KR102584685B1 - Method for transformation of grape - Google Patents

Abstract

The present invention relates to a grape transformation method for grape breeding. The grape transformation method can effectively improve transformation efficiency by immersing grape water in Agrobacterium inoculum and pressurizing it, making it useful for grape breeding. It can be utilized effectively.

Description

{Method for transformation of grape}

The present invention relates to a grape transformation method for grape breeding.

Grape is a vine plant belonging to the grape genus ( Vitis ) in the plant taxonomic family ( Vitaceae ) and is distributed in temperate and subtropical regions of the Northern Hemisphere. Depending on its origin, it is classified as native to western Asia, native to eastern Asia, and native to North America. In Korea, Vitis amurensis , a native of eastern Asia, is distributed, but most of the grapes grown in Korea are American varieties, native to the North American continent, cultivated in the U.S. and Japan. In particular, Campbell's Earley is more than 70% of the cultivation area. occupies. Therefore, there is a need to cultivate high-quality grape varieties that meet the diverse tastes of consumers.

Grape breeding is expanding the range of mutations created through cross breeding using wild grapes, but there are problems with polyploidy being diverse, offspring inferiority phenomenon occurring, and seeds not being formed like in inucleate varieties in some cases, making cross breeding not easy. (Gray DJ and Merdith CP. CAB International Wallingford . 1992). In addition, cultivating new grape varieties requires more than 10 years of time and labor, and it is difficult to introduce target traits.

A method proposed to solve this problem is a transformation method that directly introduces foreign useful genes using plant biotechnology technology. Plant transformation methods include the Agrobacterium method using bacteria of the genus Agrobacterium , the protoplast method using plant cells in a protoplasmic state, and the gene injection of DNA, etc. directly into cells by projecting a strong external force. There are gene gun laws, etc. The Agrobacterium method is a method of co-culturing grape cells or tissues subject to transformation and Agrobacterium. There have been no reports of successful transformation in Korea, and although there are differences depending on the variety, according to reports from overseas, 5 It is known to be around % (Han Ji-hak et al. Plant transformation. Jeong Moon-gak . 2007). The gene gun method can be applied to a variety of plant tissues and has the advantage of having a short experimental period, but its transformation efficiency is very low, about 1 to 3%, so it is difficult to apply to developing new grape varieties (R. Scorza, et al. Plant Cell Reports . 1995).

Accordingly, much research is still required to develop effective transformation methods for grapes.

Republic of Korea Patent Publication No. 10-2012-0066872

The purpose of the present invention is to provide a new grape transformation method using the Hwasu reduced-pressure soaking method.

One aspect of the present invention includes the steps of a) preparing an inoculum by culturing Agrobacterium into which an expression vector containing a target gene has been introduced; and b) immersing grape water in the inoculant and reducing pressure to transform the grape.

The above steps are described in detail as follows.

Step a) is a process for producing Agrobacterium containing the gene to be introduced into grapes.

First, an expression vector containing the target gene can be introduced into Agrobacterium.

“Target gene” as used in the present invention refers to various genes that can be introduced into plants. These target genes are not particularly limited, and genes known in the art can be used, but are not limited thereto. Examples may be genes derived from plants, including grapes, insects, bacteria, molds, viruses, etc.

The "expression vector" used in the present invention refers to a vector optimized for delivering and expressing a gene of interest and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert, and is also referred to as a recombinant vector. Such expression vectors can be constructed through various methods known in the art.

The expression vector may include expression control elements such as a promoter, start codon, stop codon, polyadenylation signal, and enhancer. Additionally, the expression vector contains genes encoding reporter molecules (e.g., luciferase and β-glucuronidase) and antibiotics as selection markers (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, and hygronucleus). mycin) or its resistance gene may be further included.

The type of the expression vector is not particularly limited, and plant transformation vectors known in the art can be used, but are not limited thereto. Examples include plasmid vectors such as pBI, pPZP, pSMA, pUC, and pBR, or binary vectors such as pBI.

The Agrobacterium serves as a carrier to deliver an expression vector containing a target gene to grapes, and Agrobacterium known in the art can be used without limitation.

According to one embodiment of the present invention, the Agrobacterium is Agrobacterium tumefaciens ( A. tumefaciens ), Agrobacterium rhizogenes ( A. rhizogenes ), Agrobacterium vitis ( A. vitis ), Agrobacterium 1 selected from the group consisting of A. nepotum, A. pusense , A. salinitolerans , and A. skierniewicense . It may be more than one species. In the present invention, considering transformation efficiency, Agrobacterium tumefaciens is preferred.

The process of introducing an expression vector into such Agrobacterium is not particularly limited and may be performed according to common methods known in the art, but is not limited thereto.

According to one embodiment of the present invention, an inoculation strain was prepared by introducing the GUS gene and the NPT II gene into the pBI121 vector as an expression vector and then transforming it into the Agrobacterium tumefaciens LBA4404 strain according to the Freeze-thaw method.

Thereafter, the inoculum can be prepared by culturing the Agrobacterium into which the expression vector has been introduced under appropriate conditions.

The culture can be carried out according to appropriate media and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be YEP (Yeast Extract Peptone) medium as a culture medium for Agrobacterium, but is not limited thereto. Additionally, the medium may further contain various carbon sources, nitrogen sources, and trace element components. Cultivation methods may include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but are not limited thereto. The culture temperature may be in a temperature range at which Agrobacterium can sufficiently grow, for example, 20 to 35°C, 20 to 30°C, or 25 to 35°C. The culturing period may continue until the desired yield of useful material is obtained, for example, 12 to 48 hours, 12 to 36 hours, or 12 to 24 hours.

“Inoculum” used in the present invention refers to a solution containing Agrobacterium or its culture medium into which the expression vector has been introduced for transformation of grapes, and in the present invention, it can be used interchangeably as inoculum or Agrobacterium inoculum. You can. The inoculum may further include a buffer solution capable of stabilizing Agrobacterium.

According to one embodiment of the present invention, the inoculated strain was cultured in liquid YEP medium supplemented with antibiotics at 28°C and 200 rpm for about 18 hours to prepare a culture medium with an OD ₆₀₀ value of 0.8 to 1.0 as an inoculum.

Step b) is a process of performing transformation of grape water using Agrobacterium inoculum.

“Transformation” as used in the present invention refers to introducing a gene into a host or host cell so that it can be expressed within the host, and if the transformed gene can be expressed within the host, it is inserted into the host's chromosome or Anything located other than a chromosome may be included without limitation. In the present invention, grape water can be used as a host.

The grape flower tree is a group of flowers forming a cluster of grapes, and the flowers of the grape flower tree bloom sequentially for 1 to 3 days during the flowering period. Grape flowers are mainly bisexual flowers with pistils and stamens in the same flower, so they do not require insect vectors and are capable of self-fertilization. Transformation targeting such grape blossoms or grape flowers may represent a transformant in which the target gene is introduced into the reproductive cells of grapes through pistils and stamens and passed on to the next generation. In order to increase transformation efficiency, it is desirable to use flowering plants at the full bloom stage when pistils and stamens are exposed.

According to one embodiment of the present invention, the grape flower number may be one in which more than 70% of all flowers have bloomed. More specifically, the flowering rate of the grape flower number may be 70 to 100%, 75 to 100%, 80 to 100%, 85 to 100%, 90 to 100%, or 95 to 100%, but is not limited thereto.

The transformation method is not particularly limited and may be performed according to an Agrobacterium-mediated transformation method known in the art, but is not limited thereto.

According to one embodiment of the present invention, the transformation method may be a reduced-pressure soaking method in which grape water is immersed in an Agrobacterium inoculum and pressure is reduced.

The reduced pressure immersion method involves immersing the grape flower water in the Agrobacterium inoculum so that it is completely submerged, applying a vacuum of a certain pressure for a certain period of time, and then momentarily removing the pressure, allowing the Agrobacterium to effectively penetrate into the stamen of the flower water due to the pressure difference. This is a way to make it happen.

The pressure reducing device used in the present invention is not particularly limited, and any pressure reducing device known in the art may be used, but is not limited thereto.

According to one embodiment of the present invention, the decompression may be applying a vacuum of 10 to 25 atmospheres for 3 to 15 minutes and then removing the pressure. At this time, if the pressure and time conditions do not fall within the above range, transformation does not occur properly, and if they exceed the above range, transformation efficiency decreases and the plant may be damaged, making normal growth difficult. In order to improve transformation efficiency, it is preferable to apply a vacuum of 15 to 20 atmospheres for 5 to 10 minutes and then remove the pressure.

According to one embodiment of the present invention, the eggplant Erlenmeyer flask containing the Agrobacterium inoculum is completely immersed in fire water, sealed, and vacuum applied under various pressure and time conditions (1 to 20 minutes and 0 to 25 atm). As a result of inducing the invasion of Agrobacterium by removing the pressure, when a vacuum of 10 to 25 atmospheres was applied to the water for 5 to 15 minutes and then removed, the transformation rate was about 3% or more. When the vacuum was applied for 5 to 10 minutes and then removed, the transformation rate was found to be at least 17% and up to 31%, confirming that the transformation rate was improved compared to the conventional grape transformation method.

The grape transformation method according to the present invention can effectively improve transformation efficiency by immersing grape water in Agrobacterium inoculum and pressurizing it, and can be usefully used in grape breeding.

Figure 1 is a schematic diagram of the pBI121 expression vector used for grape transformation according to an embodiment of the present invention.
Figure 2 is a photograph showing the sequence of transformation using a reduced pressure soaking method for grape water according to an embodiment of the present invention. (a) is a photograph in which the upper part of the flower pot that does not touch the Agrobacterium inoculant was removed, (b) is a photograph in which the stem is placed between the flask stopper with a center groove and the flower pot is immersed in the Agrobacterium inoculant fluid, (c) is a photo of a flask containing Agrobacterium inoculum being sealed with a stopper and subjected to reduced pressure treatment, and (d) to (f) are photos of fruit bearing 11, 31, and 97 days after the reduced pressure treatment.
Figure 3 is a photograph confirming the transformation of Campbell's Early Flower Water according to an embodiment of the present invention by GUS expression analysis.
Figure 4 is a photograph showing the sequence of cultivating transformed grape seeds according to an embodiment of the present invention. (a) is a photograph of seeds sown in a test tube containing grape growth medium and grown until they grow into new shoots, (b) is a photograph of plants with developed roots transplanted into the top soil, and (c) is a photograph of seedlings in pots. This is a photo of a plant grown for 6 months in .
Figure 5 is a photograph confirming the transformation of Campbell's Early Flower Water according to an embodiment of the present invention by gDNA PCR analysis.

Hereinafter, the present invention will be described in more detail. However, this description is merely provided as an example to aid understanding of the present invention, and the scope of the present invention is not limited by this example description.

Example 1. Campbell Early transformation

1-1. Preparation of Agrobacterium inoculum

For the transformation of Campbell's Early, an expression vector and Agrobacterium containing it were prepared.

The desired gene was introduced using the pBI121 vector, a plant transformation vector, as an expression vector. Referring to Figure 1, an expression cassette containing the NPT II gene (Vancanneyt et al. Mol Gen Genet . 1990) as a selection marker in the T-DNA inserted into the plant of the pBI121 vector, CaMV (Cauliflower Mosaic Virus) The 35S promoter (Jach et al. Plnat J. 1995) and the β-glucuronidase (GUS) reporter gene were introduced.

As Agrobacterium, Agrobacterium tumefaciens LBA4404 strain was used (Hoekema et al. Nature . 1983).

The pBI121 expression vector was introduced into strain LBA4404 according to the Freeze-thaw method (Burrow et al. Plant Mol Biol Rep . 1990). In detail, 10 μl of the pBI121 expression vector was added to 100 μl of the LBA4404 strain and reacted on ice for 30 minutes. Afterwards, it was rapidly frozen in liquid nitrogen for 1 minute and immediately reacted in a heat-block at 37°C for 5 minutes. After completion of the reaction, 1 ㎖ of liquid YEB medium (containing Nutrient broth 13.3 g/L, Yeast extract 1.0 g/L, Sucrose 5 g/L, and MgSO ₄ 0.24 g/L (pH 7.5)) was added to culture Agrobacterium at 28%. Cultured for 4 hours at ℃ and 200 rpm. After incubation, the supernatant was removed by centrifugation at 3,000 rpm for 2 minutes, and the recovered Agrobacterium was resuspended in 100 ㎕ of liquid YEP medium. The resuspended Agrobacterium was cultured on solid YEP medium (Nutrient broth 13.3 g/L, Yeast extract 1.0 g/L, Sucrose) supplemented with antibiotics (Rifampicin 100 mg/L, Streptomycin 300 mg/L, and Kanamycin 50 mg/L). It was applied to 5 g/L, MgSO ₄ 0.24 g/L (pH 7.5) and agar 10 g/L) and cultured in an incubator at 28°C for 2 days to select Agrobacterium colonies that grew in colonies.

The selected Agrobacterium was added to 50 mL of liquid YEP medium supplemented with antibiotics (containing 100 mg/L of Rifampicin, 300 mg/L of Streptomycin, and 50 mg/L of Kanamycin) and cultured at 28°C and 200 rpm for approximately 18 hours. A culture medium with an OD ₆₀₀ value of 0.8 to 1.0 was prepared as an inoculum.

1-2. transformation

Transformation was performed using the reduced pressure immersion method on Campbell's Early Flowers grown in a greenhouse (temperature 25 to 28°C, humidity 40 to 60%) and having an overall flowering rate of more than 70%.

Referring to Figure 2, 200 mL of the prepared inoculum was added to the eggplant Erlenmeyer flask, the cork stopper of the flask was cut in the center, a groove was made, a flower stem was inserted, the flower flower was completely submerged in the inoculum, and the flask was sealed by inserting a stopper. At this time, the upper part of the flower that did not touch the solution was removed. Afterwards, connect a hose to the branch of the flask and apply a vacuum to 0, 5, 10, 15, 20 or 25 atm for 1, 5, 10, 15 or 20 minutes, then remove the pressure momentarily to allow Agrobacterium to enter the stamen. Enabled effective penetration.

Afterwards, the flower clusters were taken out of the flask, the excess inoculum was removed with a paper towel, and the plants were grown using standard Campbell's Early management methods until the fruit was harvested. Fruits were confirmed 11 days after inoculation.

Experimental Example 1. Confirmation of Campbell Early gene introduction

After harvesting fruit from grape water in which transformation was induced, 1 to 5 fruits were used to confirm GUS expression, and the seeds of the remaining fruits were separated, washed at least three times with sterilized water, and stored in a refrigerator (layered storage method at 4°C). It was stored for more than 2 months and used to cultivate plants.

1-1. GUS expression analysis

Fruits were immersed in 5-bromo-4-chloro-3-indole-glucuronidase solution at 37°C (Jefferson et al. EMBO J. 1987). After 24 hours of immersion, fruits that showed a positive GUS reaction were examined by decolorizing them with a destaining solution (containing 70% EtOH and 1% glacial acetic acid). As a control, fruits from plants that were not used in the reduced pressure soaking method were used.

As a result, as shown in Figure 3, in the case of transformation, the entire tissue and seed were GUS stained and appeared bluish green, whereas in the case of non-transformation, the entire tissue was discolored to white.

Based on these GUS expression analysis results, transformation efficiency according to pressure and time conditions was calculated.

0 atmosphere 5 atmospheres 10 atmospheres 15 atmospheres 20 atmospheres 25 atmospheres 1 min 0% 0% 0% 0% 0% 0% 5 minutes 0% 3.6±3.17% 9.3±3.28% 17.8±7.96% 31.7±9.43% 11.1±4.71% 10 minutes 0% 3.7±3.23% 9.8±4.13% 18.9±1.18% 21.3±8.71% 5.9±0.35% 15 minutes 3.5±3.03% 5.4±5.27% 7.6±2.61% 8.2±3.07% 6.2±0.56% 3.4±2.96% 20 minutes 5.3±0.28% 7.0±2.58% 5.5±0.45% 5.4±0.45% 5.5±0.45% 1.7±2.89%

As a result, as shown in Table 1, when a vacuum of 10 to 25 atmospheres was applied to the water for 5 to 15 minutes and the pressure was removed, the transformation rate was similar to or higher than the transformation rate of conventional grapes (1 to 3%). In particular, excellent transformation rates were observed without damage to plants due to pressure under conditions of 15 to 20 atmospheres and 5 to 10 minutes.

1-2. gDNA PCR analysis

Ten seeds from the remaining fruit that were not used for GUS expression analysis were selected, grown, and subjected to gDNA PCR.

First, put grape growth medium (MS 4.4 g/L, Sucrose 30 g/L, BA 0.1 mg/L, IBA 0.1 mg/L, and plant agar 10 g/L, pH 5.8) in a test tube and then plant the transformed seeds. Each was sown and cultured at 25°C, and shoot growth was observed after germination. Plants with developed roots were taken out of the test tube, washed with sterilized water to ensure that no growth medium remained on the roots, then transplanted into medium soil and grown at room temperature for a month, then repotted and grown for 6 months. As a control, seeds from plants that were not used in the reduced pressure soaking method were used (see Figure 4).

200 g of leaves were collected from the cultivated plants, rapidly frozen in liquid nitrogen, pulverized using Tissue LyserⅡ (QIAGEN), and genomic DNA of each plant was extracted according to the manufacturer's manual of the DNeasy Plant Minikit (QIAGEN). PCR was performed using genomic DNA as a template and the primers shown in Table 2 below (Dyad Thermal cycler, BIO-RAD). PCR performance conditions were 5 minutes at 95°C (pre-denaturation), then repeated 30 times at 95°C for 30 seconds, 55°C for 20 seconds, and 72°C for 30 seconds, and 5 minutes at 72°C (post-extension). did. PCR amplification products were confirmed by electrophoresis using a 0.8% agarose gel.

Primer name Primer sequence (5' > 3') PCR amplification product size NPT II-F GAG GCT ATT CGG CTA TGA CTG 600 bp NPT II-R ATC GGG AGC GGC GAT ACC GTA

As a result, as shown in Figure 5, all plants germinated from transformed seeds were found to contain the NPT II gene.

Example 2. Transformation of black gemstones

Transformation was performed in the same manner as in Example 1, except that Heukgeum was used instead of Campbell Early as the grape variety.

Grape fruits in which transformation was induced were harvested, and GUS expression analysis and gDNA PCR analysis were performed in the same manner as in Experimental Example 1.

As a result, the transformation rate of Black Jewel showed a similar pattern to Campbell Early, with the best transformation rate (30.0 ± 2.93%) under the conditions of 20 atmospheres and 5 minutes.

Example 3. Transformation of Geobong

Transformation was performed in the same manner as in Example 1, except that Geobong was used instead of Campbell Early as the grape variety.

Grape fruits in which transformation was induced were harvested, and GUS expression analysis and gDNA PCR analysis were performed in the same manner as in Experimental Example 1.

As a result, the transformation rate of Geobong showed a similar pattern to that of Campbell Early, and showed the best transformation rate (23.3 ± 5.11%) under the conditions of 20 atmospheres and 5 minutes.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (4)

a) preparing an inoculum by culturing Agrobacterium into which an expression vector containing a target gene has been introduced; and
b) immersing grape water in the inoculant so as to completely submerge it and performing transformation by reducing pressure,
The decompression method in step b) involves applying a vacuum of 15 to 20 atmospheres for 5 to 10 minutes and then instantly removing the pressure.
In claim 1,
The Agrobacterium in step a) is Agrobacterium tumefaciens ( A. tumefaciens ), Agrobacterium rhizogenes ( A. rhizogenes ), Agrobacterium vitis ( A. vitis ), Agrobacterium nepotium ( A nepotum ), Agrobacterium pusense ( A. pusense ), Agrobacterium salinitolerans ( A. salinitolerans ) and Agrobacterium skierniewicense ( A. skierniewicense ). At least one species selected from the group consisting of, method.
In claim 1,
The method in which the number of grape flowers in step b) is one in which more than 70% of all flowers have bloomed.
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