JP3053609B2 - Genetic transformation of orchids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to genetic engineering of general plants, and more particularly, to a method for transforming orchid species using particle-mediated transformation techniques.

[0002]

BACKGROUND OF THE INVENTION There is substantial interest in the genetic improvement of orchid species. Orchids are commercially grown worldwide, and 80
It is the largest family of flowering plants with more than 0 genera and more than 25,000 species. The potential for genetic and genetic modification of orchids for disease and stress tolerance, induction of early flowering, and development of varieties with altered flower color and morphology is of commercial importance. Conventional plant breeding methods for orchid improvement have been largely limited by very long reproductive cycles (years), slow seed maturation (months), and difficulty in seedling analysis. Thus, the possibility of utilizing genetic engineering techniques using orchids appears to be an attractive alternative.

[0003] Genetic manipulation is a means for inserting new genes that confer traits that are not readily available in conventional breeding. In contrast to sexual crossing, genetic engineering can add new genes, while the genotype of the elite clone is retained intact. To have a successful genetic engineering approach, it is inevitable to have an efficient gene transfer system for a given species. There are two means for plant transformation: mainly through the soil plant pathogenic bacterium Agrobacterium, the use of biological vectors to deliver DNA to cells; and mainly through particle bombardment. Direct delivery of naked DNA to plant cells.

[0004] Currently, the most widely used transformation techniques are those large endogenous Ti plasmids (tumor induction).
Alternatively, it is based on the use of the soil plant pathogen Agrobacterium, which has an innate ability to transfer DNA segments from the Ri plasmid (root-derived) plasmid into infected plant cells. However, a successful application of this technology is
It depends on the host specificity range of the cterium. In the study of species and genotype independent transformation methods, several techniques have been developed based on the direct delivery of naked DNA to plant cells. These methods include electroporation,
Microinjection, PEG-mediated DNA uptake, and liposome-mediated DNA uptake, silicon carbide whiskers
(whisker), and particle bombardment. Particle bombardment technology is biolistic
(biolistic) or microprojectile (micropro
Also known as jectile), based on coating on small carrier particles of DNA, the particles are then physically accelerated into plant cells. This method has several advantages. First, removal of cell walls is not required for DNA entry. Second, DNA can be introduced into organized and differentiating cell masses such as meristems and adventitious buds. Third, no special biological plasmid manipulation is required. Finally, DNA transfer does not depend on the recognition and binding of biological vectors to cell membranes.

[0005] The current growth and breeding of orchids is based on protocorm-like bodies (protocorm
-like body) (PLB) very dependent on both cultures. The storage organ, the protocomb, is formed from germinating embryos and has apical meristem and leaf primordia. PLB, also known as somatic protocomb, is derived from in vitro culture of apical meristem and axillary bud meristem, and resembles both functionally and structurally seedling protocomb. Such meristems are suitable for particle bombardment and regeneration of transgenic plants. It has been demonstrated in herbaceous plant species that these bombarded cells can be transformed in a manner similar to Agrobacterium transformation (Klein et al., Proc. Natl. Acad.
Sci. USA., 88, 8502-8505, 1988). Soybean germline cells have been transformed with this particle-mediated transformation technique (McCabe et al., Bio / Technology, 6, 923-926, 198).
8). U.S. Pat. No. 5,015,580 describes this technique. U.S. Pat. No. 5,681,730 also discloses that somatic embryos of the gymnosperm species tree (so
matic embryo) and genetic engineering of plants. The advantage of this approach is that shoots develop directly from primary and secondary meristems without an intervening explant-organogenesis phase. This minimizes treatment with plant hormones, and thus minimizes the opportunity for somaclonal mutations.

[0006] Particle bombardment-mediated transformation methods
Little information is available on how it can be applied to the transformation of orchids, especially members of the genus Cymbidium. Currently, only two research papers on orchid transformation are available for Dendrobium members, but not for Cymbidium, which has higher commercial value. Kuehnle and Sugii, Plant Cell R
eports, 1992 reported obtaining transgenic Dendrobium. However, they did not show expression of a screenable marker gene (eg, GUS). Furthermore, it was not known whether the transgenic plants were chimeric. Chia et al., Plant Journal, 1
994 transformed Dendrobium orchids using particle bombardment. Instead of antibiotic selection, they have developed another selection method that relies on the expression of the introduced firefly luciferase gene. In this system, the light-emitting cell mass is screened and isolated using a photon counting video camera-photomultiplier tube system and a sophisticated detection microscope. All screening and isolation processes are
Repeat until a pure transgenic cell line is isolated. The isolated cell line is then induced to regenerate into a whole plant. Due to the need for expensive equipment, time-consuming and microscopic screening of transgenic cells, and the characteristic slow regenerative growth of orchids, this technique makes practical use of genetic transformation approaches for orchid improvement. Seems unrealistic for proper use.

[0007] Orchids differ substantially from other plants in their need for transformation systems. First, orchid cells grow slowly. Second, orchid cells are cumbersome for tissue culture operations. Third, plant regeneration from undifferentiated cells has not been achieved for orchids. Fourth, orchid cells have little sensitivity to antibiotic selection. Orchids are commonly used aminoglycosides (eg,
Kanamycin). More than 500 mg / l kanamycin is often required to select for transgenic cells (Chia et al., Plant
Journal, 1994). Fifth, orchid cells in tissue culture
Exudes large amounts of phenols, the oxidation products of which are toxic to cells. Finally, due to the multicellular structure of the meristem, the resulting transgenic plants can sometimes become chimeric with transformed and untransformed regions. One approach to obtaining uniformly transformed individuals from bombarded meristems is by selfing the treated generation and selecting for added traits. The other approach is
Insert DNA into meristems at an early stage of organization, then
To stimulate continuous meristemoid development during antibiotic selection. The application of these approaches to orchid transformation is mainly limited by long development times and slow growth in tissue culture. Therefore, runs, especially Cymbid
No protocols are available for practical use of genetic engineering techniques in the genus ium.

[0008]

Accordingly, it is an object of the present invention to provide an efficient method for transforming orchids, especially members of the genus Cymbidium, using particle bombardment.

It is another object of the present invention to provide a method for stimulating meristemoid development that is continuous before and after particle bombardment to transform a developing protocorm-like body.

[0010] Another object of the present invention is to provide a method for transforming protocomb-like bodies, wherein the tissue may subsequently undergo morphogenesis.

Another object of the present invention is to provide a methodology that has broad application to genetic engineering of orchids, such that run improvement can be facilitated.

[0012]

SUMMARY OF THE INVENTION The present invention is a method of genetically engineering a run using a DNA construct comprising an expression cassette, wherein the construct comprises, in a 5 'to 3' direction, a transcription initiation region and It comprises a peptide coding region located downstream of the transcription initiation region and under its transcriptional control, and comprises the following (a)
(E) providing a primary protocomb-like body (PL) of the orchid in a liquid medium;
Culturing B) to induce a secondary PLB; (b) placing a PLB containing both the primary PLB and the secondary PLB on a target surface; (c) in the PLB much more than cells of the PLB Physically accelerating a small microprojectile, said microprojectile having a copy of a foreign gene construct comprising at least one foreign gene of interest and one selectable marker gene; (d) Inducing bombarded PLB to form proliferative PLB capable of forming shoots in a liquid medium, wherein during the inducing step the selectable marker gene confers resistance. Culturing the PLB in a medium containing the selection agent, thereby selecting for a PLB that is all transformed and expresses the gene of interest; and (e) Playing the door, thereby producing a clonal transgenic orchid plants step.

[0013] In one embodiment, the orchid tissue is of the genus Acianthus, Aerides, Anoectochilus, Ansel.
genus lia, genus Aplectrum, genus Arethusa, genus Calanthe, Cattle
Genus ya, Coelogyne, Corallorhiza, Cymbidium, Cy
genus pripedium, Crytopodium, Dendrobium, Epidendr
Genus um, genus Goodyera, genus Listera, genus Codes, Oncidium
Genus, Phalaenopsis, Pholidota, Rhynchostylis,
And a class consisting of the genus Vanda.

In one embodiment, the run is Cy.
A member of the genus mbidium.

[0015] In one embodiment, the PLB comprises meristems and tissues that can be induced into meristems and regenerated into plants or plantlets.

In one embodiment, the primary PLB
Is a meristem from the apical meristem of the orchid, and secondary PLB
And tissues that can be regenerated into plants or plantlets.

In one embodiment, the secondary PLB
Include meristem tissue from the primary PLB of the orchid and tissue that can be induced into proliferative PLB and regenerated into plants or plantlets.

In one embodiment, the proliferative PL is
B includes meristems from PLB explants, including both primary and secondary PLBs of orchids, and tissues that can regenerate into plants or plantlets.

In one embodiment, the foreign gene construct comprises a plasmid.

In one embodiment, the selectable marker gene encodes an expression of an antibiotic resistance trait.

[0021] In one embodiment, the foreign gene construct also comprises a screenable marker gene, wherein the expression of the screenable marker gene is PL.
PLB that can be detected in B, so that it survives under selection
Can be confirmed.

[0022] In one embodiment, the microprojectile comprises metal particles having a diameter of about 0.5 µm to about 3 µm.

In one embodiment, a plurality of the microprojectiles are provided, each of which has the foreign gene construct immobilized thereon, and wherein each of the microprojectiles is Driven in plant tissue targets.

In one embodiment, the method further comprises a step of rooting from the transformed plant tissue.

[0025] The methods disclosed herein can be used to produce transformed orchid plants, including transformed orchid cells, wherein the transformed orchid cells contain a heterologous expression cassette containing an expression cassette. A DNA construct, the construct comprising:
It includes, in the 5 'to 3' direction, a transcription initiation region and a region encoding a peptide located downstream of and under the transcriptional control of the transcription initiation region. Genetic manipulation of orchids is accomplished through the use of particle-mediated plant transformation techniques, in which tissue cultures of plant regenerable orchid species are transformed with DNA retained on small carrier particles. The culture techniques described herein produce a relatively large number of meristem cells to be bombarded, stimulate the continuous development of bombarded meristems, and It has been shown to be useful in increasing the frequency of transformation. Bombarded meristems are expanded and selected for the presence of the gene product encoded by the introduced gene. Shoot regeneration is then induced from the meristem and transgenic seedlings are generated. Preferred plants for practicing the invention are members of the genus Cymbidium.

The objects, advantages and features of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings.

[0027]

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, it has been found that the general approach of particle-mediated transformation of plant tissue can be successfully applied to orchids in tissue culture. This approach has enabled the generation of transgenic Cymbidium runs. Since the techniques used herein are equally applicable to other orchids, they are generally advantageous for manipulating disease resistance, altered flowering time, flower color and morphology, and other ornamental qualities. It is now possible to engineer orchids into transgenic orchid plants with unique characteristics. This process applies to all runs, especially Cymbidium
It is considered applicable to runs.

As used herein, the term “run” refers to Sp
An Orchidales member of the ermatophyta phylum. An exemplary run that can be used in the practice of the present invention is Orchid
members of the aceae, for example, Acianthus,
Aerides, Anoectochilus, Ansellia, Aplectrum, Areth
usa, Calanthe, Cattleya, Coelogyne, Corallorhiza,
Cymbidium, Cypripedium, Crytopodium, Dendrobium, E
pidendrum, Goodyera, Listera, Codes, Oncidium, P
halaenopsis, Pholidota, Rhynchostylis, and Vanda
Including species of the genus. Preferred for the practice of the present invention is Cy.
He is a member of mbidium.

[0029] The process described herein involves the introduction of an exogenous genetic construct into the genome of an orchid plant. Such an exogenous genetic construct is preferably DNA from another organism, whether homologous or heterologous, introduced into orchid cells through human manipulation. Exogenous genetic constructs usually include a coding sequence that encodes the production of a transcript or protein of interest in the orchid's cells. DNA constructs typically contain flanking regulatory sequences effective to effect expression of the protein, ie, the transcript encoded by the coding sequence, in transformed orchid cells.
Examples of flanking regulatory sequences are a promoter sufficient to initiate transcription in plant cells, and a terminator and / or polyadenylation sequence sufficient to terminate the gene product, either by termination of transcription or translation. It is also possible to include a translation enhancer located between the promoter and the coding sequence, which aids in the efficiency of expression of the gene product, especially of the protein product. Any of these regulatory regions should be able to operate in cells of the tissue to be transformed, either in a constitutive or tissue-specific manner. The DNA construct may optionally include flanking DNA sequences involved in chromatin organization. Examples of such DNA sequences include matrix attachment regions (Spiker and Thompson, Plant Physiol., 1996),
Transformation booster seq
uence) (Meyer et al., Proc. Natl. Acad. Sci. USA, 1988). The use of transformation booster sequences results in a several-fold increase in the frequency of transformation using biolistics (Buising and Benbow, Mol. Gen. Genet.,
1994). Use of adjacent matrix attachment regions enhanced expression of genes introduced into poplar (Han et al., Transgeni
c Research, 1997). It is also envisioned that gene products other than proteins can also be expressed in the inserted genetic construct. For example, the inserted construct may express an antisense RNA strand that is effective either to suppress the expression of endogenous genes in orchids or to inhibit disease processes by pathogenic organisms.

Genes of interest for use in orchids encompass a wide variety of phenotypic and non-phenotypic traits. Among the phenotypic characteristics are enzymes that provide resistance to disease, altered flowering time, control of plant color and morphology, manipulation of plant aroma, regulation of growth, and the like. The gene is
It can be obtained from prokaryotes or eukaryotes (bacteria, fungi, yeast, viruses, plants, and mammals), or can be wholly or partially synthetic.

[0031] One or more cassettes may be included, where:
This cassette can be used in tandem for independent gene expression. The genes may express the products independently of each other or may be regulated simultaneously. here,
The products can act independently or together. If multiple cassettes are used, they can be on the same plasmid or on different plasmids. If the cassettes are on different plasmids, these plasmids can be carried by the same microprojectile, or they can be carried on different microprojectiles, and these microprojectiles are mixed together and Driven by organizational targets.

An expression cassette can be constructed that includes a transcription initiation region, start codon, gene coding sequence, translation stop codon, followed by a transcription termination region. This direction is 5'-3 'in the direction of transfer. Cassettes are usually less than about 10 kilobases (kb) in length, frequently less than about 6 kb, and usually at least about 1 kb in length. The expression cassette can be provided in a DNA construct that also has at least one replication system. For convenience, it is common to have a functional replication system in Escherichia coli. In this manner, at each stage after each operation, the resulting construct can be cloned, sequenced, and the accuracy of the operation determined. In addition, or in place of the E. coli replication system, a broad host range replication system, such as a P-1 incompatible plasmid replication system, can be used. In addition to the replication system, there is often at least one marker that may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be used for selection in a prokaryotic host, while another marker may be used for selection in a eukaryotic host, especially an orchid host. Markers include biocides (eg, antibiotics,
Toxins, heavy metals, etc.); provide complementation by conferring auxotrophic hosts on prototrophy; or provide a visible phenotype through the production of novel compounds in plants. obtain. An exemplary gene that can be used is neomycin phosphotransferase (NP
TII), hygromycin phosphotransferase (H
PT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, a non-limiting example of a suitable marker is a β-protein that provides indigo production.
Glucuronidase, luciferase that provides visible light production, provides kanamycin resistance or G418 resistance
NPTII, HPT providing hygromycin resistance, and mutated aroA gene providing glyphosate resistance. Various fragments, including various constructs, expression cassettes, markers, etc., can be introduced sequentially by restriction enzyme cleavage of the appropriate replication system and insertion of the particular construct or fragment into an available site. After ligation and cloning, the DNA construct can be isolated for further manipulation. All of these techniques are fully exemplified in the literature, and specific examples are given in Sambrook.
Et al., Molecular Cloning: A Laboratory Manual, Cold S
pring Harbor Laboratory, Cold Spring Harbor, NY,
Found in 1987.

Some of the instruments or devices for introducing such carrier particles into plant cells are known to those skilled in the art. Any ballistic cell transformation device can be used in the practice of the present invention. An exemplary device is Bio-Rad Laboratories, 2000 Alfred Nobel Driv
e, Hercules, CA 94547 (phone number 510-741-1000), currently available from the Biolistics Particle Accelerator (model Biol
istic PDS-1000 / He). The device comprises a bombardment chamber, which is separated into two separate compartments by an adjustable height stopping plate. The acceleration tube is attached to the upper end of the bombardment chamber.
The macroprojectile is propelled by a stopping plate with an adjustable release of pressurized helium down an acceleration tube. The stopping plate has a bore hole formed therein. This perforation is smaller in diameter than the macroprojectile. Macroprojectiles carry microprojectiles, and macroprojectiles are aimed at and fired at perforations. When the stopping plate stops the macroprojectile, the microprojectile is propelled through the perforations. The target tissue is placed in a bombardment chamber such that the microprojectiles propelled through the perforation penetrate the cell walls of cells in the target tissue and deposit DNA constructs carried into the cells of the target tissue . Exemplary materials that can form a microprojectile include metals, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (eg, graphite, diamond). Metal particles are presently preferred. Non-limiting examples of suitable metals include tungsten, gold, and iridium. The bombardment chamber is partially evacuated before use to prevent atmospheric drag from excessively slowing down the microprojectile. The chamber is only partially evacuated so that the target tissue does not dry out during its bombardment. A vacuum between about 400-800 millimeters of mercury is suitable. Any target tissue that can later regenerate the shoot can be used in the practice of the present invention. The particular tissue selected will vary, depending on the clonal propagation system available and most appropriate for the particular species to be transformed.
Exemplary tissue targets are embryos, callus tissue, stems, existing meristems (eg, apical meristem and root meristem),
As well as induced meristems (eg, protocorm-like bodies). Preference is given to tissues selected from the class consisting of meristems (both existing and derived) and tissues capable of being induced into meristems. Protocomb-like bodies are more preferred. The target tissue is placed against the bombardment so that the microprojectile enters one or more cells to be transformed. A preferred clone propagation method for practicing the present invention is the following method. The method includes several steps: 1) A step of treating the excised apical meristem on a solid PLB induction medium for a time sufficient to induce the formation of a protocorm-like body on the tissue. 2) transferring the treated target tissue to a liquid PLB growth medium to stimulate continuous growth of meristems. 3) a step of treating the protocomb-like body (PLB) with a shoot regeneration medium for a time sufficient to induce shoot formation. 4) transferring the treated shoots to a nutrient medium that does not contain exogenous growth factors until roots develop. This method results in the production of vegetative propagules that can be transferred to soil and grown to form adult plants.

The tissue culture system described immediately above has a growth window (de
velopmental window). Meanwhile, the greater number of cells in the tissue to be bombarded are in certain phases of their cell cycle, where they are more amenable to integration or expression of foreign DNA. Thus, the higher the proportion of cells in this phase in the bombarded tissue, the greater the likelihood of obtaining stable expression in the bombarded tissue. The tissue is preferably after treating the tissue in liquid PLB growth medium for at least a time sufficient to induce the formation of superficial meristems from the peripheral division of the first and second subepidermal cell layers on the target tissue. , DNA
Bombarded with construction. Also, the tissue is preferably bombarded with a DNA construct and then transferred to a shoot regeneration medium for shoot production.

The DNA coated on the small carrier particles delivered into the cells is incorporated into the genomic DNA of any part of the treated cells. Thereafter, with appropriate selection or screening, the entire tissue or organism is generally regenerated from the transformed cells. When applying the general process of particle-mediated transformation of plant tissue to obtain transgenic plants, avoid regeneration of transgenic plants from chimeric tissues consisting of both transformed and non-transformed cells is important.
Generally, it is possible to obtain non-chimeric transgenic plants by bombarding the gene into differentiated tissue (eg, meristem). It can then be regenerated directly into plantlets, as described in US Pat. No. 5,015,580. The present invention contemplates stimulating continuous development of meristems by culturing the protocorm-like body in a liquid medium prior to particle bombardment, and subjecting proliferating meristems to particle bombardment. I do. In the present invention, it is also found that stably transformed orchid plants can also be obtained by culturing bombarded meristems in a combination of liquid and solid media.

The use of a dominant selectable marker is an integral part of the transformation strategy. Important factors for efficient selection of transgenic cells are the types of selectable markers, their expression levels, and the time and intensity of selection after transformation (for a review, see Angenon
Et al., Angenon et al., Plant Molecular Biology Manual C1: 1
-13, Kluwer Academic Publishers, Boston, Gelvin and Schilperoort eds., 1994). Available markers include genes encoding antibiotic resistance, antimetabolites resistance, and herbicide resistance, as well as genes that confer resistance to toxic levels of amino acids or analogs. Neomycin phosphotransferase II (NPTI
I) is the most widely used selectable marker for plant transformation. Through phosphorylation, this can be achieved with aminoglycoside antibiotics (eg, kanamycin, neomycin,
Geneticin (G418) and Paromocin (paromoci)
n)) inactivate. The sensitivity of plant cells to a selectable marker depends on the genotype; the physiological conditions, size and type of explants; and tissue culture conditions. Therefore, the minimum level of selection agent that can completely inhibit the growth of non-transformed cells should be determined for each transformation and regeneration system. Kanamycin is generally 50-
Used at a concentration of 200 mg / l. Selection should minimize the production of bombarded cells because the selection agent should effectively suppress the growth of non-transformed cells while minimizing the toxicity of dying cells to neighboring transformed cells. It can be skipped for a period sufficient to ensure sexual growth.

[0037] In contrast, a screenable marker gene is a gene that encodes a product that is easy to detect and therefore easy to screen for transgenic tissue. One useful screenable marker is GUS (ie, the β-glucuronidase gene). This is where a convenient colorimetric assay system exists. The use of introns containing the GUS gene
Genes with introns are generally preferred because they are expressed only in eukaryotic (ie, plant) cells and not in any contaminating microorganisms. GUS expression can be observed for both transient gene expression after particle-mediated transformation and stable gene expression from virtually all plant tissues after selective regeneration. The level of transient gene expression using the GUS assay system has a general correlation with the efficacy of obtaining stable transformation, but this correlation is not a strong correlation. Once the plant or plantlet has been shown to have been transformed, the plant cells can then be used repeatedly in tissue culture, followed by propagation of the plantlet.
Thus, the modified plants can be regenerated repeatedly through the use of cell and tissue culture. In some cases, reproduction can be maintained from the seed, but monitoring for loss of the introduced gene is prudent.

The following examples are provided by way of illustration, not by way of limitation. A description of the key terms used in specific examples is as follows: meristem-undifferentiated plant tissue that gives rise to new cells; protocomb-derived from embryo, apical meristem and leaf primordia. Protocomb-like body (PLB)-tissue structure derived from vegetative tissue and resembling a seedling protocomb; primary PLB-PLB induced by aseptically culturing the tip of apical meristem; 2 Next PLB-1 during culture
PLB formed on the surface of the next PLB; proliferating PLB-growing on the surface of PLB explants containing both primary and secondary PLBs
PLB.

[0039]

EXAMPLES Example 1 Preparation of Cymbidium PLB for Bombardment The meristem tips of Cymbidium orchids were isolated under sterile conditions, and Paek et al., Proc. NIOC, Nagoya, Japan (1
997) to form primary PLB. Next, primary PLB younger than 12 months was replaced with 5% sucrose, 2 mg / l, α-naphthaleneacetic acid, 0.1% charcoal (charco
al), and Knud supplemented with 0.7% Bacto-agar (pH 5.4)
son Orchid medium (Duchefa Biochmie BV, Izaak Enchede
weg 40,2031 CS Haarlem, The Netherlands). Before bombardment, gently shake PLB (50
100100 rpm) in liquid medium for 7-10 days, and then in a 60 mm Petri dish serving as the target surface, 5
Knudson Orchi supplemented with 100% sucrose and 0.7% agar
d Placed in a 1-2 cm diameter circular area on the medium. Controls (non-bombardment tissue) were plated as well. All plant material incubations were performed using cool white fluorescent light.
It was 25 ± 2 ° C. in the 16-hour light period under 〜45 μE / m ² · sec. Example 2 Determination of the effect of liquid culture on PLB growth before bombardment Murashige and Skoog (MS), 15 Physiologia Plan with 3% sucrose and no phytohormones
Primary PLBs were derived and formed secondary PLBs prior to bombardment using three different culture techniques using tarum 31 (1962) medium; shaking liquid culture, static liquid culture, and solid culture. . For solid culture, 40 PLBs
Plated in 125-mm Petri dishes containing 25 ml MS medium supplemented with 0.7% agar. For liquid culture, shake (50-
Forty PLBs were cultured in a 250-ml Erlenmeyer flask (Earlymeyer-flask) containing 50 ml of MS liquid medium without shaking. Controls (non-bombardment tissue) were plated as well. Cultured PLB
Were evaluated for secondary PLB production. FIG. 1 shows the data obtained from this procedure. The y-axis shows the growth rate (average number of PLBs grown per PLB) and the x-axis shows the number of days in culture before bombardment (or "shooting"). Slowly shaking liquid cultures resulted in the highest growth of PLB. Example 3 Gene construction The plant gene expression vector used in this example was
Shown in FIG. 2 and in Han et al., Transgenic Research,
PKH200 according to 1997. This vector contains, in addition to one antibiotic resistance marker (kanamycin resistance) useful in bacterial hosts, separate plant expression cassettes for two different genes that can be expressed in plant cells. One of the plant gene expression cassettes contains the gene NPTII, which confers resistance to the antibiotic kanamycin. Kanamycin has been shown to be useful as a selectable marker in several plant species. The NPT-II expression cassette contains a nopaline synthase promoter and octopine synthase polyadenylation sequence flanking the coding sequence. The second expression cassette encodes the β-glucuronidase enzyme, GUS. It serves as a screenable marker since its expression can be detected by a simple histochemical assay. GUS
The gene is 3 'to the cauliflower mosaic virus 35s promoter (CaMV35s) and 5' to the nopaline synthase polyadenylation region. 2
One expression cassette contains a matrix attachment region (MAR) fragment from a tobacco genomic clone (Hall et al., Proc.
Natl. Acad. Sci. USA, 1991). Example 4 Microprojectile Bombardment Parameters The parameters used for the actual particle acceleration process were model Biolistic PDS-1000 / He configured as described in the manual provided by the manufacturer.
(Bio-Rad Laboratories, 2000 Alfred Nobel Drive, H
ercules, CA 94547). Bio-Rad Laboratories also includes stopping plates, rupture disks and microprojectiles
Powered by Plasmid DNA is essentially converted to Klein
Precipitated in the presence of microprojectiles as described in 327 Nature 70 (1987). Microprojectiles were prepared from gold particles having a diameter of about 0.6 mm. Each bombardment contains approximately 5 to 5 particles of bound 0.42 mg of plasmid DNA in a total slurry volume of 6 ml.
00 μg was delivered. The DNA preparation used for microprojectile bombardment contained a mixture of supercoiled, open circular, and linear molecules. The bombardment chamber was evacuated at a pressure of 28 inches of mercury. Remove all bombardments from the stopping plate to the surface of the tissue to be treated
Performed with pressurized helium in the range of 1,100 to 1,550 psi, with a range of mm. Example 5 Tissue Culture After Bombardment Tissue culture after bombardment consists of two phases (proliferative PLB
Induction and selective culture): 1) proliferative PLB
Induction-One-half strength M with 3% sucrose and no phytohormone to promote growth of proliferative PLB for 10-45 days after bombardment
urashige and Skoog, Physiologia Plantarum, 1962
Two (liquid vs. solid) culture techniques using media were used. For solid culture, 25 ml MS supplemented with 0.7% agar
Forty bombarded PLBs were plated in 125-mm Petri dishes containing medium. For liquid culture, shake (5
(0-100 rpm) or forty bombarded PLBs were cultured without shaking in a 250-ml Erlenmeyer flask containing 50 ml of MS liquid culture. Cultured PLB
The production of proliferative PLB was evaluated. Table 1 shows the results of this experiment. Slowly shaken liquid cultures were superior to solid cultures in stimulating the continued development of PLB. No kanamycin selection was used during the liquid culture. The culture technique was evaluated for the production of proliferating PLB. 2) Selective culture-with multiple proliferating PLBs on the surface 4
Zero secondary PLBs were transferred to 125-mm Petri dishes containing 25 ml MS medium supplemented with 0.7% agar and 100-200 mg / l kanamycin. Cultures were maintained by passage weekly into fresh medium. The environmental conditions in the growth chamber were the same as those used in Example 1. Example 6 Bombarded Tissue Assay for GUS Activity A subset of PLB was sampled at various times to determine the frequency of β-glucuronidase (GUS) activity. PLB was converted to 2.0 mM 5-bromo-4-chloro-3-indoyl-β
-D- glucuronic acid (X-gluc), 0.1M NaPO 4 buffer, pH 7.
_{0,0.01M Na 2 EDTA, pH7.0,0.5mM K 4} Fe (CN) 6 · 3H 2 O, pH
7.0 and 0.5 mM K ₃ Fe (CN) ₆ and 0.1% Triton X-1
Incubate for 18 hours at 37 ° C. in a substrate solution containing 00. The GUS focus on PLB was scored under a dissecting stereo microscope.

FIG. 3 graphically illustrates the results achieved in a repeated assay of bombarded PLB in an assay for transient GUS expression. This assay, which is destructive to tissues, is used for 24, 48, 72
Went after hours. Example 7 Selection and Analysis of Transformed PLB Kanamycin selection was used to screen for stable integration events that were relatively rare from transient activity.
PLB cultured in liquid medium for 10-45 days as described in Example 5 was replaced with the same medium (but 0.7% Bacto agar and 100-20%) to select for transformed PLB.
(Supplemented with 0 mg / l kanamycin). Cultures on kanamycin were passaged weekly to fresh medium to minimize the number of escapes due to reduced kanamycin toxicity. Transformed PLB was first identified by its ability to grow on kanamycin medium. Subsequently, stable integration and expression were performed, respectively, on PC
Confirmed by R analysis and GUS expression assay. In fact, hundreds of PLBs were produced from continuous PLB breeding. More than 100 such PLBs survived the kanamycin selection. Two
More than 0 plantlets were obtained from kanamycin resistant PLB. Three randomly selected seedlings were sacrificed for the GUS assay, and all three turned blue overall. This indicates clonal expression. FIG. 3 shows GUS expression from bombarded PLB. In essence, long-term and stable expression of the introduced foreign gene has been achieved. These plantlets are expected to be easily grown on rooted orchids. Based on past experience with other plants,
The inserted gene must generally segregate normally to the next generation and be inherited through the normal rules of Mendelian inheritance. In general, the process can reproducibly produce transformed PLB, which can give rise to whole orchid plants.

[0041]

[Table 1] ¹ liquid culture: 40 bombarded PLBs, 0.7%
Agar, supplemented with 3% sucrose, and without phytohormone to promote proliferative PLB development, half concentration of Mu
rashige and Skoog (MS), 15 Physiologia Plantarum
31 Plated in 125-mm Petri dishes containing 25 ml of 1962 medium. For liquid culture, 40 bombardments
PLB was cultured in 250-ml Erlenmeyer flasks containing 50 ml MS liquid culture with shaking (50-100 rpm) or without shaking. ^Two cultured PLBs were evaluated for production of proliferative PLB. Growth rate is expressed as the total number of PLBs produced, divided by the number of initial PLBs. Data was collected from three independent experiments.

[0042]

The present invention can provide an efficient method for transforming orchids, particularly members of the genus Cymbidium, using particle bombardment.

The method of the present invention is used to stimulate meristem development before and after bombardment, while reducing the toxic effects of oxidized phenols and resulting in successful transformation. The bombarded PLB is propagated and selected for the presence of the gene product encoded by the introduced gene. Short regeneration is then derived from PLB and transgenic seedlings are produced.

[Brief description of the drawings]

FIG. 1. 3 against PLB expansion before particle bombardment.
FIG. 3 shows the effect of two different culture techniques. Solid: PLB
Was cultured on agar coagulation medium; liquid: steady-state liquid culture;
LSL: liquid culture with low speed shaking (50-100 rpm). Growth rate is expressed as the rate of increase in the number of PLBs during the culture period.

FIG. 2 is a schematic diagram of the T-DNA region of plasmid pKH200. The relative positions of the GUS-INT and NPTII genes are indicated for the left border (LB) and right border (RB) of T-DNA, flanked by the tobacco matrix region (MAR). The T-DNA region has been drawn to scale.

FIG. 3 is a chart showing the effect of two culture methods on PLB proliferation after bombardment. Proliferation rate is shown as total number of PLBs generated / number of initial PLBs. The average growth rate is calculated from three separate experiments.

FIG. 4 is a chart showing long-term and stable expression of the marker (GUS) gene in three transgenic orchid strains after active selection and regeneration from transformed PLB.

FIG. 5 shows PCR amplified fragments of the introduced selectable marker (NPTII) gene from two transgenic run strains. Lane 1: 750 bp size marker; Lane 2: plasmid pKH200 DNA; Lane 3: untransformed orchid plant; Lanes 4 and 5: transgenic strains KTO9 and 12, respectively.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 15/09 C12R 1:91) (56) References JP-A-7-255300 (JP, A) Plant Science, 102 [1 P. 81-89 (1994) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01H 1/00 A01H 5/00 BIOSIS (DIALOG) JICST file (JOIS)

Claims (8)

(57) [Claims] 1. A method for genetically engineering a run using a DNA construct comprising an expression cassette, wherein the construct is located in a 5 ′ to 3 ′ direction at a transcription initiation region and downstream of the transcription initiation region. And containing the peptide coding region under its transcriptional control, comprising the following steps: (a) The primary protocomb-like body of the orchid (PL)
Culturing B) to induce secondary PLB; (b) PLB comprising both primary and secondary PLB
Disposing at least one foreign object of interest in said PLB.
Gene and at least one selectable marker gene.
Microprojector containing a copy of the DNA construct
Yl enough physically accelerating Engineering; the PLB from (d) (c) cultured in a liquid medium, increasing
(E) combining the proliferative PLB with at least one selective agent.
Induced to form shoots in liquid medium containing
Te, one selectable marker gene wherein the at least, the selection
Confer resistance to the proliferative PLB in the presence of a drug
Confer a prototrophy or a visible phenotype
Forming a selectable PLB having a shoot
Wherein said selectable PLB is a transformed plant tissue
In it, the process; (f) Engineering culturing the selectable PLB having the chute
And (g) transferring the shoots of the selectable PLB to a clonal plasmid.
A step of reproducing the scan transgenic orchid plants including, a method, wherein the orchid is a member of Cymbidium genus methods. 2. The method of claim 1, wherein said PLB comprises meristem tissue and tissue capable of inducing meristem tissue and regenerating into plants or plantlets. 3. The method of claim 1, wherein the primary PLB comprises meristems from apical meristems of orchids and tissues that can be induced into secondary PLBs and regenerated into plants or plantlets. Wherein including said secondary PLB is meristem primary PLB from the run, and able to induce a proliferative PLB and tissue may be regenerated into a plant or plantlet The method of claim 1. 5. The primary PLB of a run, wherein the proliferative PLB is a primary PLB of a run.
And meristems from PLB explant containing both secondary PLB, and a tissue capable of regeneration into a plant or plantlet The method of claim 1. 6. The DNA construct comprises a plasmid.
The method of claim 1. 7. The method of claim 1, wherein said selectable marker gene encodes an expression of an antibiotic resistance trait. Wherein said microprojectiles comprises metal particles having a diameter of about 0.5μm~ about 3 [mu] m,
The method of claim 1.