KR101209121B1 - Method for producing herbicide-resistant lily plants and herbicide -resistant lily plants produced by the method - Google Patents

Abstract

The present invention relates to a method for producing herbicide-resistant lilies and to herbicide-resistant lilies, and more specifically, (1) Picloram is added by slicing the scales of the incubation device of the lily to a size of 2 to 3 mm along the horizontal axis. Inducing embryogenic callus by culturing in the solid MS solid medium; (2) co-culturing the recombinant vector into which the herbicide resistance gene is inserted into the induced callus by particle bombardment; (3) selecting the co-cultured plant cells for 12 to 16 weeks in a selection medium to which herbicide is added; (4) a method for producing herbicide resistant lilies comprising regenerating the selected lily callus in a regeneration medium, a herbicide resistant lily plant prepared according to the above method, and a seed of the plant.

Lily, particle shock, transformation, herbicide, resistant, seed

Description

Method for producing herbicide-resistant lily and herbicide-resistant lily plants produced by the method

The present invention A method for producing herbicide resistant lilies and thus herbicide resistant lilies and seeds thereof.

Liliaceae belonging to the genus lilium, about 130 indigenous species, including 71 species in Asia, including Korea, China and Japan, 22 species in Europe and Russia, and 37 species in North America, temperate and temperate regions of the Northern Hemisphere (10 It is widely distributed in ° ~ 60 °), and including more than 600 varieties are known. The international lily register, published by the Royal Horticultural Society (RHS) in 1967, categorized lilies into seven groups according to their hybridization affinity: Asiatic group, Martagon group, Candidum group, American group, Longiflorum group, and Trumpet. Asiantic, Longiflorum, and Oriental group, which are distributed in Asia, have been used for classical breeding (Shimizu, 1987, Seibundo Shinkosha, Tokyo pp 148-165).

Since 1960, more than 7,000 varieties have been registered worldwide. Currently, breeding business is actively carried out in fever and Japan. In Korea, Nari is recognized as a high-value-added crop in the early 1990s. As a result, full-scale breeding research began, and in 1998, Nari varieties were cultivated at the Horticultural Research Institute. By early 2005, more than 40 varieties were registered in Korea such as Seonyum, Mir, and Ayr. Commentary). However, most of the cultivars grown in Korea are Asiatic strains grown through the classical breeding method, and the varieties raised through the recent transformation methods have not been reported.

Recently, with the development of genetic engineering techniques, a transformation technology for introducing useful genes (viral resistance genes, herbicide resistance genes, color-related genes, etc.) has been developed, and many studies have been made, and transgenic plants have been reported.

For research related to the transformation of Lilium, Hoshi et al. (2004, Plant Cell 9: 1251-1264) attempted to transform Agrobacterium into the cell-derived cell mass of Oriental Liri, Expression was confirmed, and PCR was reported to confirm the introduction of the gene into plant genomic DNA.

Korean Patent Registration No. 0529005 discloses a transformed lily plant expressing a chalcone synthase antisense gene and a method of preparing the same through agrobacterial mediated transformation, but is different from the method by the particle impact method of the present invention.

The present invention has been made in accordance with the requirements as described above, the present invention is to produce a herbicide-resistant lily by transforming the herbicide resistance gene to lily using Particle Bombardment (Particle Bombardment).

In order to solve the above problems, the present invention (1) to induce embryogenic callus by incubating the scales of the incubation device sphere of the lily to a size of 2-3mm in the horizontal axis and incubated in MS solid medium to which Picloram (Picloram) is added step; (2) co-culturing the recombinant vector into which the herbicide resistance gene is inserted into the induced callus by particle bombardment; (3) selecting the co-cultured plant cells for 12 to 16 weeks in a selection medium to which herbicide is added; (4) it provides a method for producing herbicide-resistant lily comprising the step of re-differentiating the selected lily callus in a regeneration medium.

The present invention also provides a herbicide resistant lily plant prepared according to the above method.

The present invention also provides seeds of the herbicide resistant lily plant.

According to the present invention, by providing herbicide-resistant lilies can reduce the herbicide effort and costs that are a problem in lily cultivation can contribute to farm income.

In order to achieve the object of the present invention,

(1) inducing the embryogenic callus by slicing the scales of the in-vehicle culture element sphere of the lily to a size of 2 to 3 mm in the horizontal axis and culturing it in MS solid medium to which Picloram was added;

(2) co-culturing the recombinant vector into which the herbicide resistance gene is inserted into the induced callus by particle bombardment;

(3) selecting the co-cultured plant cells for 12 to 16 weeks in a selection medium to which herbicide is added;

(4) it provides a method for producing herbicide-resistant lily comprising the step of re-differentiating the selected lily callus in a regeneration medium.

First, in order to obtain embryogenic callus, the scales of lilies are cultured in Murashige & Sukog (Murashige & Skoog, MS) medium containing plant growth regulators and the like to induce plant somatic embryo callus. At this time, as a plant growth regulator, it is preferable to use picloram 0.5 ~ 2mg / L, preferably 1mg / L.

In the method according to an embodiment of the present invention, the step of inducing lily callus of step (1) is 0.5-2 mg / L Picloram, 4-6% sucrose, 0.2-1 g / L casein in MS medium It is preferred to add 0.3-0.5% gelite, more preferably Picloram 1 mg / L, 5% sucrose, 0.5 g / L casein, 0.4% gelite. In addition, it is more preferable to incubate the scales in advance in the plane to obtain the device spheres, and then to separate the pieces from the device spheres and cut them to a size of 2 to 3 mm in the horizontal axis, and the induction period is preferably 8 to 12 weeks.

In the method according to an embodiment of the present invention, the callus obtained from the scale of step (1) is propagated by shaking culture in a liquid medium in which 1 mg / L of picloram and 3% sucrose are added to MS medium, and 5 to 10 It is preferred to subculture once per day, more preferably 7 days.

In the method according to an embodiment of the present invention, the particle impact of the lily callus of the step (2) is finely chopped to a diameter of 1 ~ 3mm 1 day before performing shaking culture in MS liquid medium to which 1mg / L picloram added It is more preferable to carry out particle impact by transferring the solid medium to which 0.125M of sorbitol, an osmotic pressure regulator, was added 1 hour ago.

In the method according to an embodiment of the present invention, the plant expression vector of step (2) is a herbicide resistance gene is inserted, herbicide resistance gene may be used bar, aroA, bxn, etc., preferably the bar gene . The lily transformed with the bar gene may select a transformant by using a herbicide, Vasta. More preferably, the plant expression vector is a pDM302 vector described in FIG. 2. The vector is a pDM302 vector consisting of the bar gene under the Actl promoter.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminators of the octopine gene of Bacterium tumefaciens ( Agrobacterium tumefaciens ), but are not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

In the method according to an embodiment of the present invention, the particle impact experiment of step (2) was performed using PDS-1000 / He System (BidRad, Richmond, Calif.), And the plasmid DNA of the recombinant plant expression vector was added to the gold particles. In the coating method, after washing the particles using ethanol and distilled water, 50% glycerol was added to prepare a microcarrier. Plasmid DNA (1 μg / μl), 2.5M CaCl ₂ and 0.1M spermidine were added to the prepared micro-carrier, mixed, and the supernatant was removed by centrifugation and washed by adding 70% ethanol. Finally, resuspended with 100% ethanol and dispensed into 10ul per micro-carrier and dried in sterile culture.

In the method according to an embodiment of the present invention, the particle impact of step (2) was used 1g embryogenic callus and 2μg plasmid DNA per impact, 500μg gold particles, 1,100psi helium, 9cm flight distance It is preferable to carry out.

In the method according to one embodiment of the invention, the co-culture period of step (2) is 6-8 days, preferably 7 days. The co-culture period refers to a period for introducing the vector into the lily callus after coating the plasmid DNA of the recombinant plant expression vector pDM302 with gold particles on the lily callus and performing particle impact.

In the method according to an embodiment of the present invention, the period for selecting the co-cultured lily cells in the selection medium of step (3) is preferably 12 to 16 weeks, the antibiotic is applied to the antibiotic resistance gene used in the recombinant plant expression vector Accordingly phosphinothricin was used.

In the method according to an embodiment of the present invention, the selection of the step (3) is picloram 0.5 ~ 2mg / L, 2 ~ 4% sucrose, 2 ~ 5mg / L phosphinothricin and 0.3 ~ in MS medium 8 weeks in solid medium with 0.5% gelite, subcultured at intervals of 2 weeks in medium with 4-6 mg / L phosphinothricin, picloram 0.5-2 mg / L, 2-4% sucrose and Six weeks can be passaged at 7-day intervals in a liquid medium supplemented with 4-6 mg / L phosphinothricin and rotationally shaken at 50-150 rpm. More preferably, the selection of step (3) is carried out in a solid medium in which 1 mg / L of picloram, 3% sucrose, 2-5 mg / L phosphinothricin, and 0.4% gelite are added to MS medium. The period of time is preferably 5 to 8 weeks in a liquid medium that does not contain 8 to 10 weeks of zeolite, more preferably 8 weeks in a medium to which 2 mg / L phosphinothricin is added, and a medium to which 5 mg / L phosphinothricin is added. Passage at two-week intervals at 6 weeks in a liquid medium supplemented with picloram 1 mg / L, 3% sucrose and 5 mg / L phosphinothricin at 7-day intervals and shaken at 100 rpm for phosphinotri Obtained callus showing resistance to the scene.

In the method according to an embodiment of the present invention, the re-differentiation of step (4) is 5 ~ 10mg / L phosphinothricin and 0.3 ~ 0.5% gelite (preferably 0.4% gel in MS medium without hormones) It is preferable to transfer to a solid medium to which light) is added, and to perform selection and re-differentiation for 8 weeks. More preferably, it is cultured in a dark room temperature to obtain lily domains that exhibit resistance to phosphinothricin.

The present invention also provides a herbicide resistant lily plant prepared according to the above method. The prepared lily plants will be resistant to various herbicides depending on herbicide resistance genes. For example, if the herbicide resistance gene is bar, it will be resistant to herbicide batha. Therefore, the herbicide-resistant lily produced by the method of the present invention may be usefully used to reduce the herbicidal effort which is a problem in lily cultivation.

The present invention also provides seeds of the herbicide resistant lily plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

From plants Callus  Formation and Plant Regeneration

Peel off each of the incubation spheres of Marco Polo, an Oriental Lilium 'Marco Polo', into slices of 2 to 3 mm in the horizontal axis, where 1 mg / L of picloram, 5% sucrose, 0.5 g / L casein and 0.4% gelrite Embryonic callus was induced by culturing in the added MS solid medium. The embryogenic callus derived from the scales was removed and subcultured in the same medium at two week intervals, and some of the induced embryogenic callus were 1 mg / L, 3% sucrose, 0.5 g of picloram without addition of zeolite. / L casein was transferred to the liquid medium and cultured by rotating shaking at 100 rpm for growth. Subculture of embryogenic callus was used for transformation by mass proliferation, replacing 4/5 of the medium with the same amount of fresh medium every week.

2. Transformation method and selection

Particle impact experiments were performed using the PDS-1000 / He System (BidRad, Richmond, Calif.), And gold particles were coated with plasmid DNA using the method of Sanford et al. (1991, Technique 3: 3-16). After washing the particles using ethanol and distilled water to prepare a micro-carrier by adding 50% glycerol. Plasmid DNA (1 μg / μl), 2.5M CaCl ₂ and 0.1M spermidine were added to the prepared micro-carrier, mixed, and the supernatant was removed by centrifugation and washed by adding 70% ethanol. Finally, resuspended with 100% ethanol and dispensed into 10ul per micro-carrier and dried under laminar air flow. Particle impact of Lilium callus was finely chopped to 1 ~ 3mm in diameter one day before shaking and cultured in MS liquid medium containing 1mg / L picloram and treated in solid medium added with 0.125M of sorbitol, osmotic pressure control agent 1 hour ago. Particle shock was then performed. 1g embryogenic callus and 2µg plasmid DNA were used per impact, 500µg of gold particles, 1,100psi of helium, and 9cm flight were performed.

After particle impact, callus was cocultured without picking for 1 week in MS solid medium containing 1 mg / L of picloram, and the selection culture was 2-5 mg / L phosphino in 1 mg / L of picloram and 3% sucrose. Incubate for 8 to 10 weeks in solid medium with trisine and 0.4% gelite, for 5 to 8 weeks in liquid medium without gelite, and for 6 weeks in liquid medium with 5 mg / L phosphinothricin. Callus showing resistance to pinotriscin was obtained. The regeneration medium was transferred to a solid medium containing 5-10 mg / L phosphinothricin and 0.4% gelite added to the MS medium containing no hormone, and selected for 8 weeks in the dark and redifferentiated.

3. Transformant Analysis

For transformant analysis, 100 mg of the leaves of the growing plant in the greenhouse were purified and crushed with pestle, and DNA was isolated using a DNA kit (G-spin For Plant Genomic DNA Extraction Kit from iNtRON). PCR using Bar gene forward primer 5'-GTC AAC TTC CGT ACC GAG CCG CAG-3 '(SEQ ID NO: 1) and reverse primer 5'-CAT GCC AGT TCC CGT GCT TGA AG-3' (SEQ ID NO: 2) Analysis was performed. DNA amplification reaction was denatured at 95 ° C for 5 minutes using Palm cycler (Corbett Research, 2003), then 1 minute (denature) at 94 ° C, 1 minute (annealing) at 62 ° C, and 1 minute at 72 ° C. ) Was repeated 40 cycles and last extended for 10 minutes at 72 ℃ to complete the PCR reaction at 25 ℃ and stored at -20 ℃.

PCR products were loaded with 5 μl with 1.5 μl loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol) and electrophoresed at 80V on 1% agarose gel with ethidium bromide. After running for 2 hours using 0.5X TBE buffer (pH 8.0) as a running buffer, separated DNA bands were photographed using a digital camera under a U.V lamp.

< Example  1. from in-plant foods Callus  Induction and Plant Regeneration>

Callus organics from lily cultivars cultured on board took about 8 weeks in medium containing 1 mg / L of picloram, 5% sucrose, and 0.4% gelite in MS medium, and the induced callus was picloram in MS medium. In a liquid medium to which 1 mg / L and 3% sucrose were added, the culture was grown by rotating shaking at 100 rpm and cultured for 4 weeks to obtain a sufficient amount of callus for transformation. Plant regeneration was observed in the case of cancer culture for 2 to 3 months in the MS medium without hormones to generate the device spheres from callus.

< Example  2. Transformation Methods and Selection>

Lily callus to be used in the particle impact experiment was finely chopped to a diameter of 1 ~ 3mm 1 day before the particle impact, shaken incubated in MS liquid medium containing 1mg / L picloram, 1 hour before the osmotic pressure control agent sorbitol 0.125M was added Particle shock was carried out by transferring from the solid medium. The plasmid DNA pDM302 of the recombinant vector is bar Having a gene, the DNA was extracted and subjected to particle shock. 1g embryogenic callus and 2µg plasmid DNA were used per impact, 500µg of gold particles, 1,100psi of helium, and 9cm flight were performed.

After particle impact, callus was co-cultured for 7 days without selection in MS solid medium containing 1 mg / L piccloram, and the selection culture was 2-5 mg / L phosphino in 1 mg / L piculoram and 3% sucrose. Incubate for 5 to 8 weeks in a liquid medium containing 8 to 10 weeks of zeolite in a solid medium containing trisine and 0.4% gelite, and incubate for 6 weeks in a liquid medium containing 5 mg / L phosphinothricin. Callus showing resistance to phosphinothricin was obtained. Callus resistant to phosphinothricin was bright yellow in color and continued to grow, and callus without resistance was found to be brown or dark in color and unable to grow. Regeneration medium is a solid medium containing 5 ~ 10mg / L phosphinothricin and 0.4% gelite in hormone-free MS medium.The callus was cut into 2 ~ 4mm in diameter and selected for 8 weeks in the dark. The calligraphy of a lily was formed right on the callus.

< Example  3. Transformant Analysis>

Genetic analysis and packaging assays were carried out using the 'Marco polo' transformant 126 strain transformed with the bar gene through particle shock. The system moved into the verification of continuous bar gene confirmed a total of 126 strains transgenic plants for PCR analysis and 1,000 mg / L phosphino Tree transfer and expression of the bar gene herbicide-resistant packaging black spray gods in the result, 2 years The strains were 105 strains, 102 strains showed herbicide resistance for 2 years, and 85 strains were found to match the PCR and field test results.

1 is a photograph of the lily callus and elemental sphere GUS expression of transgenic callus and in-flight selection.

A: GUS expression of callus after particle impact,

B: lily callus on board

C: nontransformant lily bulb of selection medium,

D: transgenic lily bulb of selection medium

2 is a schematic diagram of the pDM302 vector.

3 is a picture of greenhouse growth after purification.

4 is a test photograph for confirming phosphinothricin titer concentration.

FIG. 5 is a herbicide resistance test performed by spraying Vaster with 1,000 mg / L of phosphinothricin active ingredient.

Figure 6 is a photograph showing a PCR analysis for identifying the bar gene of the transformant. Lane 1: 100 bp DNA ladder marker

7 shows PCR and herbicide resistance results of the lily transformant 126 strain.

<110> Industry-Academic Cooperation Foundation, Dankook University <120> Method for producing herbicide-resistant lily plants and          herbicide -resistant lily plants produced by the method <130> PN09241 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 gtcaacttcc gtaccgagcc gcag 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 catgccagtt cccgtgcttg aag 23  

Claims (9)

(1) inducing the embryogenic callus by slicing the scales of the in-vehicle culture element sphere of the lily to a size of 2 to 3 mm in the horizontal axis and culturing it in MS solid medium to which Picloram was added; (2) co-culturing the recombinant vector containing the bar gene into the induced lily callus by performing particle bombardment; (3) selecting the co-cultured plant cells for 12-16 weeks in a selection medium to which phosphinothricin was added; And (4) The selected lily callus was transferred to a solid medium to which 5 to 10 mg / L phosphinothricin and 0.3 to 0.5% zeolite were added to MS medium containing no hormone, and selection and re-differentiation were carried out for 8 weeks in the dark state. A method of making a herbicide resistant lily comprising the step of performing. The method of claim 1, wherein step (1) comprises adding picloram 0.5-2 mg / L, 4-6% sucrose, 0.2-1 g / L casein and 0.3-0.5% gelite to MS solid medium. How to. delete The method of claim 1, wherein the coculture period of step (2) is 6 to 8 days. delete The method of claim 1, wherein the selection of the step (3) comprises piclaram 0.5-2 mg / L, 2-4% sucrose, 2-5 mg / L phosphinothricin and 0.3-0.5% gelite in MS medium. 8 weeks, 4-6 mg / L phosphinothricin-added subcultured at 2-week intervals, picloram 0.5-2 mg / L, 2-4% sucrose and 4-6 mg / L Method for subculture of 6 weeks at 7 days intervals in a liquid medium to which phosphinothricin is added and rotating shaking culture at 50 ~ 150rpm. delete delete delete

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