KR101174491B1 - Method for increasing transformation efficiency of Chinese cabbage plant using Agrobacterium and Chinese cabbage plant produced by the method - Google Patents

Abstract

The present invention relates to a method of increasing the transformation efficiency of Chinese cabbage using Agrobacterium and to a cabbage plant prepared by the above method, the suspension treatment method of co-cultivation, callus organic and re-differentiation from cabbage hypocotyl section, and culture temperature at the time of regeneration The present invention relates to a method for increasing the transformation efficiency of Chinese cabbage by different treatments, and to a high-efficiency transformed cabbage plant produced by the above method and seeds thereof.

Description

Method for increasing transformation efficiency of Chinese cabbage plant using Agrobacterium and Chinese cabbage plant produced by the method

The present invention relates to a method for increasing the transformation efficiency of Chinese cabbage using Agrobacterium and to a highly efficient transformed cabbage plant prepared by the above method.

Since plant transformation technology was first developed in 1983, a great deal of investment and research has been carried out worldwide, and several traits have been introduced into plants. According to various statistics, the global market for agricultural products developed by plant biotechnology is estimated to be about $ 54 billion in 2005. However, in view of the reality of the world, the development of transgenic crops in Korea is still insufficient, and we need to develop our own high-tech technology as soon as possible before it is too late to leap into an advanced agricultural country with agricultural competitiveness in the 21st century. In 2006, Chinese cabbage production amounted to 634 billion won, which is one of the four most grown vegetables after red pepper. Successful cases of redifferentiation and transformation using sliced tissue of Chinese cabbage, a major vegetable crop, have been reported, but it is difficult to obtain large amounts of transformants due to low regeneration and transformation rate. Therefore, the establishment of high efficiency and stable cabbage transformation system and securing transformants will enable the development of new varieties through shortening of breeding period and molecular breeding by creating new mutations of cabbages that have introduced foreign useful genes.

Korean Patent Registration Nos. 10-0523758 and 10-0375674 disclose a method of transforming cabbage using Agrobacterium, but is different from the transformation method of the present invention.

The present invention was derived by the above requirements, while researching to develop an environmentally resistant transgenic cabbage varieties, suspension treatment method during co-cultivation, cabbage hypocotyl to increase the transformation efficiency of the cabbage using Agrobacterium The present invention was completed by revealing that the cabbage transformant of high frequency can be obtained by enhancing the transformation efficiency according to the culture temperature treatment during callus organic regeneration, regeneration, and regeneration from sections.

In order to solve the above problems, the present invention provides a method of increasing the transformation efficiency of Chinese cabbage by varying the suspension treatment method during co-culture, callus organic and redifferentiation from cabbage hypocotyl segments, and incubation temperature during regeneration.

The present invention also provides a highly efficient transgenic cabbage plant and seeds thereof prepared according to the above method.

According to the present invention, the establishment of a high-efficiency stable cabbage transformation system and securing a transformant will enable the development of new varieties through short breeding and molecular breeding by creating new mutations of cabbages introduced with foreign useful genes.

1 is a diagram illustrating a step-by-step transformation method using the hypocotyl segment of Chinese cabbage seedlings.
Figure 2 is a pCAMBIA1304 vector picture containing the hygromycin resistance and GFP gene used for transformation.
3 is Agrobacterium After 2 and 7 weeks in co-cultivation starter medium I, callus and shoots are induced, and then the roots are induced in MS medium to obtain re-differentiated transgenic plants.
Figure 4 shows the appearance of GFP expression in the leaves and roots of plants after obtaining the Chinese cabbage transformants.
Figure 5 shows the results of PCR analysis of Chinese cabbage transformant, showing a 500bp amplification band showing the introduction of hygromycin gene (M: size marker, PC: vector, NC: non-transformed plant, lanes 1 to 28: transformation Plants).

In order to achieve the object of the present invention, the present invention, after pre-cultivation of the hypocotyl section of cancer germination 4 days old Chinese cabbage seedlings Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) containing a recombinant plant expression vector suspended in the inoculation medium 1 Transformation efficiency of Chinese cabbage, comprising the step of obtaining a cabbage transformant by immersion, inoculation and co-cultivation after time of transformation and selection of antibiotic-added antibiotics, pH, solid concentration conditions and temperature treatment A method of increasing is provided (FIG. 1).

The method of the present invention specifically

(1) germinating the cabbage seeds for 3 to 5 days at 24 to 26 ℃;

(2) 22-24 in pretreatment medium consisting of 0.5 ~ 1.5 mg / L NAA (naphthalene acetic acid) and 3 ~ 5 mg / L BA (benzyladenine) added to the hypocotyl segment of the cancer germinated cabbage seedlings Photoculture at 1 ° C. for 1 to 3 days;

(3) suspending Agrobacterium tumefaciens comprising a recombinant plant expression vector in a bacterial inoculation medium;

(4) immersing the pretreated cabbage hypocotyl segment of step (2) in a medium suspended in Agrobacterium tumerfaciens of step (3) for 5 to 15 minutes, followed by co-culture and transformation;

(5) the transformed cabbage hypocotyl segment was prepared using NAA, BA, AgNO ₃ And inducing shoots for 2-3 months in selection medium I to which antibiotics have been added; And

(6) may include the step of inducing the renal elongation of the selected Chinese cabbage shoot selection medium II.

The method of the present invention includes the step of germinating the cabbage seed for 3 to 5 days at 24 ~ 26 ℃. The step may preferably germinate the cabbage seeds for 4 days at 25 ℃.

In the method of the present invention, the hypocotyl section of the cancer germinated Chinese cabbage seedlings is prepared in a pretreatment medium consisting of MS basal medium to which 0.5 to 1.5 mg / L NAA and 3 to 5 mg / L BA are added for 1 to 3 days. Photoculturing. The step may preferably be photocultured at 23 ° C. for 2 days in a pretreatment medium consisting of MS basal medium supplemented with 1 mg / L NAA and 4 mg / L BA by cutting the hypocotyls of the germinated cabbage seedlings at 1 cm intervals. have.

The method includes regenerating from cotyledon and hypocotyl segments of Chinese cabbage seedlings. The step may preferably be used for callus organic and regeneration by cutting the seedling hypocotyl section at intervals of 1 cm except for 1 cm of growth points near the cotyledons and roots. The lower hypocotyl region had good callus formation rate and shoot regeneration.

The method comprises suspending Agrobacterium tumefaciens containing a recombinant plant expression vector in a bacterial inoculation medium. The inoculation medium may be MS basal liquid medium of pH 5.0 to 5.4 containing glucose 3-4%, preferably MS basal liquid medium of pH 5.2 containing 3.6% glucose. The step is preferably Agrobacterium tumerfaciens ( Agrobacterium) comprising a recombinant plant expression vector tumefaciens ) and the absorbance (OD) is concentrated by centrifugation when the absorbance (OD) is 0.7 ~ 0.9 and then suspended in the inoculation medium at 22 ~ 24 ℃ temperature conditions for 0.5 to 1.5 hours, more preferably recombinant plants Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens) to be cultured to a suspension for 1 hour, it was concentrated with centrifugation at 23 ℃ temperature conditions in the bacteria inoculated medium when the absorbance (OD) was 0.8, including the expression vector . Inoculation of Agrobacterium tumerfaciens 1 hour after suspension than inoculation immediately after suspension significantly increased the transformation efficiency of the cabbage (see Table 2).

In the method of the present invention, a preferred example of the recombinant plant expression vector is a Ti-plasmid vector capable of transferring a portion of itself, a so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

Recombinant plant expression vectors will preferably include one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the recombinant plant expression vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. 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 recombinant plant expression vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens ( Terminator of the octopine gene of Agrobacterium tumefaciens, but is not limited thereto. Regarding 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 of the present invention, the pretreated cabbage hypocotyl segment of step (2) is immersed in the medium in which Agrobacterium tumerfaciens of step (3) is suspended for 5 to 15 minutes, and then co-cultured and transformed. Include. The immersion may be preferably 10 minutes, the co-culture may be cancer culture for 1 to 3 days at 22 ~ 24 ℃, preferably cancer culture for 2 days at 23 ℃.

According to the method of the present invention, the transformed cabbage hypocotyl segment is divided into NAA, BA, AgNO ₃ And inducing shoots for 2 to 3 months in selection medium I to which antibiotics have been added. The selection medium I may be preferably MS basal medium to which 0.5 to 1.5 mg / L NAA, 3 to 5 mg / L BA, 3 to 5 mg / L AgNO ₃ and antibiotics are added, more preferably 1 mg / L NAA, 4 mg / L BA, 4 mg / L AgNO ₃ And MS basal medium with antibiotics added.

The method of the present invention comprises the step of inducing shoot elongation in the selected Chinese cabbage shoot selection medium II. The selection medium II is preferably 0.05 to 0.15 mg / L NAA, 3 to 5 mg / L BA, 3 to 5 mg / L AgNO ₃ And MS basal medium with antibiotics added, more preferably 0.1 mg / L NAA, 4 mg / L BA, 4 mg / L AgNO ₃ And MS basal medium with antibiotics added.

In the method of the present invention, the steps (5) and (6) is treated with a culture temperature of 22 ~ 24 ℃, pH in the medium of 5.0 ~ 5.4, and as a solid to include 0.9 ~ 0.95% plant agar (plant agar) Preferably, the culture temperature is 23 ℃, the pH in the medium is treated with 5.2, and may contain 0.95% of plant agar (solid) as a solid. 0.9 ~ 0.95% of plant agar, which has a higher solidity than the general medium, lowered the vitrification rate of Chinese cabbage shoots, resulting in a high plant regeneration rate of 79% (see Example 2). In addition, it was confirmed that the re-differentiation and transformant acquisition rate increased significantly at 23 ° C lower than the general 25 ° C culture temperature and pH 5.2 lower than the general culture condition (see Example 3).

It will be readily appreciated by those skilled in the art that the method of the present invention includes a step of inducing rooting, followed by a step of inducing rooting and a purifying treatment procedure.

Therefore, the method of increasing the transformation efficiency of the cabbage of the present invention is preferably

(1) germinating the cabbage seeds for 3 to 5 days at 24 to 26 ℃;

(2) photoculturing the hypocotyl segments of the cauliflower cabbage seedlings at 22-24 ° C. for 1-3 days in MS basal medium to which 0.5-1.5 mg / L NAA and 3-5 mg / L BA were added;

(3) pH containing Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens), the culture and the absorbance (OD) is glucose 3-4% this was concentrated by centrifugation when the 0.7 ~ 0.9 containing a recombinant plant expression vector Suspending for 0.5-1.5 hours at a temperature of 22-24 ° C. in an MS basic liquid medium of 5.0-5.4;

(4) the pretreated cabbage hypocotyl section of step (2) is immersed in the medium in which Agrobacterium tumerfaciens of step (3) is suspended for 5 to 15 minutes, and then at 22 to 24 ° C. for 1 to 3 days. Transforming by co-culture by cancer culture;

(5) the transformed cabbage hypocotyl section 0.5 ~ 1.5 mg / L NAA, 3 ~ 5 mg / L BA, 3 ~ 5 mg / L AgNO ₃ And induction of shoots for 2-3 months by treating the plant agar as a solid at 0.9-0.95% with a culture temperature of 22-24 ° C. in MS basal medium of pH 5.0-5.4 with antibiotics added. And

(6) Culture temperature of the selected Chinese cabbage shoots in MS basal medium of pH 5.0-5.4 with addition of 0.05-0.15 mg / L NAA, 3-5 mg / L BA, 3-5 mg / L AgNO ₃ and antibiotics Treating plant agar 0.9 to 0.95% as a solid to induce shoot kidney,

More preferably

(1) germinating the cabbage seeds at 25 ° C. for 4 days;

(2) photoculturing the hypocotyl segments of the cancer germinated cabbage seedlings at 23 ° C. for 2 days in MS basal medium to which 1 mg / L NAA and 4 mg / L BA were added;

3 of Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens), the pH of the culture and the absorbance (OD) contains glucose 3.6%, it was concentrated by centrifugation when a 0.8 5.2, containing a recombinant plant expression vector MS basal Suspending for 1 hour at 23 ° C. in a liquid medium;

(4) the pre-treated cabbage hypocotyl segment of step (2) was immersed in a medium containing Agrobacterium tumerfaciens of step (3) for 10 minutes, and then co-cultured by cancer culture at 23 ° C. for 2 days. Transforming;

(5) The transformed Chinese cabbage hypocotyl slices were grown in MS basal medium at pH 5.2 to which 1 mg / L NAA, 4 mg / L BA, 4 mg / L AgNO ₃ and antibiotics were added. (plant agar) 0.9 to 0.95% treatment to induce shoots for 2-3 months; And

(6) the selected Chinese cabbage shoot 0.1 mg / L NAA, 4 mg / L BA, 4 mg / L AgNO ₃ And incubating plant agar 0.9 to 0.95% as a solid at a culture temperature of 23 ° C. in a MS basal medium of pH 5.2 to which antibiotics were added.

The present invention also provides a highly efficient transgenic cabbage plant and its seeds produced by the above method.

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.

end. Pretreatment method of Chinese cabbage for transformation

Plants for cabbage transformation were used to cut the lower hypocotyl segments of seedlings in which 'Seoul cabbage' varieties were germinated for 4 days on board, except 1cm and around 1cm in the growth point. It was used for transformation after 2 days photoculture in MS basal medium to which 1 mg / L naphthalene acetic acid (NAA), 4 mg / L BA (benzyladenine) was added.

I. Agrobacterium Agrobacterium Chinese cabbage transformation using different coculture

Transformation was used by constructing a vector inserted with the NDPK2 gene linked to the enhanced CaMV 35S promoter (E35S) and 35S terminator (35S Ter) in pCAMBIA1304 containing the hygromycin and GFP gene (Fig. 2). The strain used was LBA4404, a monoclonal clone of Agrobacterium containing the vector maintained in YEP solid medium was inoculated into 50 ml of liquid YEP medium. This was incubated in a shake incubator at a speed of 28 ° C. and 150 rpm for about 20 hours, concentrated by centrifugation when the bacterial concentration (OD) was about 0.8, suspended in 50 ml inoculation medium and sectioned after 1 hour. Transformation was performed by soaking the tissue for 10 minutes. Coculture was selected for transformants after transfer to selection medium I containing 5 mg / L of hygromycin and 250 mg / L of cefotaxime after treatment for 2 days in a dark room at 23 ° C. Transformed shoots were selected in the selection medium containing hygromycin, and the shoots formed from the explants were well cut in the mother part except for the cells with complete shoots, and then passaged to selection medium II for shoot extension and plant regeneration. Induced. Subsequently, subcultures were performed at intervals of about 2 weeks to induce shoot kidneys, and then, to some extent, shoots were induced on hormone free MS medium to induce roots (FIG. 3).

All. cabbage Hypocotyl  Transformation from Section Callus  Formation and Plant Regeneration

Cabbage hypocotyl sections were inoculated with callus and shoots from selection medium I after inoculation with Agrobacterium, and then replanted shoots were cut to induce plant regeneration in selection medium II. The pH in pretreatment, co-culture and selection medium was maintained at 5.2, lower than normal culture conditions, and the medium solids were re-differentiated by minimizing vitrification of re-differentiated plants with high agar content of 0.4% phytagel or 0.9 ~ 0.95% plant agar. The efficiency was enhanced (Table 1).

Chinese cabbage transformation stages Step (badge) Medium composition (mg / L) Agar (%) pH Inflight sowing 1/2 MS phtagel 0.4 5.8 Pretreatment medium MS + BA 4 + NAA 1 plant agar 0.9 5.2 Inoculation medium MS 36 g of glucose (liquid) 5.2 Co-culture MS + BA 4 + NAA 1 plant agar 0.9 5.2 Selection Badge I MS + BA 4 + NAA 1 + AgNO3 4
+ Celltaxime 250 + Hygromycin 5 plant agar 0.95 5.2 Selection Badge II MS + BA 4 + NAA 0.1 + AgNO3 4
+ Celltaxime 250 + Hygromycin 5 plant agar 0.95 5.2 Rooting badge MS plant agar 0.9 5.2

※ Basic medium: MS basic medium + sucrose 2%, 23 ℃ incubation temperature

la. Purification and Analysis of Cabbage Transformants

Root-derived transformants were rinsed with sterile water to rinse the medium, and then transplanted into pots containing vermiculite: perlite: humus = 3: 2: 1 and purified. In order to confirm gene insertion using purified leaves of plants, genomic DNA was collected and the conversion rate was confirmed by PCR and Southern analysis using hygromycin resistance genes as primers and probes.

Example  1: Agrobacterium ( Agrobacterium Chinese cabbage transformation efficiency with different coculture

As a result of evaluating the conditions of the transformation stage of the Chinese cabbage, when the hypocotyl section was photocultured in the pretreatment medium for 2 days, when the bacterial concentration (OD) was about 0.8, 1 hour elapsed after inoculating immediately in the centrifugal bacterial inoculation medium. Later, when the transfection was performed by immersing the section tissue for 10 minutes, the transformation rate was 4.1%, which was more than doubled (Table 2).

Agrobacterium Transformation Rate of Chinese Cabbage with Different Inoculation Methods Inoculation method Number of slices (a) hpt PCR analysis (b) Efficiency (b / a,%) Inoculation after suspension  1,326  19 1.4 Inoculate 1 hour after suspension  725  30 4.1 290% increase in efficiency

※ 4 days of germination seedling hypocotyl section, pretreatment 2 days, co-culture 2 days, 23 ℃ culture treatment

Example  2: Chinese cabbage Hypocotyl  Transformation from Section Callus  Formation and Plant Regeneration Efficiency

Cut the cabbage seedlings of the cabbage seedlings 'Seoul cabbage', which were germinated for 4 days on board, to 1 cm in length, and then callus and MS in medium medium supplemented with NAA 1 mg / L, BA 4 mg / L and AgNO ₃ 4 mg / L. Shoots were organic. After shoot abandonment, subcultured with medium lowering NAA concentration to 0.1 mg / L, and 0.9 ~ 0.95% of plant agar, which had higher solidity than normal medium, lowered vitrification rate of Chinese cabbage shoots, resulting in a high percentage of normal plant regeneration. . Transformation was carried out by pretreatment and Agrobacterium co-cultivation using the high-efficiency regeneration system established by the above method to increase the cabbage transformant regeneration and transformant acquisition rate.

Example  3: Chinese cabbage transformant regeneration and analysis

Cabbage transformants were seeded from selection medium I after 2 days of coculture of hypocotyl segments. At this time, it was confirmed that the regeneration and transformant acquisition rate increased significantly at 23 ° C. lower than the general 25 ° C. culture temperature. The expression of GFP in leaf and root tissues of re-differentiated plants was confirmed, and the results of PCR analysis confirmed insertion of hygromycin resistance genes (FIGS. 4 and 5).

Claims (10)

delete delete delete delete delete delete delete (1) germinating the cabbage seeds for 3 to 5 days at 24 to 26 ℃;
(2) photoculturing the hypocotyl segments of the cauliflower cabbage seedlings at 22-24 ° C. for 1-3 days in MS basal medium to which 0.5-1.5 mg / L NAA and 3-5 mg / L BA were added;
(3) Agrobacterium containing a recombinant plant expression vector tumefaciens tyumeo Pacific Enschede (Agrobacterium tumefaciens), the culture and the absorbance (OD) of 0.7 ~ 0.9 that is when centrifuged and concentrated after this glucose 3 ~ 4% (w / v Suspending for 0.5-1.5 hours at 22-24 ° C. temperature condition in MS basic liquid medium at pH 5.0-5.4;
(4) the pretreated cabbage hypocotyl section of step (2) is immersed in the medium in which Agrobacterium tumerfaciens of step (3) is suspended for 5 to 15 minutes, and then at 22 to 24 ° C. for 1 to 3 days. Transforming by co-culture by cancer culture;
(5) The transformed cabbage hypocotyl sections were cultured in MS basal medium at pH 5.0-5.4 with 0.5-1.5 mg / L NAA, 3-5 mg / L BA, 3-5 mg / L AgNO ₃ and antibiotics added. Inducing shoots for 2 to 3 months by treating plant agar 0.9 to 0.95% (w / v) as a solid at a temperature of 22 to 24 ° C .; And
(6) Culture temperature of the selected Chinese cabbage shoots in MS basal medium of pH 5.0-5.4 with addition of 0.05-0.15 mg / L NAA, 3-5 mg / L BA, 3-5 mg / L AgNO ₃ and antibiotics Method of increasing the transformation efficiency of the Chinese cabbage comprising the step of inducing the shoot kidney by treating the plant agar 0.9 ~ 0.95% (w / v) as a solid, ~ 24 ℃. delete delete

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