was found to be caused by the presence of virus or virus-like particles. (Brill, 1981) . Possibly a case similar to a cms-system occurs in garlic. Pollen degeneration typical of sporophytic cms plants was found, but electron microscope studies showed richettsia-like inclusions in the anthers, which could be eliminated with antibiotics, causing the pollen to become fertile (Konvicha et al., Z. Pfanzenzychtung 80:265 (1978). A third method male sterile traits could be formed is by introducing an altered protein, using a transit peptide sequence so that it will be transported into the mitochondria, and perturb the mitochondrial functions. This protein could work to overwhelm a normal mitochondrial function or reduce a metabolite required in a vital pathway. It is widely believed that slight perturbations in the mitochondria will lead to male sterility. Remy et al., Theor. Appl. Genet. 64:249 (1983) conducted a two dimensional analysis of chloroplast proteins from normal and cytoplasmic male sterile Bassica napus lines. Chloroplast and mitochondrial DNA's of N and cms lines of B__ napus were characterized and compared using restriction enzyme analysis. Identical restriction patterns were found for chloroplastic DNA's from the cms B__ napus lines and the cms lines of the Japanese radish used to transfer the cms trait into B__ napus. In Re y's study, chloroplast proteins from stroma and thylakoids of N and cms lines of B. napus were characterized and compared using a 2-D polyacrylamide gel separation. It was shown that 1) stromal compartments of the two lines were very similar and 2) the lines could be distinguished by the spots corresponding to the β subunits of the coupling factor CF, from the ATPase complex.

A fourth method is by inducing or inhibiting a hormone that will alter normal gametophytic development. For example, inhibiting the production of gibberellic acid prior to or at the flowering stage to disturb pollen formation or modify production of ethylene prior to or at the flowering stage to alter flower formation and/or sex expression.

A fifth method is by inhibiting a developmental gene required for the normal male gametophytic tissue. For example, using anti-sense RNA that is complementary to the developmental signal RNA or mRNA. Padmaja et al. Cytologia 53:585 (1988) discusses cytogenetical investigations on a spontaneous male sterile mutant isolated from the Petunia inbred lines. Male sterility was found to be associated with atypical behavior of tapetum, characterized by prolonged nuclear divisions and untimely degeneration as a result of conversion from glandular to periplasmodial type.

A sixth method is by isolating a self- incompatibility gene and using this gene in the Geneware vector. Self-incompatibility (S) gene systems that encourage out-breeding are prseent in more than 50% of the angiosperm plant families Ebert et al., Cell 56:255 (1989) . Multiple S gene systems are known in some species. In several systems, abundant style glycoproteins (S glycoproteins) have been identified. These glycoproteins are polymorphic and can be correlated with identified S alleles. S genes, corresponding to the style glycoproteins of N___ alaba and B. oleraceae have been cloned and sequenced. Amino acid substitutions and deletions/insertions though present throughout the sequences, tend to be clustered in regions of hypervariability that are likely to encode allelic specificity.

A seventh method is by blocking self incompatibility by engineering a protein that will bind and inactivate the compatibility site or turn off self- compatibility by engineering an anti-sense RNA that will bind with the mRNA to a self-compatibility protein.

Specific effects resulting in male sterility can range from the early stages of sporogenous cell formation right through to a condition in which anthers containing viable pollen do not dehisce. some or all developmental stages within this range may be affected. Some of the more obvious specific effects include, the following examples:

1) Meiosis is disrupted, leading to degeneration of the pollen mother cells or early microspor^s in which case pollen aborts and anther development is arrested at an early stage.

2) Exine formation is disrupted and microspores are thin-walled, perhaps distorted in shape, and nonviable. Anthers are generally more developed than above but still not normal.

3) Microspore vacuole abnormalities, decreased starch deposition, and tapetum persistence are evidence. Pollen is nonviable and anthers are still not normal. 4) Pollen is present and viable, and anthers appear normal but either do not dehisce or show much delayed dehiscence.

5) Self incompatibility mechanisms which disrupt or prevent enzymatic digestion of the style by the pollen grain. Male sterility may be induced by the mechanisms listed above at any plant stage prior to pollen shed. The male sterility mechanism selected may be applied to plants in the field (or in the greenhouse) at any time after seedling emergence and before pollen shed. The exact time of application will depend on the male sterility mechanism used and the optimum effectiveness in producing male sterile plants.

EXAMPLES

The following examples further illustrate the present invention. In these examples, enzyme reactions were conducted in accordance with the manufacturer's recommended procedures unless otherwise indicated. Standard techniques such as those described in Maniatis, T. et al., supra; Meth.in Enzvmol. -Vol. 68, 100, 101, 118, 152-155, supra; and DNA Cloning. Vol. I, II, III, supra, were utilized for vector constructions and transformation unless otherwise specified.

EXAMPLE 1 Preparation of Chimeric TMV DNA

Comprising Tyrosinase Gene in Place of the Coat Protein Gene

The 1.2 kb tyrosinase gene was derived from the pIJ702 streptomyces plasmid (available to the public) using a SacI/PVUII cut. It was inserted into the SacI/EcoRV site of the Stratagene bluescript vector (KS) , which is 2.9 kb in size. The resulting plasmid was maintained as pBGllO (4.1 kb) . The tyrosinase gene was removed from pBGllO by a Sacl/Hindlll cut and inserted into pUC19 (2.7 kb) giving pBG115 (3.9 kb) . The tyrosinase gene was removed from pBG115 by a EcoRI/Hindlll cut and inserted into the Stratagene bluescript vector (KS+; 2.9 kb) giving pBG120 (4.1 kb) . The tyrosinase gene was removed from pBG120 with a Xhol/Smal cut and Xhol linkers were added to the Smal end. This tyrosinase gene with Xhol ends was inserted into the Xhol site of the Stratagene bluescript vector (KS+; 2.9 kb) to give pBG130 (4.1 kb) . Since there is an unwanted PstI site carried over in a poly-linker region, the pBG130 was modified by removing some of the non-coding bases and the poly-linker region. This was performed using a Hindlll/SacI digest to release the tyrosinase from pBG130, then treating the 1.2 kb fragment with ExoIII to digest approximately 200 bases from the Hindlll 3' end. After blunting the ends with T₄ DNA Polymerase, Xhol linkers were added and the 1.0 kb fragment was inserted into the Stratagene bluescript vector (Ks+; 2.9 kb) giving pBG132. The 1.0 kb tyrosinase gene was inserted into p803 (6.3 kb) , which is a 3.6 kb subclone of the TMV plasmid, pS3-28. (available from W. 0. Dawson, University of California, Riverside), in pUC19, giving plasmids pBG21 and pBG22 (depending on the direction of the tyrosinase gene; 7.3 kb each) . The plasmid S3-28 (11.1 kb) is a clone of TMV that has had the coat protein gene removed with an Xhol site at that position. Dawson, W. 0. et al., Phytopathology 78, 783 (1988) . A NcoI/EcoRV cut of the pBG21 and pBG22 released a 2.4 kb fragment containing the tyrosinase gene. This fragment replaced a 1.4 kb piece in pS3-28 that had been removed. The resulting plasmids, pBG23 and pBG24 (depending on the direction of the tyrosinase gene; 12.1 kb each) were the final DNA constructions that were used to make infectious RNA that were introduced into tobacco plants to introduce a mRNA for tyrosinase. This mRNA can be detected using northern blot procedures. EXAMPLE 2

Preparation of Chimeric TMV DNA Comprising GUS Gene in Place of the Coat Protein Gene

The 1.8 kb GUS gene was derived from the pRAJ275 (4.5 kb; Clonetech Laboratories) using a EcoRI/Ncol cut. The sticky ends were filled in using the Klenow fragment. Xhol linkers were added and the fragment was inserted into the Stratagene bluescript vector (KS+) , which is 2.9 kb in size. The resulting plasmid was maintained as pBG150 (4.7 kb) . Using the techniques of Example 1, this GUS gene with the Xhol linkers was moved into the p803, giving pBG25 and pBG26 (depending on the direction of the GUS gene; 8.1 kb each). A Sall/Ncol cut of the pBG25 and pBG26 released a 3.8 kb fragment containing the GUS gene. These fragments replaced a 2.0 kb piece in pS3-28 that had been removed. Th3 resulting plasmids, pBG27 and pBG28 (depending on the direction of the GUS gene; 12.9 kb each) were the final DNA constructions that were used to make infectious RNA that were introduced into tobacco plants to introduce a mRNA for the GUS gene. This mRNA can be detected using northern blot procedures. The activity of the GUS enzyme is also detectable.

EXAMPLE 3 Preparation of Chimeric TMV DNA Comprising

GUS/Coat Protein Gene in Place of the TMV Coat Protein Gene

The 1.8 kb GUS gene from pBG150 (4.7 kbk) , described in Example 2 above, was used for this construction. A Pstl/Ncol cut of p35-5 released a 0.7 kb fragment that contains a portion of the 3* end and a portion of the 5¹ end of the TMV coat protein gene. The plasmid p35-5 (11.3 kb) is a clone of TMV that has had most of the coat protein gene removed and contains an Xhol site at the site where the internal portion of the coat protein gene is removed. Dawson, W. O. et al., Phytopathology 78, 783 (1988). This 0.7 kb fragment replaced a 0.5 kb piece in p803 that had been removed, giving pBG29 (6.5 kb) . The 1.8 kb GUS gene was inserted into the Xhol site of pBG29, giving pBG31 and pBG32 (depending on the direction of the GUS/coat protein fusion gene; 8.3 kb each) . A Sall/Ncol cut of the pBG31 and pBG32 released a 4.0 kb fragment containing the GUS/coat protein fusion gene. These fragments replaced a 2.0 kb piece in pS3-28 that had been removed. The resulting plasmids, pBG33 and pBG34 (depending on the direction of the GUS/coat protein fusion gene; 13.1 kb each) were the final DNA constructions that were used to make infectious RNA. pBG33, pBG34 and pS3-28 (as control) are transcribed into RNA which is used to infect tobacco plants by rubbing on the plants the RNA, an abrasive and a RNase inhibitor. Spots are noted on the plants indicating a successful infection. The infected plant tissue is macerated and fluorescence examined. Tissue infected with pS3-28 did not fluoresce whereas tissue infected with either pBG33 or pBG34 did fluoresce.

EXAMPLE 4 Preparation of a Non-Transmissible

TMV Nucleotide Sequence

A full-length DNA copy of the TMV genome is prepared and inserted into the Pst I site of pBR322 as described by Dawson, W.O. et al., Proc. Nat. Acad. Sci. USA 83, 1832 (1986) . The viral coat protein gene is located at position 5711 of the TMV genome adjacent the 30k protein gene. The vector containing the DNA copy of the TMV genome is digested with the appropriate restric¬ tion enzymes and exonucleases to delete the coat protein coding sequence. For example, the coat protein coding sequence is removed by a partial digestion with Clal and Nsil, followed by relegation to reattach the 3'-tail of the virus. Alternatively, the vector is cut at the 3' end of the viral nucleic acid. The viral DNA is removed by digestion with Bal31 or exonuclease III up through the start codon of the coat protein coding sequence. A synthetic DNA sequence containing the sequence of the viral 3'-tail is then ligated to the remaining 5'-end. The deletion of the coding sequence for the viral coat protein is confirmed by isolating TMV RNA and using it to infect tobacco plants. The isolated TMV RNA is found to be non-infective, i.e. biologically contained, under natural conditions.

EXAMPLE 5 Preparation of a Non-Transmissible

OMV Nucleotide Sequence

A full-length DNA copy of the OMV genome is prepared as described by Dawson, W.O. et al., (1986), supra. The vector containing the DNA copy of the OMV genome is digested with the appropriate restriction enzymes or suitable exonucleases such as described in Example 4 to delete the coat protein coding sequence. The deletion of the coding sequence for the viral coat protein is confirmed by isolating OMV RNA and using it to infect germinating barley plants. The isolated OMV RNA is found to be biologically contained under natural conditions.

EXAMPLE 6

Preparation of a Non-Transmissible RNV Nucleotide Sequence

A full-length DNA copy of the RNV genome is prepared as described by Dawson, W.O. et al., (1986), supra. The vector containing the DNA copy of the RNV genome is digested with the appropriate restriction enzymes or suitable exonucleases such as described in Example 4 to delete the coat protein coding sequence. The deletion of the coding sequence for the viral coat protein is confirmed by isolating RNV RNA and using it to infect germinating barley plants. The i.r.olated RNV RNA is found to be non-infective under natural conditions.

EXAMPLE 7 Preparation of a Non-Transmissible

MSV Nucleotide Sequence

A full-length DNA copy of the maize streak virus (MSV) genome is prepared as described by Dawson, W.O. et al., (1986), supra. The vector containing the DNA copy of the MSV genome is digested with the appropriate restriction enzymes or suitable exonucleases such as described in Example 4 to delete the coat protein coding sequence. The deletion of the coding sequence for the viral coat protein is confirmed by isolating MSV RNA and using it to infect potato plants. The isolated MSV RNA is found to be biologically contained under natural conditions.

EXAMPLE 8

Preparation of a Non-Transmissible TGMV Nucleotide Sequence

A full-length DNA copy of the TGMV genome is prepared as described by Dawson, W.O. et al., (1986), supra. The vector containing the DNA copy of the TGMV genome is digested with the appropriate restriction enzymes or suitable exonucleases such as described in Example 4 to delete the coat protein coding sequence. The deletion of the coding sequence for the viral coat protein is confirmed by isolating TGMV RNA and using it to infect potato plants. The isolated TGMV RNA is found to be biologically contained under natural conditions.

EXAMPLE 9

Preparation of Chimeric Nucleotide Sequence Containing the CMS-T Coding Sequence

The coding sequence for.CMS-T is isolated from a BamHI maize mtDNA library as described by Dewey, R.E., et al., Cell 44. 439 (1986). The ORF-13 coding sequence is isolated by restriction endonucleuse digestion followed by 5'-exonuclease digestion to the start codon. Alternatively, a restriction site is engineered adjacent the start codon of the ORF-13 coding sequence by site- directed oligonucleotide mutagenesis. Digestion with the appropriate restriction enzyme yields the coding sequence for ORF-13. The fragment containing the ORF- 13 coding sequence is isolated and cloned adjacent the promoter of the viral coat protein gene in the vectors prepared in Examples 5, 6 and 8.

EXAMPLE 10 Preparation of Virus Containing

CMS-T Coding Sequence

A lambda P_R promoter is attached to the chimeric nucleotide sequence of Example 9 in accordance with the technique described in Dawson, W.O. et al., (186), supra. The resulting vector is used to transform E. coli, the production cell in this instance.

A second vector is prepared by inserting the viral coat protein coding sequence, isolated in Examples 5, 6 or 7, adjacent the lac promoter in the vector pBR322. This vector is used to transform the production cells having a vector with the corresponding compatible chimeric nucleotide sequence. The production cells are grown and the resultant viruses are isolated. Alternatively, the second vector is used to transform a second strain of E. coli which produces the coat protein. The coat protein and viral vector are then combined to form the virus.

EXAMPLE 11

Induction of Male Sterility in Maize

The viruses isolated in Example lore used to infect maize plants (viruses based on OMV, RNV or TGMV) prior to tassel formation. The infected plants are grown under normal growth conditions. The plants produce cms- T which induces male sterility in the infected maize plants.

EXAMPLE 12

Preparation of a Chimeric Nucleotide as Amended Sequence Containing a

Self-Incompatibility Coding Sequence

The coding sequence of S₂-protein (for self- incompatibility) is isolated from Nicotiana alata as described in EPA 0 222 526. The S_₂-protein coding sequence is isolated by restriction endonucleuse digestion followed by 5'-exonuclease digestion to the start codon. Alternatively, a restriction site is engineered adjacent the start codon of the S.₂-protein coding sequence by site-directed oligonucleotide mutagenesis. Digestion with the appropriate restriction enzyme yields the coding sequence for S_₂-protein. The fragment containing the S_₂-protein coding sequence is isolated and cloned adjacent the promoter of the viral coat protein gene in the vectors prepared in Example 4. EXAMPLE 13

Preparation of Virus Containing a Self-Incompatibility Coding Sequence

A lambda P_R promoter is attached to the chimeric nucleotide sequence of Example 12 in accordance with the technique described in Dawson, W.O. et al., (1986), supra. The resulting vector is used to transform E. coli, the production cell in this instance.

A second vector is prepared by inserting the viral coat protein coding sequence, isolated in Example 4, adjacent the lac promoter in the vector pBR322. This vector is used to transform the production cells having a vector with the corresponding compatible chimeric nucleotide sequence. The production cells are grown and the resultant viruses are isolated.

EXAMPLE 14 Induction of Male Sterility In Tobacco

The viruses isolated in Example 13 are used to infect tobacco (___ tobacum) prior to pollen formation. The infected plants are grown under normal growth conditions. The plants produce S_-protein which induces male sterility via the self-incompatibility mechanism.

EXAMPLE 15

Preparation of a Chimeric Nucleotide Sequence of TMV and Chloramphenicol

Acetyltransferase

A non-transmissible TMV nucleotide sequence (pTMVS3-28) is prepared as described in Example 4. A chimeric nucleotide sequence containing the chloramphenicol acetyltransferase (CAT) gene substituted for the previously removed coat protein gene {S3-CAT-28) is then prepared. The CAT gene is removed from pCMl (Pharmacia) with Sal I and ligated into the nucleotide sequence from Example 4 which has been cleaved by Xho I. This construction produces pTMVS3-CAT-28 from which the mutant cp S3-CAT-28 is transcribed. Correct sequence and orientation is confirmed by sequencing as described by Sagursky, R.J. et al., Gene Anal. Technol. 2., 89 (1985) .

This chimeric DNA sequence is then transcribed using the PM promoter (Ahlquist, P. et al., Mol. Cell Biol. 4., 2876 (1984)) and RNA polymerase of I _ coli (Dawson, W.O. et al., Proc. Nat. Acad. Sci. USA, 83, 1832 (1986) ) to produce infectious chimeric RNA. The infectivity is assayed by grinding leaves in cold 1% sodium pyrophosphate buffer, pH9.0, plus 1% bentonite and 1% Celite with immediate inoculation. Alternatively, the infectivity may be assayed using phenol-extracted RNA in the same buffer (Dawson, W.O., Virology. 1__, 319 (1976)). The chimeric RNA is found to replicate effectively in tobacco leaves. The RNA can be propagated serially from plant to plant or may be stored after phenol extraction. However, no virions are produced in the infected plants. The chimeric RNA fails to produce a band during

SDS-PAGE that can be positively identified as chloramphenicol acetyltransferase. However, functional enzyme activity can be detected in plant extracts that acetylated ¹⁴C-chloramphenicol with acetyl CoA.

EXAMPLE 16

Preparation of a Virus Containing Chloramphenicol Acetyltransferase

A lambda P_R promoter is attached to the chimeric nucleotide sequence of Example 15 in accordance with the technique described by Dawson, W. 0. et al., Proc. Nat. Acad. Sci. U.S.A. 83, 1832 (1986) . The resulting vector is used to transform the production cell, ______ coli

A second vector is prepared by inserting the viral coat protein coding sequence (isolated in Example 4) , adjacent the lac promoter in the vector pBR322. This vector is used to transform the E___ coli production cells having the first vector with the chimeric nucleotide sequence. The production cells are grown, and the resultant viruses are isolated. Alternatively, the second vector is used to transform a second strain of E. coli which produces the coat protein. The coat protein and viral vectors are then combined to form the virus.

EXAMPLE 17

Production of Chloramphenicol Acetyltransferase

The viruses isolated in Example 16 are used to infect tobacco plants. The infected plants are grown under normal growth conditions. The plants produce the enzyme, chloramphenicol acetyltransferase, which is isolated from the plants using conventional techniques.

EXAMPLE 18

Transformation of Nicotiana Tobacum with Capsid Protein Gene

The capsid protein of tobacco mosaic virus (TMV) is encoded by a nucleotide sequence between 5712 and 6190. A 3'-untranslated region of the RNA genome extends to nucleotide 6395. A double stranded (ds) complementary DNA (cDNA) of the cistron containing nucleotide 5701 to 6395 and coding for capsid protein, is generated from TMV-RNA using reverse transcriptase. The transcriptase contains an oligonucleotide primer which is complementary to an oligonucleotide sequence at the 3 ' end of the viral RNA and contains a BamHI site. A Klenow fragment of DNA polymerase is used to synthesize the second DNA-strand. The Hindlll-BamHI fragment of cDNA is cloned into the plasmid pUC9 (Vieira, J. et al., Gene 19, 259 (1982)). Thα resulting plasmid is digested with Alalll at nucleotide 5707 (Goelet, P. et al., Proc. Natl. Acad. Sci. USA 79, 5818 (1982)) and with BamHI to remove the sequence for capsid protein. This fragment is ligated into the vector pMON237. pMON237 is a derivative of pMON200 (Horsch, R.B. et al., Science 227, 1229 (1985)). This vector contains a 19S CaMV promoter, a polylinker sequence and the sequence for nopaline synthese (NOS) at the 3' end. Ligation occurs at the Xbal site which is made blunt, and at the BamHI site. The capsid protein coding sequence is removed from the plasmid by digestion with Xbal and BamHI and given a Bglll site at the 5• end and an EcoRI site at the 3 ' end by transfer to a suitable plasmid. The Bglll-EcoRI fragment is transferred into the expression cassette vector pMON316 between the CaMV 35S promoter and the NOS 3' untranslated region (Rogers, S.G. et al., in BioTechnology in Plant Science: Relevance to Agriculture in the Nineteen Eighties, M. Zaitlin, P. Day, A. Hollaender, Eds., Academic Press, N.Y., p. 219 (1986). The resulting chimeric gene contains the 35S promoter from CaMV and a polyadenylation signal from the NOS gene. The resulting plasmid is mated into Agrobaσterium tumefac ens strain GV 3111 carrying the disarmed pTi BGS3-SE plasmid (Fraley, R.T. et al., Bio/Technology 3, 629 (1985)). A cointegrate plasmid results by recombination between LIH regions.

Transformed A_^_ tumefaciens are selected for antibiotic resistance. Selected colonies are used to transform N. tobacum leaf disks which are then regenerated into whole plants (Horsch, R.B. et al. , supra; Abel, P.P. et al., Science 232, 738 (1986)).

A. tumefaciens transformed with the sequence for TMV capsid protein is used as a production cell. Likewise tobacco cells that have been transformed with the sequence for TMV capsid protein are used as production cells for producing capsid protein monomers or multimers. Capsid protein numbers and timing of production can be controlled by methods well known in the arts (Maniatis, T. et al., supra) . Proper selection of a temperature sensitive repressor and a strong promoter results in high copy numbers of plasmids and high translation rates only when desired.

EXAMPLE 19 Production of Encapsidated Virus Particles

Protoplasts of transformed tobacco cells obtained in the Example 18 are resuspended in a 0.5 M mannitol solution containing 12-30 nM MgCl₂. A heat shock of 45°C for five minutes is given. The protoplasts are distributed in aliquots for transformation in centrifuge tubes, 0.3 ml of suspended protoplasts per tube. During the next 10 minutes the following are added. Plasmids obtained in Example 1 containing cDNA coding for the TMV genome but missing the gene for capsid protein, and which contains a chimeric gene construct; and polyethylene glycol (PEG) solution (MW 6000, 40% (w/v) containing 0.1M Ca(N0₃)₂ and 0.4M mannitol; pH 8-9 with KOH) to give a final concentration of 20% PEG. The aliquots are incubated for 30 minutes with occasional gentle shaking, and then the protoplasts are placed in petri dishes (0.3 ml original protoplast suspension per 6 cm diameter dish) and cultured. Encapsidated virus particles are isolated from the protoplasts using conventional techniques. Alternatively, the transformed plants produced in Example 49 are infected with virus nucleic acid, e.g. a chimeric nucleotide sequence of Example 3. The nucleic acid is replicated and coat protein is produced which encapsidates the nucleic acid. The encapsidated virus particles are isolated using conventional techniques.

EXAMPLE 20

Transformation of Nicotiana Tobacum with Replicase Gene

Two replicase subunits of tobacco mosaic virus

(TMV) are encoded by a nucleotide sequence between 70 and 4919. The nucleotide sequence for the 130-kD protein terminates at an amber codon which can be suppressed to produce a 180-kD protein. , A double stranded (ds) complementary DNA (cDNA) of the cistron containing nucleotides 70 to 4919 and coding for replicase, is generated from TMV-RNA using reverse transcriptase.

The transcriptase contains an oligonucleotide primer which is complementary to an oligonucleotide sequence at the 3' end of the viral RNA and contains a BamHI site. A Klenow fragment of DNA polymerase is used to synthesize the second DNA-strand. The Hindlll-BamHi fragment of cDNA is cloned into the plasmid pUC9 (Viera, J. et al., Gene 19. 259 (1982)). The resulting plasmid is digested with Alalll at nucleotide 4919 (Goelet, P. et al., Proc. Natl. Acad. Sci. USA 79. 5818 (1982)) and with BamHI to remove the sequence for capsid protein.

This fragment is ligated into the vector pMON237. pMON237 is a derivative of pMON200 (Horsch, R.B. et al., Science 227, 1229 (1985)). This vector contains a 19S CaMV promoter, a polylinker sequence and the sequence for nopaline synthase (NOS) at the 3' end. Ligation occurs at the Xbal site which is made blunt, and at the BamHI site. The capsid protein coding sequence is removed from the plasmid by digestion with Xbal and BamHI and given a Bglll site at the 5* end and EcoRI site at the 3' end by transfer to a suitable plasmid. The Bglll-EcoRI fragment is transferred into the expression cassette vector, pMON316 between the CaMV 35S promoter and the NOS 3' untranslated region (Rogers, S.G. et al. , in BioTechnology in Plant Science: Relevance to Agriculture in the Nineteen Eighties, M. Zaitlin, P. Day, A. Hollaender, Eds., Academic Press, N.Y., p. 219, 1986). The resulting chimeric gene contains the 35S promoter from CaMV and a polyadenylation signal from the NOS gene. The resulting plasmid is mated into Agrobacterium tumefaciens strain GV 3111 carrying the disarmed pTi BGS3-SE plasmid (Fraley, R.T. et al., Bio Technology 3. 629 (1985)). A cointegrate plasmid results by recombination between LIH regions. Transformed A. tumefaciens are selected for antibiotic resistance. Selected colonies are used to transform ___ tobacum leaf disks which are then regenerated into whole plants (Horsch, R.B. et al., supra. Abel, P.P. et al., Science 232, 738 (1986)). The transformed hosts can be used as production cells or hosts for the infection with TMV based viruses. The stably incorporated replicase will function to enhance replication of the viral nucleic acid.

EXAMPLE 21

Transformation of Tobacco Cells with Transport Protein

A nucleotide sequence of TMV from nucleotide 4903 to 5709 encodes a 30-kD protein which is implicated in facilitating cell to cell movement of the virus. Leaf disk cells of _ _ tobacum are transformed by Agrobacterium carrying a cointegrate plasmid made by the method of Example 49. ___ tobacum cells so transformed are used as host cells for encapsidated defective virus particles.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known and customary practice within the art to which the invention pertains.

Claims

WHAT IS CLAIMED IS:

1. A chimeric nucleotide sequence comprising a first nucleotide sequence which has substantial sequence homology to a plant viral nucleotide sequence which is capable of replication and is biologically contained, and a second nucleotide sequence which is adjacent a plant viral promoter and which is capable of inducing male sterility in a plant.

2. The chimeric nucleotide sequence of claim 1, wherein said chimeric nucleotide sequence is selected from the group consisting of DNA, RNA and cDNA.

3. The chimeric nucleotide sequence of claim 1 or 2, wherein said viral nucleotide sequence lacks a biologically functional viral coat protein coding sequence.

4. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said viral nucleotide sequence further lacks a biologically functional viral transmissibility factor coding sequence.

5. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said viral promoter is a viral coat protein promoter.

6. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence is a coding sequence of a male sterility gene.

7. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said coding sequence is obtained from plant nuclear DNA.

8. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said coding sequence is obtained from a virus or virus-like particle.

9. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence is a coding sequence of a self- incompatibility gene.

10. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence codes for a protein that binds to and inactivates the compatibility site.

11. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence codes for a protein which perturbs mitochondial functioning.

12. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence is capable of altering the production of a hormone to interfere with normal gametophytic development.

13. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said second nucleotide sequence is capable of inducing male sterility by an antisense RNA.

14. The chimeric nucleotide sequence of any one of the preceeding claims, wherein said antisense RNA is complementary to a developmental gene necessary for normal male gametophytic tissue.

15. A virus comprising the chimeric nucleotide sequence of any one of the preceeding claims.

16. A vector comprising the chimeric nucleotide sequence of any one of claims 1 to 15 adjacent a nucleotide sequence selected from the group consisting of a production cell promoter and an origin of replication compatible with said production cell.

17. A plant having the chimeric nucleotide sequence of any one of claims 1 to 15.

18. The plant of claim 17 which^" has been stably transformed to contain a coding sequence for a replicase useful for the replication of said chimeric nucleotide sequence.

19. The plant of claim 17 which has been stably transformed to contain a coding sequence for a plant viral transport protien useful for the transport of said chimeric nucleotide sequence within the plant.

20. A process for inducing male sterility in a plant which comprises (a) infecting a plant with a virus comprising the chimeric nucleotide sequence of any one of claims 1 to 15 and (b) growing the infected plant, whereby said plant becomes male sterile.

21. A process of inducing male sterility in a transgenic plant containing one or more coding sequences for: (i) a plant viral replicase, (ii) a plant viral transport protein or (iii) both (i) and (ii) which comprises (a) infecting a plant with an infectious virus, said virus comprising the chimeric nucleotide sequence of any one of claims 1 to 15 and (b) growing the transgenic plant, whereby said transgenic plant becomes male sterile.

22. A male sterile, transgenic plant comprising cells which are host to a chimeric nucleotide sequence of an infectious virus and which contain a coding sequence for a viral replicase compatible with said chimeric nucleotide sequence, said virus comprising the chimeric nucleotide sequence of any one of claims 1 to 15.