Whisker-mediated transformation of embryogenic cotton suspension cultures

This application is a continuation of prior application Ser. No. 09/975,238, filed Oct. 11, 2001, now abandoned, which claimed priority from Ser. No. 60/239,511 filed Oct. 11, 2000.

This invention relates to a method of using elongated, needle-like microfibers or “whiskers” to transform embryogenic cotton suspension cultures.

Until recently, genetically manipulated plants were limited almost exclusively to those events created by application of classical breeding methods. Creation of new plant varieties by breeding was reserved primarily for the most agronomically important crops, such as corn, due to the cost and time needed to identify, cross, and stably fix a gene in the genome, thus creating the desired trait. In comparison, the advent of genetic engineering has resulted in the introduction of many different heterologous genes and subsequent traits into diverse crops including corn, cotton, soybeans, wheat, rice, sunflowers and canola in a more rapid manner. However, the intergression of a new transgene into elite germplasm is still quite a laborious task due to the tissue culturing and back-crossing needed to produce a commercially viable, elite, line.

Several techniques exist which allow for the introduction, plant regeneration, stable integration, and expression of foreign recombinant vectors containing heterologous genes of interest in plant cells. One such technique involves acceleration of microparticles coated with genetic material directly into plant cells (U.S. Pat. No. 4,945,050 to Cornell; U.S. Pat. No. 5,141,131 to DowElanco; and U.S. Pat. No. 5,538,877 and U.S. Pat. No. 5,538,880, both to Dekalb). This technique is commonly referred to as “microparticle bombardment” or “biolistics”. Plants may also be transformed using Agrobacterium technology (U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max Planck, European Patent Applications 604662, 627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus). Another transformation method involves the use of elongated needle-like microfibers or “whiskers” to transform maize cell suspension cultures (U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca). In addition, electroporation technology has been used to transform plant cells from which fertile plants have been obtained (WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb; U.S. Pat. Nos. 5,679,558, 5,641,664, WO9209696 and WO9321335 to Plant Genetic Systems).

Despite all of the technical achievements, genetic transformation and routine production of transgenic plants in a commercially viable, elite, germplasm is still a laborious task. For example, microparticle bombardment, while capable of being used either on individual cells, cell aggregates, or plant tissues, requires preparing DNA-attached gold particles and optimization of an expensive and not yet widely available, “gun” apparatus. Techniques involving Agrobacterium are extremely limited because not all plant species or varieties within a given species are susceptible to infection by the bacterium. Electroporation techniques are not preferred due to the extreme difficulties and cost typically encountered in routinely making protoplast from different plant species and tissues thereof and the concomitant low viability and low transformation rate associated therewith.

Heterologous DNA can be introduced into regenerable plant cell cultures via whiskers-mediated transformation. While a general description of the process can be found in U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca, no protocols have been published to date for whisker-mediated transformation of embryogenic cotton cultures.

WO 99/38979 describes whisker-mediated transformation of cotton callus, but does not disclose or suggest whisker-mediated transformation of embryogenic cotton suspension cultures.

The present invention relates to the production of fertile, transgenic, Gossypium hirsutum L. plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.

Another aspect of the present invention relates to Gossypium hirsutum L. plants, plant parts, plant fibers, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA. The invention produces the fertile transgenic plants described herein by means of whisker-mediated cell perforation and heterologous DNA uptake, said whisker-mediated cell perforation being performed on embryogenic cotton suspension cultures.

Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.

The following phrases and terms are defined below:

By “antisense” is meant an RNA transcript that comprises sequences complementary to a target RNA and/or mRNA or portions thereof and that blocks the expression of a target gene by interfering with the processing, transport, and/or translation of its primary transcript and/or mRNA. The complementarity may exist with any part of the target RNA, i.e., the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. Antisense RNA is typically a complement (mirror image) of the sense RNA.

By “cDNA” is meant DNA that is complementary to and derived from a mRNA.

By “chimeric DNA construction” is meant a recombinant DNA containing genes or portions thereof from one or more species in either the sense or antisense orientation.

By “constitutive promoter” is meant promoter elements that direct continuous gene expression in all cell types and at all times (i.e., actin, ubiquitin, CaMV 35S, 35T, and the like).

By “cosuppression” is meant the introduction of a foreign gene having substantial homology to an endogenous gene, and in a plant cell causes the reduction in activity of the foreign gene and/or the endogenous gene product. Cosuppression can be sometimes achieved by introducing into said plant cell either the promoter sequence, the 5′ and/or 3′ ends, introns or the coding region of a gene.

By “developmental specific” promoter is meant promoter elements responsible for gene expression at specific plant developmental stages, such as in early or late-embryogenesis and the like.

By “enhancer” is meant nucleotide sequence elements which can stimulate promoter activity such as those from maize streak virus (MSV), alfalfa mosaic virus (AMV), alcohol dehydrogenase intron 1 and the like.

By “expression” as used herein, is meant the transcription of enzymatic nucleic acid molecules, mRNA, and/or the antisense RNA inside a plant cell. Expression of genes also involves transcription of the gene and may or may not involve translation of the mRNA into precursor or mature proteins.

By “foreign” or “heterologous gene” is meant a gene having a DNA sequence that is not normally found in the host cell, but is introduced by whisker-mediated transformation.

By “gene” is meant to include all genetic material involved in protein expression including chimeric DNA constructions, genes, plant genes and portions thereof.

By “genome” is meant genetic material contained in each cell of an organism and/or virus.

By “inducible promoter” is meant promoter elements which are responsible for expression of genes in response to a specific signal, such as: physical stimuli (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites, stress and the like.

By “modified plant” is meant a plant wherein the mRNA levels, protein levels or enzyme specific activity or a particular protein have been altered relative to that seen in an unmodified plant. Modification can be achieved by methods such as antisense, cosuppression, or over-expression.

By “plant tissues” is meant organized tissues including but not limited to meristems, embryos, pollen, cotyledons, germ cells, and the like.

By “promoter regulatory element” is meant nucleotide sequence elements within a nucleic acid fragment or gene which controls the expression of that nucleic acid fragment or gene. Promoter sequences provide the recognition for RNA polymerase and other transcriptional factors required for efficient transcription. Promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express sense and antisense gene constructs. Promoter regulatory elements are also meant to include constitutive promoters, tissue-specific promoters, developmental-specific promoters, inducible promoters and the like. Promoter regulatory elements may also include certain enhancer sequence elements that improve transcriptional or translational efficiency.

By “tissue-specific” promoter is meant promoter elements responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (i.e., zein, oleosin, napin, ACP, globulin and the like).

By “whiskers” is meant elongated needle-like bodies capable of being produced from numerous substances as described in “The Condensed Chemical Dictionary, Seventh Edition, Ed. Arthur & Elizabeth Rose, Reinhold Publishing Corp., New York (1966). The invention is not meant to be limited to the material from which the whiskers are made but instead is meant to define a needle-like shaped structure wherein said whisker is smaller than the cell for which it is intended to be used in the transformation thereof. It is within the scope of this invention for whiskers to be shaped in a manner whereby DNA entry into a cell is facilitated. It is also intended that the scope of said invention include any material having a needle-like shape, said needle-like shaped material being able to perforate a plant cell with or without cell walls and thus facilitate DNA uptake and plant cell transformation. It is also intended that the scope of this invention not include microinjection techniques, such as wherein a DNA molecule is inserted into a cell by passing said DNA through an orifice intrinsic to a needle, said needle being first inserted into said cell. Preferably, whiskers are metal or ceramic needle-like bodies, with those most preferred being made of either silicon carbide or silicon nitride and being 30×0.5 μm to 10×0.3 μm in size.

By “whisker-mediated transformation” is meant the facilitation of DNA insertion into plant cells and/or plant tissues by whiskers and expression of said DNA in either a transient or stable manner.

In producing plant cell lines, tissues of interest are aseptically isolated and placed onto solid initiation medium whereby processes associated with cell differentiation and specialization occurring in organized plant cell tissues are disrupted, thus resulting in said tissues becoming dedifferentiated. Typically, initiation medium is solidified by adding agar or the like because callus cannot be readily initiated in liquid medium. Media are typically based on the N6 salts of Chu et al., (1978, Proc. Symp. Plant Tissue Culture, Peking Press, p 43–56) being supplemented with sucrose, vitamins, minerals, amino acids, and in some cases, synthetic hormones. However, callus tissues can also proliferate on media derived from the MS salts of Murashige and Skoog, (1962 Physiol. Plant. 15: 473–497). Cultures are generally maintained in a dark, sterile environment at about 28° C.

The heterologous DNA used for transformation herein may be circular, linear, double-stranded or single-stranded. Generally, said DNA is a recombinant vector plasmid and contains coding regions therein which serve to promote expression of the heterologous gene of interest as well as provide a selectable marker whereby those tissues containing said gene can be identified. Preferably, these recombinant vectors are capable of stable integration into the plant genome where selection of transformed plant lines is made possible by having said selectable marker expression driven either by constitutive, tissue-specific, or inducible promoters included therein. One variable present in a heterologous DNA is the choice of the chimeric gene. Chimeric genes, either in the sense or antisense orientation, are expressed in plant cells under control of a constitutive, tissue-specific, developmental, or inducible promoter and the like. Preference for a particular chimeric gene is at the discretion of the artisan; however, chimeric genes can be, but are not limited to, those from plants, animals, or bacteria and the like and can used to express proteins either not found in a non-transformed cell or found in a transformed cell. Chimeric genes can be also used for, but are not limited to, up-regulation or down-regulation of an endogenous gene of interest. The chimeric gene may be any gene that it is desired to express in plants. Particularly useful genes are those that confer tolerance to herbicides, insects, or viruses, and genes that provide improved nutritional value or processing characteristics of the plant. Examples of suitable agronomically useful genes include the insecticidal gene from Bacillus thuringiensis for conferring insect resistance and the 5′-enolpyruvyl-3′-phosphoshikimate synthase (EPSPS) gene and any variant thereof for conferring tolerance to glyphosate herbicides. As is readily understood by those skilled in the art, any agronomically important gene conferring a desired trait can be used.

Another variable is the choice of a selectable marker. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirable to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli , the chloramphenicol acetyl transferase gene from Tn9 of E. coli , the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria , and the luciferase genes from firefly Photinus pyralis . An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17–19) to identify transformed cells.

Another variable is a promoter regulatory element. In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express heterologous genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402 published Apr. 17, 1997) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters.

Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, stability of the mRNA and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.

Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and may also be used.

Promoter regulatory elements may also be active during a certain stage of the plants' development as well as active in specific plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress. Other desirable transcription and translation elements functional in plants may also be used. Numerous plant-specific gene transfer vectors are known and available to the skilled artisan.

In whisker-mediated transformation, DNA uptake into plant material is facilitated by very small, elongated, needle-like particles comprised of a biologically inert material. When said particles are agitated in the presence of DNA and plant cell lines, one or more of the particles produce small punctures in the regenerable plant cell aggregates thereby allowing said aggregates to uptake the DNA. Cells which have taken up the DNA are considered to be transformed. Some transformed cells stably retain the introduced DNA and express it.

The elongated needle-like particles used in plant cell transformation are termed “whiskers” and are preferably made of a high density material such as silicon carbide or silicon nitride; however, any material having a needle-like structure wherein the size of said structure is smaller than the cell intended to be transformed is within the scope of the invention. More preferably, whiskers are made of silicon carbide and are either Silar SC-9 or Alfa Aesar as described herein.

For transformation, whiskers are typically placed in a small container, such as a conical or microfuge tube and the like, wherein is placed a mixture comprising the DNA construct of interest and embryogenic cotton suspension culture. Thereafter, the container is sealed and agitated. Unlike particles used in biolistic transformation of plant tissue (Sanford et al., 1990 Physiol. Plantarum, 79:206–209; and U.S. Pat. No. 5,100,712), whiskers do not require any special pretreatment with DNA carriers or precipitants prior to use such as CaCl 2 , spermidine, sheared salmon sperm DNA and the like.

Agitation time used in the transformation process can vary and is typically from between about 10 sec to about 160 sec. The amount of whiskers added per transformation can also vary from between about 1 mg to about 4 mg per tube. An inverse relationship is observed between the amount of whiskers added and the agitation time needed to obtain optimal transformation. Therefore, the amount of whiskers added and the agitation time needed to achieve transformation is determinable by one having skill in the art. In addition, the volume of liquid medium added can vary from about 200 μL to about 1000 μL, with about 200 μL being preferred. Moreover, the amount of heterologous DNA added can vary from a preferred amount of about 10 μL to about 100 μL of 1 mg/mL solution. The volume of DNA added is not as critical of factor to the invention as disclosed herein as the final DNA concentration. However, preferred final DNA concentrations are from about 0.03 μg/μL to about 0.14 μg/μL. The scope of the present invention is not intended to be limited to said container size, the amount or concentration of heterologous DNA added, the volume of heterologous DNA added, the amount of the liquid medium added, the amount of suspension culture added or the amount of whiskers added as disclosed herein. The scope of the invention is also not intended to be limited by the instrumentation used to agitate the mixture or whether agitation is accomplished by manual or mechanical means.

Once the plant cell lines have been perforated and the heterologous DNA has entered therein, it is necessary to identify, propagate, and select those cells which not only contain the heterologous DNA of interest but are also capable of regeneration. Said cells and plants regenerated therefrom can be screened for the presence or absence of the heterologous DNA by various standard methods including but not limited to assessment of reporter gene expression. Alternatively, transmission of a selectable marker gene along with or as part of the heterologous DNA allows those cells containing said DNA to be identified by use of a selective agent.

Selection of only those cells containing and expressing the heterologous DNA of interest is a critical step in production of fertile, transgenic plants. Selection conditions must be chosen in such a manner as to allow growth of transformed cells while inhibiting growth of untransformed cells, which initially, are far more abundant. In addition, selection conditions must not be so severe as to cause transformed cells to lose their plant regenerability, future viability or fertility. A skilled artisan can easily determine appropriate conditions for selecting transformed cells expressing a particular selectable marker by performing growth inhibition curves. Growth inhibition curves are generated by plotting cell growth versus selective agent concentration. Typically, selective agent concentrations are set at a concentration whereby almost all non-transformed cells are growth inhibited but yet are not killed. Preferred are selective agent concentrations wherein 90–99% of non-transformed cells are growth inhibited but yet not killed. Most preferred are selective agent concentrations wherein 97–99% of non-transformed cells are growth inhibited but yet not killed.

Transformed cells transferred and exposed to selective agents are generally incubated on solid or liquid medium supportive of growth. The medium preferred for each type of tissue has been well defined in the art. After initial exposure to selective agents, the cells are transferred periodically to fresh medium while maintainina selective agent concentrations. After transformed cell mass has essentially doubled in size, masses showing the most growth and appearing to be healthy are selected and transferred to fresh medium having-selective agent concentrations wherein non-transformed cells will be killed. Repeated selection and transference of growing cells to fresh medium result eventually in a selected group of cells comprised almost exclusively of transformed cells containing the heterologous DNA of interest.

Regeneration, while important to the present invention, may be performed in any conventional manner available to the skilled artisan. If cells have been transformed with selectable marker gene, the selective agent may be incorporated into the regeneration media to further confirm that the regenerated plantlets are transformed. After subsequent weeks of culturing, regenerated plantlet immune to the selective agent can be transferred to soil and grown to maturity.

Cells and plant derived therefrom can be identified as transformants by phenotypic and/or genotypic analysis. For example, if an enzyme or protein is encoded by the heterologous DNA, enzymatic or immunological assays specific for the particular enzyme or protein can be used. Other gene products may be assayed by using suitable bioassays or chemical assays. Other techniques include analyzing the genomic component of the plant using methods as described by Southern ((1975) J. Mol. Biol., 98:503–517), polymerase chain reaction (PCR) and the like.

Plants regenerated from transformed cells are referred to as the RO generation or RO plants. Seed produced by various sexual crosses from plants of this generation are referred to as R1 progeny. R1 seed are then germinated to produce R1 plants. Successful transmission and inheritance of heterologous DNA to R1 plants and beyond should be confirmed using the methods described herein.

Particular embodiments of this invention are further exemplified in the Examples. However, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.