EP1108010A4 - Herbe a gazon transgenique halophile - Google Patents

Herbe a gazon transgenique halophile

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Publication number
EP1108010A4
EP1108010A4 EP99951422A EP99951422A EP1108010A4 EP 1108010 A4 EP1108010 A4 EP 1108010A4 EP 99951422 A EP99951422 A EP 99951422A EP 99951422 A EP99951422 A EP 99951422A EP 1108010 A4 EP1108010 A4 EP 1108010A4
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EP
European Patent Office
Prior art keywords
transgenic
plant
salt
transgenic plant
transgene
Prior art date
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EP99951422A
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German (de)
English (en)
Other versions
EP1108010A1 (fr
Inventor
Tseh An Chen
Shou-Yi Chen
Geng-Yun Zhang
Faith C Belanger
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Institute of Genetics and Developmental Biology of CAS
Rutgers State University of New Jersey
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Institute of Genetics of CAS
Rutgers State University of New Jersey
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Publication of EP1108010A1 publication Critical patent/EP1108010A1/fr
Publication of EP1108010A4 publication Critical patent/EP1108010A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/8273—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 drought, cold, salt resistance

Definitions

  • transgenic turfgrass is provided, which is salt and drought tolerant by virtue of expressing a transgene encoding betaine aldehyde dehydrogenase .
  • glycine betaine is accumulated in cells of some higher plants and animals as an osmoprotectant (Hanson and Grumet, 1985, In
  • the invention provides transgenic turfgrasses having increased salt tolerance by virtue of expressing a gene involved in the biosynthesis of glycine betaine, an osmoprotectant .
  • a transgenic cell of a Graminaceous plant which comprises a transgene encoding an enzyme of the glycine betaine biosynthetic pathway.
  • the transgene encodes betaine aldehyde dehydrogenase from Atripl ex hortensis, and in a most preferred embodiment, plasmid pRTT120 comprises the transgene.
  • the transgenic cell is preferably a turfgrass, and most preferably selected from the group consisting of Creeping Bentgrass, Perennial Ryegrass, Kentucky Bluegrass, and Bermudagrass .
  • a salt-tolerant Graminaceous plant produced from the aforementioned transgenic cell is also provided.
  • this salt-tolerant turfgrass is also drought- tolerant. Seeds produced from the salt-tolerant turfgrass are also provided.
  • a salt-tolerant transgenic Graminaceous plant which expresses a transgene encoding an enzyme of the glycine betaine biosynthetic pathway.
  • the plant expresses a transgene encoding betaine aldehyde dehydrogenase from Atripl ex hortensi s, and most preferably pRTT120 comprises the transgene.
  • the salt-tolerant transgenic Graminaceous plant is also preferably drought-tolerant, and preferably is selected from the group consisting of Creeping Bentgrass, Perennial Ryegrass, Kentucky Bluegrass and Bermudagrass . Seeds of the transgenic plant are also provided.
  • transgenic Graminaceous plant with improved phenotypic characteristics.
  • This transgenic plant has at least one of the following characteristics: higher BAH activity, a higher concentration of glycine betaine and a higher growth rate under salt stress, and a higher level of drought tolerance.
  • Figure 1 Schematic representation of the BADH expression vector pRTT120.
  • FIG. 2A Comparison of the salt damage effect between transgenic and non-transgenic plants, expressed as the percentage of dead, damaged and healthy leaves after 3 weeks treatment.
  • Fig. 2A 0.8% NaCl stress.
  • the term ⁇ tolerant” or * tolerance means the ability of a plant to overcome, completely or to some degree, the detrimental effect of an environmental stress or other limiting factor.
  • the transgenic plants are tolerant to high environmental salt concentrations by virtue of producing an abundance of betaine glycine, which functions in the cell as an osmoprotectant .
  • selectable marker refers to a gene product that confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant.
  • Selectable markers are encoded by expressible DNA sequences, which are sometimes referred to herein as ⁇ N selectable marker genes.”
  • promoter region refers to the 5' regulatory regions of a gene, including promoters per se, as well as other transcriptional and translational regulatory sequences.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequences are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.
  • transcription control elements e.g. promoters, enhancers, and termination elements
  • NN transgene refers to an artificial gene which is used to transform a cell of an organism, such as a bacterium or a plant.
  • the term "about” means within a margin of commonly acceptable error for the determination being made, using standard methods.
  • concentrations of various components initially added to culture media may change somewhat during use of the media, e.g., by evaporation of liquid from the medium or by condensation onto the medium.
  • concentrations of the macronutrients, vitamins and carbon sources are less critical to the efficacy of the media than are the micronutrient , hormone and antibiotic concentrations.
  • statistical significance of quantified differences is determined using one-way analysis of variance (ANOVA) .
  • ANOVA analysis of variance
  • This statistical test is well known to those in the art, and computer programs that carry out this test are commercially available.
  • the level of probably (P) used is 0.05 in a preferred embodiment, 0.01 in a more preferred embodiment, and 0.001 in a most preferred embodiment .
  • ⁇ equivalent plants are ones of the same genotype or cultivar, at the same age, and having been grown under the same conditions.
  • the equivalent plant may be transformed by a similar DNA construct but without the critical transgene, or may not be transformed but regenerated from tissue culture.
  • transgenic turfgrasses are provided that have increased salt tolerance.
  • this invention provides transgenic bentgrass carrying the betaine aldehyde dehydrogenase transgene, which are highly salt tolerant.
  • BADH and BADH we use the abbreviation BADH and BADH to indicate the betaine aldehyde dehydrogenase gene and gene product, respectively.
  • glycine betaine in Graminaceae (also known as Poaceae) has been reported (Hitz and Hanson, 1980, Phytochemistry 19:2371-2374). Some species show glycine betaine accumulation under salt or drought stress and the amount of accumulation is sufficient to produce effective osmotic protection (Marcum and Murdoch, 1994, J. A er. Soc. Hort. Sci. 119:779-84). Recently, the genes conferring the two enzymes in the biosynthetic pathway of glycine betaine have been isolated. The BADH gene has been cloned from Mountain Spinach, Atripl ex hortensis , which grows on the shore of a salt lake in western China. In an exemplary embodiment of the present invention, we have successfully transformed Creeping Bentgrass with a Mountain Spinach BADH transgene, and have shown that the ratio of BADH activity and betaine content increased in the transgenic bentgrass.
  • glycine betaine concentration in the transgenic plants reached 5, 891 ⁇ g/g dry weight, which was 74.5% higher than that in the nontransgenic plants. Glycine betaine is considered to be located entirely in the cytoplasm (Gorham and Jones, 1983, Planta 157:344-349). The relative contribution of glycine betaine to osmotic adjustment can be estimated by assuming that the cytoplasmic volume in mature bentgrass mesophyll cells is about 10% of total cell volume (Leigh et al., 1981, Planta 153:34-41), and that the osmotic coefficient of glycine betaine is 1.
  • the contribution of glycine betaine in the transgenic plants is estimated to be 360 mOsmol-kg "1 higher than that in the non-transgenic plants (830 mOsmol-kg "1 for the transformants and 470 mOsmol-kg -1 for the controls) . This is likely to be the reason for the improved in salt tolerance of the B4 transformants at both the cellular and whole plant levels.
  • BADH activity was not induced by stress .
  • betaine accumulation itself is nontoxic in plants, its synthesis is energetically expensive and may cause possible side effects, such as susceptibly to attack by certain pathogenic fungi and aphids (Strange et al . , 1974, Physiol. Plant Path.
  • salt stress tests in vi tro demonstrated that the BADH- transformed callus lines generated by the aforementioned methods showed improved salt tolerance on 0.8% NaCl medium as compared to the control callus tissues.
  • one transgenic line showed better salt tolerance than did the others. Both leaves and roots of these plants grew stronger than control plants under saline conditions, particularly under 1.2% NaCl stress.
  • position effects of the transgene played a role in the differential salt tolerance observed among transgenic plants, thus emphasizing the importance of selecting and evaluating several transformants from a particular transformation procedure.
  • the salt-tolerant transgenic cells of the invention grow significantly faster that equivalent untransformed cells on a 0.8% NaCl medium.
  • a transgenic plant of the invention grows significantly faster than the equivalent untransformed plant under 1.2% NaCl stress.
  • the transgenic plant of the invention is significantly more tolerant to drought than equivalent plants.
  • the transgenic plant of the invention has at least 1.5 X, 2.0 X or 3.0 X more BAH activity than equivalent untransformed plants in preferred, more preferred and most preferred embodiments, respectively.
  • the transgenic plant of the invention has at least 2 X, 4 X, or 6 X more glycine betaine per dry weight in leaf tissue than equivalent untransformed plants in preferred, more preferred and most preferred embodiments.
  • the transgenic plant of the invention grows 1.2 X, 1.5 X or 2.0 X faster than the equivalent untransformed plant in preferred, more preferred and most preferred embodiments.
  • Preferred turfgrasses of the invention i.e. transgenic and salt tolerant
  • Creeping Bentgrass, Agrosti s pal ustri s Huds . Perennial Ryegrass (genotype R821) ⁇ Loli um spp . )
  • Bermudagrass cv. Tifeagle Cynodon dactyl on
  • the salt tolerance of these grasses can also be improved by transformation with the BADH transgene.
  • Other turfgrasses contemplated for use in the invention include, but are not limited to, Velvet Bentgrass, Hard Fescue, Chewings Fescue, Strong Creeping Fescue and Colonial Bentgrass.
  • grass species of particular interest include, but are not limited to, Dichondra micrantha, Pennisetum clandestinum, Stenotaphrum secundatum, Zoysia japonica, Agrostis spp., Festuca spp., Lolium spp., Avena spp., Triticum spp., Secale spp., Hordeum spp., Oryza spp., Panicum spp., Saccharum spp., Sorghum spp., Zea spp., Cynodon spp., Zizania spp., Andropogon spp., Schizachyrium spp., Bouteloua spp.
  • the spinach BADH gene (Weretilnyk and Hanson, 1990, PNAS 87:2745-2749) may also be used, as may other BADH genes isolated in the future (especially preferred are BADH genes from monocotyledonous species) .
  • this invention contemplates the use as transgenes of any gene in the glycine betaine biosynthetic pathway (e.g., choline monooxygenase) , or any combination of those genes .
  • Biolistic delivery of transforming DNA is exemplified herein for transformation of turfgrass. However, other transformation techniques can be used.
  • Plant transformation methods include Agrobacteri um- mediated delivery, PEG treatment of protoplasts, UV laser microbeam, ge ini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like.
  • Agrobacteri um- mediated delivery PEG treatment of protoplasts, UV laser microbeam, ge ini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like.
  • the transgenic plants of the present invention may be made by the following transformation/regeneration protocol, based on biolistic delivery of transforming DNA according to the method of Hartman et al . (1994, Bio/Technology 12:919-923).
  • Embryogenic callus is initiated from germinating seeds of a selected turfgrass. After surface sterilization, seeds are planted on a callus medium.
  • the callus medium may contain plant growth regulators such as 2,4-D, BAP, DiCamba and zeatin riboside. The selection, concentrations and ratios of these growth regulators is varied to suit a particular species of plant. The selection of type and concentration of growth regulators is a matter of routine experimentation and is well know to those skilled in the art of tissue culture.
  • samples for bombardment consisting of embryogenic callus on filter discs in a plate of callus medium containing 0.6 M mannitol. The samples are kept in the mannitol medium for an appropriate time (e.g., 16 hours) prior to bombardment.
  • DNA-coated gold particles are prepared as described, e.g., by Christou et al . (1991) . Samples are co-transformed with a selected expression vector containing a coding sequence of interest operably linked to appropriate 5' and 3' regulatory regions, and a vector that expresses a selectable marker.
  • the bombardment is carried out using a standard biolistic delivery system.
  • the day following bombardment the calli are transferred to callus medium without mannitol. After an appropriate period of time (e.g., 1 week), the calli are transferred to callus medium supplemented with the selectable marker (e.g., hygromycin) .
  • the selectable marker e.g., hygromycin
  • the resistant calli After several weeks of growth on the selection medium, the resistant calli are transferred to regeneration medium and placed in the light. The regenerated shoots from the calli usually appear within 4-8 weeks. The young plants are then transferred to standard growth medium, and the regenerating shoots appear in the order of 3 weeks thereafter.
  • tissue culture methods and media are well known to persons skilled in the art of plant tissue culture.
  • the aforementioned cultures generally are performed at about room temperature, e.g., 22-26°C, under a light regimen of between about 30 and 70 ⁇ mol • m ⁇ 2 • sec '1 . It should be recognized that the amount as well as the tissue specificity of expression of the transgene of interest in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such position effects are well known in the art.
  • the transformation/regeneration formulations and protocol of the present invention can be used to introduce the salt tolerance-conferring transgene or selectable marker gene into a selected Graminaceous plant.
  • Useful transgenes may employ many useful promoters and coding sequences. Examples of useful promoters for either the transgene or selectable marker gene include, but are not limited to, the rice actin promoter, the maize ubiquitin promoter, the maize ADH 1 promoter, the rice or maize tubulin promoters ( Tub A, B or C) and the alfalfa His 3 promoter.
  • selectable marker genes may be used in this transformation procedure. In addition to hygromycin resistance, other selectable markers genes suitable for use in the present invention are known (e.g., bialaphos resistance) .
  • this invention provides salt-tolerant transgenic Graminaceous plants, and also is intended to encompass cells and tissues of those plants, including, but not limited to, leaves, stems, shoots, roots, flowers and seeds.
  • seeds of the transgenic plants are provided.
  • the plants grown from the aforementioned seeds, or seeds from other Graminaceous varieties, or the progeny thereof, all of which are considered within the scope of this invention, are used in crosses and selection methods to transfer the transgene of interest into other Graminaceous genotypes, cultivars, varieties and the like.
  • Plants grown from the transgenic seeds of the invention can also be analyzed to detect the presence of the inserted transgene and vector sequences using DNA extraction, cleavage by one or more restriction endonucleases, and analysis, e.g., Southern blotting using probes derived from the transgene. In this manner, the transfer of foreign transgenes into progeny of breeding crosses can be monitored. An example of the use of such detection and monitoring methods is described in greater detail in Example 1.
  • Transgenic turfgrass that carry and express the aforementioned BADH coding sequences are expected to be highly resistant not only to salt stress, but also to drought stress and cold stress, which are both osmotically regulated, at least in part.
  • MATERIALS AND METHODS Initiation and maintenance of embryogenic callus. Embryogenic callus was initiated from germinating seeds of Creeping Bentgrass, cv. Crenshaw. After surface sterilization, seeds were planted on Murashige and Skoog medium (MS) supplemented with 30 mM Dicamba (3, 6-dichloro-o-anisic acid), 20 mM BAP and 100 mg/1 myo-inositol, 30 g/1 sucrose and 2.0 g/1 Gell-gro, pH 5.80-5.85. After 6-8 weeks culture in the dark at 25°C, compact embryogenic calli were selected and transferred to new culture plates. The calli were subcultured every 2-3 weeks on the same medium. Transformation and regeneration of transformants .
  • MS Murashige and Skoog medium
  • Samples for bombardment consisted of 0.5 g embryogenic callus on 5.5 cm filter discs in plate of callus media containing 0.6 M mannitol. Samples were kept on the mannitol medium for 16 hours prior to bombardment (Vain et al . , 1993, Plant Cell Reports 12:84- 88) . DNA-coated gold particles (1.0 micron diameter) were prepared as described by Christou et al . (1991, Bio/Technology 9:957-962). Samples were co-transformed with pRTT120 (the BADH expression vector) , and pAcH, (the hygromycin resistance expression vector) .
  • pRTT120 the BADH expression vector
  • pAcH the hygromycin resistance expression vector
  • the bombardment was carried out using a Bio-Rad PDS-1000/He Biolistic Delivery System at 1,100 psi and at a distance of 12 cm. Each dish was bombarded twice. The day following bombardment, the calli were transferred to callus medium without mannitol. After 1 week, they were transferred to the callus medium supplemented with 200 mg/L hygromycin. After 6-8 weeks of growth on the selection medium, the resistant calli were transferred to regeneration medium and placed in the light. The regenerated shoots from the calli usually appeared within 4-8 weeks. The young plants were transferred to MS media, and the regenerated roots appeared after 3 weeks. Expression vectors.
  • the BADH cDNA used was isolated from mountain spinach (Atripl ex hortensis) and its registration number was EMBL X69770 (Xiao et al . , 1995, Chinese Science Bulletin 40 ( 8 ): 741-745) .
  • the BADH cDNA fragment was inserted into Smal/Sacl site of pAHC25 (Christensen and Quail, 1994, Transgenic Research 5:213-218) replacing the GUS coding sequence.
  • the resulting 4.0 kb Hind III DNA fragment which contained the 1.6 kb BADH coding sequence flanked by the maize ubiquitin promoter and nos-3' terminal sequence, was then cloned into the Hind III site of pBluescript.
  • the resulting plasmid was designated pRTT120 ( Figure 1) .
  • Plasmid pAcHl (provided by Dr. German Spangenberg) contains a truncated hph hygromycin coding sequence (Bilang et al . , 1991, Gene 100:247-250) under the control of the rice actin promoter (McElroy et al., 1991, Mol. Gen. Genet. 231:150-160).
  • BADH activity assay was carried out according to Weretilnyk and Hanson (1989, Arch. Biochem. Biophys . 271:55-63). Leaves or callus samples of 1-2 g were homogenized with a pestle in liquid N 2 and transferred into the extraction buffer (50 mM Hepes/KOH, pH 8.0, 1 mM EDTA, 5 mM DTT) at 1.0 g sample per 2.5 ml buffer. The homogenate was incubated at 0°C in an ice bath for 10 min and centrifuged at 13,000 X g for 10 min at 4°C. The supernatant was fractionated by 35-70% (NH 4 ) 2 S0 4 saturation.
  • the precipitate was collected by centrifugation at 15,000 X g for 15 min at 4°C and the pellet was dissolved in 1.0 ml protein buffer (10 mM Hepes/KOH pH 8.0, 1 mM EDTA, 0.2 mM DTT) .
  • the protein concentration was determined by using the Bio-Rad protein assay kit using bovine serum albumin as the standard.
  • the 1.0 ml reaction system used for BADH activity assay was consisted of 50 mM Hepes/KOH pH 8.0, 5 mM DTT, 1 mM EDTA, 1 mM NAD, 1 mM BADH sample and 0.5 mg protein.
  • the reaction was initiated with the addition of betaine aldehyde chloride and incubated at 37°C for 20 min.
  • the BADH activity was calculated after determining the absorbance at 340 nm of the reaction products.
  • One unit of enzyme activity was defined as the amount of enzyme needed to convert 1 nmol of NAD per minute under the assay conditions. Calculation of BADH activity was based on an extinction coefficient of 6200 M ⁇ c ⁇ f 1 for NADH.
  • Betaine content determination was assayed by the periodide method of Pearce et al . (1976, Phytochemistry 15:953-954). Extracted samples were fractionated by thin layer chro atography to check compositions, using the methods of Eneroth and Lindstedt (1965, Anal. Biochem. 10:479-484).
  • Salinity treatments The salt tolerance for both transgenic callus tissues and regenerated plants was tested.
  • normal callus medium and the same medium with 0.8% NaCl additive were used.
  • About 0.2 g of both non-transgenic and transgenic calli were transplanted onto a plate containing one of the callus media, and the plate was evenly divided into two parts. After 3 weeks, the calli were carefully removed from the plate and weighed. Three replicates of each treatment were made and the test was repeated three times.
  • the plants were allowed to acclimatize to hydroponic conditions for one week before salt was added to the growth solution.
  • the salt was added with an increase of 0.2% NaCl every 3 days until it reached 0.8% and 1.2%, respectively.
  • the plants were allowed to grow in the final salt level for 3 weeks. Nutrient solutions were changed every week. After 3 weeks, the leaves of tested plants were rated. Leaves were divided into dead, damaged or healthy based on whether they were completely dry or wilting, tip burning larger than 1/5 of the leaf length, or nearly no damage, respectively.
  • Transformation, selection and regeneration of bentgrass When co-transformation was used, the plasmid ratio between hygromycin resistance transgene and the BADH transgene was 1:1. From 7 transformed dishes, 9 stable hygromycin resistant cell lines were obtained after 8 weeks selection. Shoots appeared for all of the nine cell lines in 6 weeks after the calli were transferred to the regeneration medium. Roots appeared 3 weeks after the shoot were transferred to the Plantcons ® half filled with MS medium.
  • BADH gene 0.3-0.5 cm young leaf-tips were used to screen for BADH transgene insertion by PCR (Klimyuk et al . , 1993, The Plant J. 3:493-494). Three (B4, B7 and B8) of the nine hygromycin resistant transgenic lines were confirmed to also have the BADH transgene insertion. RT-PCR and BADH activity assay were used to identify the expression of BADH transgene in positive regeneration lines. The ratios of BADH activity in B4, B7 and B8 were 2.9, 2.0 and 8.0 times of the wild type ( BADH- control), respectively.
  • the ratio of BADH activity in B4 calli was always higher than that of the control. There was no indication that BADH activity in the control or B4 calli was induced, whether they were under short term (2 days) or long term (3 weeks) treatments, although glycine betaine content increased many folds under salt stress. In contrast, a loss of BADH activity was observed, especially for the control under short term treatment.
  • Non- Transgenic transgenic plants plants glycine betaine content without stress 4533.4 2746.6
  • Embryogenic callus was initiated from germinating seeds of Perennial Ryegrass, genotype R821. After surface sterilization, seeds were planted on Murashige and Skoog medium (MS) supplemented with 4.0 mg/L 2,4-D (2, 4-dichloro-phenoxyacetic acid), 0.05 mg/L BAP and 100 mg/L myo-inositol, 30 g/L sucrose and 2.0 g/L Gell-gro, pH 5.80-5.85. After 2-3 weeks culture in the dark at 25°C, compact embryogenic calli were selected and transferred to new culture plates. The calli were subcultured every 2 weeks on the same medium.
  • Transformation and regeneration of transformants Plants were transformed and regenerated by the method of Example 1.
  • Expression vectors The BADH expression vector of Example 1 was used in the transformation.
  • BADH activity assay The BADH activity assay used is described in Example 1.
  • Drought treatments For the drought tolerance test, 110 individual plants which came from 110 individual seeds, were randomly selected as control plants to represent the genetic background of genotype R821. Four to six individuals of each transgenic plant line were used as representative of that specific transgenic line. Six of the twenty-seven transgenic lines which showed better drought tolerance in a preliminary test were used in drought test. When plants grew to the 5-10 tiller stage, all the plants were transplanted into two large plastic tubs (23 x 17 inches), each of which contained 6 inches of fritted clay soaked in Hoagland solution. Holes were drilled at the bottom of the tubs for drainage. The transgenic plants and control plants were randomly arranged and planted in the tubs.
  • Soil and other organic particles adhering to the roots were washed away in tap water before transplantation. Plants were watered every two days. Two weeks after transplantation, plants were mowed to 3 inches height and allowed to grow for another week. All plants were watered thoroughly, and then water was withheld for 18 days.
  • Transformation, selection and regeneration of ryegrass From 8 dishes of transformed tissues, fifty- two stable hygromycin resistant cell lines were obtained after 8 weeks selection. Six weeks after the calli were transferred to regeneration medium, shoots appeared for forty-eight cell lines. Roots appeared 3 weeks after the shoots were transferred to PlantconS ® containing MS medium.
  • BADH gene Young leaf tips (0.3- 0.5 cm) were used to screen for BADH transgene insertion by PCR (Klimyuk et al . , 1993, The Plant J. 3:493-494). Twenty-seven of the fifty-eight hygromycin resistant transgenic lines were confirmed by Southern blotting to have the BADH transgene insertion. The BADH activity assay was used to identify the expression of BADH transgene in positive regeneration lines. For the six transgenic lines used, the ratios of BADH activity were 1.5 to 3.4 times that of the average of wild type (BADH- control) .
  • Embryogenic callus was initiated from fresh nodes of Bermudagrass, cv. Tif. Eagle. After surface sterilization, nodes were planted on Murashige and Skoog medium (MS) supplemented with 30 mM Dicamba, 20 mM BAP and 100 mg/L myo-inositol, 30 g/L sucrose and 2.0 g/L Gell-gro, pH 5.80-5.85. After 6-8 weeks culture in the dark at 30°C, compact embryogenic calli were selected and transferred to new culture plates. The calli were subcultured every 10 days on the same medium. Transformation and regeneration of transformants .
  • MS Murashige and Skoog medium
  • Samples for bombardment consisted of 0.5 g embryogenic callus on 5.5 cm filter paper discs in a plate with callus media containing 0.6 M mannitol. Samples were kept on the mannitol medium for 16 hours prior to bombardment (Vain et al . , 1993, Plant Cell Reports 12:84-88). DNA-coated gold particles (1.0 micron diameter) were prepared as described by Christou et al . (1991, Bio/Technology 9:957-962). Samples were co- transformed with pRTT120 (the BADH expression vector) , and pAcH, (the hygromycin resistance expression vector) .
  • pRTT120 the BADH expression vector
  • pAcH the hygromycin resistance expression vector
  • the bombardment was carried out using a Bio-Rad PDS-1000/He Biolistic Delivery System at 1,100 psi and at a distance of 9 cm. Each dish was bombarded twice. The day following bombardment, the calli were transferred to callus medium without mannitol. After 1 week, they were transferred to the callus selection medium supplemented with 200 mg/L hygromycin. After 4-6 weeks of growth on the selection medium, the resistant calli were transferred to regeneration medium and placed in the light. The regenerated shoots from the calli usually appeared within 6-8 weeks. The young plants were transferred to MS media, and the regenerated roots appeared after 2 weeks.
  • the BADH expression vector was used to transform Bermudagrass, cv. Tif. Eagle plants.
  • the DNA construct is described in Example 1.
  • Embryogenic callus was initiated from germinating seeds of Kentucky Bluegrass genotype 94-301. After surface sterilization, seeds were planted on
  • Murashige and Skoog medium (MS) supplemented with 20 mM Dicamba, 0.3 mg/1 zeatin riboside and 100 mg/1 myo- inositol, 30 g/1 sucrose and 2.0 g/1 Gell-gro, pH 5.80- 5.85.
  • MS Murashige and Skoog medium
  • zeatin riboside 100 mg/1 myo- inositol
  • 30 g/1 sucrose and 2.0 g/1 Gell-gro pH 5.80- 5.85.
  • Transformation and regeneration of transformants Plants were transformed and regenerated by the method of Example 1.
  • the BADH expression vector was used to transform Kentucky Bluegrass genotype 94-301 plants.
  • the DNA construct is described in Example 1.
  • Transformation, selection and regeneration of bentgrass When co-transformation was used, the plasmid ratio between hygromycin resistance transgene and the BADH transgene was 1:2. From 8 transformed dishes, thirty-six stable hygromycin resistant cell lines were obtained after 8 weeks selection. Shoots appeared for thirty-two cell lines in 6 weeks after the calli were transferred to the regeneration medium. Roots appeared 2 weeks after the shoot were transferred to the PlantconS ® containing MS medium.

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Abstract

L'invention concerne une herbe à gazon transgénique qui exprime un transgène codant la bétaïne aldéhyde déshydrogénase. Cette plante montre une tolérance fortement accrue à la salinité comparée à ses congénères non-transgéniques. Elle montre également une tolérance accrue à la sécheresse. On peut planter ce gazon transgénique halophile et xérophile dans les régions à salinité élevée, comme les bords de mer, ou dans les régions affectées par des pénuries d'eau d'irrigation.
EP99951422A 1998-08-24 1999-08-24 Herbe a gazon transgenique halophile Withdrawn EP1108010A4 (fr)

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KUO YU-JEN ET AL: "Merging callus level and whole plant microculture to select salt-tolerant Seaside creeping bentgrass", JOURNAL OF PLANT NUTRITION, vol. 17, no. 4, 1994, pages 549 - 560, XP008030132, ISSN: 0190-4167 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481347A (zh) * 2020-12-07 2021-03-12 兰州大学 一种抗盐基因的筛选方法及其应用
CN112481347B (zh) * 2020-12-07 2022-09-27 兰州大学 一种抗盐基因的筛选方法及其应用

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