KR19990025487A - Tissue Culture of Pepper Plants and Transformation Using Myocarcinoma - Google Patents

Abstract

The present invention milled the T ₁ plants from the cotyledons of red pepper plants and examined the tissue culture conditions of the regenerated plants. The present invention produces transformants of pepper plants mediated by myocarcinoma tumors, which transform or reduce the pathology for cucumber mosaic virus CMV-Y or CMY-kor strains, respectively.

Description

Tissue Culture of Red Pepper Plants and Transformation Using Myocardium Carcinoma

The present invention relates to a regeneration and transformation system of red pepper plants and a method for producing a novel transformed red pepper plant resistant to cucumber mosaic virus using the same. Hot pepper is one of the important vegetable species to eat in various forms. Nevertheless, red pepper is known to be difficult to regenerate in the laboratory because it has little research on regeneration and transformation. Therefore, genetic engineering of pepper plants has also been limited because regeneration and transformation systems are still not possible in the laboratory.

In view of the above problems, the present invention aims to generate red pepper plants of new characteristics by re-dividing pepper whole plants and expressing foreign genes.

It is another object of the present invention to improve the disease resistance of cucumber pepper virus transformed on the basis of the redevelopment and transformation system of the pepper plant in the next generation.

Accordingly, the present invention induces plant regeneration from the cotyledon of Capsicum annuum CV Golden Tower and transformation of pepper plants by using Agrobacterim and refers to Cucumber mosaic Virus (CMV). The object of the present invention was achieved by investigating the disease resistance level of the transformant passage plant expressing the expression of. In particular, the transformed novel pepper plants were self-corrected and passaged (Progeny test) to confirm the genetic stability and expression of cDNA of CMV Satellite RNA. PCR and RNA gel blot analysis confirmed that the foreign gene was stably transferred and expressed in passage plants. The symptoms of cucumber mosaic virus of later pepper plants were confirmed by inoculation with CMV-Y or CMV-kor strains.

1 is a photograph showing the whole process of cotyledon esplant regeneration and transformation of red pepper.

Figure 2 is a photograph of red pepper seedlings taken 6 weeks after germination as a T ₁ pepper plant cultured in MS medium (containing 200㎍ / ㎖ kanamycin sulfate).

N: Pepper seedling sensitive to Kanamycin Sulfate

T: Red pepper seedlings resistant to Kanamycin Sulfate

Arrows indicate untypified Cotyledon and roots sensitive to kanamycin.

Figure 3 is agarose gel electrophoresis picture of the PCR amplification product from the transformed pepper plants.

λ / HindIII marker (lane 1), PCR product amplified genomic DNA of T ₁ pepper plants (lane 3-16), untransformed pepper plants (lane 17), T ₀ pepper plants (lane 2, lane 18). The arrow indicates that the size of the amplified nptII product is 0.7 kb.

Figure 4 is a photograph showing the RNA gel blot analysis.

The cDNA clone of cucumber mosaic virus (CMV) I ₁₇ N Satellite RNA was used as a probe, and lane1-19 was T ₁ transgenic plant, Lane M was T ₀ plant, and Lane C was total RNA from untransfected plant. Was used.

The arrow indicates the position of the 1.0 kb transcript, and the figure in the square shows the cDNA insert region and the expression cassette of pRok1 / 105.

Figure 5 is a photograph showing the symptom development of the untransformed pepper plant (1) (3) and the transformed pepper plant (2) (4).

A: Three months after the inoculation of the cucumber mosaic virus CMV-Y.

B: It was photographed 3 months after inoculating the cucumber mosaic virus CMV-kor.

Figure 6 is a quantitative analysis of the draft development of transformed T ₁ pepper plants. Local lesion assays were performed with C. amaranticolor inoculated with CMV-Y (A) and CMV-kor (B).

CMV is a positive-sense RNA virus whose genome contains four major genes encoded by three genomic RNAs (palukaitis et al. 1992). Replicase protein consists of la (111KDa) and 2a (97KDa), encoded by RNA 1 and RNA2, respectively. RNA3 also encodes two proteins. The 3a protein (30KDa) is encoded by the 5'-proximal open reading frame of RNA3 and the open reading frame of the envelope protein is located in the 3'-proximal half of RNA3. The envelope protein (24.5 KDa) is then translated from subgenomic RNA designating RNA4 (Gould and Symons, 1977). In addition to this genomic RNA, certain types of viruses have separate RNA species, the so-called satellite RNA (Francki, 1985; fritsh and Mayo, 1989). The replication of these Satellite RNAs depends entirely on their helper virus (Francki, 1985, Masuta and Takanami 1989, Kuwata et al. 1991). The presence of Satellite RNA, however, regulates the symptoms caused by his helper virus (Jaegle et al. 1990, Tien and wu, 1991). Satellite RNA generally attenuates the condition, but in some cases it can aggravate the condition or show other symptoms. Thus, the shape of the condition is a triangular relationship with satellite RNA, helper virus and host (Jaegle et al. 1990, Tien and Wu, 1991; Wu et al. 1993).

The present invention is a process for producing plant transformed from the cotyledon cotyledons and transformants using Agrobacterium on the basis of this fact; It consists of a process of infecting CMV-Y and CMV-kor to investigate the viral resistance of pepper plants expressing CMV I ₁₇ N satellite RNA. Hereinafter, the configuration and operation of the present invention will be described in detail by way of examples.

Reagents and Chemicals

Restriction enzymes were purchased from promega and Taq DNA polymerase was purchased from Korea Biotech. [a- ³² P] dCTP used Amersham products and other chemicals manufactured by Sigma Chemical.

Plant material

Pepper plant used in the present invention was Golden Tower and seeds were sterilized in 2% NaCl solution for 15 minutes and rinsed 6 times in sterile distilled water. Ten seeds were placed in a 87 × 15 mm plate containing 1/2 MS salt, 1 / 2MS vitamin, 3% sucrose and 0.8% agar added germination medium (pH5.8). Next, the resultant was transferred to a 100 ml flask and irradiated with light at 25 ° C. for 16 hours, but maintained at 2,000 Lux. Cotyledon explants used in the experiment were cut from 11 days old seedlings before the first leaves.

Regeneration of cotyledons

The effects of NAA and Zeatin concentrations on the regeneration of cotyledons were examined and the auxin / cytokinin ratios were examined.

As a new superorganic medium, MS salt, vitamin B ₅ , and 2% sucrose containing various concentrations of NAA and Zeatin were used twice. After 4 weeks of culture, normal shoots and eyes developed in cotyledons, which were then transplanted into shoot growth medium. Shoot growth medium was used 100ml flask or bottle. Regeneration rate was determined after 2 months. Rooting was induced in muscle organ medium containing MS salt, vitamin and 3% sucrose.

Transformation

Recombinant DNA from E. Coli was introduced into Agrobacterium tumefaciens strain LBA4404. Agrobacterium transfected with a plasmid containing cDNA of CMV I ₁₇ N-Satellite RNA by the direct DNA uptake method (An, 1987) demonstrated the restriction plasmid DNA from restriction digestion. The transformed bacteria were named Agrobacterium tumefaciens LBA4404 / pBOK1 / 105 and deposited on September 2, 1997 at the International Depository Institute of Biotechnology (Accession No. KCTC 8828P). For the transformation, cotyledons were healed on chloroplast-free medium without Kanamycin for 2 days, and Agrovacterium and empty culture were performed for 3 days. Hemorrhoidized shoots were washed with sterile water and cultured in myocardium medium containing 100mg / L Kanamycin Sulfate and carbenicillin. Four weeks later, the cotyledons were transferred to the same shoot growth medium and cultured for about two months, and then the shoots formed on the cotyledons were cut and transplanted into root organic medium (containing 50mg / L Kanamycin Sulfate). The rooted plants were transferred to pots and further grown. Experimental results are shown in FIG.

PCR

Two specific oligonucleotides of the nptII gene were used as primers for PCR.

5'-GAGGCTATTCGGCTATGACTG-3 '

5'-ATCGGGAGCGGCGATACCGTA-3 '

Genomic DNA was amplified by a method developed by Edward et al. (1991). PCR reaction solutions were 10 mM Tris-HCl (pH8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 50 µg / ml BSA, 0.001% gelatin, 200 μM of dATP, dCTP, dTTP, dGTP and 2 units of Taq Polymerase and 50 pmol The primers were prepared in 35 cycles in a thermal cycler (Perkin Elmer Cetus) (each cycle was performed at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 2 minutes, then left for 7 minutes. Stored at 4 ° C.)

RNA gel blot analysis

Total RNA was extracted to determine whether cDNA clone of CMV Satellite RNA was expressed. 10 μg of RNA samples were treated with formaldehyde and formamide. Treated samples were electrophoresed in a 1.0% agarose gel solution containing 0.67M formaldehyde. The DNA probe was prepared by labeling the 0.3 Kb ScaI fragment with [a- ³² P] dCTP.

Mechanical inoculation test

T ₁ plants with 7-8 leaves developed were used for virus inoculation experiments. CMV-Y and CMV-kor that did not contain Satellite RNA were used for transformed pepper plants and untransformed pepper plants, respectively. Two to three leaves expressing satellite RNA were mechanically inoculated with 10 μg / ml of CMV-Y or CMV-kor dissolved in 0.01 M Na ₂ SO ₄ buffer (pH7.2) using 500 mesh Carborundum. . The inoculated leaf surface was washed with tap water and the plants were grown at 22-30 ° C. in a greenhouse. Post-inoculation symptom development was observed daily. Local lesion assay was performed to identify and quantify the symptoms. The plants were placed in the shade 12-24 hours prior to inoculation with C. amaranticolor about 1 month old and then infected with CMV-Y or CMV-kor in the same manner as above. The infection rate was measured by counting the number of local lesions per leaf.

The present invention was able to obtain the following results through the above experiments and examples. 1 is a photograph showing the whole process from the cotyledons of pepper (C.annuum L) to the development of shoots and whole plants. Most cotyledons formed shoot-bud and often callus about 3 weeks after transplantation. However, shoot-bud and callus formation was inhibited in tissue culture medium supplemented with NAA 1.0 mg / L and Zeatin 2.0 mg / L. Cultured from cotyledons from seedlings older than 11 days reduced shoot regeneration. In other words, cultured from cotyledons from seedlings about 11 days old formed Callus well, but no shoots were formed from Callus. For shoot regeneration, NNA 0.05ml / L and Zeatin 2.0mg / L mixed culture were most suitable. As shoot growth medium, NNA 0.01mg / L and Zeatin 2.0mg / L mixed culture were most suitable.

In addition, the present invention successfully achieved a transformation system for integrating an external gene into pepper plants through Agrobacterum, which has not been tried so far. In the present invention, a transgenic plant was obtained from the tissue culture of red pepper. Agrobacterium and co-cultured non-cultured cotyledons produced light green shoots, which could be distinguished from the transgenic shoot plants showing dark green color. Transformed pepper plants were recovered about 4% (4 Kanamycin-resistant regeneration plants / 98 cotyledons) in selective medium.

As a result of PCR and Southern blot analysis performed by the inventors, a number of transformed pepper plants could be searched quickly. The incorporation of transformed pepper plants was accomplished by amplifying genomic DNA from Kanamyucin resistant pepper plants by PCR using specific primers of nptII gene and fractionating the amplified DNA samples by electrophoresis.

T _One Genetic Safety of External Genes Introduced in Red Pepper Plants

The present inventors obtained four independent pepper plants by the Agrobacterium tumefaciens Ti-plasmid system as described above. Kanamycin-sensitive pepper seedlings were abnormal in medium contained in kanamycin within 6 weeks after germination, whereas resistant pepper seedlings grew normally (FIG. 2). PCR analysis was performed on 43 T ₁ lines from two different T ₀ parents to determine whether the T-DNA region containing nptII gene in Kanamycin-resistant pepper seedlings was genetically stable. The amplified DNA samples were electrophoresed on a 1.2% agarose gel, and 50% of Kanamycin-resistant T ₁ lines that tested DNA fragments contained 0.7 Kb inserts, but no Kanamycin-sensitive T ₁ plants were analyzed. (Figure 3). The reason is not clear, but a set of primers were identified as two small bands of the vector or transformant plant (Fig. 3, lan2 and Lane18). PCR analysis confirmed that the T-DNA region included in the nptII gene was present in 18 T ₁ plants. RNA gel analysis confirmed that the transcript of CMV Satellite RNA was present. Clones obtained from cDNA passages of CMV I ₁₇ N Satellite RNA full length were used as probes for total RNA (FIG. 4). As shown in Figure 4, about 1.0 kb of transcript was detected from the transgenic plants (Lane1-19). The arrow indicates the position of the 1.0 kb transcript. Experimental results showed that the cDNA clone into which CMV Satellite RNA was introduced was transcribed into T ₁ plants. However, there were also descendants who did not produce CMV Satellite RNA transcripts (Lane12).

T _One Delayed and reduced symptom development by inoculation of CMV-Y or CMV-kor in plants

Virus tolerance was measured in T ₁ plants of red pepper by CMV inoculation, and it was confirmed that cDNA of CMV Satellite RNA was expressed in 17 T ₁ plants. The symptom development was observed in T ₁ plants that did not show unique viral symptoms on a daily basis for 2 months but was significantly reduced or delayed in the transformed plants (FIG. 5). Figure 5 (A) is transformed than 5 (B), and the even Y-CMV inoculation there is a reduced or delayed symptoms in the T ₁ plants (2,4) transformed geotinde taken inoculated CMV-kor after 3 months In unconverted plants (1, 3), symptom development was rapid. In the symptom, CMV-Y inoculation showed yellow mosaic and faded leaves, but CMV-kor inoculation showed distinct mosaic and leaf drying. On the other hand, Lacal lesion analysis results are shown in FIG. Fig. 6 (A) shows the group infected with CMV-Y and Fig. 6 (B) shows the group infected with CMV-kor. Experimental results showed that the reduced or delayed effect was significantly higher in the transformed T ₁ plants.

As described above in the Examples and the experimental results, the present invention has the effect of providing the cotyledon tissue of NA pepper and zeatin added to the cotyledon tissue of the pepper which has not been attempted so far and the kidney culture culture conditions. In addition, the present invention has the effect of providing a technique for introducing and expressing cDNA of CMV I ₁₇ N-Satellite RNA into pepper plants using Agrobacterim, which can delay or reduce the symptoms of cucumber mosaic in red pepper plants. The vegetable plant is a very useful invention in the greenhouse cultivation industry.

Claims (3)

In tissue culture of cotyledons of Capsicum annuum L., shoot shoots were healed in medium supplemented with 0.05 mg / L NAA and 2.0 mg / L Zeatin, and kidney shoots grown at 0.01 mg / L NAA and 2.0 mg / L. Tissue culture method of red pepper, characterized in that the denture in the medium containing Zeatin. Agrobacterium tumefaciens LBA4404 / pBOK1 / 105 (KCTC 8828P) that has transferred Plasmid containing cDNA of CMV I ₁₇ N-Satellite RNA Obtaining Agrobacterim tumefaciens strain 4404 transfer of plasmid containing cDNA of CMV I ₁₇ N-Satellite RNA; Cultivating shoots in medium without adding Kanamycin and then performing culture of the myocarcinoma with the myocardium; Washing the dried shoots with sterile water and then culturing them in muscle-induced medium containing Kanamycin Sulfate and Carbenicillin; The shoots are transformed into pepper plants, characterized in that combined with the process of cultivating public culture for about two months after transplanting again to the same muscle growth medium.