[0001] This application claims the benefit of U.S. Provisional Application No. 60/045,121, filed Apr. 30, 1997 and U.S. application Ser. No. 09/056,418 filed April 7, 1998, both of which are incorporated by reference.
[0002] The invention relates to methods and compositions for the transformation of sorghum, particularly to methods for transformation utilizing Agrobacterium.
[0003] Sorghum is one of the most important cereal crops for subsistence farmers in arid and semi-arid portions of Africa, Asia and the Americas. The crop is essential for human life on marginal lands throughout the poorest regions of the world. However, further development of the crop is needed if food production in these areas is to keep pace with increases in population. In developed countries, sorghum is important as a feed crop and as a crop that can be grown on marginal lands as part of a sustainable agroecosystem.
[0004] Sorghum is typically the cereal grown in areas where the extremes of high temperature and low soil moisture are unsuitable for maize. In 1991, sorghum was ranked fifth in production for all cereals with 58 million metric tons harvested on 45 million hectares of land. See,
[0005] Sorghum is plagued by diseases, especially in higher yielding environments. Many of the diseases are caused by highly variable pathogens. Generally, as yield potential increases, so does the proportion of the crop lost to diseases.
[0006] Until recently, genetic improvement of sorghum for agronomic and quality traits has been carried out by traditional plant breeding methods and improved cultural management practices. Advances in tissue culture and transformation technologies have resulted in the production of transgenic plants of all major cereals, including sorghum. To date, key to this transformation was the development of microprojectile bombardment devices for DNA delivery into cells. Microprojectile bombardment circumvented two major constraints of cereal transformation. These constraints are the lack of an available natural vector such as
[0007] Agrobacterium, a natural plant pathogen, has been widely used for the transformation of dicotyledonous plants. Agrobacterium remains the most widely used vector for transformation of dicot species. Because monocotyledonous plants are rarely natural hosts for Agrobacterium, they have not been expected to be susceptible to gene transfer mediated by the bacterium.
[0008] The advantage of the Agrobacterium-mediated gene transfer is that it offers the potential to regenerate transgenic cells at relatively high frequencies without a significant reduction in plant regeneration rates. Moreover, the process of DNA transfer to the plant genome is defined. That is, the DNA does not normally undergo any major rearrangements, and it integrates into the genome often in single or low copy numbers.
[0009] Agrobacterium-mediated transformation involves incubation of cells or tissues with the bacterium, followed by regeneration of plants from the transformed cells via a callus stage. Inoculation of explants has proven to be the most effective means of creating transgenic plants.
[0010] Early work with Agrobacterium indicated that the bacterium could transfer T-DNA to monocotyledonous hosts. However, clear evidence of T-DNA integration existed only for asparagus, and even in that case, no transformed plants were produced. Because of the recalcitrant nature of monocots to Agrobacterium infection, other methods, such as particle bombardment, were developed for the transformation of monocots. More recently, the transformation of maize and rice using Agrobacterium has been reported. See, Ishida et al. (1996)
[0011] While reports indicate that some genotypes of maize and rice can be transformed with Agrobacterium, there is no report of Agrobacterium-mediated transformation of sorghum. While transgenic sorghum plants have been reported following microprojectile bombardment, transgenic plants were obtained only at very low frequencies. Further, inherent characteristics of the sorghum cells make them somewhat unresponsive for transient expression. Casas et al. (1993)
[0012] Accordingly, there is needed an efficient method for the transformation of sorghum wherein stable transformation of large inserts can be obtained. That is, there is needed a method for the transformation of sorghum utilizing Agrobacterium.
[0013] The present invention is drawn to methods and compositions for the efficient transformation of sorghum. The method involves the use of bacteria belonging to the genus, Agrobacterium, particularly those comprising a super-binary vector. In this manner, any gene of interest can be introduced into the sorghum plant. The transferred gene will be flanked by at least one T-DNA border and present in the transformed sorghum in low copy number.
[0014] Transformed sorghum cells, tissues, plants, and seed are also provided. Such transformed compositions are characterized by the presence of T-DNA borders and a low copy number of the transferred gene. The invention encompasses regenerated, fertile transgenic sorghum plants, transgenic seeds produced therefrom, T1 and subsequent generations.
[0015] Compositions and methods for the efficient transformation of sorghum are provided. The transformed sorghum plants are characterized by containing transferred nucleic acid such as a transferred gene or genes of interest flanked by at least one T-DNA border inserted within the genome of the sorghum plants. The plants are normal in morphology and fertile. Generally, the transformed plants contain a single copy of the transferred nucleic acid with no notable rearrangements. Alternatively, the transferred nucleic acid of interest is present in the transformed sorghum in low copy numbers. By low copy number is intended that transformants comprise no more than five (5) copies of the transferred nucleic acid, preferably, no more than three (3) copies of the transferred nucleic acid, more preferably, fewer than three (3) copies of the transferred nucleic acid. The transferred nucleic acid will comprise at least one T-DNA border sequence.
[0016] The methods of the invention rely upon the use of Agrobacterium-mediated gene transfer. Agrobacterium-mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes. Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti plasmid into plant cells at wound sites. The typical result of gene transfer is a tumorous growth called a crown gall in which the T-DNA is stably integrated into a host chromosome. The ability to cause crown gall disease can be removed by deletion of the genes in the T-DNA without loss of DNA transfer and integration. The DNA to be transferred is attached to border sequences that define the end points of an integrated T-DNA.
[0017] Gene transfer by means of engineered Agrobacterium strains has become routine for most dicotyledonous plants and for some monocotyledonous plants. However, there are no reports to date of producing transformed sorghum by means of Agrobacterium-mediated transformation. See, for example, Fraley et al. (1983)
[0018] The Agrobacterium strain utilized in the methods of the invention is modified to contain a gene or genes of interest, or a nucleic acid to be expressed in the transformed cells. The nucleic acid to be transferred is incorporated into the T-region and is flanked by at least one T-DNA border sequence. A variety of Agrobacterium species are known in the art particularly for dicotyledon transformation. Such Agrobacterium can be used in the methods of the invention. See, for example, Hooykaas, P. J. (1989)
[0019] In the Ti plasmid, the T-region is distinct from the vir region whose functions are responsible for transfer and integration. Binary vector systems have been developed where the manipulated disarmed T-DNA carrying foreign DNA and the vir functions are present on separate plasmids. In this manner, a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid which replicates in
[0020] Preferred vectors of the invention are super-binary vectors. See, for example, U.S. Pat. No. 5,591,616 and EPA 0604662A1, herein incorporated by reference. Such a super-binary vector has been constructed containing a DNA region originating from the virulence region of Ti plasmid pTiBo542 (Jin et al. (1987)
[0021] Super-binary vectors are known in the art and include pTOK162 (Japanese Patent Appl. (Kokai) No. 4-222527, EP-A-504,869, EP-A-604,662, and U.S. Pat. No. 5,591,616 herein incorporated by reference) and pTOK233 (Komari, T. (1990)
[0022] The T-region of the super-binary vectors and other vectors for use in the invention are constructed to have restriction sites for the insertion of the genes to be delivered. Alternatively, the DNA to be transformed can be inserted in the T-DNA region of the vector by utilizing in vivo homologous recombination. See, Herrera-Esterella et al. (1983)
[0023] As will be evident to one of skill in the art, now that a method has been provided for stable transformation of sorghum, any nucleic acid of interest can be used in the methods of the invention. For example, a sorghum plant can be engineered to express disease and insect resistance genes, genes conferring nutritional value, genes to confer male and/or female sterility, antifungal, antibacterial or antiviral genes, and the like. Likewise, the method can be used to transfer any nucleic acid to control gene expression. For example, the nucleic acid to be transferred could encode an antisense oligonucleotide.
[0024] Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. Crops of special interest include corn, soybeans, canola, sunflower, rapeseed, rice, tobacco, wheat, sorghum, and alfalfa. General categories of genes of interest include, for example, those genes involved in information, such as Zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics and commercial products.
[0025] Agronomically important traits such as oil, starch and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur and providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in WO96/38563, WO94/16078 and WO96/38562 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997, the disclosures of which are incorporated herein in their entirety by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016 and the chymotrypsin inhibitor from barley (Williamson et al.
[0026] Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm or that European Corn Borer. For example, genes from the microorganism
[0027] Genes encoding disease resistance traits may include detoxification genes, such as against fumonosin or other toxins. Fumonisin-resistance can be used to transform plant cells normally susceptible to Fusarium or other toxin-producing fungus as described in U.S. Pat. No. 5,792,931. Other examples are genes conferring viral resistance and antimicrobial peptides.
[0028] Herbicide resistance traits may include genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular, the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.
[0029] Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
[0030] The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins, described in WO96/38563, WO94/16078 and WO96/38562 which provide descriptions of modifications of proteins for desired purposes.
[0031] Commercial traits can also be encoded on a gene or genes which could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics, such as described in U.S. Pat. No. 5,602,321, issued Feb. 11, 1997. Genes such as, B-ketothiolase, PHBase (polyhydroxyburyrate synthase)and acetoacetyl-CoA reductase (see Schubert et al. (1988)
[0032] For convenience, the nucleic acid to be transferred can be contained within expression cassettes. The expression cassette will comprise a transcriptional initiation region linked to the nucleic acid or gene of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene or genes of interest to be under the transcriptional regulation of the regulatory regions.
[0033] The transcriptional initiation region, the promoter, may be native or homologous or foreign or heterologous to the host, or could be the natural sequence or a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence.
[0034] The transcriptional cassette will include the in 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of
[0035] Alternatively, the gene(s) of interest can be provided on another expression cassette. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. Where mammalian, yeast, or bacterial or dicot genes are used in the invention, they can be synthesized using monocot or sorghum preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436, 391, and Murray et al. (1989)
[0036] The expression cassettes may additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989)
[0037] The expression cassettes may contain one or more than one gene or nucleic acid sequence to be transferred and expressed in the transformed plant. Thus, each nucleic acid sequence will be operably linked to 5′ and 3′ regulatory sequences. Alternatively, multiple expression cassettes may be provided.
[0038] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) as well as genes conferring resist insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. (See DeBlock et al. (1987)
[0039] For purposes of the present invention, selectable marker genes include, but are not limited to genes encoding: neomycin phosphotransferase II (Fraley et al. (1986)
[0040] Also included are genes encoding resistance to: chloramphenicol (Herrera-Estrella et al. (1983)
[0041] The bar gene confers herbicide resistance to glufosinate-type herbicides, such as phosphinothricin (PPT) or bialaphos, and the like. As noted above, other selectable markers that could be used in the vector constructs include, but are not limited to, the pat gene, also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, the HPH or HYG gene for hygromycin resistance, the EPSP synthase gene for glyphosate resistance, the Hm1 gene for resistance to the Hc-toxin, and other selective agents used routinely and known to one of ordinary skill in the art.
[0042] See generally, G. T. Yarranton (1992)
[0043] The above list of selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the present invention.
[0044] Where appropriate, the selectable marker genes and other gene(s) and nucleic acid of interest to be transferred can be synthesized for optimal expression in sorghum. That is, the coding sequence of the genes can be modified to enhance expression in sorghum. The synthetic nucleic acid is designed to be expressed in the transformed tissues and plants at a higher level. The use of optimized selectable marker genes may result in higher transformation efficiency.
[0045] Methods for synthetic optimization of genes are available in the art. The nucleotide sequence can be optimized for expression in sorghum or alternatively can be modified for optimal expression in monocots. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in sorghum. It is recognized that genes which have been optimized for expression in maize and other monocots can be used in the methods of the invention. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991)
[0046] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
[0047] The methods of the invention are useful for transforming sorghum plant cells. Such cells include callus which can be originated from any tissues of sorghum plants. Preferably, the tissue utilized in initiating callus is immature tissue such as immature embryos, immature inflorescences, and the basal portion of young leaves. Alternatively, the callus can be originated from anthers, microspores, mature embryos, and in principal from any other tissue of sorghum capable of forming callus and/or secondary embryos. A useful tissue for producing regenerable callus is the scutellum of immature sorghum embryos. Of particular interest, are the use of immature embryos. Such embryos can be isolated from immature kernels and treated for transformation. Alternatively, the embryos can be isolated and cultured for several days, generally about 3 to about 10 days, preferably about 5 to about 8 days, prior to inoculation with Agrobacterium.
[0048] The method can also be used to transform cell suspensions. Such cell suspensions can be formed from any sorghum tissue.
[0049] Immature embryos are an intact tissue that is capable of cell division to give rise to callus cells that can then differentiate to produce tissues and organs of the whole plant. Immature embryos can be obtained from the fertilized reproductive organs of a mature sorghum plant. Exemplary methods for isolating immature embryos are described by Green and Phillips (1976)
[0050] The Agrobacterium-mediated transformation process of the invention can be broken into several steps. The basic steps include an infection step (step 1); a co- cultivation step (step 2); an optional resting step (step 3); a selection step (step 4); and a regeneration step (step 5).
[0051] An optional preculture step may be added prior to the infection step. The preculture step involves culturing the immature embryos or other target tissue prior to the infection step on a suitable medium such as N6, LSD1.5, or PHI-J medium. (See Example 2). The preculture period may vary from about 1 to about 10 days, preferably about 3 to about 7 days, more preferably about 5 to about 6 days. Such a preculture step was found to prevent transformation of maize cultures. See EP0672752A1.
[0052] In the infection step, the cells to be transformed are isolated and exposed to Agrobacterium. If the target cells are immature embryos, the embryos are isolated and the cells contacted with a suspension of Agrobacterium. As noted above, the Agrobacterium has been modified to contain a gene or nucleic acid of interest. The nucleic acid is inserted into the T-DNA region of the vector. General molecular techniques used in the invention are provided, for example, by Sambrook et al. (eds.)
[0053] Agrobacterium containing the plasmid of interest are preferably maintained on Agrobacterium master plates with stock frozen at about −80° C. As used herein, the term “Agrobacterium capable of transferring at least one gene” refers to Agrobacterium containing the gene or nucleic acid of interest, generally in a plasmid that is suitable for mediating the events required to transfer the gene to the cells to be infected. Master plates can be used to inoculate agar plates to obtain Agrobacterium which is then resuspended in media for use in the infection process. Alternatively, bacteria from the master plate can be used to inoculate broth cultures that are grown to logarithmic phase prior to transformation.
[0054] The concentration of Agrobacterium used in the infection step and co-cultivation step can affect the transformation frequency. Likewise, very high concentrations of Agrobacterium may damage the tissue to be transformed, such as the immature embryos, and result in a reduced callus response. Thus, the concentration of Agrobacterium useful in the methods of the invention may vary depending on the Agrobacterium strain utilized, the tissue being transformed, the sorghum genotype being transformed, and the like. To optimize the transformation protocol for a particular sorghum line or tissue, the tissue to be transformed, (immature embryos, for example), can be incubated with various concentrations of Agrobacterium. Likewise, the level of marker gene expression and the transformation efficiency can be assessed for various Agrobacterium concentrations. While the concentration of Agrobacterium may vary, generally a concentration range of about 1 ×10
[0055] The tissue to be transformed is generally added to the Agrobacterium suspension in a liquid contact phase containing a concentration of Agrobacterium to optimize transformation efficiencies. The contact phase facilitates maximum contact of the cells/tissue to be transformed with the suspension of Agrobacterium. The cells are contacted with the suspension of Agrobacterium for a period of at least about three (3) minutes to about 15 minutes, preferably about 4 minutes to about 10 minutes, more preferably about 5 minutes to about 8 minutes.
[0056] The liquid contact phase of the infection step takes place in a liquid solution that includes the major inorganic salts and vitamins of N6 medium referred to herein as “N6 salts” (Chu C. C. Proc. Symp. Plant Tissue Culture, Science Press Peking. pp. 43-50,1987). As used herein, medium containing “N6 salts” includes medium containing about 400-500 mg/l ammonium sulfate and preferably about 463.0 mg/l ammonium sulfate; about 1.0-2.0 mg/l boric acid and preferably about 1.6 mg/l boric acid; about 100-140 mg/l calcium chloride anhydrous and preferably about 125 mg/l calcium chloride anhydrous; about 20-50 mg/l Na
[0057] Other equivalent liquid suspensions are known in the art and can be used. See, for example, Ishida et al. (1996)
[0058] In addition, the media in the infection step generally excludes AgNO
[0059] In the co-cultivation step, the cells to be transferred are co-cultivated with Agrobacterium. For immature embryos, the co-cultivation with the Agrobacterium takes place on a solid medium. The embryos are positioned axis down on the solid medium and the medium can include AgNO
[0060] Following the co-cultivation step, the transformed cells may be subjected to a resting step. As noted above, the resting step is optional. Where no resting step is used, an extended co-cultivation step may utilized to provide a period of culture time prior to the addition of a selective agent.
[0061] For the resting step, the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase is performed in the absence of any selective pressures to permit preferential initiation and growth of callus from the transformed cells containing the heterologous nucleic acid. An antibiotic is added to inhibit Agrobacterium growth. Such antibiotics are known in the art which inhibit Agrobacterium and include Cefotaxime, timetin, vancomycin, carbenicillin, and the like. Concentrations of the antibiotic will vary according to what is standard for each antibiotic. For example, concentrations of carbenicillin will range from about 50 mg/l to about 250 mg/l carbenicillin in solid media, preferably about 75 mg/l to about 200 mg/l, more preferably about 100-125 mg/l. Those of ordinary skill in the art of monocot sulfate.7H
[0062] The resting phase cultures are preferably allowed to rest in the dark at 28° C. for about 1 to about 15 days, preferably for about 3 to about 10 days, more preferably for about 5 to about 8 days. Any of the media known in the art can be utilized for the resting step.
[0063] Following the co-cultivation step, or following the resting step, where it is used, the transformed cells are exposed to selective pressure to select for those cells that have received and are expressing polypeptide from the heterologous nucleic acid introduced by Agrobacterium. Where the cells are embryos, the embryos are transferred to plates with solid medium that includes both an antibiotic to inhibit growth of the Agrobacterium and a selection agent. The agent used to select for transformants will select for preferential growth of explants containing at least one selectable marker insert positioned within the superbinary vector and delivered by the Agrobacterium.
[0064] Generally, any of the media known in the art suitable for the culture of sorghum can be used in the selection step, such as media containing N6 salts or MS salts. During selection, the embryos are cultured until callus formation is observed. Typically, calli grown on selection medium are allowed to grow to a size of about 1.5 to about 2 cm. diameter.
[0065] After the calli have reached the appropriate size, the calli are cultured on regeneration medium in the dark for several weeks, generally about 1 to 3 weeks to allow the somatic embryos to mature. Preferred regeneration media include media containing MS salts, such as PHI-E and PHI-F media as provided in the Examples. The calli are then cultured on rooting medium in a light/dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art and preferred methods are provided by Kamo et al. (
[0066] Small plantlets are then transferred to tubes containing rooting medium and allowed to grow and develop more roots for approximately another week. The plants are then transplanted to soil mixture in pots in the greenhouse.
[0067] Now that it has been demonstrated that sorghum can be transformed utilizing Agrobacterium, alterations to the general method described herein can be used to increase efficiency or to transform elite inbred lines which may exhibit some recalcitrance to transformation. Factors that affect the efficiency of transformation include the types and stages of tissues infected, the concentration of
[0068] While any sorghum line or variety can be used in the transformation methods of the invention, examples of sorghum lines include but are not limited to public lines such as CS3541, M91051, SRN39, Shanqui red, IS8260, IS4225, Tx430, P898012, P954035, PP290 (Casas et al. supra) and commercially important Pioneer proprietary inbred lines such as PH860, PH987, PHB180, PHB123, and PHB82.
[0069] Further modifications may be utilized including providing a second infection step to increase infection by the Agrobacterium. Also, the vectors and methods of the invention can be used in combination with particle bombardment to produce transformed sorghum plants. Particle bombardment can be used to increase wounding in the tissues to be transformed by Agrobacterium. (Bidney et al. (1990)
[0070] After wounding of the cells by microprojectile bombardment, the cells are inoculated with Agrobacterium solution. The additional infection step and particle bombardment may be useful in transforming those genotypes of sorghum which are particularly recalcitrant to infection by Agrobacterium.
[0071] The following examples are offered by way of illustration and not by way of limitation.
[0072] PHP10525:
[0073] All vectors were constructed using standard molecular biology techniques (Sambrook et al. (eds.), supra). A reporter gene and a selectable marker gene for gene expression and selection was inserted between the T-DNA borders of a superbinary vector. The reporter gene included the β-glucuronidase (GUS) gene (Jefferson, R. A. et al. (1986)
[0074] For the selectable marker, a Cauliflower Mosaic Virus 35S promoter with a duplicated enhancer region (2X35S; bases −421 to −90 and −421 to +2 from Gardner et al. (1981)
[0075] The plasmid, pPHP8904 was constructed by inserting the GUS expression cassette as a HindIII/NotI fragment and the BAR expression cassette as a NotI/SacI fragment between the right and left T-DNA borders in pSB11 and HindIII and SacI sites. The GUS cassette was inserted proximal to the right T-DNA border. The plasmid pSB11 was obtained from Japan Tobacco Inc. (Tokyo, Japan). The construction of pSB11 from pSB21 and the construction of pSB21 from starting vectors is described by Komari et al. (1996)
[0076] PHP11264:
[0077] PHP11264 was basically constructed as described above. One difference noted is the selectable marker gene. For the selectable marker, a maize ubiquitin (UBI) promoter (Christensen et al. (1992)
[0078] Preparation of Agrobacterium Suspension
[0079] Single colonies were selected from matings and streaked out sequentially at least three times to ensure purity of the Agrobacterium strain. From the final streaked plates, a single colony was selected and used to initiate a liquid culture. Following growth to stationary phase, the liquid culture was used to make glycerol stocks and miniprep plasmid DNA. Sambrook et al., supra. The resulting plasmid DNA was digested with SalI to verify the co-integrate LBA4404(pPHP10525). The glycerol stocks were stored at −80° C. and were used as the source for master plates.
[0080] Agrobacterium was streaked out from a −80° frozen aliquot onto a plate containing PHI-L medium and cultured at 28° C. in the dark for 3 days. PHI-L media comprised 25 ml/l Stock Solution A, 25 ml/l Stock Solution B, 450.0 ml/l Stock Solution C and spectinomycin (Sigma Chemicals) added to a concentration of 50 mg/l in sterile ddH
[0081] The plate can be stored at 4° C. and used usually for about 1 month. Several colonies were picked from the master plate and streaked onto plate containing PHI-M medium [yeast extract (Difco) 5.0 g/l/ peptone (Difco) 10.0 g/l; Nacl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8, containing 50 mg/L spectinomycin] and incubated at 28° C. in the dark for 2 days.
[0082] Five ml of either PHI-G [100 ml/l of a 10X solution of N6 macronutrients (463.0 mg/l (NH
[0083] Embryo Isolation, Infection and Co-cultivation
[0084] Sorghum immature embryos of about 0.8 to about 1.5 mm were isolated from sterilized immature kernels of sorghum line P898012, a drought-resistant sorghum cultivar obtained from Purdue University. The immature kernels were removed from a sorghum head and placed in an autoclaved glass jar. These immature kernels were immersed in 50% bleach and 0.1% Tween 20 in this jar and the jar was shaken well to allow the solution to cover all kernels and to reach everywhere inside the jar. Apply vacuum for 10 minutes and rinse with autoclaved deionized distilled water, twice in a hood and these kernels were kept in sterile water until use. The immature embryos were isolated from sterilized kernels using a sterile spatula (Baxter Scientific Products S1565). The isolated embryos were cultured on PHI-J medium without acetosyringone in the dark at about 25° C. for 5 days and these precultured embryos were inoculated with 10
[0085] Any embryos left in the tube were transferred to the plate using a sterile spatula. The Agrobacterium suspension was drawn off and the embryos placed axis side down on the media. The plate was sealed with Parafilm tape or Pylon Vegetative Combine Tape Product named “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd., Japan), and incubated in the dark at about 25° C. for an additional 5 days of co-cultivation.
[0086] Resting Step
[0087] No resting step was utilized.
[0088] Selection and Regeneration Steps
[0089] For selection, all of the embryos were then transferred to new plates containing PHI-J medium without glucose and acetosyringone, but adding 100 mg/l carbenicillin and 5 mg/l phosphinothricin (PPT), as a selection medium, putting about 20 embryos onto each plate. The plates were sealed as described above and incubated in the dark at 25° C. for the first two weeks of selection. The embryos were then transferred to fresh selection medium at the end of two weeks. After a period of another 2 week culture, these embryos were sub-cultured onto the same PHI-J medium except PPT was increased from 5mg/l to 10 mg/l. The embryos were then transferred to fresh selection medium containing 10 mg/l PPT at 3 week intervals continuing at 25° C. in the dark. The tissue was subcultured by transferring to fresh selection medium for a total of about 3.5 months to obtain herbicide-resistant calli.
[0090] For regeneration, the callus was then cultured on PHI-E medium [MS salts 4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l; thiamine.HCl 0.1 mg/l; Pyridoxine.HCl 0.5 mg/l; Glycine 2.0 mg/l; Zeatin 0.5 mg/l; sucrose 60.0 g/l; Agar (Sigma, A-7049) 8.0 g/l; Indoleacetic acid (IAA, Sigma) 1.0 mg/l; Abscisic acid (ABA, Sigma) 0.1 μM; PPT 10 mg/l; carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3 weeks to allow somatic embryos to mature. The callus was then cultured on PHI-F medium [MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl 01. mg/l; Pyridoxine.HCl 0.5 mg/l; Glycine 2.0 mg/l; nicotinic acid 0.5 mg/l; sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at 25° C. under a daylight schedule of 16 hrs. light (270 uE m
[0091] Confirmation of Stable Transformation
[0092] 25 T0 plants have been regenerated from the callus and grown in the greenhouse. Five plants were chosen at random for PCR assay for the presence of BAR and Gus genes. The PCR result confirmed that all of the 5 plants were stably transformed with Agrobacterium.
[0093] The 25 plants, as well as non-transformed control plants, were painted with a 1% solution of the herbicide Liberty on their leaves to verify herbicide resistance due to the expression of the BAR gene. All of the 25 regenerated plants were Liberty resistant while the control plants exhibited sensitivity to Liberty.
[0094] Using the methods detailed above in Examples 1 and 2, transformation protocols can be optimized for any sorghum genotype. To demonstrate, the following protocol sets forth particular parameters that can be tested for an effect on transformation efficiency. It is recognized that other protocols can be formulated and other sorghum lines tested. The following is an illustration of steps to optimize transformation.
[0095] Materials
[0096] Agrobacterium stain: LBA4404 (PHP11264) (UBI-UBI intron-Bar-PinII).
[0097] Sorghum lines: P898012 and PH391 (a Pioneer elite inbred).
[0098] Treatments
[0099] The following table illustrates a number of treatments that will indicate which factors are important for efficient transformation of the two exemplary sorghum lines.
TABLE 1 Treatment Conditions P898012 PH391 Treat- Em- Em- ment Conditions bryos Conditions bryos 1 Fresh embryo with 50-100 Fresh embryo with 50-100 Agroinfection, co- Agroinfection, co- cultivation for 3 cultivation for 3 days & resting for 4 days and resting for days 4 days 2 Fresh embryo with 50-100 Fresh embryo with 50-100 Agroinfection, co- Agroinfection, co- cultivation for 7 cultivation for 7 days and no resting days and no resting 3 Fresh embryo 1 50-100 Fresh embryo 1 50-100 infection and 2 infection and 2 infection at the 3 infection at the 3 day of co-cultiva- day of co-cultiva- tion, additional 4 tion, additional 4 days for 2 days for 2 tivation and no tivation and no resting resting 4 Pre-culture 7 days 50-100 Pre-culture 7 days 50-100 and then and Agroinfection, Agroinfection, co- co-cultivation for 7 cultivation for 7 days and no resting days and no resting 5 Pre-culture 7 days, 50-100 Pre-culture 7 days, 50-100 bombarding and bombarding and Agroinfection, co- Agroinfection, co- cultivation for 7 cultivation for 7 days and no resting days and no resting 6 no Agroinfection 20 no Agroinfection 20 control control
[0100] Detailed Procedure of These Experiments
[0101] A brief description of the treatments set forth in Table 1 is provided below. Generally, the treatment protocols will be performed for each of the sorghum lines and will follow the details given in Examples 1 and 2 with the following differences.
[0102] Treatment −1: Fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. The isolated embryos are inoculated with 10
[0103] Treatment −2: Fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. The isolated embryos are inoculated with 10
[0104] Treatment −3: Fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. The isolated embryos are inoculated with 10
[0105] Treatment −4: Fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. The isolated embryos are cultured on PHI-J medium plus 10 mg/l ascorbic acid at 25° C. for about 7 days. The precultured embryos are inoculated with 10
[0106] Treatment −5: Fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. The isolated embryos are cultured on PHI-J medium plus 10 mg/l ascorbic acid at 25° C. for about 7 days. The precultured embryos are bombarded with gold or tungsten particles at 200 mm Hg to increase wounding. The bombarded embryos are inoculated with 10
[0107] Treatment −6: To provide a negative control, fresh sorghum immature embryos of 0.8 to 1.5 mm are isolated from sterilized immature kernels. No infection or co-cultivation with Agrobacterium is used. The embryos are cultured on PHI-J plus 10 mg/l ascorbic acid without glucose and acetosyringone at 25° C. in the dark, but adding 100 mg/l carbenicillin and 5 mg/l PPT or 1.5 mg/l Bialaphos. No callus growth is expected.
[0108] Analysis of Results:
[0109] The regenerated T0 plants are assayed by Southern or PCR methods to confirm stable transformation. Additionally, the plants are painted with the herbicide Liberty to verify BAR gene expression. The T1 generation is analyzed to confirm transmission of the transgene. The following comparison will determine the specificity of this technology.
[0110] The results of the treatments will provide guidance on the factors which are important for transformation of the sorghum lines being tested. Similar methods can be used to efficiently transform any sorghum line. A comparison of the data from: Treatments 1 and 2 will indicate the importance of the resting step on transformation; Treatments 2 and 3 indicate whether a second infection increases transformation; Treatments 2 and 4 indicate the importance of the pre-culture of the isolated embryos; and Treatments 4 and 5 indicate whether bombardment can improve transformation efficiency with Agrobacterium.
[0111] Using the methods of the invention any sorghum line can be stably transformed. The transformed plants of the invention are an improvement over the transformed sorghum plants produced by bombardment. In contrast to the plants produced by bombardment, the plants of the invention contain transferred nucleic acid which has not undergone any major rearrangements. Furthermore, the transferred nucleic acid integrates into the genome in low copy numbers, usually as a single copy.
[0112] P898012 was used as a model line to test the treatment conditions set forth in Example 3. Embryos were harvested, cultured and transformed as described in Treatments 1-6, respectively. The transformation frequency for each treatment is set forth in Table 2.
TABLE 2 Transformation Results Treat- Expt. Embryo # ment Condition # # Event Frequency 1 Fresh embryo with G-1 155 2 1.3% Agroinfection (1 × 10 G-2 128 1 0.8% cfu/ml),co- G-3 123 1 0.8% cultivation for 3 days G-4 123 2 1.6% & resting for 4 days. G-5 162 1 0.6% G-6 148 1 0.7% G-7 122 2 1.6% G-8 130 1 0.8% G-9 76 5 6.6% G-10 51 2 3.9% G-11 144 1 0.7% G-12 30 2 6.7% Total 1392 21 1.5% (0.6-6.6%) 2 Fresh embryo with G-1 88 1 1.1% Agroinvection G-2 104 1 1.0% (1 × 10 G-3 90 2 2.2% co-cultivation for 7 G-4 95 1 1.1% days & no resting G-5 133 1 0.8% G-6 47 2 4.3% G-7 53 1 1.9% G-8 117 1 0.9% G-9 108 2 1.9% G-10 118 2 1.7% G-11 229 2 0.9% G-12 121 2 1.7% G-13 96 1 1.0% Total 1,399 19 1.4% (0.8-4.3%) 3 Fresh embryo 1st G-1 76 0 0% infection (0.001 × 10 G-2 84 0 0% 0.1 × 10 G-3 100 0 0% and 2nd infection G-4 84 1 1.2% (0.5 × 10 G-5 85 0 0% cfu/ml) at the 3rd G-6 93 0 0% day of co-cultivation, G-7 100 0 0% additional 4 days for Total 622 1 0.2% 2nd co-cultivation (0-1.2%) and no resting 4 Pre-culture 3-7 days G-1 48 1 2.1% and then G-2 70 1 1.4% Agroinfection (1 × 10 G-3 92 1 1.1% cfu/ml), co- G-4 110 3 2.7% cultivation for 7 days G-5 227 1 0.4% and no resting G-6 94 1 1.1% Total 641 8 1.2% (0.4-2.7%) 5 Pre-culture 5-7 G-1 119 0 0% days, bombarding & G-2 120 0 0% Agroinfection (1 × 10 G-3 103 1 1.0% cfu/ml), co- G-4 100 0 0% cultivation for 7 days Total 442 1 0.2% and no resting (0-1.0%) 6 no Agroinfection G-1 21 0 0% control G-2 10 0 0% G-3 20 0 0% G-4 53 0 0% G-5 17 0 0% G-6 35 0 0% G-7 46 0 0% G-8 36 0 0% G-9 30 0 0% G-10 28 0 0% Total 296 0 0%
[0113] The data showed that for P89012, stable transgenic events were readily produced with treatment-1, treatment-2 and treatment -4. In treatment-1, 21 stable events were generated from 1,392 embryos in 12 experiments and the average transformation frequency was 1.5%, ranging from 0.6- 6.6%. In treatment-2, 19 events were generated from 1,399 embryos in 13 experiments and the average frequency was 1.4%, ranging from 0.8- 4.3%. In treatment-4, 8 events were generated from 641 embryos in 6 experiments and the average frequency was 1.2%, ranging from 0.4- 2.7%.
[0114] However, in experimental practice, treatment-2 is preferred because it is a simplified procedure. One does not have to move the embryos in different media.
[0115] In treatment-4, a pre-culture period of 3 days, 4 days, 5 days and 7 days was tested. The transformation frequencies were in the same range in these different days pre-culture treatment. The data from these different days pre-culture were pooled together to represent the results of treatment-4.
[0116] One event was produced from 442 embryos in treatment-5. Bombardment (treatment-5) actually reduced the transformation frequency from 1.2% (treatment-4) to 0.2% (treatment-5). Pre-culture for 5 days and 7 days were tested in this treatment and the data were pooled together because no obvious difference could be observed in these two pre-culture conditions.
[0117] No event was produced in the non-Agrbacterium infection control (treatment-6).
[0118] Using P898012, further optimization of the transformation conditions was tested. Some conditions such as the use of an antioxidant and the source of sorghum embryos were tested. The results indicate that an antioxidant and source of embryos (sorghum plant growth conditions) played a role in sorghum transformation efficiency.
[0119] Antioxidant:
[0120] Antioxidants have been used in Agrobacterium-mediated transformations in at least one plant in the art. Those results indicated that antioxidants were very critical for Agrobacterium-mediated transformation of grape (Vitis vinifera L.) (Avihai Peri et al. (1996)
[0121] To test the role of antioxidants in sorghum, the protocol of treatment 2 was followed with the addition of 1% PVPP (polyvinylpolypyrrolidone) in the co-cultivation medium (710P).
TABLE 3 Treatment PVPP Total Embryos Stable Events Frequency 2 NO 1,399 19 1.4% 2 1% in PHI-T 501 12 2.4%
[0122] The preferred conditions of Example 4 were repeated using field grown sorghum PH391. PH391 is an elite line in the sorghum breeding program. The transformation methodologies developed using model line P898012 were also tested in PH391 to verify the common application of these methologies in general sorghum transformation. The results are given in Table 4.
TABLE 4 Treatment Condition Plant # Embryo # Stable Event # Frequency (%) 1 Fresh embryo with T-1 115 9 7.8% Agroinfection (0.5 × 10 T-2 90 2 2.2% cfu/ml), co-cultivation Total (2) 205 11 5.4% for 3 days and resting for 4 days, 2 Fresh embryo with T-1 20 4 20.0% Agroinfection (0.5 × 10 T-2 60 1 1.7% cfu/ml), co-cultivation Total (2) 85 5 6.3% for 7 days (1% PVPP) & no resting,
[0123] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0124] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.