KR101149171B1 - Method for production of virus-free plants and seeds from virus infected plants - Google Patents

Abstract

The present invention comprises the steps of separating the tissue from the virus infected plant; Inoculating the separated tissue on a callus organic medium to induce callus; Separating the separated callus into a predetermined size using a sieve; And culturing the separated callus in a callus culture medium and then re-differentiating to form a redifferentiated body, thereby providing a method for producing virus free strains and seeds from a virus infected plant. According to the present invention described above, a large amount of virus-free bottles and seeds can be produced from virus-infected plant tissues using a plant tissue culture technique having a simple process and high production efficiency.

Description

Method for production of virus-free plants and seeds from virus infected plants}

The present invention relates to a method for producing virus free strains and seeds from virus infected plants. More specifically, the present invention relates to a method for producing a large amount of virus-free bottles and seeds from virus-infected plant tissues using a simple process and high production efficiency plant tissue culture techniques.

Plant viruses cause disease in many plants and cause serious economic damages throughout agricultural production, including reduced yields of various crops and flowers, deterioration of quality and varietal degradation. In recent years, as the international trade volume of grains and ornamental plants, such as food, has increased, foreign pathogenic plant viruses have been introduced into the problem. Thus, virus prevention, treatment and quarantine of cultivated plants is one of the important challenges in the plant industry.

Conventional methods for producing virus-free plants are largely methods using growth point culture (Gregory C., Phillips, GB Collins., 1979. Virus Symptom-Free Plant of Red Clover Using Meristm Culture.Crop Sci 19: 213-216.N. Verma, R. Ram, V. Hallan, K. Kumar, AA Zaidi, 2004, Production of Cucumber mosaic virus -free chrysanthemums by meristem tip culture.Crop Protection 23: 469-473), Methods using grafting (N. Katoh, M. Yui, S. Sato, T. Shirai, H. Yuasa, M, Hagimori, 2004, Production of virus-free plants from virus-infected sweet pepper by in vitro grafting.Sciientia Hort., 100: 1-6 J. Ju, E. Camarasa, C. Ortega, JM Arregui, M. Cambra, G. Li, L. Navarro. 1992. RECOVERY OF VIRUS-FREE ALMOND PLANTS BY SHOOT-TIP GRAFTING IN VITRO.Acta Hort. (ISHS) 309: 393-400), methods using heat treatment and tissue culture (H. Lozoya-Salda, O. Merlin-Lara, 1984, Thermotherapy and tissue culture for elimination of potato virus X (PVX) in Mexican potato cultivars resist ant to late blight.Am. J. Pot.Res., 61: 735-739 Bruna, A. 1997. EFFECT OF THERMOTHERAPY AND MERISTEM-TIP CULTURE ON PRODUCTION OF VIRUS-FREE GARLIC IN CHILE. Acta Hort. (ISHS) 433: 631-634), methods using chemical treatment and heat treatment (Kim, MS, Lee, YR, Song, CH, Kim, BH, Kyun, SB, Eun, JS, 1997, Elimination of Odontoglossum ring spot virus (ORSV) from orchid ( Cymbidium spp.) By chemotherapy and thermotherapy.- RDA J. Crop Protect. 39: 19-24.Vcelar, BM, Ferreira, DI, Niederwieser, JG 1992, Elimination of Ornithogalum mosaic virus in the Ornithogalum cv Rojel through meristems tip culture and chemotherapy . Plant Cell Tissue Organ Cult. 29: 51-55.), But the production efficiency is low and the process has a complex problem.

More specifically, the method of using the growth point is limited to the available tissue because there is only one apical part in the plant, low production efficiency, low efficiency of extracting the apical part, and using a variety of samples to produce a genetically uniform plant There was a problem that, if this method failed, limited genetic resources were reduced. In addition, the method using the grafting method is made by hand and there is a high risk of continuous infection if there is an infected plant during the operation. Further, the heat treatment and chemical treatment method has a problem that the germination rate of the plant during the heat treatment is reduced by about 3 to 5%, problems such as genetic modification by chemicals, and the process is complicated.

The present invention has been made to solve the problems of the prior art, it is an object of the present invention to provide a method for mass production of virus-free bottles or seeds in a high efficiency by a simple process in plants or seeds infected with viruses.

Another object of the present invention is to provide a method for producing genetically homogeneous virus free strains or seeds with minimal genetic modification from plants or seeds infected with the virus.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the present invention comprises the steps of separating the tissue from the virus infected plant; Inoculating the separated tissue on a callus organic medium to induce callus; Separating the separated callus into a predetermined size using a sieve; And culturing the separated callus in a callus culture medium to re-differentiate to form a re-differentiated body.

As described above, the present invention has an advantage in that the callus derived from the virus-infected plant tissue is crushed finely using a sieve to secure a large amount of tissues in which virus migration is not developed between cells. Therefore, according to the present invention, virus-free bottles or seeds can be mass-produced at high efficiency by a simplified process. The recovery rate of virus free strains according to the present invention was as high as 30% among the redifferentiated individuals. In addition, problems such as a decrease in germination rate or genetic modification due to heat treatment or chemical treatment by the conventional method can be prevented.

In the present invention, the tissue is not particularly limited, any tissue may be used. That is, one or more may be selected from the group consisting of leaves, stems, roots and seeds. As described above, in the present invention, since all tissues of the plant can be used, the production efficiency and work efficiency are high, and the production cost can be reduced, and the limited gene source can be efficiently used, compared to the conventional method using the apical part. There is an advantage that can produce a large amount of genetically uniform plants.

In the present invention, the sieve is not limited thereto, but is preferably 150 ~ 1000㎛ size. When the callus is separated, a small sieve of 150 μm or less results in damage to the cells, resulting in a decrease in callus culture rate. When using a large sieve of 1000 μm or more, the recovery of disease free strains is low due to virus migration between callus cells. You lose.

In the present invention, the redispers can be separated after 10 weeks from the separated callus. Therefore, according to the present invention, there is an advantage in that a large amount of re-differentiated body can be produced in a relatively short time.

In the present invention, the plant is not particularly limited, but may be selected from the group consisting of tobacco, tomatoes, peppers, carrots, and gourd plants having a high virus damage, and tobacco is preferable.

In the present invention, the callus organic medium is 3% sucrose (sucrose) in MS solid medium, plant-agar 0.8%, BAP (Benzylamino purine) 1mg / L, NAA (1-Naphthylacetic) acid) 0.1mg / L, and the callus culture medium contained 3% sucrose to MS solids, 0.3% plant-agar, 1 mg / L BAP to 0.1% NAA It is composed by adding mg / L. The MS medium is preferably a semi-solid medium for smooth absorption of hormones after sieving.

In the present invention, the isolated tissue may be separated from the callus 6 to 8 weeks after being healed in the callus organic medium. Therefore, according to the present invention, there is an advantage in that a large amount of re-differentiated body can be produced in a relatively short time.

According to another aspect of the present invention, the present invention provides a method for treating a virus, comprising: separating tissue from a virus infected plant; Sterilizing the separated tissue; Organically separating the separated tissues with callus; Separating the separated callus into a predetermined size using a sieve; Re-differentiating the separated callus to form a re-differentiated body; And the re-differentiated body provides a method for producing virus-free strains and seeds from virus-infected plants, characterized in that comprising the step of performing the RT-PCR to determine whether the virus infected.

The sterilization step is a sterilization step for tissue culture and is not particularly limited and may be performed by a conventionally known method. In addition, the RT-PCR is visually observed to confirm that the re-differentiation is a virus-free strain, such as when the virus-infected plants can not be distinguished.

Although not limited thereto, in the present invention, the plant is tobacco, the virus is a PMMoV virus ( Pepper Mild Mottle Virus ), and the RT-PCR extracts RNA from a redistribute and quantifies it using a Tabacco 18S rRNA sequence. It is carried out using PMMoV primers having base sequences of Nos. 3 and 4.

According to the present invention having the above-described configuration, it is possible to achieve the effect of mass production of virus-free strains or seeds with high efficiency by a simple process using all tissues of the virus-infected plant or seed.

In addition, according to the present invention, it is possible to minimize genetic modification from a virus-infected plant or seed and to produce a large amount of genetically homologous virus-free strains or seeds.

Therefore, the present invention will be said to be a very useful invention that can produce a large amount of virus-free strains or seeds having homogeneous genes even when all excellent gene sources are infected with viruses.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Virus Infected Tobacco Individual Formation

1) Tobacco seed sowing

sunshine mix No. 5 (Ihobio) Sowing Samsun nn varieties in a greenhouse using soil and transplanting them into a greenhouse after 4 weeks, transplanting them into one pot per tobacco individual, the length of the seeded tobacco is about 5cm, tobacco leaves are more than 4 leaves If so, it was infected with a virus.

2) virus infection

To form the virus infected plants, the plants were Tobacco Samsun nn and the viruses were PMMoV ( Pepper Mild Mottle Virus ). In a mortar and pestle, 0.1 g of virus-infected plants were mixed with 100-fold 0.1 M PBS buffer (NaCl 136 mM, KCl 2 mM, Na ₂ HPO _{4 10} mM, KH ₂ PO ₄ 1.76 mM). Except for one or two leaves close to the growth point, three sequential leaves were lightly scattered with caborundum. The leaves sprinkled with Caborundum were smeared with PBS buffer and PMMoV virus on the fingers and applied slightly to the sides of the middle vein.

3) Confirmation of Virus Infection

Virus infection was confirmed by RT-PCR using a PMMoV-CP primer (see SEQ ID NOs: 3 and 4 in Table 1) to a visually identified individual (see FIG. 2).

Example 2 Regeneration of Organs Using Virus Infected Cigarettes

1) tobacco callus organic

In cigarettes infected with PMMoV virus, mosaic symptoms appeared after about 10 days, and when virus infection was confirmed by RT-PCR, sterilized (70% EtOH 30 seconds shake->) using the third and fourth leaves from the growth point for tissue culture. 3 times washing with sterile water-> 15 minutes soaking in 50% Lax-> 3-4 times washing with sterile water-> dry). Subsequently, cut to 1 cm in width and then callus organic medium (MS solid, sucrose 3%, plant-agar 0.8%, BAP (Benzylamino purine) 1mg / L, NAA (1-Naphthylacetic acid) 0.1mg / L: see Table 3) Was wounded). Every two weeks, the medium was transferred to induce callus, and the callus was inoculated and re-differentiated individuals were removed. As described above, the callus was used to obtain a callus suitable for separation after about 6 to 8 weeks after toothache in the callus induction medium.

2) Virus Blacking of Organic Callus

RT-PCR was performed by extracting ELISA and RNA as follows to determine the change of virus in callus induced by tissue culture.

* ELISA method (using Kisan Bio)

① Add 100 μl of coating antibody (well) to each well in a 96 well plate.

② Incubate for 4 hours in a humid place at 37 ℃.

③ Wash 3 ~ 4 times with 1X PBST buffer and shake off with towel to remove bubbles.

④ After grinding the sample and control leaves in 1.5ml E-tube finely using liquid nitrogen, add 1m of 1X General Extract Buffer and put 100µl of the solution into each well.

⑤ After incubating at room temperature for about 2 hours, wash 3 ~ 4 times.

⑥ Dilute 1/500 enzyme conjugate in 1 × ECI buffer and add 100 μl to each well.

⑦ After incubation at room temperature for about 2 hours, wash (washing) five times and wash off with a towel to remove bubbles.

⑧ Add 100 μl of 1 × PNP substrate to each well.

⑨ Put in the humid box (humid box) after 30 minutes the first measurement, 1 hour after the second measurement.

⑩ Measured at 405nm using a Microplate Reader.

* RNA extraction using Tri-reagent

① Put 0.1g of leaf of redispers into 1.5ml E-tube, add liquid nitrogen, and grind it using a grinder.

② Vortex vigorously for 15 seconds after adding 1 ml of Tri-reagent.

③ After 5 minutes at room temperature, 12000R.P.M. Centrifuge at 4 ° C for 10 minutes.

④ After centrifugation, separate the supernatant carefully into a new E-tube, add 0.2 ml of chloroform, and vortex again for 15 seconds.

⑤ Leave at room temperature for 15 minutes and once again press 12000R.P.M. Centrifuge at 4 ° C for 10 minutes.

⑥ After centrifugation, separate the supernatant carefully into a new E-tube, and add 0.5 ml of isopropanol and 3M NaOAc and leave it at room temperature for 10 minutes. And 12000R.P.M. Centrifuge for 8 min at 4 ° C.

⑥ Discard the supernatant, add 1 ml of 75% EtOH, and add 12000R.P.M. Centrifuge at 4 ° C for 5 minutes.

⑦ Discard EtOH and dry.

⑧ After drying, add 50µl of DEPC-water to dissolve RNA.

The primer sequences used at this time are shown in Table 1.

Tobacco 18S rRNA
F: 5'- TGGTACGGCCGTTCTTAGT-3 '
 SEQ ID NO: 1 R: 5'-TCGGCCAAGGCTATATAAACTCGT-3 '
 SEQ ID NO: 2
PMMoV-cp
F: 5 '-CTTAAGGAGTTGTAGCCCAG-3'
 SEQ ID NO: 3 R: 5 '-ATGGCTTACACAGTCTCC-3'
 SEQ ID NO: 4

RT-PCR was used INTRON BIOTECHNOLOGY Maxime ^TM RT-PCR PreMix Kit, RT-PCR conditions are shown in Table 2.

Reverse Transcription Reaction 45 ℃ 30min
Inactivation of Rtase 94 ℃ 5min 1cycle Denaturation 94 ℃ 45sec
Annealing 55 ℃ 45sec
Extension 72 ℃ 1min
32cycle Final Extension 72 ℃ 5min
4 ℃ ~

As described above, in order to determine the virus migration and virus change in the callus state, the result of confirming the virus by RT-PCR by extracting DAS-ELISA and RNA in the callus state is shown in FIG. 3. As a result, as shown in FIG. 3, the tobacco callus (No 1-27) was higher than the negative control group because the DAS-ELISA result showed 0.15 to 0.42 in the OD ₄₀₅ . Therefore, virus was identified in all callus by DAS-ELISA and RT-PCR method, and the more swelling callus, the lower DAS-ELISA result was measured (FIG. 3).

3) Segmentation of tobacco callus

If the tobacco callus grows to 1 cm or more after about 6 weeks, the plant is separated into 300 mesh using a sieve (cell dissociation sieve-Tissus Grinder kit (SIGMA-ALDRICH, Germany)) and plant agar (plant) agar) was wound on a semi-solid medium containing 0.3% (see c of Figure 4b). While visually confirming that the cells of the separated callus grew, when each cell became more than 0.4 cm, it was transferred to a solid medium and healed, and the medium was changed every two weeks. The callus grew actively after about 4-6 weeks, and the callus was visually observed (see d of FIG. 4B). Table 3 shows the medium conditions for inducing callus to regenerate tobacco plants from leaf explants and isolate callus.

Medium compounds induction of callus from leaf Induction of callus from segmented callus Medium MS solid MS solid Carbohydrate 3% Sucrose 3% Sucrose Hormone BAP 1mg / l, NAA 0.1mg / l BAP 1mg / l, NAA 0.1mg / l Agar Plant-agar 0.8% Plant-agar 0.3% pH 5.6-5.8 5.6-5.8

4) Regeneration and Root Induction of Isolated Tobacco Callus

When regeneration begins after more than 10 weeks from the separated callus, the redispers are separated from the callus and individually healed in petri-dish, and when the leaves are differentiated more than 3-4 sheets, the medium containing IAA hormone (IAA) Root was induced at 0.1 mg / l) (see e of FIG. 4b).

5) Purification to Greenhouse Greenhouse

When roots are induced, they are transferred to pots and purified in greenhouses.

Example 3 Analysis of Redifferentiated Body

1) visual inspection

After acclimatization to the greenhouse, it was visually observed to separate the first and second appearance of the mosaic pattern from each individual leaf (Figs. 5a and 5b).

2) Extraction of RNA and RT-PCR

RNA was extracted from each redifferentiator using Tri-reagent, quantified using 18S rRNA, and then isolated and confirmed by virus-infected and virus-infected individuals by RT-PCR using PMMoV-cp primer. The results are shown in Figures 6a and 6b. As a result, the recovery rate of virus free strain was 30%.

Example 4 Conducting a Second Experiment and Obtaining Seeds of Redifferentiated Body

1) Second experiment

RT-PCR Results A second experiment (RT-PCR) was prepared using relatively blurred banded individuals. The method was the same as the first experiment.

2) Harvesting Seeds of Regeneration

After RT-PCR results, seeds of plants not infected with virus were obtained (FIG. 7).

The present invention described above is not limited to the above-described embodiments and drawings, and various substitutions, modifications, and changes can be made without departing from the technical spirit of the present invention. It will be apparent to those who have

1 is a schematic diagram of a method for producing virus free strains and seeds from a virus infected plant according to the present invention.

Figure 2 is a photograph showing the tobacco plants infected with the PMMoV virus ( Pepper Mild Mottle Virus ).

FIG. 3 shows the accumulation of coat proteins in selected viral infected callus as measured by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Infected tobacco callus (No. 3-27) showed 0.15 to 0.42 at OD ₄₀₅ . P: positive control, N: negative control, 3-27: infected tobacco callus

Figures 4a and 4b is a view showing the various stages of isolated callus induction and apical regeneration in tobacco by the present invention. (a) Callus derived from leaves (b) Two-month-old callus (c) Isolated callus implanted on semi-solid medium (d) Semi-solid medium (medium was exchanged every two weeks and only large-sized callus by tweezers) Callus induced for 6 weeks from isolated callus on fresh medium) (e) Re-differentiated apical (wounded in apical medium when the size of callus was 0.8 mm or more, and apical medium after re-differentiated apical (IAA 0.1mg / l) )).

5A and 5B are photographs of plants re-differentiated from isolated callus virus infected by the present invention. (a) Phenotypes of virus-free R1 plants (left) and R1 plants (right) infected with PMMoV (b) Viral-free R1 plant leaves (bottom) and R1 plant leaves (top) infected with PMMoV. Leaf of R1 plant infected with PMMoV shows mosaic pattern and leaf malformation (c) Picture of virus-free R1 plant (d) Picture of R1 plant infected with PMMoV

Figure 6a and 6b is an electrophoresis picture (RT1: redifferentiation of infected tobacco plants) of RT-PCR of R1 regeneration plants according to the present invention. M: 100 bp DNA marker (Bionix Co.); P: positive control (RNA of infected tobacco with PMMoV); N: negative control (RNA of virus-free tobacco); 1-27: Regenerated Tobacco Plants

7 is a plant photograph for obtaining tobacco R1 seed according to the present invention.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Method for production of virus-free plants and seeds from virus          infected plants <130> P11-091120-01 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 1 tggtacggcc gttcttagt 19 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 tcggccaagg ctatataaac tcgt 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 cttaaggagt tgtagcccag 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 4 atggcttaca cagtctcc 18  

Claims (8)

Separating the tissue from the virus infected plant; Inoculating the separated tissue on a callus organic medium to induce callus; Separating the separated callus into a predetermined size using a sieve; And And culturing the separated callus in a callus culture medium and then re-differentiating to form a redifferentiation method. The method of claim 1, wherein said tissue is selected from the group consisting of leaves, stems, roots, and seeds. The method of claim 1, wherein the sieve is 150-1000 μm in size. The method of claim 1, wherein the regeneration is separated after 10 weeks from the isolated callus. The method of claim 1, wherein the plant is selected from the group consisting of tobacco, tomatoes, peppers, carrots, and nectarines. The method of claim 1, wherein the callus is separated 6 to 8 weeks after the isolated tissue is placed on callus organic medium. The method of claim 1, wherein the redifferentiated body further comprises the step of performing RT-PCR to determine whether the virus is infected. The method according to claim 7, wherein the plant is tobacco, the virus is a PMMoV virus ( Pepper Mild Mottle Virus ), the RT-PCR extracts RNA from the re-differentiation and quantified using Tabacco 18S rRNA and then SEQ ID NO: 3 and A method of producing a virus free strain from a virus infected plant, characterized by using a PMMoV primer having a nucleotide sequence of 4.

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