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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8214—Plastid transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

• This application pertains to the field of genetic engineering of plant plastid genomes, particularly chloroplasts, and to methods of and engineered plants without the use of antibiotics.
• This application relates in particular to a method of selecting genetically engineered or transformed plants without the use of antibiotics as a selectable marker.
• the application also relates to a method of transforming plants to confer drought tolerance and to the transformed plants which are drought tolerant.
• the transformed plants will be capable of producing HC-toxin reductase.
• the pathogen namely Cocholiobolus carbonum Nelson race 1
• the lethal selection of transformed plants will result.
• Ursin in U.S. patent 5,633,153 (May 27, 1997) entitled "Aldehyde dehydrogenase selectable markers for plant transformation," proposed a method of using an aldehyde dehydrogenase as a selectable marker for nuclear transgenic plant cells.
• GM crops containing antibiotic resistant genes have been banned from release (Peerenboom 2000).
• Plastid genetic engineering as an alternative to nuclear genetic engineering. Plastid genetic engineering, particularly chloroplast genetic engineering, is emerging as an alternative new technology to overcome some of the environmental concerns of nuclear genetic engineering (reviewed by Bogorad, 2000).
• One common environmental concern is the escape of foreign gene through pollen or seed dispersal from transgenic crop plants to their weedy relatives creating super weeds or causing genetic pollution among other crops (Daniell 1999B).
• Keeler et al. (1996) have summarized valuable data on the weedy wild relatives of sixty important crop plants and potential hybridization between crops and wild relatives. Among sixty crops, only eleven do not have congeners and the rest of the crops have wild relatives somewhere in the world.
• Bt Bacillus thuringiensis
• Plant-specific recommendations to reduce Bt resistance development include increasing Bt expression levels (high dose strategy), expressing multiple toxins (gene pyramiding), or expressing the protein only in tissues highly sensitive to damage (tissue specific expression). All three approaches are attainable through chloroplast transformation (Daniell 1999C). For example, hyperexpression of several thousand copies of a novel B.t. gene via chloroplast genetic engineering, resulted in 100% mortality of insects that are up to 40.000-fold resistant to other B.t. proteins (Kota et al. 1999).
• chloroplast genetic engineering in higher plants may be the utilization of the antibiotic resistance genes as the selectable marker to confer streptomycin/spectinomycin resistance. Initially, selection for chloroplast transformation utilized a cloned mutant 16S rRNA gene that does not bind the antibiotic and this conferred spectinomycin resistance (Svab et al. 1990). Subsequently, the aadA gene product that inactivates the antibiotic by transferring the adenyl moiety of ATP to spectinomycin /streptomycin was used (Svab and Maliga 1993). These antibiotics are commonly used to control bacterial infection in humans and animals.
• the probability of gene transfer from plants to bacteria living in the gastrointestinal tract or soil may be enhanced by the compatible protein synthetic machinery between chloroplasts and bacteria, in addition to presence of thousands of copies of the antibiotic resistance genes per cell. Also, most antibiotic resistance genes used in genetic engineering originate from bacteria.
• chloroplast genetic engineering Because of the presence of thousands of antibiotic resistant genes in each cell of chloroplast transgenic plants and the use of the most commonly used antibiotics in the selection process, it is important to develop a chloroplast genetic engineering approach without the use of antibiotics. Non-obviousness of antibiotic free selection.
• chloroplast genetic engineering Despite several advantages of plastid transformation, one major disadvantage with chloroplast genetic engineering in higher plants is the utilization of the antibiotic resistance genes as the selectable marker. Initially, selection for chloroplast transformation utilized a cloned mutant 16S rRNA gene that did not bind the antibiotic and this conferred spectinomycin resistance. Subsequently, the aadA gene was used as a selectable marker.
• Aminoglycoside 3 ' -adenylylfransferase inactivates the antibiotic by transferring the adenyl moiety of ATP to spectinomycin /streptomycin.
• bacterial infections in humans and animals are also controlled by using these antibiotics.
• the probability of gene transfer from plants to bacteria living in the soil or gastrointestinal tract may be enhanced by the compatible protein synthetic machinery between chloroplasts and bacteria, in addition to presence of thousands of copies of the antibiotic resistance genes per cell. Also, most antibiotic resistance genes used in genetic engineering originate from bacteria.
• BADH betaine aldehyde dehydrogenase
• BADH enzyme in the stroma within plastids to be fully functional. It was not known whether the BADH enzyme would be catalytically active without proper cleavage within plastids.
• the nuclear BADH cDNA with high GC content was never anticipated to express well in the AT rich prokaryotic plastid compartment because the codon usage is very different between the prokaryotic chloroplast compartment and the eukaryotic nuclear compartment. Therefore, it was not obvious to express a nuclear gene in the plastid compartment.
• the invention provides for a method to circumvent the problem of genetic pollution through plastid transformation and the use antibiotic-free selectable markers.
• Antibiotic-free phytotoxic agents and their corresponding detoxifying enzymes or proteins are used as a system of selection.
• the betaine aldehyde dehydrogenase (BADH) gene from spinach has been used as a selectable marker.
• BADH betaine aldehyde dehydrogenase
• This enzyme is present only in chloroplasts of a few plant species adapted to dry and saline environments.
• the selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to nontoxic glycine betaine (GB), which also serves as an osmoprotectant.
• the preferred embodiment of this invention provides a method of selecting plant transformants using a plastid vector that includes a promoter targeted to the plastid, a DNA sequence encoding a gene of interest, another DNA sequence encoding a selectable marker such as an aldehyde dehydrogenase, and a terminator sequence.
• the transformed plants are selected by allowing transformed plants to grow in medium with the effective amount of a phytotoxin which is detoxified by the encoded aldehyde dehydrogenase. Lethal selection of the plants transformants will result.
• the vector is targeted to plant chloroplasts. This embodiment can be carried out using both the universal chloroplast vector and a vector which is universal.
• the vector includes a ribosome binding site and a 5' untranslated region (5' UTR.
• a promoter functional in green or non-green plastids is to be used in conjunction with the 5'UTR.
• the invention provides the application of a wide variety of plants species and plant parts, including flowers, fruits, cereals, and all major crop plants.
• the invention also provides for the plants transformants engineered and selected a antibiotic- free selectable marker with preferably a target heterologous DNA sequence.
• the invention also provides for a method of conferring drought tolerance to plants with a antibiotic-free selectable marker.
• the plants or plant cells are transformed through the chloroplast by a vector containing a promoter targeted to the chloroplast, a DNA sequence encoding betaine aldehyde dehydrogenase, DNA sequences encoding at least one gene of interest, and a terminator sequence.
• the transformed plants are selected by allowing transformed plants to grow in medium with the effective amount of a phytotoxin which is detoxified by the encoded aldehyde dehydrogenase. Lethal selection ofthe plants transformants will result.
• the plants so transformed will be capable of glycine betaine production that leads to enhanced drought tolerance.
• Figure 1 shows the chloroplast universal vector pLD BADH.
• Primer 3P lands on the native chloroplast genome (in the 5' end region of 16-S r DNA gene).
• 3M lands on the aadA gene generating a 1.6 kb fragment. Restriction enzyme cut site are located on the map.
• Figure 2 shows BADH enzyme activity in E.coli. Cells harvested from overnight grown cultures were resuspended in a minimal volume ofthe assay buffer. Sonicated cell homogenate was desalted in G-25 columns and 50 ⁇ g total protein was used fr each assay. NAD+ dependent BADH enzyme was analyzed for the formation of NADH by increase in the absorbency at 340 nm.
• Figure 3 shows a comparison of betaine aldehyde and spectinomycin selection.
• A. N. tabacum Petit Havana control in RMOP medium containing spectinomycin after 45 days.
• Figure 4 shows the PCR analysis of DNA extracted from transformed plants run on a 0.8% agarose gel. Lane M lkb ladder, lane 1, untransformed Petit Havana control, lane 17 is positive control and lanes 2through 16 are transgenic clones. Except lanes 10, 13, 15 and 16 all other lanes show the integration of aadA gene into the chloroplast genome.
• Figure 5 shows the Southern analysis of transgenic plants. A: Probe PI was used to confirm chloroplast integration of foreign genes. The 0.81 kb fragment was cut with BamHl and Bglll contains the flanking sequence used for homologous recombination. Untransformed control plants shuold generate 4.47 kb fragment and transformed plants should generate a 7.29 kb fragment.
• FIG. 1 Lanes 1, untransformed Petit Havana; Lanes 7 pLD-BADH plasmid DNA or purified DNA or purified 1.0 kb Eco Rl BADH gene fragment. Lanes 2 through 6 of transgenic plants. Probe (P2) was used t confirm the integration of BADH gene.
• Figure 6 shows BADH enzyme activity in different ages of leaves of transgenic tobacco plant. Proteins were extracted from 1-2 g leaves. Extracts were centrifuged at 10,000xG for 10 minutes and the resulting supernatant was desalted in small G-25 columns, and tested for assay (50 ⁇ g protein per assay). NAD+ dependent BADH enzyme was analyzed for the formation of NADH. Y, D, M and O represent young, developing, mature and old leaves, respectively.
• Figure 7 shows the phenotypes of control (A) and chloroplast transgenic plants (B).
• Figure 8 shows the germination of control untransformed (a) and chloroplast transgenic (b) seeds on MS medium containing 500 ⁇ g/ml spectinomycin.
• Figure 9 A and B show the vectors for BADH selection in other plants.
• Table 1 shows the comparison of spectinomycin and betaine aldehyde as the selectable marker for the first round of selection.
• the invention discloses a novel way of selecting transformed plants, wherein the plant's plastid genome is transformed via a vector targeted to the plastid, and the selectable markers used for such transformation is a antibiotic-free marker.
• the invention further consists of the plants transformed and selected using the present method.
• the invention also discloses a method to confer osmoprotection to plants through chloroplast transformation.
• the present invention is applicable to all plastids of plants. These include chromoplasts which are present in the fruits, vegetables and flowers; amyloplasts which are present in tubers like the potato; proplastids in roots; leucoplasts and etioplasts, both of which are present in non-green parts of plants.
• the Vectors This invention contemplates the use of vectors capable of plastid transformation, particularly of chloroplast transformation. Such vectors would include chloroplast expression vectors such as pUC, pBR322, pBlueScript, pGEM, and all others identified by Daniell in U.S. patent no. 5,693,507 and U.S. patent no. 5,932,479. Included are also vectors whose flanking sequence is located outside the inverted repeat of the chloroplast genome.
• a preferred embodiment of this invention utilizes auniversal integration and expression vector competent for stably transforming the chloroplast genome of different plant species (Universal Vector).
• Universal Vector A universal vector is described in WO 99/10513 which was published on March 4, 1999, which is herein incorporated in its entity.
• the vector pLD-B ADH was constructed by generating a PCR product using spinach cDNA clone as the template.
• the 5' primer also included the chloroplast optimal ribosome binding site (GGAGG).
• PCR product was subcloned into the EcoRl site of pLD-CtV, resulting in pLD-BADH.
• BADH is one of the few proteins targeted to the chloroplast that lacks a definite transit peptide (Rathinasabapathi et al 1994). Authors suggest that information for transport may be contained within the mature protein. Even if a transit peptide was present, it should be cleaved in the stroma by the stromal processing peptidase (Keegstra and Cline, 1999). Furthermore, nuclear encoded cytosolic proteins with transit peptides have been successfully expressed within chloroplasts and found to be fully functional (Daniell et al. 1998). Therefore there was no need to delete any transit peptide.
• the universal vector, pLD-BADH integrates the aadA and BADH genes into the 16S-23S-spacer region of the chloroplast genome.
• Expression cassettes of the chloroplast integration vector contain the chimeric aadA gene and the BADH gene driven by the constitutive 16S rRNA promoter and regulated by the 3 ' untranslated region ofthe plastid psbA gene.
• the chimeric aadA gene encoding aminoglycoside 3'adenyltransferase confers spectinomycin resistance in chloroplasts enabling selection ofthe transformants on spectinomycin dihydrochloride.
• BADH converts the toxic betaine aldehyde in cells to glycine betaine.
• this pathway is compartmentalized within chloroplasts (Nuccio, et al. 1999).
• SD Shine-Dalgarno
• Other plant specific vectors can be used to transform the plastids, particularly chloroplast, of various crops for betaine aldehyde selection.
• Some examples of these include the pLD-Alfa-BADH is for transforming the chloroplast genome of Alfalfa using betaine aldehyde selection; the pLD-Gm- utr-BADH is for transforming the chloroplast genome of Soybean (Glycine max) with betaine aldehyde; this contains the psbA promoter and untranslated region (UTR) for enhanced expression; the pLD-St-B ADH is for transforming the chloroplast genome of potato (Solanum tuberosum) using betaine aldehyde selection; pLD-St-utr-BADH is for transforming the chloroplast genome of potato (Solanum tuberosum) with betaine aldehyde; this contains the psbA promoter and untranslated region (UTR) for enhanced expression; and the pLD
• Promoters For transcription and translation ofthe DNA sequence encoding the gene of interest, the entire promoter region from a gene capable of expression in the plastid generally is used.
• the promoter region may include promoters obtained from green and non-green chloroplast genes that are operative upon the chloroplast, such as the psbA gene from spinach or pea, the rbcL, atpB promoter region from maize, the accD promoter and 16S rRNA promoter. Competent promoters are also described in U.S. patent 5,693,507, and the other literature sources contained therein. These publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. Selectable markers.
• the preferred embodiment of this invention teaches the use ofthe spinach BADH gene as a selectable marker; wherein a plant is transformed via the chloroplast with the spinach BADH gene along with another nucleotide sequence encoding a desirable trait.
• the BADH gene product - betaine aldehyde dehydrogenase - will oxidize the betaine aldehyde in the growth medium allowing for the lethal selection of transformed plants.
• Enzymes and proteins that function in plastids can be used as antibiotic-free phytotoxic agents.
• the synthesis is regulated by the substrate. When adequate amino acid is made, it binds to one ofthe enzymes in the pathway to block further synthesis (feed back inhibition).
• Mutant genes are available for many enzymes that are insensitive to such feed back inhibition.
• Such enzymes are expressed in the chloroplast by engineering feed back insensitive mutant genes via the chloroplast genome. Putative transgenic shoots are regenerated in a growth medium lacking specific amino acids. True transgenic plants will be regenerated in the growth medium. Thus, antibiotic free selection is accomplished.
• Pigment biosynthesis can also be used in antibiotic free selection in plastids. While ancient plants (including pines) have the ability to synthesize chloroplhyll in the dark, flowering plants lost this capacity. This is because ofthe last step in chlorophyll biosynthesis is controlled by the enzyme protochlorophyllide reductase. This enzyme can function in the dark in primitive land plants and certain algae but is light dependent in higher plants. That is why ornamental plants kept inside the house requires light to synthesize chlorophyll.
• chloroplast gene (chlB) for protochlorophyllide reductase in the green alga Chlamydomonas is required for light independent protochlorophyllide reductase activity (Plant Cell 5: 1817-1829). Therefore, chlB gene from the Chlamydomonas chloroplast is introduced into the chloroplast genome of higher plants and transgenic green shoots appearing in the dark is selected. Thus, pigment biosynthesis genes are used as antibiotic free selectable markers.
• herbicide selection S everal methods can be used to genetically engineer herbicide resistance via the chloroplast genome.
• the target enzyme or protein is overproduced with 10,000 copies of foreign genes per transformed cell. This results in binding of all herbicide molecules thereby facilitating regeneration of transgenic shoots.
• Another approach is the use of modified enzyme or proteins (mutant) that does not bind the herbicide.
• the third approach is to use enzymes or proteins to breakdown the herbicide.
• Drought tolerance likewise can be used as a selectable marker.
• Expression ofthe BADH enzyme or trehalose phosphate synthase via the chloroplast genome enables cells to tolerate drought. Drought conditions are created in culture plates by the addition of polyethylene glycol to the growth medium (3-6%). Only cells that express BADH or TPS are capable of drought tolerance and grows in the presence of polyethylene glycol. Thus, antibiotic free chloroplast transgenic plants are obtained.
• Other Aldehyde Dehydrogenases Other genes that code for an aldehyde dehydrogenase capable of detoxifying other phytotoxic aldehydes can be used in this novel selection system.
• the transformation of this invention maybe accomplished by any methods of transformation known in the art. Such methods include, but are not limited to PEG treatment, Agrobacterium treatment, and microinjection. Methods of transfomiation are described by Daniell et. al., "New Tools for Chloroplast Genetic Engineering,” Nat. Biotechnology, 17:855-857 (1999). This publication is hereby incorporated by reference in its entirety.
• the method for transformation is by bombardment.
• the BADH gene expression was tested in E. coli cell extracts by enzyme assays before proceeding with bombardment.
• the universal vector pLD-B ADH was transformed into the E. coli strain XL-1 Blue and grown in Terrific Broth (Guda et al.
• Tobacco (Nicotiana tabacum var. Petit Havana) was grown aseptically by germination of seeds in MSO medium.
• This medium contains MS salts (4.3 g/liter), B5 vitamin mixture
• leaves were chopped into small pieces of ⁇ 5 mm 2 in size and placed on the selection medium (RMOP containing 500 ⁇ g/ml of spectinomycin dihydrochloride or 5-10 mM betaine aldehyde) with abaxial side touching the medium in deep ( 100x25 mm) petri plates .
• the regenerated resistant shoots were chopped into small pieces ( ⁇ 2mm 2 ) and subcloned into fresh deep petri plates containing the same selection medium.
• Resistant shoots from the second culture cycle were transferred to the rooting medium (MSO medium supplemented with IBA, 1 mg/liter containing appropriate selectable marker). Rooted plants were transferred to soil and grown at 26 ° C under 1 hour photoperiod. Selection and heightened, rapid regeneration of homoplasmic transgenic plants.
• Leaf disks in Figure 3 under betaine aldehyde selection appear partially green because they were photographed 12 days after the initiation ofthe selection process whereas the disc photographed on spectinomycin were 45 days after initiation ofthe selection process. In spite ofthe short period of selection one leaf disk was almost bleached (Fig 3D) and all of them were killed after 30 days. Under lOmM betaine aldehyde selection, control untransformed samples were killed (turned black, 3G-1) whereas transgenic leaves produced new shoots ( Figure 3G, 2-4).
• Southern blot analysis was performed using total DNA isolated from transgenic and wild type tobacco leaves. Total DNA was digested with a suitable restriction enzyme. Presence of a BgUI cut site at the 3' end ofthe flanking 16S rRNA gene and the trnA intron allowed excision of predicted size fragments in the chloroplast transformants and untransformed plants. To confirm foreign gene integration and homoplasmy, individual blots were probed with the flanking chloroplast DNA sequence (probe 1 , Figure 5 A). In the case ofthe BADH integrated plastid transformants, the border sequence hybridized with a 7.29 kbp fragment while it hybridized with a native 4.47 kbp fragment in the untransformed plants ( Figure 5B).
• the copy number ofthe integrated BADH gene was also determined by establishing homoplasmy in transgenic plants (Daniell et al. 1998; Guda et al. 2000). Tobacco chloroplasts contain about 10,000 copies of chloroplast genomes per cell. If only a fraction ofthe genomes was transformed, the copy number should be less than 10,000. By confirming that the BADH integrated genome is the only one present in transgenic plants, it could be established that the BADH gene copy number could be as many as 10,000 per cell.
• DNA gel blots were also probed with the BADH gene coding sequence (P2) to confirm specific integration into the chloroplast genomes and eliminate transgenic plants that had foreign genes also integrated into the nuclear genome.
• the BADH coding sequence hybridized with a 7.29 kbp fragment which also hybridized with the border sequence in plastid transformant lines ( Figure 5B). This shows that the BADH gene was integrated only into the chloroplast genome and not the nuclear genome in transgenic lines examined in this blot. Also, this confirms that the tobacco transformants indeed integrated the intact gene expression cassette into the chloroplast genome and that no internal deletions or loop outs during integration occurred via homologous recombination. Osmoprotection.
• osmoprotectants help to protect plant organelles from osmotic shock as well as the cellular membranes from damage during stress (Nuccio et al. 1999).
• glycine betaine is the most effective and is commonly present in a few families, including Chenopodiaceae and Poaceae. But most of the crop species including tobacco do not accumulate glycine betaine. Since synthesis and localization of glycine betaine is compartmentalized in chloroplasts, engineering the chloroplast genome for glycine betaine synthesis may provide an added advantage for chloroplast transgenic plants.
• BADH converts toxic betaine aldehyde to non-toxic glycine betaine which is the second step in the formation of glycine betaine from choline.
• BADH enzyme activity By analyzing BADH enzyme activity, the expression of introduced BADH gene can be monitored. Since BADH is a NAD+ dependent, enzyme activity is analyzed for the formation NADH. The reaction rate is measured by an increase in absorbency at 340 nm resulting from the reduction of NAD+.
• BADH enzyme activity was assayed in crude leaf extracts of wild type and transgenic plants. Unlike previous reports, no purification with ammonium sulfate was necessary in order to perform the BADH assay. Crude extracts from chloroplast transgenic plants showed elevated activity (15-18 fold) compared to the untransformed tobacco ( Figure 6). The wild type tobacco showed low endogenous activity as reported previously (Rathinasababathy et al. 1994). BADH enzyme activity was investigated from young (top 3-4 leaves), mature (large well developed), developing leaves (in between young and mature) and bleached old leaves from transgenic plants.
• This invention applies to any higher plants, such as monocotyledonous and dicotyledonous plant species.
• the plants that may be transformed via the universal vector with a antibiotic-free selectable marker maybe solanacious plants or plants that grow underground.
• this invention is applicable to the major economically important crops such as maize, rice, soybean, wheat, and cotton.
• a non-exclusive list of examples of higher plants which maybe so transformed include cereals such as barley, com, oat, rice, and wheat; melons such as cucumber, muskmelon, and watermelon; legumes such as bean, cowpea, pea, peanut; oil crops such as canola and soybean; solanaceous plants such as tobacco; tuber crops such as potato and sweet potato; and vegetables like tomato, pepper and radish; fruits such as pear, grape, peach, plum, banana, apple and strawberry; fiber crops like the Gossypium genus such as cotton, flax and hemp; and other plants such as beet, cotton, coffee, radish, commercial flowing plants, such as carnation and roses; grasses, such as sugar cane or turfgrass; evergreen trees such as fir, spruce, and pine, and deciduous trees, such as maple and oak.
• cereals such as barley, com, oat, rice, and wheat
• melons such as cucumber, muskmelon, and
• this invention can be practiced upon other monocotyledonous and dicotyledonous plants, including maize, rice, soybean, wheat, cotton, oat, barley, cucumber, muskmelon, watermelon, bean, cowpea, pea, peanut, canola, potato and sweet potato; tomato, pepper, radish, pear, grape, peach, plum, banana, apple, strawberry, flax, hemp, beet, coffee, radish, commercial flowing plants, such as carnation and roses; grasses, such as sugar cane or turfgrass; fir, spruce, and pine, maple and oak.
• monocotyledonous and dicotyledonous plants including maize, rice, soybean, wheat, cotton, oat, barley, cucumber, muskmelon, watermelon, bean, cowpea, pea, peanut, canola, potato and sweet potato; tomato, pepper, radish, pear, grape, peach, plum, banana, apple, strawberry, flax, hemp, bee
• Other targeted genes of interest This invention provides that genes of interest expressing desirable traits are encoded by the targeted DNA sequence in the expression cassette.
• phytotoxic aldehydes such as acetaldehyde, formaldehyde, proprionaldehyde, and butyraldehyde
• herbicides such as triazines and cyanamide, including those listed in Molecular Biotechnology by Glick and Pasternak, page 459, Table 18.4. Also useful is light selection.
• Genes of interest maybe isolated from other organisms such as Sugar Beet and E. Coli.
• Promoters can be used to drive expression ofthe genes, including the psbA promoter, the accD promoter, the 16SrRNA promoter, and those listed in U.S. patent 5,693,507 and International Publication No. WO99/10513, both to Daniell.
• Example 5 Other chloroplast vectors may be used in lieu ofthe universal vector, including those listed in U. S. patents 5693507 and 5932479 to Daniell.
• Targeted Genes of Interest include: Polypepide pro-insulin, PBP synthetic polymer, Insulin, Human Serum Albumin, and Herbicide glyphosate.
• Other genes of interest include, but are not limited to the aminoglycosides listed in "Aminoglycosides: A Practical Review" by Gonzalez, L. S. and Spencer, J.P., American Family Physician, No. 8, 58:1811.
• Sidorov VA Kasten D, Pang SZ, Hajdukiewicz PTJ, Staub JM, Nehra, NS (1999) Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant Journal 19: 209-216. Sijmons, P.C, Cekker, B.M.M., Schrammeijer, B., Verwoerd, T.C, van den Elzen, P.J.M., Hoekema, A. (1990) Biotechnology 8: 217 - 221.

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