WO2016084965A1

WO2016084965A1 - Method for creating virus-free plant individual

Info

Publication number: WO2016084965A1
Application number: PCT/JP2015/083501
Authority: WO
Inventors: 吉川　信幸; 紀子山岸
Original assignee: 国立大学法人岩手大学
Priority date: 2014-11-28
Filing date: 2015-11-27
Publication date: 2016-06-02
Also published as: JPWO2016084965A1; JP6698999B2

Abstract

A method for creating a virus-free plant individual, said method comprising: step (1) for growing an rALSV-infected plant individual for 2 to 6 weeks at a temperature which is higher than the optimum temperature thereof and at which rALSV is inactivated; step (2) for, after step (1), growing the rALSV-infected plant individual at the optimum temperature thereof and thus allowing the elongation of an rALSV-absent site from the rALSV-infected plant individual; and step (3) for grafting the rALSV-absent site elongated in step (2) as a scion and growing the same.

Description

Production method of virus-free plant

The present invention relates to a technique for producing a plant individual having a trait of a recombinant ALSV (rALSV) -infected plant individual and being virus-free.

The present inventors have obtained from a propagation host (quinoa) infected with a recombinant apple small spherical latent virus (ALSV) expressing the flowering promoting gene FLOWERINGERLOCUS T (AtFT) of Arabidopsis thaliana (At). By inoculating the concentrated virus (ALSV-AtFT) RNA into the cotyledons of rose fruit fruit seedlings immediately after rooting using the particle gun method, for example, in the case of apples until flowering that requires 6-12 years after germination The period has been successfully shortened to 1.5 to 3 months (Patent Document 1, Non-Patent Document 1).

ALSV is symptomatically infected without causing illness in apples and maintains stable systemic infection, but the seed transmission rate is low, so the recombinant ALSV expressing AtFT gene (ALSV-AtFT) It is possible to select virus-free individuals from the next generation of Rosaceae fruit trees whose flowering has been promoted by infection with concentrated RNA, and the selected virus-free individuals will be different from fruit trees that have not been transfected It can be used as it is as a breeding material (Patent Document 1).

Furthermore, the present inventors grew seeds that were infected with rALSV that expresses AtFT and induces gene silencing of the Rosaceae flowering suppression gene MdTFL1, and then pollinated the flowered flowers for 12 months after sowing. Has succeeded in fruiting fruits containing next-generation seeds (Patent Document 2).

JP 2010-183891 JP 2014-183754 A

As described above, it is possible to select virus-free individuals from the next generation of Rosaceae fruit trees whose flowering has been promoted by infection with recombinant ALSV (rALSV). It takes six to twelve years to grow to a fruit tree that can. In addition, since apples and pears are cross-fertilized plants, plant individuals germinated from next-generation seeds obtained by pollination may have different traits from the plant individuals of the present generation. Therefore, in fruit tree plants such as apples and pears, “grafting” is used as a means for growing individuals having favorable traits. However, even if an individual having a favorable trait is obtained by rALSV infection, a new plant individual grown by grafting using a part of this individual is safe even if rALSV is infected systemically. Even if secured, it is difficult for producers and consumers to accept, and in practice it is difficult to sell or ship.

For this reason, if at least a part of rALSV-infected plant individuals having favorable traits can be made virus-free, it is possible to produce a new variety by grafting the part as a spike.

As a technique for making a virus-infected plant virus-free, a growth point culture or a method combining growth point culture and high-temperature treatment is also known (for example, Non-Patent Documents 3 and 4). However, in the case of growth point culture, the virus-free part is very small and it is not easy to produce a 100% virus-free strain. In addition, in the case of growth point culture, it takes time and labor for tissue culture and individual regeneration, and further, it takes 6 to 12 years to grow to fruit trees.

The present invention has been made in view of the circumstances as described above, and it is possible to obtain a virus-free plant individual (fruit tree) having a trait of an rALSV-infected plant individual in a simple and short period of time. It aims at providing the production method of a plant individual.

In order to solve the above problems, a method for producing a plant individual of the present invention is a method for producing a plant individual having a trait of a recombinant ALSV (rALSV) -infected plant individual and being virus-free. Process of:
(1) A process of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks;
(2) After step (1), a rALSV-infected plant individual is grown at the optimum temperature, and a rALSV-free site is extended from the rALSV-infected plant individual; and (3) is extended in step (2). It is characterized by including a step of grafting and growing a non-existing portion of rALSV as a spike.

In this production method, the temperature during the growth period in step (1) is preferably 35 ° C. to 42 ° C.

The growing method of the present invention is a growing method for extending a rALSV non-existing site from a plant individual infected with recombinant ALSV (rALSV),
(1) A step of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature of the rALSV-infected plant individual and at which rALSV is inactivated for 2 to 6 weeks;
(2) The method includes a step of growing an rALSV-infected plant individual at the optimum temperature and extending a rALSV-free site from the rALSV-infected plant individual.

In this growth method, the temperature during the growth period in step (1) is preferably 35 ° C. to 42 ° C.

The rALSV-infected plant individual of the present invention is an rALSV-infected plant individual grown by the above-described growing method, and has an extended rALSV non-existing site.

In the present invention, the “plant individual” is a multicellular organism generated from a seed, and means the whole plant composed of a plurality of organs (roots, trunks, branches, leaves, etc.). “Virus-free” in the present invention means a state in which at least rALSV is not present in a plant individual. The “rALSV non-existing site” in an rALSV-infected plant individual is a site where ALSV genomic RNA is not detected by molecular biological techniques such as hybridization and PCR.

According to the plant individual production method of the present invention, a virus-free plant individual (fruit tree) having a trait of an rALSV-infected plant individual can be obtained easily and in a short period of time. According to the growing method of the present invention, a rALSV non-existing site can be easily extended. According to the rALSV-infected plant individual of the present invention, it has an extended rALSV-free site, and by using this rALSV-free site as a graft for grafting, it has a trait of the rALSV-infected plant individual, and a virus A free plant individual (fruit tree) can be obtained.

It is the figure which showed the result of the virus test by RT-PCR from the infected apple seedling (ALSV, ACLSV) before and after a high temperature (37 degreeC) process. The most significant leaf of each seedling (seedling number 1-9) (7th to 10th leaves before high temperature treatment, 11th to 14th leaves after high temperature treatment, and a seedling treated at room temperature for 2 months) The 16th to 20th leaves were tested. NC represents an uninfected leaf, PC represents an ALSV infected leaf, and M represents a 100 bp ladder DNA marker. It is a figure which shows the result of the virus test by dot hybridization and RT-PCR from the apple seedling which grew at normal temperature (25 degreeC) for 2 months after high temperature (37 degreeC) treatment. (A) is a photograph showing apple seedlings grown at room temperature (25 ° C.) for 2 months after high temperature (37 ° C.) treatment. (B) is a diagram showing the results of dot hybridization using RNA extracted from each leaf shown in (A) and a probe for detecting ALSV-RNA. (C) is a diagram showing the results of virus testing by RT-PCR using primers for detecting ALSV and ACLSV. The first to fifth and eighth leaves indicate the leaves that were developed before the high temperature treatment. NC indicates non-infected leaves and PC indicates infected samples. It is a figure which shows the result of the virus test by real-time RT-PCR from the apple seedling grown at normal temperature (25 degreeC) for 2 months after high temperature (37 degreeC) treatment. NC indicates an uninfected leaf, PC indicates an infected sample, ΔRn indicates fluorescence intensity amplified by PCR, and Ct indicates a cycle value. The figure which shows the result of the ALSV test by RT-PCR from the highest leaf in each time point of the 1st week, the 2nd week, the 3rd week, and the 4th week about the apple processed for 1 to 4 weeks at high temperature (37 degreeC). is there. NC indicates non-infected leaves and PC indicates infected samples.

The present invention is a production method for producing a plant individual having a trait of a recombinant ALSV (rALSV) -infected plant individual and being virus-free.

The rALSV-infected plants are not specifically limited, but are preferably fruit trees of the Rosaceae family, particularly apples and pears that belong to the Rosaceae family of Maloideae.

The polynucleotide to be incorporated into rALSV may be various genes or fragments thereof according to the purpose (introduction of foreign genes, induction of silencing of endogenous genes, etc.). Specifically, as already proposed by the present inventors, for example, AtFT gene and MdTFL1 gene for promoting flowering can be preferably exemplified. In addition to this, as described in Non-Patent Document 5, for example, as described in Non-patent Document 5, as a foreign gene to be incorporated into rALSV, a phytoene desaturase (PDS) gene, a sulfur (SU) gene, and a proliferating cell nuclear An antigen (PCNA) gene etc. can be illustrated.

Further, the method of infecting plant individuals with rALSV is not limited, but for example, particle gun inoculation (for example,

Patent Documents

1 and 2, Non-Patent Documents 1 and 2) is suitable.

In the production method of the present invention, it is important to extend a rALSV non-existing site from a plant individual infected with rALSV. Therefore, first, a growing method for extending a rALSV-free site from a plant individual infected with rALSV will be described.

The growing method of the present invention includes the following steps:
(1) A process of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks;
(2) After the step (1), a step of growing an rALSV-infected plant individual at the optimum temperature and extending a rALSV-free site from the rALSV-infected plant individual is included.

Hereinafter, each process will be described in detail.

In step (1), an rALSV-infected plant individual is grown for 2 to 6 weeks at a temperature higher than the optimum temperature and at which rALSV is inactivated (high temperature treatment).

“Optimum temperature” refers to the normal growth temperature range suitable for plant growth. The optimum temperature varies depending on the type and variety of the plant, but for example, in the case of apples and pears, a temperature range of about 15 ° C. to 30 ° C. can be exemplified. The temperature at which rALSV is inactivated is preferably in the range of 35 ° C. to 42 ° C., more preferably in the range of 36 ° C. to 39 ° C., particularly preferably about 37 ° C. (36.5 ° C. to 37.4 ° C.). Can be illustrated. For example, rALSV may not be inactivated when the temperature is lower than 35 ° C., and if it is higher than 42 ° C. (about 37 ° C. depending on the plant species), survival of the plant may be difficult. Specifically, for example, in the case of apples and pears, the temperature of the high-temperature treatment is about 37 ° C. (36.5 ° C. to 37.4 ° C.) in order to achieve both inactivation of rALSV and plant survival. Is preferred. In addition, the growth of rALSV-infected plant individuals at such a temperature can be performed in a room maintained at a predetermined temperature.

The growth period (high temperature treatment period) of rALSV-infected plants in step (1) is 2 to 6 weeks, and the progress of inactivation of rALSV, plant type, growth rate, temperature of high temperature treatment, etc. It can be set as appropriate in consideration. If the growing period (high temperature treatment period) is shorter than 2 weeks, the inactivation of rALSV may be insufficient. In addition, if the growth period (high temperature treatment period) is 6 weeks at the maximum, rALSV is surely inactivated, so it is impractical to set a longer growth period and it is difficult to survive the plant. There is a fear. More specifically, for example, in apples and pears, in order to achieve both inactivation of rALSV and survival of the plant, the high temperature treatment period is 4 weeks when the temperature of the high temperature treatment is 36 ° C. or more and less than 37 ° C. When the temperature of the high-temperature treatment is 37 ° C. or more and 39 ° C. or less for 6 weeks from the beginning, it can be set in the range of 3 weeks to 6 weeks. Particularly in the case of apples and pears, the high temperature treatment period is particularly preferably about 37 ° C. (36.5 ° C. to 37.4 ° C.) and about 4 weeks (25 days to 31 days).

The rALSV-infected plant individual is raised at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks, so that the movement of rALSV within the plant individual stops. For this reason, the rALSV-infected plant individual may not have rALSV moved to the part extended during the growing period of step (1). In this case, in step (1), a rALSV-infected plant individual has a site where rALSV is not present (rALSV non-existing site).

In step (2), an rALSV-infected plant individual is grown at the optimum temperature, and a rALSV-free site is extended from the rALSV-infected plant individual.

The rALSV-infected plant individual that has undergone the step (1) is deactivated due to inactivation of rALSV in the plant individual due to the high temperature treatment. The state in which the movement of rALSV is stopped by such high-temperature treatment is considered irreversible. Even if the rALSV-infected plant individual is grown at the optimum temperature in the subsequent step (2), the movement of rALSV once stopped. Will not resume movement within the plant. This phenomenon has not been confirmed with other viruses, and is a phenomenon peculiar to ALSV (rALSV) newly found by the present inventors.

Therefore, by growing the rALSV-infected plant individual at the optimum temperature in step (2), rALSV does not move to the site extended from the rALSV-infected plant individual within this period. For this reason, the site | part (rALSV non-existing site | part) in which rALSV does not exist expand | extends from a rALSV infection plant individual by the growth in a process (2). Therefore, the rALSV-infected plant individual that has undergone the steps (1) and (2) has a site where rALSV exists (a site developed before step (1)) and a site that does not have rALSV extended from that site. It has a completely new feature.

It should be noted that the growing period of the step (2) is not particularly limited, and can be appropriately set in consideration of, for example, fruit setting or a size as a hogi described later. Specifically, the growing period of the step (2) can be exemplified by a period of about 3 to 24 months as a guide.

As described above, by passing through steps (1) and (2), a rALSV-free site can be extended from a plant individual infected with rALSV. For this reason, the plant individual which has the character of a rALSV infection plant individual, and is virus free can be created using a rALSV absence site.

That is, the plant individual production method of the present invention includes the following steps:
(1) A process of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks;
(2) After step (1), a rALSV-infected plant individual is grown at the optimum temperature, and a rALSV-free site is extended from the rALSV-infected plant individual; and (3) is extended in step (2). It includes a step of grafting and growing a non-existing portion of rALSV as a spike.

Since steps (1) and (2) have been described above as a method for extending the rALSV non-existing site, description thereof is omitted.

In step (3), the rALSV non-existing portion extended in step (2) is grafted and grown as a scion.

Specifically, a rALSV nonexisting site extended to an appropriate length as a hogi is collected from an rALSV-infected plant individual, and is adhered to a cut surface of a desired rootstock to grow as one plant individual. According to this method, since the rALSV non-existing site of the current rALSV-infected plant individual infected with rALSV is grafted, regardless of the selection of next-generation seeds, etc., the trait of the rALSV-infected plant individual, and Virus-free plant individuals (fruit trees) that are not infected with rALSV can be obtained easily and quickly. In addition, a virus-free plant individual (fruit tree) can be obtained easily and much faster than conventional methods such as growth point culture.

Then, as a method for producing a plant individual of the present invention, the above-described step (1) (for the rALSV-infected plant individual infected with rALSV incorporating AtFT gene, MdTFL1 gene, etc. and early flowering (1.5 to 3 months after germination) ( 2) When performing (3), a virus-free plant individual that is not infected with rALSV in a very short time can be obtained from an rALSV-infected plant individual that has been confirmed to flower early.

The method for producing an individual plant of the present invention is not limited to the above embodiment.

Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.
(Example 1)
<1> Materials and Methods (1) Virus Apple small spherical latent virus (ALSV) vector and apple chlorotic leaf spot virus (ACLSV) were tested.

(2) Test plant Apple seeds were wrapped in a paper towel moistened with water and germinated by placing them at 4 ° C for about 3 months, and the germinated seeds were inoculated with ALSV vector.

(3) Preparation of concentrated viral RNA Concentrated viral RNA was prepared as follows. Seven to ten days after inoculation of quinoa with the ALSV vector, the inoculated leaves and the upper leaves showing disease symptoms were sampled. To 10 g of this infected leaf, 30 ml of extraction buffer (0.1 M Tris-HCl (pH 7.8), 0.1 M NaCl, 5 mM MgCl, 1% mercaptoethanol) was added and ground, and then filtered through double gauze. And centrifuged at 9,000 rpm for 10 minutes (4 ° C.). To the resulting solution, 600 μl of bentonite solution (30 mg / ml) was gently added while stirring, and centrifuged at 9,000 rpm for 10 minutes (4 ° C.) to clarify. Next, polyethylene glycol 6000 was added to the supernatant to 8%, and the mixture was stirred in ice for 1 hour. After centrifugation for 10 minutes (4 ° C.), the precipitate was dissolved in 5 ml of extraction buffer and centrifuged for 10 minutes (4 ° C.), and then the supernatant was transferred to a new tube. Subsequently, 3 ml of water-saturated phenol and 3 ml of chloroform were added to the supernatant and vigorously stirred. After centrifugation for 10 minutes (4 ° C.), the aqueous layer was ethanol precipitated to obtain concentrated virus RNA. The concentrated virus RNA and apple chlorotic leaf spot virus (ACLSV) RNA were coated on gold particles, and apple seedlings were inoculated with a particle gun by the following method.

(4) ALSV vector inoculation by particle gun inoculation (Helios Gene Gun System (BIO-RAD)) Microcarriers (RNA; 5 μg / gold particles; 0.4 mg / shot) were prepared by the following procedure. First, 8 mg of gold particles (0.6 μm) were weighed into a 1.5 ml tube, added with 40 μl of sterilized water, and thoroughly mixed with a vortex mixer. The tube was placed in an ultrasonic cleaner and sonicated for 5 minutes. The tube was set in a vortex mixer, and 100 μl concentrated ALSV RNA solution (1 μg / μl) and 60 μl ACLSV genome transcription RNA solution (1 μg / μl) were gently added while stirring. Similarly, 20 μl of 5M ammonium acetate was added gently followed by 440 μl of isopropanol. After stirring for a while, the mixture was allowed to stand at -20 ° C for 1 hour or longer. The supernatant was removed and the gold particle precipitate was stirred for a moment with a vortex mixer. To the gold particle precipitate, 1 ml of 100% ethanol was added, and gently shaken so as not to break the precipitate, and then the supernatant was removed. After this operation was repeated 4 times, the gold particles were transferred to a 15 ml tube using 100% ethanol, and finally the gold particles were suspended in 1200 μl of 100% ethanol and used for the preparation of a gold coated tube. A gold coated tube (BIO-RAD) was set in a tubing prep station (BIO-RAD), and pure nitrogen gas was passed for 20 minutes to completely dry the inside of the gold coated tube. Subsequently, the gold particle suspension is filled uniformly in the gold coat tube and left for 5 minutes to deposit the gold particles in the gold coat tube. Then, 99.5% ethanol of the supernatant is placed in the gold coat tube. Removed from. Subsequently, the tubing prep station was rotated, and pure gold gas was passed through the gold coat tube while uniformly diffusing the gold particles into the gold coat tube, thereby completely drying the gold particles. Subsequently, the gold-coated tube was cut into 20 pieces using a tube cutter (BIO-RAD), and introduced into a plant using a particle gun. The seed coat of the apple seed immediately after germination was removed with a scalpel and inoculated into its cotyledons. Inoculation was performed with 4 shots per individual using Helios Gene Gun System (BIO-RAD) at a helium pressure of 400 psi.

(5) Breeding of inoculated individuals The inoculated individuals were kept for 2 days in the dark at 4 ° C, and then grown at 25 ° C (16 hours light period-8 hours dark period). The seedlings were replanted with seedling culture soil (Takii seedlings) and grown until the 7-10 leaf stage.

(6) Method of extending ALSV-free site from ALSV-infected plant individuals infected with ALSV vector In order to investigate the effect of high temperature (37 ° C) treatment on the growth and distribution of viruses in ALSV-infected apple seedlings, ALSV And apple plants infected with ACLSV were grown under the following conditions.

After growing apple seedlings (7-10 leaves) co-infected with ALSV and ACLSV for 1 week in a 25 ° C incubator (light period 16 hours-dark period 8 hours), a 37 ° C incubator (light period 16) Time-dark period 8 hours) and nurtured for 4 weeks. After that, it is transferred to an incubator at 25 ° C (within the optimum temperature range) (16 hours light period-8 hours dark period) and grown, and virus tests are performed on each leaf before treatment, immediately after treatment, and 2 months after treatment. went.

(7) ALSV and ACLSV assay by RT-PCR Apple RNA was extracted as follows. Leaves (0.05 g) frozen at −80 ° C. and stainless steel beads of 4.8 mm size (Tomy Seiko) were placed in a 2.0 ml sample tube (Assist) and crushed using Micro Smash MS-100R (Tommy Seiko). Add 750 μl of RNA extraction buffer [2% CTAB, 2% PVP K-30, 0.1 M Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), 2 M NaCl, 2% mercaptoethanol] and mix well. And then kept warm at 65 ° C. for 20 minutes with occasional mixing. After treatment, add 750 μl of chloroform, stir for 2 minutes, centrifuge at 14,000 rpm, 4 ° C. for 10 minutes, transfer the aqueous layer to a new 1.5 ml tube, and add 1/3 volume of 7.5 M LiCl to complete the treatment. Was mixed. After standing at −20 ° C. for 1 hour, the mixture was centrifuged at 14,000 rpm and 4 ° C. for 20 minutes, and the resulting precipitate was washed with 1 ml of 70% ethanol. After almost completely removing 70% ethanol, the precipitate was dissolved in 15 μl of RNase free H ₂ O and quantified with a spectrophotometer (ND-1000 v3.1.2).

The reverse transcription reaction was performed as follows. 1 μl RNA solution, 0.5 μl 10 μM Oligo (dT) ₁₈ primer, 4 μl 2.5 mM dNTP mixture (TaKaRa), 2 μl 5 X RT Buffer (TOYOBO), 0.5 μl ReverTra Ace (TOYOBO), 2 μl sterile water The mixture was mixed to 10 μl, and reacted with TaKaRa Thermal Cycler Dice (TaKaRa) at 42 ° C. for 60 minutes, 99 ° C. for 5 minutes, and finally at 4 ° C. for 5 minutes.

PCR primers amplify ALSV
ALSR2 3687 (+) [5'-gccacttcagtgcaactctg-3 '] (SEQ ID NO: 1)
ALSR2 3802 (-) [5'-gaaaaggactcaaagatagcag-3 '] (SEQ ID NO: 2)
For primers that amplify ACLSV,
ACLSV (+) [5'-agatctgaaagcgttcctg-3 '] (SEQ ID NO: 3) and
A combination of ACLSV (−) [5′-ctaaatgcaaagatcagttgtaac-3 ′] (SEQ ID NO: 4) was used.

1 μl cDNA solution, each 4 μM synthetic primer (2 μl), 2 μl 10 μX PCR buffer (TaKaRa), 1.6 μl 2.5 mM dNTP mix (TaKaRa), 0.2 μl Ex-Taq (2.5 U / μl TaKaRa), 13.2 μl of sterilized water was mixed and set in TaKaRa Thermal Cycler Dice (TaKaRa). First, heat denaturation at 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 20 seconds, annealing at 55 ° C for 10 seconds, and DNA complementary strand extension at 72 ° C for 1 minute. Thereafter, it was treated at 72 ° C. for 7 minutes. After completion of PCR, mix 10 μl of the reaction solution and 1 μl of 10X loading buffer (1% SDS, 50% glycerol, 0.05% bromophenol blue), and then perform electrophoresis (100V) on 2.5% agarose gel. The presence or absence was confirmed.

(8) Detection of ALSV by dot blot hybridization RNA was extracted by the above method from each leaf position of the apple at the 26th leaf stage after 2 months from the high temperature treatment. To 3 μl RNA solution containing 270 ng RNA, add RNA denaturation buffer (10xSSC, 50% formaldehyde solution), mix, and after denaturation treatment at 65 ° C for 20 minutes, quench in ice water for dot blot hybridization. A sample was provided. The denatured RNA solution was dot blotted onto a nylon membrane Hybond-N + (GE Healthcare), and the membrane after the dot blot was treated with 20 × SSC, followed by UV irradiation to fix the RNA to the membrane. The membrane is immersed in a hybridization solution (50% formamide, 5xSSC, 0.1% N-lauroyl sarcosine sodium, 0.02% SDS, 2% Blocking reagent (Roche)), prehybridized at 68 ° C for 1 hour, and then the ALSV genome. Hybridization was performed at 68 ° C. for 18 hours using a Vp20 (−) probe, which is a digoxigenin (DIG) -labeled RNA probe having a complementary sequence in the upper Vp20 region. After completion of hybridization, the membrane was washed twice with washing solution 1 (2xSSC, 0.1% SDS) for 5 minutes (room temperature) and then twice with washing solution 2 (0.1xSSC, 0.1% SDS) for 15 minutes (68 ° C). . Next, the membrane was treated with buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 5 minutes (room temperature) and then with blocking buffer (10% Blocking reagent: buffer 1 = 1: 9) for 1 hour (room temperature). Processed and blocked the membrane. After the blocking, the membrane was reacted for 30 minutes (room temperature) in an antibody solution in which Anti-Digoxigenin.AP.Fab fragments (Roche) was diluted 1/10000 with a blocking buffer. After completion of the antibody reaction, the membrane was washed twice with washing solution 3 (buffer 1 containing 0.45% Tween 20) for 20 minutes (room temperature) and further in buffer 4 (0.1 MTris-HCl, 0.1 M NaCl, 50 mM MgCl ₂ , pH 9.5). Gently shaken twice for 3 minutes (room temperature). Subsequently, CDP-Star Detection Reagent (GE Healthcare) was reacted on the membrane for 5 minutes, wrapped in the membrane, and a signal was detected by ImageQuantLAS4000 (GE Healthcare).

(9) Detection of ALSV by real-time RT-PCR Real-time RT-PCR was performed by the following method using RNA extracted from the top leaves of 11-14 leaf apples immediately after the high temperature (37 ° C) treatment. 1 μl (100 ng) of RNA solution from the test sample, 10 μM Oligo (dT) 1 ₈ 0.5 μl, 2.5 mM dNTP mixture (TaKaRa) 4 μl, 5 × RT Buffer (TOYOBO) 2 μl, ReverTra Ace (TOYOBO) 0.5 μl, sterile water Mix 2 μl to 10 μl, react with TaKaRa Thermal Cycler Dice Version III (TaKaRa) at 42 ° C. for 60 minutes, 99 ° C. for 5 minutes, and finally at 4 ° C. for 5 minutes to obtain the cDNA solution used for the next qPCR method. did. For amplification by SYBR method, the sequence between 6150-6279 of ALSV-RNA1 can be amplified.
primer4F 6150 (+) [5'-cgatgaatctccctgataga-3 '] (SEQ ID NO: 5),
primer4R 6279 (−) [5′-agagtagtggtctccagcaa-3 ′] (SEQ ID NO: 6) was used as a positive strand primer and a negative strand primer, respectively. Mix 1 μl of 10 μl of cDNA solution obtained by reverse transcription reaction with 0.2 μl of 15 μM plus strand primer and 15 μM minus strand primer, 8.6 μl of sterilized water, and 10 μl of SYBR ^(R) Premix Ex Taq ^TM (TaKaRa). , ECO ^™ Real-Time PCR System (Illmina) was used for 30 seconds at 95 ° C, followed by 35 cycles of [95 ° C, 5 seconds → 60 ° C, 30 seconds], followed by [95 ° C, 15 Second → 55 ° C., 15 seconds → 95 ° C., 15 seconds] was processed in one cycle to complete qPCR.
<2> Results (1) Detection of ALSV and ACLSV from virus-infected apple seedlings before, immediately after, and 2 months after treatment The highest of apple seedlings (individual numbers 1 to 9) inoculated with ALSV and ACLSV When RT-PCR test was performed on the upper leaves (7th to 10th leaves), ALSV and ACLSV were detected from all seedlings (Fig. 1), and it was clear that both viruses were systemically infected into apple seedlings Became. When these seedlings were treated with high temperature (37 ° C., 4 weeks) and the topmost leaves (11th to 14th leaves) were subjected to RT-PCR assay, neither ALSV nor ACLSV was detected (FIG. 1). This was thought to be because the replication of both viruses was inhibited by high-temperature treatment. When these plants were grown at room temperature (25 ° C) and grown for 2 months, the topmost leaf was subjected to RT-PCR test. ACLSV was detected in all individuals, but ALSV was not detected in any individuals. (FIG. 1).
(2) Distribution of ALSV and ACLSV in apple seedlings grown at room temperature for 2 months after high-temperature treatment To investigate the virus distribution in seedlings grown at room temperature for 2 months after high-temperature treatment, as shown in Figure 2A From the first leaf to the top leaf, the virus was assayed by dot blot hybridization and RT-PCR. As a result, as shown in FIG. 2B, in the dot hybridization method, a signal indicating ALSV infection was detected in the first to fifth leaves, but no ALSV was detected in the leaves of the tenth and higher leaves. . In the RT-PCR assay, ACLSV was strongly detected in all leaves from the 1st to 10th and 15th and higher leaves, whereas ALSV was detected weakly (thinly) in the 1st to 8th leaves. However, it was not detected at all in the leaves higher than the 10th leaf (FIG. 2C). From the above, it is clear that low concentration of ALSV is present in the 1st to 8th leaves developed before the high temperature treatment, but ALSV is not present in the upper leaves developed after the high temperature treatment. Became.
(3) ALSV detection by real-time RT-PCR from apple seedlings grown at room temperature for 2 months after high-temperature treatment To further confirm the RT-PCR assay results, real-time detection sensitivity about 1000 times that of RT-PCR The ALSV assay of apple seedlings was performed by RT-PCR. As a result, as shown in FIG. 3, the fluorescence signal of the infected apple sample started to increase from 10 cycles, whereas the increase in the fluorescence signal was not observed in the seedlings grown for 2 months at room temperature after the high temperature treatment, It was proved that ALSV was not infected.
<3> Conclusion When an apple seedling infected with an ALSV vector is treated at high temperature (37 ° C), no ALSV is found in the branches and leaves grown after the treatment, and the individual has a site where ALSV is not present (ALSV non-existing site) Can be created. Therefore, by grafting the ALSV-free site, which is extended using a recombinant ALSV (rALSV) -infected plant individual, as a spikelet, it has the characteristics of a recombinant ALSV (rALSV) -infected plant individual and is virus-free. It is possible to easily grow a plant individual.

(Example 2)
Since high-temperature treatment is also a great stress for plants, in order to obtain virus-free plant tissue by high-temperature treatment, it is necessary to set a high-temperature treatment period in which virus growth is stopped and the temperature at which plants can survive is compatible. is there. Therefore, the high temperature treatment period necessary to form a site where no ALSV is present (ALSV non-existing site) was examined.
Specifically, in the case of apples, at 37 ° C., individuals that die when grown for 4 weeks or more appear, so the high temperature treatment period was set to 4 weeks. 37 ° C is a temperature near the limit for survival of apples and pears, and there is a risk that it will die at temperatures higher than this.
Subsequently, the treatment period at 37 ° C. and the fluctuation of ALSV were investigated (FIG. 4). Specifically, the highest leaf at that time was sampled from each individual for each week of high temperature treatment, and ALSV test was performed by RT-PCR. ALSV was found in all samples after 1 week and 2 weeks of high temperature treatment. was detected. This is thought to be the result of the leaf primordium that ALSV invaded before the high temperature treatment developed as leaves during the high temperature treatment.
Furthermore, when high temperature treatment was continued, ALSV was not detected from 4 out of 7 individuals at 3 weeks and from 5 out of 7 individuals at 4 weeks. This is thought to be because ALSV was removed from the shoot apical meristem by high-temperature treatment and could not enter the leaf primordium. In addition, after treatment for 4 weeks at 37 ° C, these individuals were transferred to 25 ° C and grown for 2 months, and the developed uppermost leaves were tested, including 2 individuals in which ALSV was detected in the 4th week of high temperature treatment, ALSV was not detected in the individual. This means that after high-temperature treatment for 4 weeks, ALSV is removed from the apple shoot apical meristem, and as a result, there is no ALSV in the tissue that develops thereafter.

In Examples 1 and 2, the apples were subjected to a high-temperature treatment at 37 ° C., but the temperature is not limited to this. 35 to 42 ° C. (preferably depending on the type of plant) In the range of 36 ° C. to 39 ° C.), an individual having a site where no ALSV is present (ALSV non-existing site) can be produced.

Claims

A method for producing a plant individual having a trait of a recombinant ALSV (rALSV) -infected plant and virus-free, comprising the following steps:
(1) A process of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks;
(2) After step (1), a rALSV-infected plant individual is grown at the optimum temperature, and a rALSV-free site is extended from the rALSV-infected plant individual; and (3) is extended in step (2). A production method comprising the step of grafting and growing a non-existing portion of rALSV as a spike.
2. The production method according to claim 1, wherein the temperature during the growth period of step (1) is 35 to 42 ° C.
From a plant individual infected with recombinant ALSV (rALSV), a growth method for extending a rALSV-free site,
(1) A process of growing an rALSV-infected plant individual at a temperature higher than the optimum temperature and at which rALSV is inactivated for 2 to 6 weeks;
(2) A growing method comprising a step of growing an rALSV-infected plant individual at the optimum temperature and extending a rALSV-free site from the rALSV-infected plant individual.
4. The method according to claim 3, wherein the temperature during the growth period in step (1) is 35 ° C. to 42 ° C.
An rALSV-infected plant individual grown by the method according to claim 3, wherein the plant has an extended rALSV-free site.