WO2000006757A1

WO2000006757A1 - Improved plant transformation process by scaffold attachment regions (sar)

Info

Publication number: WO2000006757A1
Application number: PCT/US1999/007598
Authority: WO
Inventors: Michael E. Horn; Gerald E. Hall, Jr.
Original assignee: Mycogen Plant Science, Inc.
Priority date: 1998-07-31
Filing date: 1999-04-06
Publication date: 2000-02-10
Also published as: AR019267A1; AU3476399A

Abstract

The subject invention pertains to the use of scaffold attachment regions (SARs), also known as matrix attachment regions (MARs), in expression cassettes to improve the transformation efficiency of such expression cassettes in a transformation process.

Description

DESCRIPTION

IMPROVED PLANT TRANSFORMATION PROCESS BY SCAFFOLD ATTACHMENT REGIONS ( SAR )

Cross-Reference to a Related Application

This application is related to and claims the benefit of U. S. Serial No. 09/127,080 filed July 31, 1998.

Field of the Invention The present invention relates to the use of scaffold attachment regions (SARs), also known as matrix attachment regions (MARs), in transformation of cells and tissues.

Background of the Invention SARs/MARs (hereinafter collectively referred to as "SARs") are AT-rich genomic DNA sequences that occur in eukaryotic genomes (see Boulikas [1993] J. Cell Biochem. 52:14). SARs are non-coding DNA sequences that flank structural genes and stabilize the transcription process. It is also known that SARs bind to certain components of the proteinaceous scaffold material that encompasses nuclear DNA. SARs have been found to improve the expression of heterologous genes in transformed plants (Allen et al. [1993] Plant Cell 5:603).

Brief Summary of the Invention

It has been unexpectedly discovered that the use of SARs in plant expression cassettes increases the frequency of recovery of stable transformation events of such expression cassettes in a plant transformation process. In particular, stable transformation events have been observed to increase up to about 7-fold or more while, at the same time, in the case of GUS expression, the transient transformation events have been observed to decrease. This startling observation makes the increase in stable transformation events all the more surprising. This invention is particularly useful in improved electroporation transformation techniques.

It has also been unexpectedly discovered that the use of SARs in plant expression cassettes advantageously decreases the time required to recover a stably transformed plant. The SAR-positive clones invariably appear in culture faster as compared to SAR-negative clones which, in turn, allows for quicker regeneration. Shorter times in culture leads to easier regeneration, less abnormalities in the plants, higher fertility, better seed set, etc. Quicker transformation regeneration processes are highly desirable from a commercial standpoint due to the fact that shorter time periods allow for dramatically increased throughput of events which result in quicker identification of commercial candidates.

Additionally, the use of SARs in a plant expression cassette employed in electroporation transformation processes results in a high proportion of low copy number transformants which is very desirable from a commercial standpoint. High copy number transformants are associated with high frequencies of gene silencing. Complex Southern blot patterns that are indicative of high copy number insertions have been observed rarely (approximately 10%) in SAR-positive maize transformants from an electroporation process of whole intact maize cells.

Brief Description of the Drawings

Figure 1 shows Southern blots of maize callus samples transformed with SAR-positive plant expression cassettes compared with controls. Treated callus was plated on bialaphos selection and bialaphos-resistant colonies appeared after 7 to 12 weeks. Callus was extracted for DNA and separated on an agarose gel as per Materials and Methods. Lane 1 : DNA sizing standards; Lanes 2-12: DNA from selected colonies arising on bialaphos selection; Lanes 13: DNA from PAT+ control callus known to contain the PAT gene; Lane 14: DNA from PAT- control callus known not to contain the PAT gene; Lane 15: piasmid DNA corresponding to the equivalent of 1 copy; Lane 16: piasmid DNA corresponding to the equivalent of 3 copies.

Figure 2 shows Southern analysis of DNA from regenerated T₀ plants from callus (Fig. 1) derived from transfection with pAGM 607 piasmid DNA. Lane 1 : DNA sizing standards; Lane 2: piasmid DNA correspondingto the equivalent of 1 copy; Lanes 3-1 1 : not relevant; Lane 12: DNA from PAT+ control leaf tissue known to contain the PAT gene; Lane 13: DNA from PAT- control leaf tissue known not to contain the PAT gene; Lanes 15-27: DNA from leaves of T₀ plants regenerated from Southern positive callus.

Figure 3 shows Southern analysis of DNA from leaf tissue from plants segregating for the PAT gene in the ST2-1 derived T, generation. Lane 1 : DNA sizing standards; Lane 2: blank; Lanes 3-11 : DNA from leaf tissue of plants judged to be sensitive to the herbicide; Lane 22: DNA from PAT- control leaf tissue known not to contain the PAT gene; Lane 23 : Lane 12: DNA from PAT+ control leaf tissue known to contain the PAT gene; Lane 24: piasmid DNA corresponding to the equivalent of 1 copy; Lane 25: piasmid DNA corresponding to the equivalent of 3 copies; Lanes 26-28: not relevant.

Figure 4 shows a nucleotide sequence of a SAR polynucleotide which can be used according to the present invention. Figure 5 shows a restriction map of piasmid pAGM243. Figure 6 shows a restriction map of piasmid pAGM285A. Figure 7 shows a restriction map of piasmid pAGM607. Figure 8 shows a restriction map of piasmid pAGM608.

Detailed Disclosure of the Invention

The present invention concerns methods and materials for increasing frequency of recovery of stable transformation events in plant transformation processes and for increasing the number of low copy number transformants, as well as reducing or eliminating the occurrence of gene silencing throughout subsequent generations descended from the original transformant.

In one embodiment of the present invention, SARs are used in a plant expression cassette to provide a plant transformationprocess that produces a greater percentage of stable transformants and a greater percentage of low copy number transformants than are obtained with SAR-negative plant expression cassettes. In one embodiment, a method of the subject invention comprises transforming a cell or tissue with a SAR polynucleotide sequence operably linked to a polynucleotide of interest that encodes a protein, polypeptide, or peptide. SARs useful with the subject invention include, but are not limited to, SARs originating from plants and animals. SAR-encoding polynucleotides useful with the subject invention include, for example, SAR isolated from tobacco (Hall et al., [ 1991 ] Proc. Natl. Acad. Sci. USA 88:9320). In a preferred embodiment, a SAR polynucleotide useful in practicing the present invention comprises the nucleotide sequence shown in Figure 4, or a functional fragment or mutant thereof. Preferably, the SAR polynucleotide sequence is provided in the form of an "expression cassette" on a suitable vector. Any vector suitable for

DNA mediated transformation can be used and such vectors are known in the art. In a highly preferred embodiment, a SAR polynucleotide sequence is operably linked at either and or both ends of the polynucleotide of interest. Transformation can be accomplished using known methods, including, for example, particle bombardment or biolistics transformation with DNA coated microparticles, Agrobacterium- ediaied transformation, electroporation,microinjection, magnetophoresis, silicon carbide whiskers, PEG mediated transformation, and protoplast transformation. In a preferred embodiment, plant cells are transformed with the polynucleotides of the invention by electroporation according to standard techniques known in the art. See, for example, Pescitelli, S. M., K. Sukhapinda (1995) "Stable Transformation via Electroporation

Into Maize Type II Callus and Regeneration of Fertile Transgenic Plants" Plant Cell Reports

14: 712-716 (hereby incorporated by reference in its entirety). Transformed plant cells can be selected and then cultured under suitable conditions according to routine practice to generate transformed plantlets and plants.

It is well known in the art that the nucleotide sequences of the subject invention can be truncated and/or mutated such that certain of the resulting fragments and/or mutants of the original full-length sequence can retain the desired characteristics of the full-length sequence. A wide variety of restriction enzymes are well known by ordinarily skilled artisans which are suitable for generating fragments from larger nucleic acid molecules. In addition, it is well known that Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA. See, for example, Maniatis et α/.(1982) Molecular Cloning: A Laboratory Manual, Coldspring Harbor Laboratory, New York, pages 135-139, incorporated herein by reference. See also Wei et al. (1983) J. Biol. Chem., 258:13006-13512. By use of Bal3 \ exonuclease (commonly referred to as "erase-a-base" procedures), the ordinarily skilled artisan can remove nucleotides from either or both ends of the subject acids to generate a wide spectrum of fragments which are functionally equivalent to the subject nucleotide sequences. One of ordinary skill in the art can, in this manner, generate hundreds of fragments of controlled, varying length from locations all along the original SAR molecule in one afternoon. The ordinarily skilled artisan is able to routinely test or screen the generated fragments for their characteristics for determiningthe utility of the fragments as taught herein. It is also well known that mutant sequences of the full length sequence, or fragments thereof, can be easily produced with site directed mutagenesis. See, for example, Larionov, O.A. and Nikiforov, V.G. (1982) "Directed Mutagenesis" Genetika 18(3):349-59; Shortle,D., DiMaio,D., and Nathans, D. (1981) "Directed Mutagenesis" Annu. Rev. Genet. 15:265-94; both incorporated herein by reference. The skilled artisan can routinely produce deletion-, insertion-, or substitution-typemutations and test whether the resulting mutants contain the desired characteristics of the full length wild-type sequence, or fragments thereof.

In one embodiment of the invention, a polynucleotide comprising a polynucleotide sequence that has substantially the same sequence as a SAR polynucleotide is operably linked to a polynucleotide that encodes a protein, polypeptide, or peptide. Any desired polynucleotide sequence can be employed to transform cells or tissue according to the present invention. Of particular importance in this regard are genes used, for example, as (i) selective markers (antibiotic and/or herbicide resistance genes), (ii) reporter genes (e.g.,GUS), (iii) insecticide resistance genes (B.t. delta endotoxins) and (iv) any other genes that improve the value or use of a plant. Such genes can include, but are not limited to, glucuronidase, phosphinothricin N- acetyltransferase,green fluorescent protein (GFP), luciferase, Pat/bar, and glyphosate resistance genes (NPTII, HPT, biomoxila resistance gene, AHAS, ALS, cyanomide hydrolase, adenine deaminase, 2,4-D monooxygeanse). The encoded protein, polypeptideor peptide can be one that is naturally present in the transformed cell or it can be heterologous to the transformed cell. Optionally, a regulatory sequence, such as, for example, a promoter sequence that can regulate transcription of the polynucleotide sequence, can be included in a polynucleotide of the invention. In a preferred embodiment, the SAR polynucleotide sequence is a polynucleotide component of an expression cassette on a suitable vector. Vectors useful with the SAR polynucleotides of the invention are known in the art and can be prepared and/or selected according to standard techniques. The methods and materials of the present invention can be used to transform cells or tissue from any organism, and preferably from a eucaryotic organism. In a preferred embodiment, the cells are plant cells. Any plant cell competent to be transformed can be employed in the present invention. It is preferred to employ plant cells that are readily regenerable into whole plants. Suitable plant cells include embryogenic suspension cells, non-embryogenic suspension cells (except in corn where these cells are not regenerable), plant explants, germline cells ( pollen, ovules, meristem domes, megaspore cells, embryos cells, egg cells and embryosacs),microspore cells and callus tissue cells, both compact callus and friable callus. Preferred cells include embryogenic callus, suspension cells (embryogenic suspension) and callused immature zygotic embryos. Particularly preferred plant cells are early embryogenic suspension and young callus (still attached to the zygotic embryo) cells of from about 3-14 and preferably from about 5-10, days old. Wounding the plant cells and/or treating the plant cells prior to being subjected to electroporation is unnecessary and is in fact expressly avoided. When embryogenic suspension tissues are employed it is preferred to gently break up the tissue into small clumps or into fine aggregates as small as possible without damaging the embryogenic suspension cells. This can be done by sieving the tissue through a screen, such as, for example, pushing tissue through a 1 ,000 micron (u) sieve with a spatula or pestle. The finer the aggregates or suspension of undamaged cells, the more efficient the present process.

Plant tissue useful with the invention includes, but is not limited to, callus, meristematic, leaf, shoot, root, and embryonic tissue. The present invention is applicable to any plant species including angiosperms (dicots, monocots) and gymnosperms. Suitable crops include corn, wheat (especially Type C wheat callus), sorghum, rice, pearl millet, sugar cane, orchardgrass and other Gramineae plants; soybean, peanuts, alfalfa and other members of the Luguminoseae family; cotton, kenaf, and other members of the Malvaceae family; poppy and other members of the Papavaraceae family; cannabis and other members of the Cannabinaceae family; tea and other members of the Theaceae family; rape (canola), vegetables and oilseed crops and other members of the Cruciferae family; sunflower, safflower and other members of the Compositae family; coffee and other members of the Rubiaceae family; cacao, theobroma and other members of the Byttneriaceae family; fruits and vegetables, trees, orchard crops, and turf grass. Preferred crops include cotton, tomato, sugarbeet, potato, peanut, alfalfa, rice, wheat and especially corn (maize).

In a preferred embodiment, the polynucleotide sequences employed in the present invention comprise any sequences which have a 5' promoter region, a structural gene region and a 3' nontranslated region (polyadenylation site) which can be expressed in plants. The polynucleotide sequences can be modified in any manner (extra codons, deletion of codons, changed codons, etc.) as long as gene expression is not prohibited. The polynucleotides inserted into the plants according to the present invention can include any desired gene whether eukaryotic or procaryotic in nature. Usually, more than one gene will be inserted into plant cells which are transformed for agronomic purposes. One gene will typically be a selective marker gene (antibiotic resistance gene or an herbicide resistance gene) in order to easily detect transformants from non-transformedcells. Additional genes can also be added to the plant cell genome to impart an additional property, to suppress an existing property (via "antisense" mechanism) or to amplify a known property of the plant cells and the whole plants regenerated therefrom. The genes can be expressed in specific tissues by the use of tissue specific promoters. The genes can be constructed according to techniques well known to one skilled in the art.

Gene constructs may exist as single gene expression cassettes comprised of a promoter, a structural gene coding sequence and a sequence to permit the addition of poly-adenine (poly- A) residues. The promoter is necessary to initiate transcription of the DNA coding for the structural gene into RNA. The promoter may be derived from a variety of sources, as long as it is functional in the cells to be transformed, and may be modified to enhance expression by the addition or deletion of sequences. The DNA may contain intron sequences, either outside or within the coding region for the protein. The removal of these introns and the addition of the poly-A sequence results in the production of a mature messenger RNA (mRNA) which can be translated into the corresponding protein. Gene expression cassettes may be linked in groups of two or more. Polycistronic expression cassettes, in which a single mRNA may code for more than one protein, may also be used. In addition, expression cassettes may be used to produce an "antisense" RNA from the transcription of a strand of DNA which is opposite to the strand of DNA coding for a protein. Examples of promoters active in plants include maize ubiquitin promoter (Christensen et al., [1992] Plant Molecular Biology 18. 675-689). The untranslated leader sequence, includingthe first intron, of the maize ubiquitin gene may be incorporated, particularly for use in monocot cells. The 35S promoter of Cauliflower Mosaic Virus (Murray et al, [1991] Plant Molecular Biology 16: 1035-1050) or the T-DNA Mas2 promoter of the mannopine synthase gene (Leung et al. , [ 1991 ] Molecular & General Genetics 230: 463-474) may also be used. The 35 S promoter may contain a deletion with the addition of an upstream enhancer sequence and an intron in the untranslated leader region (Last et al., [1991] Theoretical & Applied Genetics 81 : 581-588) and the Mas2 promoter may also contain a deletion to enhance expression (Leung et al., supra).

Examples of structural genes include reporter genes such as that coding for GUS, or -glucuronidase, (Jefferson et al, [1987] EMBO Journal 6:3901-3907),a selectable marker gene such as that coding for PAT, or phosphinothricin N-acetyltransferase, which confers resistance to the active ingredient of the commercial herbicide Basta (Droge et al, [1992] Planta 187:142- 151 ), or genes which result in expression of a value-added phenotypic trait. Examples of the latter type gene includes those derived from Bacillus thuringiensis (B.t.) which confer resistance to insects such as lepidoptera (Adang et al, [1985] Gene 36:289-300) or coleoptera (Bradfisch et al, EP 0 50031 1 A2 and U.S. Patent No. 5,208,017)and designated B.t.2 and B.t.3 in Figure 1. The B.t. genes may be reconfigured to enhance their expression in plant cells (Adang et al, U.S. Patent No. 5,380,831 issued January 10, 1995).

While herbicide resistance genes serve the purpose of selecting transformants, they also serve the valuable agronomic purpose of allowing herbicide use in the field in otherwise sensitive crops and/or preventing damage to otherwise sensitive crops planted to fields wherein those herbicides were used earlier in the field for weed control (Herbicide carryover). Additional types of genes of value for use in plants include genes isolated from Bacillus thuringiensis that code for delta-endotoxins as well as truncated and/or synthetic derivatives thereof; fungal resistance genes; oil biosynthesis genes; anti-sense genes and genes responsible for nutritional and/or fiber quality.

The DNA and cells are reacted according to the present invention in a suitable buffered medium that is preferably iso-osmotic. See for example, Wong and Neumann's F-medium, Biochemistry and Biophysics Research Communications, Vol. 107, pp. 584-587, 1982; Krens et al. [1981] Nature 296:72-74. A preferred buffer medium is EPR Buffer (555 mM glucose, 4 mMCaCl₂, 10 mM Hepes buffer, pH 7.2). When conductingthe present process the fresh plant cells are added to a reaction vessel followed by the addition of a mixture of DNA and buffer medium. The order of addition of the materials is not critical.

The temperature at which the electroporation process takes place is not critical although it is preferred to cool the DNA/plantcell mixture immediately prior to and after the application of the electric field. Any culturing or regenerating steps are conducted under conditions

(including temperature) well known to those of ordinary skill in the art. Heat shock treatments, i.e., 37° C for 10 minutes, of the DNA and plant cell mixture can also be employed.

The electroporation step of the present invention is accomplished by applying an electric field to the DNA/cell mixture according to well-known techniques. Any electric field can be employed. Electric pulses can be from 25-5,000 volts (V) or more depending on the current employed. Preferred methods include rectangular pulse generating systems and capacitor discharge systems. The capacitor discharge system creates pulses of exponentially decaying voltages.

In practicing the present invention, DNA and the plant cells are incubated together at room temperature for at least about 10 minutes and preferably for 20-90 minutes. The DNA/cell mixture is then transferred in aliquots to electroporation cuvettes and optionally cooled on ice prior to applying an electric field to it. The electric field strength can vary depending on a variety of factors, such as, for example, the particular plant species being transformed, the particular type (including age) of cells being employed in the transformation process, the type of electric field being employed including the length of exposure time of the plant cells to the electric field, the concentration and type of DNA, etc. One of ordinary skill in the art can easily determine the optimum process conditions by employing routine titration experiments.

With a capacitor discharge system in the transformation of corn callus cells preferred electroporation conditions include a 250-1500 μF capacitor, 25-500 or more volts and a pulse time of from 50-500 msec. Discharge should be from 25-250 volts. Especially preferred conditionsare 850 μF, 150 V and a pulse time of 200 msec. Immediately after application of the electric field the electroporation cuvettes can be optionally placed on ice for about 10 minutes. The cuvettes are then allowed to stand at room temperature for at least about 5 minutes and a small aliquot of cell culture medium is added thereto. Samples are then pipetted from the cuvettes and placed in 2 ml of culture medium in a well of a six-well plate. The treated cells are maintained in culture and regenerated employing standard culturing and plants regenerating techniques.

SAR polynucleotidescontemplated within the scope of the present invention encompass known SARs, including functional fragments and allelic variants of a SAR, as well as any SAR that may be identified in the future so long as the SAR retains substantially the same biological^" activity as SARs exemplified herein. SARs can be prepared from natural sources or synthesized using standard techniques known in the art, such as an automated DNA synthesizer. The SAR polynucleotides of the subject invention also encompass variant sequences containing mutations in the natural sequences. These mutations can include, for example, nucleotide substitutions, insertions, and deletions as long as the variant SAR sequence retains substantially the same biological activity as the natural SAR sequences of the present invention.

The subject invention also concerns cells and tissue transformed using the methods of the invention. Plants, plantlets, and plant seeds transformed to express heterologous genes according to the methods of the described herein are also contemplated within the scope of the invention.

The disclosure of all references cited throughoutthe present specification is incorporated herein by reference.

Materials and Methods

PAT-ELISA

Nunc Maxisorb Microtiter plates were coated with lOOμl of lμg/ml protein A purified rabbit anti-PAT IgG in Coating Buffer for two hours while gently shaking at room temperature. After discarding the coating solution, 400μl blocking solution was added and the plates were blocked overnight at 4°C while gently shaking. The plates were then washed using a Skatron Skan Washer plate washer and 100ml of antigen or cell extract was added to the wells and allowed to incubate for two hours at room temperature while gently shaking. Extracts were made by grinding ~100 mgfw of tissue in five volumes Plant Extraction Buffer (PEB) for callus or ten volumes for leaf material and microfuging the crude extract for 5 min. Then the supernatant was transferred and microfuged again for 5 min. Extracts were then diluted 10-fold with grinding buffer prior to adding to the microtiter plate. After washing the plates, lOOμl of lμg/ml protein A purified goat anti-PAT IgG in Ab Buffer was added and the plates incubated for one hour at room temperature with gentle shaking. The plates were washed and 1 OOμl of 1 :30,000 dilution of anti-goat antibody conjugated to alkaline phosphatase (Pierce) was added and the plates incubated for one hour at room temperature with gentle shaking. After washing, 200ml of SIGMA FAST pNPP substrate was added and the plates were allowed to sit without shaking for one to three hours with 595nm (reference 405nm) absorbency readings taken every hour. Quantitation was achieved by comparingthe absorbencieswith a standard curve made by spiking pure PAT protein into a 10-fold dilution of negative control extract.

Southern Blotting and Hybridization Procedure Southern Blotting and Pre-hybridization: Southern blotting methodology was performed essentially as described in Murray, M., et α/., [1992] Plant Molecular Biology Reporter, vol. 10(2). Briefly, genomic DNA (5- 1 Oug) which has been digested with the appropriate restriction enzyme(s) and resuspended in IX loading buffer is loaded into an agarose/TAE gel (0.85%). The DNA is separated by electrophoresis(75 W/4h) and the gel is then stained (0.1 ug/ml EtBr in 10 mM NaPO) for 30 min. and photographed. The gel is then denatured for 20 min. (150 mM NaPO). The separated DNA is then transferred onto nylon membrane via capillary action overnight. The nylon membrane is then baked for 2 h at 80°C, blocked for 2 h (2% SDS, 0.5% BSA. 1 mM EDTA, 1 mM Orthophenanthroline)and allowed to pre- hybridize for 2 h (100 mM Na phosphate buffer (pH 7.8), 20 mM Na pyrophosphate, 5mM EDTA, 1 mM 1,10 orthophenanthroline,0.1 % SDS, 10% dextran sulfate 500 ug/ml heparin sulfate, 50 ug/ml yeast RNA, 50 ug/ml herring sperm DNA).

Hybridization: DNA template to be used as a probe is labeled with P dCTP using a Prime-It RmT Random Priming Labeling Kit (Stratagene). Labeling efficiency of the probe was measured and approximately 1 X 10⁶ CPM ML is added to the prehybridized membrane. The membrane is hybridized with the probe at 65°C for 12-16 h. The membranes are then washed 3X to remove unbound probe (5mM Na phosphate (pH 7.8), 1.25 mM Na pyrophosphate, 0.25 mM EDTA, 0.1% SDS) and exposed to Kodak scientific imaging film.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 Several experiments were conducted to determine the transformation efficiency, both transient and stable, for maize cells transformed with and without SARs in an expression cassette. The transformation process conducted was an electroporation process of whole intact maize cells. The results of this experiment are listed in Table 1 below. As can be seen, the SAR-positive clones (+SARs) had a lower transient transformation efficiency (19.7% vs. 29.49%) but a much higher stable transformation efficiency (3.62% vs. 0.51%).

Table 1

Transient Transient

Expt. # GUS+SARs GUS-SARs Stable +SARs Stable - SARs

ST-1 NA NA 4/150 - ST-2 NS NA 14/135 1/135 ST-3 12.6 29.3 1/25 0/25 ST-4 10.4 32.7 0/20 0/20 ST-5 16.1 37.2 0/25 0/25 ST-6 19.5 28.9 3/90 1/90 ST-7 38.8 28.7 0/50 0/50 ST-8 31.6 36.9 3/155 1/150 ST-9 8.8 12.7 2/95 0.95

TOTALS 137.8 206.4 27/745 5/390

Average 19.7 29.49 3.62% 0.51%

Example 2

ELISA for the detection of phosphinothricinacetyltransferase(P AT) was conducted on several SAR-negative and SAR-positive clones that contained the PAT gene as described in the Materials and Methods section herein. Clone T34-1 represents a positive control that was one of the highest PAT-expressing SAR-negative clones. The results of this analysis are shown in Table 2. It can be seen that several SAR-positive clones produce high levels of PAT protein.

Table 2

Callus # Prot. Adj. ng PAT Adj. Value % of Protein

ST9 neg C 0.017 0.000 0.000 0.0000%

ST9-2 (-SARs) 0.149 -0.010 -0.644 -0.0001%

ST9-3 (-SARs) 0.145 -0.019 - 1.314 -0.0001%

ST9-4 (+SARs) 0.099 0.128 13.001 0.0013%

ST9-5 (+SARs) 0.158 0.097 6.140 0.0006%

ST2-2 (+SARs) 0.1 10 0.025 2.286 0.0002%

ST2-3 (+) 0.169 0.095 5.626 0.0006%

ST2-4 (+) 0.307 0.1 16 3.772 0.0004%

ST2-5 (+) 0.093 0.039 4.140 0.0004%

ST2-6 (+) 0.160 0.070 4.355 0.0004%

ST2-7 (+) 0.179 0.053 2.934 0.0003%

ST2-8 (+) 0.015 0.018 12.146 0.0012%

ST2-9 (+) 0.165 0.028 1.678 0.0002%

ST2-10 (+) 0.212 0.029 1.381 0.0001%

^^ T3^^^^^ 0.041 0.009 2.210 0.0002%

Example 3

A Southern analysis of several transformed maize callus clones was conducted for the detection of the PAT gene as described in the Materials and Methods section herein. The restriction enzyme used was BamHl and the probe was PAT (PCR). Results of Southern blotting experiments, including controls, are shown in Figures 1-3. The results in Fig. 1 show that all the selected calli contained the PAT gene since bands appeared when incubated with a PAT-specific radioactive probe. Figure 2 shows that plants regenerated from selected calliform plants which also contain the PAT gene. Most of the lines show one or a few inserted copies of the PAT gene while one, Lane 24 or ST8- 1 , showed a complex banding pattern indicative of many PAT gene insertions. Southern analysis of DNA from leaf tissue from plants segregating for the PAT gene in the ST2-1 derived T, generation indicated that, with two exceptions, all plants thought to be herbicide sensitive were negative for the presence of the PAT gene (Figure 3). With no exceptions, all plants thought to be herbicide resistant did show the presence of the PAT gene. This data also shows that the inserted gene was capable of passing through meiosis and into the T, progeny.

Example 4 Immature zygotic embryos were isolated from 12-day old cobs of the genotype Hill.

They were plated on callus initiation medium (CIM), scutellum side up, which contained N₆ salts, 1 mg/L 2,4-dichlorophenoxyaceticacid (2,4-D), and lOμM AgNO₃, incubated in the dark for 8 days at 27-29°C and then examined for Type II callus formation. 250 callused embryos were chosen and placed into sterile 1.5ml micro-cuvettes, at five embryos per cuvette. 20μl of AGM285A piasmid DNA (lμg/μl) was introduced, followed by 200μl of EPM buffer (80 mM KC1, 5 mM CaCl₂, 10 mM hepes, 0.425 M mannitol, pH 7.2). One cuvette was inoculated with pAGM243 (comprising the gene construct Ubi-pαt-Nos::Ubi-g«s-Nos). Another cuvette contained no piasmid DNA. The contents of the cuvettes were agitated and then allowed to stand for 1 hr at room temperature. Electroporation was carried out using the gene ZAPPER 450/2500 (IBI) at 850μF, 150V for one pulse. The cuvettes were then allowed to stand for 20 min at room temperature before removing the embryos and replating on CIM, scutellum side up, and returned to the dark at 27-29°C. Three days later, the embryos treated with pAGM243 were placed into GUS stain and 9 days later were observed for GUS+ events. Those embryos showed 242 GUS+ events or 48.4 GUS+ events per embryo. Seven days after treatment, all pAGM285 A-treated embryos and embryos subjected to electroporation in the absence of piasmid DNA were transferred to callus maintenance medium (CMM) containing N₆ salts, 1 mg/L 2,4-D and 5μM bialaphos. These were transferred again to the same medium type after 22 days. About three weeks later, the tissue was transferred again.

Example 5

Maize embryos were transformed using the pAGM 607 and pAGM608 constructs. Hill (B X A) or Hill S, immature zygotic embroyos of maize were isolated 9-12 days after pollination using standard techniques and plated on callus induction medium (CIM). They were then incubated in the dark at 28°C. Seven or eight days later, the embryos showing pre-embryogenic callus formation were selected and placed into sterile spectrophotometer cuvetttes (MFGR) at five callused embroyos per cuvette. These cuvettes had been prepackaged and sterilized by gassing. Then 20μl of piasmid DNA was added to the cuvette followed by 200μl of EPM buffer. The cuvettes were flicked lightly to distribute the embryos in the cuvette for better coverage by the DNA/buffer solution. The cuvettes were then allowed to incubate for one hour at room temperature.

We utilized the IBI gene ZAPPER 450/2500 at the electroporation step. Electroporation was conducted at 150V and 850μF. Following electroporation,the cuvettes were allowed to sit at room temperature for 20 min. The callused embryos were then removed and placed on CIM medium, callus side up. It is importantthat the callus remain above the surface of the medium.

The callused embryos were cultured for one week on CIM medium (N6 sals, 2.88g/l proline, lOOmg/l casamino acids, 20 g/1 sucrose, modified B5 vitamins, 1 mg/1 2, 4-D, lmg/L

AgNO₃, pH 5.8) and then transferred to callus maintenance medium (CMM; CIM medium with no AgNO₃) containing 5μM to lOμM Bialaphos. Non-DNA-treated controls were always included and these were split into two groups, one on CMM with Bialaphos and one on CMM without Bialaphos. All callused embryos were transferrred to the same medium every three weeks or so.

Bialaphos-resistant colonies began to appear approximately 10 weeks or so after treatment. These were isolated and put on CMM with 5μM for one more round of selection. Any colonies which grew and had a healthy appearance, i.e. good cream color and embryogenic morphology, were then given a clone number and allowed to proliferate for the purposes of analysis and plant regeneration.

Most of the clones produced from pAGM 607 treatments appeared on selection much earlier than clones produced from pAGM 608 treatment. Clones were observed in as little as 5-6 weeks (Table 2) after treatment whereas normally we see clones between 9 and 12 weeks after treatment (SAR-). Moreover, the majority of SAR+ clones continued to grow rapidly and maintain a desirable phenotype even under high (lOμM Bialaphos) selection pressure.

Table 2. Cumulative data concerning SAR+ and SAR- transgenic maize clones

Clone # +/- Date Date Iso. d to iso Southern Pat- Plants? SARs Init. ELISA Southern

ST2-1 + 6/28/96 9/10/96 73 ? ND Yes Yes

ST2-2 6/28/96 9/10/96 73 Simple 0.0002% Yes Yes

ST2-3 6/28/96 9/10/96 73 Simple 0.0006% Yes Yes

ST2-4 6/28/96 9/10/96 73 Simple 0.0004% Yes Yes

ST2-5 6/28/96 9/10/96 73 Simple 0.0004% Yes Yes

ST2-6 6/28/96 9/10/96 73 Simple 0.0004% Yes Yes

ST2-7 6/28/96 9/10/96 73 Simple 0.0007% Yes Yes

ST2-8 6/28/96 9/30/96 93 Simple 0.0012% Yes Yes ST2-9 6/28/96 11/5/96 129 Simple 0.0002% Yes Yes

ST2-10 6/28/96 11/5/96 129 Simple 0.0001% Yes Yes

ST2-12 6/28/96 12/18/96 172 ND 0.0507% Yes Yes

ST2-13 6/28/96 11/5/96 129 ND ND not Yes tested

ST2-14 6/28/96 12/18/96 172 ND ND not Yes tested

ST8-1 8/13/96 46 Complex 0.0871% Yes Yes

ST8-2 8/13/96 46 Simple 0.0194% Yes Yes

ST9-4 8/16/96 69 Medium 0.0013% Yes Yes

ST9-5 8/16/96 69 Medium 0.0006% Yes Yes

Average 92.06

Std. 2.83

Dev.

ST2-15 6/28/96 12/18/96 172 ND ND No

ST8-4 8/13/96 11/13/96 92 Simple ND No

Average 132

Std. 40

Dev.

ND=Not done

All but two of the SAR+ clones were highly regenerable, more so than any previous Transfection-derived clones without SARs. Plant recovery was very rapid and several dozen plants were obtained from each clone with little effort. The two pAGM 607 clones that were not readily regenerable, appeared much later than the other 607 clones, did not show a particularly good phenotype and were relatively slow growing. We postulate that the SARs on these inserts had been partially degraded prior to incorporation into the cell's chromosome and thus were not able to exert their positive effects. The plants derived from pAGM 607 were quite normal in appearance, were very vigorous but flowered and set seed early. The S, progeny however, were normal in every way, segregated for Liberty resistance with expected segregation ratios (data not shown) and showed very high Liberty resistance. Example 6

Field trials with the maize-SAR+ material showed that 13 out of 19 events gave segregation ratios consistent with heterozygous insertions (3: 1 ratio in self crosses; 1 : 1 ratio in outcross to wild type).

SAR Event Mendelian Ratios

ST2-02 Yes

ST2-04 No

ST2-05 Yes ST2-06 Yes

ST2-09 Yes

ST2-10 No

ST8-01 Yes

ST8-02 No ST9-04 Yes

ST9-05 No

ST2-04*CQ715 Yes

ST2-06*CQ715 Yes

ST2-10*CQ715 No ST8-01 *CQ715 Yes

ST9-04*CQ715 No

ST2-1 *CQ806 Yes

ST2-05*CQ806 Yes

ST2-06*CQ806 Yes ST9-05*CQ806 Yes

Moreover, the only SAR+ line tested showed excellent resistance to the Liberty herbicide in the third generation even at eight times the normal rate of field application (27.3 oz acre of herbicide Remove). This result shows the extraordinary stability of high Pat gene expression even after several generations. Normally, transgenic plants exhibit a relatively high frequency of gene silencing of the^" transgene as a given transgenic line is advanced from one generation to the next. Gene silencing is a major problem in the commercializationof transgenic crops. It occurs at a rate of less than 0.1% up to 3% and sometimes more. SARs have now been shown to prevent this phenomenon when used at the 3 ' and 5' ends of the inserted transgene. Transgenic maize lines containing Ubiquitin promoter-phosphinothricin acetyl transferase-now terminator (Ubi-pat-nos) with the Rb7 SAR regions attached at both ends prevent gene silencing in transgenic com produced by the methods described in this patent or other known transformation methods over several generations in the field. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims

1. In a method for transformation of plant cells comprising inserting a polynucleotide of interest into plant cells and maintaining said cells under conditions whereby successfully transformed cells are identified and selected, the improvement comprising providing at least one scaffold attachment region polynucleotide sequence operably linked to said polynucleotide of interest whereby the frequency of recovery of stable transformation events is increased.

2. The improved method according to claim 1, wherein the polynucleotide of interest has at least one scaffold attachment region operably linked to each of its 5' and 3' ends.

3. The improved method according to claim 1 , wherein said scaffold attachment region polynucleotide comprises the nucleotide sequence of SEQ ID NO. 1, or a functional fragment or mutant thereof.

4. The improved method according to claim 1, wherein said scaffold attachment region polynucleotide is provided in the form of a DNA expression cassette which can be expressed in a plant.

5. The improved method according to claim 1, wherein said plant transformation process is selected from the group consisting of bombardment with DNA coated microparticles, Agrobacterium-mediated transformation, electroporation, microinjection, magnetophoresis, silicon carbide whiskers, PEG mediated transformation, and protoplast transformation.

6. The improved method according to claim 1 , wherein said gene of interest is selected from the group consisting of antibiotic resistance genes, herbicide resistance genes, reporter genes, marker genes, disease resistance genes, oil biosynthesis genes, anti-sense genes, insecticide resistance genes, and nutritional enhancement genes.

7. The improved method accordingto claim 1 , wherein said plant cells are selected from the group consisting of embryogenic suspension cells, non-embryogenic suspension cells, plant explant cells, germ line cells, microspore cells, and callus tissue cells.

8. The improved method accordingto claim 1 , wherein said plant cells are of a species selected from the group consisting of cotton, tomato, peanut, alfalfa, rice, wheat, com, trees, orchard crops, fruits, vegetables, soybean, canola, turfgrass, cannabis, sunflower, sugarcane, sugarbeet, and potato.

9. The improved method according to claim 4, wherein said expression cassette comprises a 5'- promoter region, a structural gene region comprising said polynucleotide of interest, a 3'- nontranslated region, and a SAR flanking either or both sides of the said structural gene region.

10. The improved method accordingto claim 6, wherein said reporter gene is selected from the group consisting of glucuronidase, phosphinothricin N-acetyltransferase, green fluorescent protein, and luciferase.

11. The improved method according to claim 6, wherein said marker gene is selected from the group consisting of Pat/bar, glyphosate resistance genes, NPTII, HPT, bromoxil resistance genes, AHAS, ALS, cyamamide hydrotase, adenine deaminase, 2, 4-D monooxygenase.

12. A transformed plant cell produced by the method of claim 1.

13. The transformed cell according to claim 12, said cell comprising a scaffold attachment region polynucleotide having the nucleotide sequence of SEQ ID NO. 1, or a functional fragment or mutant thereof.

14. A transgenic cell descended form the cell of claim 12.

15. A descendanttransgeniccell of claim 14, said descendant cell comprising a scaffold attachment region having the nucleotide sequence of SEQ ID NO. 1, or a functional fragment or mutant thereof.

16. A transgenic plant regenerated from a cell according to claim 12.

17. The transgenic plant according to claim 16, said plant comprising cells which comprise a scaffold attachment region having the nucleotide sequence of SEQ ID NO. 1, or a functional fragment or mutant thereof.

18. A method for increasing the number of low copy number transformants in a plant transformation process, said method comprising transforming a plant cell or tissue with at least one scaffold attachment region polynucleotide sequence operably linked to a structural gene of interest.

19. The method according to claim 18, wherein said gene of interest has at least one scaffold attachment region operably linked to each of its 5' and 3' ends.

20. The method according to claim 18, wherein said scaffold attachment region polynucleotide comprises the nucleotide sequence of SEQ ID NO. 1 , or a functional fragment or mutant thereof.

21. The method according to claim 18, wherein said scaffold attachment region polynucleotide is provided in the form of a DNA expression cassette comprising a structural gene region which can be expressed in a plant.

22. The method according to claim 18, wherein said plant transformation process is selected from the group consisting of bombardment with DNA coated microparticles, Agrobacterium mediated transformation, and electroporation.

23. The method according to claim 18, wherein said gene of interest is selected from the group consisting of antibiotic resistance genes, herbicide resistance genes, reporter genes, fungal resistance genes, oil biosynthesis genes, anti-sense genes, and insecticide resistance genes.

24. The method according to claim 18, wherein said plant cells are selected from the group consisting of embryogenic suspension cells, non-embryogenic suspension cells, plant explant cells, germ line cells, microspore cells, and callus tissue cells.

25. The method according to claim 18, wherein said plant tissue is selected from the group consisting of callus, meristematic tissue, leaf tissue, shoot tissue, root tissue, and embryonic tissue.

26. The method according to claim 18, wherein said plant cell or tissue is of a species selected from the group consisting of cotton, tomato, peanut, alfalfa, rice, wheat, and com.

27. The method according to claim 21, wherein said expression cassette comprises a five-prime promoter region, a structural gene region and a three-prime nontranslated region, and a SAR flanking either or both ends of said structural gene.

28. The method according to claim 23, wherein said reporter gene is select from the group consisting of glucuronidase and phosphinothricin N-acetyltransferase.