KR20140040513A - Production of virus free plants from in vitro shoot tips through in vitro meristem culture - Google Patents

Abstract

The present invention relates to: a method of producing an in vitro plant body by tissue-culturing a shoot tip for collecting a growing point for apical meristem culture; a proliferation medium for the method; a production method of virus and disease-free stocks through the apical meristem culture using the growing point collected from the in vitro plant body which is produced by the method; and a medium for the production method. According to the present invention, a user can collect the growing point without the limitation of the seasons, and mass-produce the plant bodies with low virus contamination rate and high survival rate, thereby contributing to the increase of income in farmhouses by easily supplying high quality varieties, and being expected to be able to export the high quality varieties by improving international competitiveness through enhancing the quality of products.

Description

TECHNICAL FIELD The present invention relates to a method for producing a virus free disease through cultivation of a growing point obtained from an in vitro plant,

The present invention relates to a method for producing a virus-free disease through a growth point culture, and a propagation and rooting medium for use in such a production method.

When a plant is infected with a virus, the yield decreases (20-30%), the appearance becomes worse, the sugar content decreases, and the color is faded. Once the virus is infected, the plant is not healed and a method is needed to remove the virus and produce virus-free plants.

Representative methods for producing virus-free disease plants include callus culture, growth-point culture, and in-flight grafting.

First, the growth point culture refers to a culture method in which a growth point of a plant grown in vitro or a surrounding tissue including the growth point is separated and cultured in vitro. The growth point is separated and cultured in a tissue containing a growth point, for example, in a calf, a root, or a bud by a method belonging to a tissue culture. Therefore, the growth point culturing is usually carried out by separating the tissue including the growth point from the outpatient plant, disinfecting the separated tissue, separating the growth point from the disinfected tissue, culturing the separated growth point in the cabin, . By using the growth point culture method, it is possible to reproduce the same individual, and therefore, the uniformity of the product is guaranteed, and mass production of excellent varieties is possible. However, since the plant is harvested from the plant, the disinfection process is essential. Thus, plants that have undergone disinfection prior to the growth stage and which are nevertheless difficult to remove are subject to heat treatment of the plants before the growth stage to lower the virus contamination rate and extend the range of the virus non-toxicity. However, there is a problem that the heat treatment process is complicated and the growth point is difficult to disinfect and low survival rate after disinfection. In addition, there is a difference in the degree of difficulty of cultivation depending on the size of the growing point. The smaller the size of the growing point, the higher the probability of virus removal, but the difficulty of culturing becomes difficult. In addition, to remove the virus, the growth point is cut to a small size of about 0.2-0.5 mm, but it is very difficult to cut this small size.

The callus cultivation is similar to the growth point culturing, but in the case of the growth point culture, the plant stem and leaf are differentiated directly at the growth point, but the callus culturing differentiates the callus in the callus after organizing and propagating the callus at the growing point . In the case of callus cultivation, there is a difficulty in maintaining excellent varieties because of high probability of genetic variation, and in many plants, there is a problem that plant differentiation is not easy in callus.

The in-flight grafting method is a method of grafting the rootstock of seedlings using a grafted tissue to receive grafts and grafting the grafts in the same manner as the grafting and grafting. It requires a technique of grafting and has a low survival rate .

In addition, since the growth points, callus cultures, and in-flight grafting methods used in the above-mentioned grafting points are obtained from out-of-plant plants, the concentration of growth inhibitory substances (for example, ABA, penic compound) There is a limit to the time of harvesting the growing point that the survival rate is high only in spring when the content of gibberellin, dioxin, and cytokinin is high.

Therefore, there is a need to develop a method for producing virus-free disease plants by eliminating the disadvantages of the conventional growth-point culture method by producing plants according to the growth-point culture method.

Accordingly, a problem to be solved by the present invention is to provide a method for producing a virus-free disease plant having a low rate of contamination and a high plant survival rate, capable of harvesting the growth point without seasonal limitation, and a medium for use in such a method.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a method for producing an in-flight plant for cultivating a growing point for culturing a plant in a vitro, comprising the step of cutting and disinfecting the plant in vitro and the step of tissue culture in the cabin do.

The method according to the present invention can be applied to the production of plants by the growth point culturing, in which various problems caused by harvesting the growth points in the out-of-plant plants, such as low plant survival rate, high fungal and bacterial contamination rates, The present invention relates to a method for producing a plant by in-vitro tissue culture for harvesting a growing point for growing a growing point.

Describing the above method in each step,

(S1) a step of cutting and disinfecting the plant in an ex vitro condition

The length of the calf cut to be cut can be appropriately adjusted in consideration of the cutting site, the culturing time, the kind of the plant, and the like, as long as a person skilled in the art is aware of the length of the calf. In the case of apples or apricots, it may be cut to a length of 5-10 mm, but is not limited thereto. The cutting time is preferably the spring, in which the concentration of the growth inhibitory substance (for example, ABA, penolic compound) in the plant is low and the content of the growth promoting substance (for example, gibberellin, oleic acid, cytokinin) is high.

In addition, the excised vertebrae are sterilized. The disinfection may be accomplished by a variety of methods known in the art, for example, by washing with sterile water or washing with a sterilizing agent, and the sterilizing agent may further include an electrodeposition agent. As the bactericide, sodium hypochlorite (NaOCl) or calcium hypochlorite {Ca (ClO) ₂ } may be used, but the present invention is not limited thereto.

(S2) Step of tissue culture in sterilized air

In the context of the present invention, the term "tissue culture" refers to culturing a tissue and propagating it into a complete plant having the same genetic trait as the maize.

In the specification according to the present invention, the term "in vitro culture" means culturing a plant in a sterilized container, and "in-flight plant" means a plant cultured by in-vitro culture. Therefore, in order to culture the plant in the cabin, a suitable medium is necessary.

It is preferable to perform tissue culture in an MS medium supplemented with a growth regulator, and it is preferable to add BA and IBA in the growth regulator. The BA and IBA are plant growth hormone and induce a significant difference in plant growth response depending on the species.

In the case of seedlings, tissue culture can be preferably performed on MS medium containing 1 to 5 mg / L of BA, 0.2 to 1 mg / L of IBA and 15 to 30 g / L of sucrose, and most preferably BA 2 to 3 mg / L, IBA 0.5 mg / L, and sucrose 30 g / L. When added at less than 1 mg / L of BA, less than 0.2 mg / L of IBA, or less than 15 g / L of sucrose, there is a problem that proliferation is slowed, and BA is more than 5 mg / L, IBA is more than 1 mg / g / L, there are problems such as appearance of poor shoots.

In the case of apple trees, tissue culture can be preferably performed in an MS medium containing 0.5 to 2 mg / L of BA, 0.2 to 1 mg / L of IBA and 15 to 30 g / L of sucrose, and most preferably, BA 1 mg / L, IBA 0.5 mg / L, and sucrose 30 g / L. When BA is added in an amount of less than 0.5 mg / L, less than 0.2 mg / L of IBA or less than 15 g / L of sucrose, there is a problem in that proliferation is slowed and more than 2 mg / L of BA, g / L, there are problems such as appearance of poor shoots.

The incubation temperature, incubation time, and illuminance of the present invention may be appropriately adjusted by a person skilled in the art, for example, at 25 ± 1 ° C under a light condition (3000 lux, 16 Time-of-day) for at least 8 weeks, but not limited thereto.

According to another aspect of the present invention, there is provided a method for producing a virus-free disease plant comprising the steps of: collecting a growth point in an in-flight plant produced according to the production method; and culturing the collected growth point in a cabin without disinfection.

Describing the above method in each step,

(S1) collecting a growth point in an on-board plant produced according to the production method

Since the in-flight plant produced according to the above production method is cultivated in the cabin, it is possible to control the growth environment in a proper environment for harvesting the leaves, for example, by artificially adding a growth regulator in the medium to promote plant growth, It is possible to maintain an aseptic condition which is not contaminated by the bacteria. Even if the polluted plant is contained in the diarrhea or the infant plant, contaminated on-flight plant through the disease observation can be easily removed, and the present invention can be carried out on the uncontaminated plant. In addition, since the in-flight plant for harvesting leaves is secured, it is possible to collect a growing point from various tissues of the inflorescence at any time whenever necessary, and it is possible to repeatedly collect the growing point from the same in-flight plant after harvesting. On the other hand, according to the method of collecting the growth point from the out-of-plant plant, if the contamination probability of the plant is high, the whole process is repeated from the growing point collection step to not only being economical, There is a problem in that the harvesting point is limited to one-time harvesting, so that it is distinguished from the present invention.

As a method of harvesting the growth point, a method of separating a growth point directly from an in-flight plant, a method of separating a growth point after cutting a tissue including a growth point, and the like can be used, and a method of separating the growth point . In addition, there is no restriction on the time of harvesting, and it is possible during the year.

(S2) culturing the harvesting point in the cabin without disinfection

Since the growth point is taken from the inflorescence plant, the growth point can be cultivated immediately without any disinfection process.

Growth points can be cultured by various methods and conditions known in the art for the growth of the growing point, and may be subjected to a propagation process (a shoot formation process or a multi-shoot formation process) and a rooting process.

For growth and rooting, medium composition is important. It is preferable to grow and root in MS medium supplemented with growth regulator. BA and IBA are preferably added to the growth regulator for growth, and only IBA is added for rooting desirable.

Growth can be proliferated in MS medium containing BA 1-5 mg / L, IBA 0.2-1 mg / L and sucrose 15-30 g / L, and most preferably BA 2 - 3 mg / L, 0.5 mg / L of IBA and 30 g / L of sucrose. When added at less than 1 mg / L of BA, less than 0.2 mg / L of IBA, or less than 15 g / L of sucrose, there is a problem that proliferation is slowed, and BA is more than 5 mg / L, IBA is more than 1 mg / g / L, there are problems such as appearance of defective shoots, and in particular, reverse osmosis is caused due to the addition of excess sucrose, or strangulation is not caused by excessive transpiration or elongation.

In the case of apple trees, it can be propagated in MS medium containing preferably 0.5 to 2 mg / L of BA, 0.2 to 1 mg / L of IBA and 15 to 30 g / L of sucrose, mg / L, IBA 0.5 mg / L, and sucrose 30 g / L. When BA is added in an amount of less than 0.5 mg / L, less than 0.2 mg / L of IBA or less than 15 g / L of sucrose, there is a problem in that proliferation is slowed and more than 2 mg / L of BA, g / L, there is a problem such as appearance of defective shoots. In particular, there is a problem in that excessive shoots are not enlarged or elongated due to reverse osmosis or transparency due to excess of excess sucrose.

In addition, rooting can be rooted in 1/4 -1/2 MS medium containing 1-5 mg / L of IBA and 7.5-30 g / L of sucrose, and more preferably, IBA 2 -5 mg / L and sucrose 7.5-15 g / L in 1 / 4MS medium. Most preferably, it can be rooted in 1 / 4MS medium containing 2 mg / L IBA and 15 g / L sucrose. When MS medium containing less than 1/4 times of salt or sucrose of less than 7.5 g / L is added, there is a problem that rooting is slowed, and MS medium containing more than 1/2 times of salt or sucrose of more than 30 g / L There is a problem that rooting is poor and callus appears.

In the case of apple trees, rooting can be carried out in 1/4 -1/2 MS medium containing 0.5-5 mg / L IBA and 7.5-30 g / L sucrose, more preferably IBA 0.5- 2 mg / L and sucrose 7.5-15 g / L in 1 / 4MS medium. Most preferably, it can be rooted in 1 / 4MS medium containing 1 mg / L IBA and 15 g / L sucrose. When MS medium containing less than 1/4 times of salt or sucrose of less than 7.5 g / L is added, there is a problem that rooting is slowed, and MS medium containing more than 1/2 times of salt or sucrose of more than 30 g / L There is a problem that rooting is poor and callus appears.

In addition, the virus may be subjected to a step of removing the virus infection through the virus assay. Viral assays can be performed according to various methods known in the art and can be performed by, for example, screening, serological assays, or Multiplex RT-PCR methods.

According to the above method, a virus-free disease plant can be produced. In the specification of the present invention, the term "virus" includes all viruses that are infected with plants and are capable of proliferating. In addition, the term " virus-free disease "refers to a plant that is not contaminated with viroid, mycoplasma analogue, bacteria, or fungi, together with the virus.

According to the present invention, it is possible to collect a growing point without seasonal restriction, to produce a plant having a low virus contamination rate and a high survival rate, and to supply a good variety smoothly, thereby contributing to an increase in farm household income, It is expected that exports of excellent varieties will be possible.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Production of in-flight plants for harvesting the growing point for cultivation of the growing point and the medium used therein

First, the shrimp of the pear and apple tree branches were cut off and washed with running water for 24 hours to remove all attached soil and foreign matter. Then, the plate was immersed in 70% ethanol for 30 to 60 seconds, disinfected, and then immersed in 3% sodium hypochlorite (NaOCl) solution containing Tween 20 for 15 minutes and washed three times with sterilized water. The seedlings were inoculated into the growth medium of the composition shown in Table 1 at a temperature of 25 ± 1 ° C (3,000 lux, 16 hours per day) and cultured in vitro.

fruit tree badge Apple MS medium + BA 1.0 mg / L + IBA 0.5 mg / L + sucrose 30 g / L ship MS medium + BA 2.0 - 3.0 mg / L + IBA 0.5 mg / L + sucrose 30 g / L

< Experimental Example  1> BA  And IBA  Depending on concentration Growth rate  compare

Since the composition of medium, especially the concentration of growth regulator, is very important factor for the production of inflorescence for harvesting, the growth rate of BA and IBA was compared.

Table 2 compares the effect of BA on the proliferation of apple tree M9 shoots. Table 1 shows the best results in the number of shoots and fresh weight / flakes at 1.0 mg / L of BA. BA and BA Were found to be adversely affecting the growth of shoots.

process
(mg / L) Number of shoots
(dog) Shinko length
(cm) Fresh weight (mg)
/ Fragment Callus BA 0.0 1.2 5.3 212      0.5 3.3 4.7 386 -      1.0 4.4 3.6 494 -      2.0 3.1 3.4 435 -      3.0 2.7 2.7 467 +      5.0 2.4 2.4 489 ++

 * Use medium containing MS + sucrose 30 g / L

* Callus -: None, +: Low, ++: Medium

Table 3 below compares the effect of IBA and BA on the proliferation of apple tree M9 shoots. When the concentration of BA is 1.0 mg / L, especially when the IBA concentration is 0.5 mg / L, the number of shoots, shoot length and fresh weight / The best results were obtained.

process
(mg / L) Number of shoots
(dog) Shinko length
(cm) Fresh weight (mg)
/ Fragment Callus Control 1.1 5.6 257 - BA 0.5 + IBA 0.2 4.0 4.7 436 - BA 0.5 + IBA 0.5 4.3 4.1 435 - BA 0.5 + IBA 1.0 3.7 3.8 460 - BA 1.0 + IBA 0.2 5.3 4.0 494 - BA 1.0 + IBA 0.5 6.1 4.2 540 - BA 1.0 + IBA 1.0 4.9 3.8 524 - BA 2.0 + IBA 0.2 4.1 3.4 485 - BA 2.0 + IBA 0.5 4.8 3.3 477 - BA 2.0 + IBA 1.0 4.4 2.9 498 -

 * Use medium containing MS + sucrose 30 g / L

* Callus -: None

Table 4 below shows the comparison of the effect of BA on the proliferation of shoots of &quot; Bai Yeon 3 &quot; of Baejangbok, and it was confirmed that the best results were obtained in the number of shoots and fresh weight / broth at BA 2.0-3.0 mg / L .

process
(mg / L) Number of shoots
(dog) Shinko length
(cm) Fresh weight (mg)
/ Fragment Callus BA 0.0 1.0 5.1 154 - 0.5 2.3 4.3 268 - 1.0 3.2 3.4 249 - 2.0 4.3 3.7 354 - 3.0 4.0 3.2 317 - 5.0 3.1 2.7 263 +

 * Use medium containing MS + sucrose 30 g / L

* Callus -: None, +: Less

Table 5 compares the effect of IBA and BA on the growth of the ginseng root "Bai Yeon 3". Table 5 shows the effects of IB 2.0-3.0 mg / L and especially 0.5 mg / L IBA concentration on shoot growth, shoot length and fresh weight / Fragment. The results are shown in Fig.

process
(mg / L) Number of shoots
(dog) Shinko length
(cm) Fresh weight (mg)
/ Fragment Control 1.0 4.6 165 BA 1.0 + IBA 0.2 3.4 3.4 276 BA 1.0 + IBA 0.5 4.0 3.1 254 BA 1.0 + IBA 1.0 4.7 2.3 260 BA 2.0 + IBA 0.2 6.2 3.5 332 BA 2.0 + IBA 0.5 6.8 3.1 354 BA 2.0 + IBA 1.0 6.1 3.2 346 BA 3.0 + IBA 0.2 5.5 3.4 309 BA 3.0 + IBA 0.5 6.1 3.6 321 BA 3.0 + IBA 1.0 4.9 2.8 329

* Use medium containing MS + sucrose 30 g / L

< Example  2> Growth points collected from in-flight plants were cultured to obtain virus Disease-free state  Plant production

The shoots were cut from a pear and an apple tree, which were produced by the method of Example 1, and immediately transferred to a clean bench (sterile workbench) without being subjected to a separate disinfection process from the material. Using a surgical knife and tweezers, Lt; / RTI &gt; The harvested leaves were placed in a sterilized test tube (diameter 24 mm, height 150 mm) containing MS medium (Murashige and Skoog) medium containing sucrose 30 g / L and plant growth regulators (BA, IBA) And cultured for 8 weeks in a culture room maintained at ± 1 ° C under a light condition (3,000 lux, 16 hours one day).

After attaching a confirmation label to each of the cultured pear and apple tree, a part of the leaf tissue for the assay was sampled, and the same specimen was juiced to collect the juice. Viral assays were carried out in accordance with Multiplex RT-PCR, which was carried out by the Assaying Team of the Agricultural Practical Use Foundation. As a result, Byeonju was removed and only the remaining specimens were retained.

< Experimental Example  2> Plant virus Contamination rate  And Survival Rate Comparison

The following experiments were conducted to compare the fungal contamination rate and the survival rate between the plant production method according to the conventional growth point culture and the plant production method according to the above Examples 1 and 2.

A method used in the experiment was to disinfect the outpatient plant to obtain the growth point and then to proliferate according to the method of Example 2. In contrast, Method B, which is the method according to Example 1, And the growth point was removed from the shoot of the in-flight plant grown by in-flight culture in the cabin, and cultivation was carried out according to the method of Example 2 without disinfection.

The method A was carried out in April-May by cutting off the shrimp of the pear and apple tree branches and rinsing with flowing water for 24 hours to remove all attached soil and foreign matter. Then, the plate was immersed in 70% ethanol for 30 to 60 seconds, disinfected, and then immersed in 3% sodium hypochlorite (NaOCl) solution containing Tween 20 for 15 minutes and washed three times with sterilized water. After the disinfection process, the calves were transferred to a clean bench (sterile workbench), and the growth points were removed using a surgical knife and tweezers, and cultured according to the method of Example 2. The B method was the same as that of Examples 1 and 2.

After 8 weeks of cultivation, the survival rate and the growth rate of the plants according to the growth point method of apples M9 and Mt.

As shown in Table 6, the survival rate of the plants cultured according to the method A was low and the contamination rates of viruses, fungi and bacteria were high as compared to the plants cultured according to the method B.

According to the method A, the disinfectant solution is likely to be deposited at the growth point due to the disinfection process just before the growth point, and the growth point according to the present method is separated from the outpatient plant, so that the growth inhibitory substances accumulated in the out- The rate was considered high. On the other hand, according to the method B, the disinfection process is carried out before the cultivation, but the disinfection process is not performed when the growth point is separated from the inflorescence plant, so that the disinfectant solution is not likely to be deposited at the growth point, . As shown in Table 6, the survival rate of the B method was about 50% higher than that of the A method, and the survival rate of the apple was 40% higher than that of the A method.

According to Table 6, according to Table 6, compared with the plants according to the method A, both the survival rate and the survival rate of the B method were superior to those of the plants according to the method A and the shoot length and shoot length, Was found.

According to the method A, 100% fungus and bacteria were difficult to remove in the process of disinfection of the growth point, and the contamination rate was high. The smaller the size of the growth point, the higher the probability of virus removal, but the difficulty of culturing became difficult. On the other hand, according to method B, the disinfection process is carried out before the cultivation, and when the growth point is separated from the inflorescence plant, the disinfection process is not performed and the disinfectant solution is not likely to be deposited at the growth point. It is possible to maintain the condition, and contamination rate of fungus and bacteria is low. As shown in Table 6 below, it was confirmed that the contamination rate of apples and pears cultured by method B was remarkably higher than that of method A, which had contamination rate of about 20% at 0%.

In addition, according to the method A, the growth point is extracted from the out-plant plant. In the case of the out-plant plant, the concentration of growth inhibitory substance (for example, ABA, penolic compound) in the plant is low and the content of growth promoting substance (for example, gibberellin) There was a time limitation in that it could be harvested only in spring, which is a high period. On the other hand, according to method B, there was no limitation on the stage of harvesting since the harvesting point was obtained from the in-flight plant cultured in a suitably controlled environment.

Growth Point Culture crops Survival rate
(%) Contamination rate
(%) Number of shoots
(dog) Shinko length
(cm) A method
(Using external plants) Apple 45.8 25.0 4.4 2.7 ship 55.3 22.5 3.6 2.2 Method B
(Using tissue cultured plants) Apple 92.5 0 4.7 2.6 ship 95.0 0 3.4 2.4

< Example  3> Virus Disease-free state  Bulk growth of plants and the medium used therefor

The viral disease-free plants according to Example 2 were transferred to a culture bottle (diameter 81 mm, height 132 mm) containing the growth medium of the composition shown in Table 1 at 25 ± 1 ° C under a light condition (3,000 lux, 16 hours per day) They were proliferated for 8 weeks and subcultured at 4-5 week intervals. Then, the roots were transferred to the rooting medium shown in Table 7 below to form roots before soil purification, and subcultured again at 25 ± 1 ° C under light conditions (3,000 lux, 16 hours per day). At this stage, a culture bottle cap with an air hole was used, and air holes were sealed with a Millipore tape to maintain the sterilized condition and to purify the outside air.

fruit tree badge Apple 1 / 4MS medium + IBA 1.0 mg / L + sucrose 15 g / L ship 1 / 4MS medium + IBA 2.0 mg / L + sucrose 15 g / L

As a result, it was confirmed that the root was induced and the complete plant was formed after 1-2 months.

< Experimental Example  3> MS  Depending on media concentration and sucrose concentration Rooting rate  compare

Rooting rate was an important factor for the mass propagation of viral disease-free plants because it was difficult to breed in the cabin. Therefore, rooting rate studies were conducted according to MS medium salt concentration, sucrose, BA, and IBA concentrations.

Table 8 below shows the rooting rate of apple rootstock M9 shoots. It was found that when the salt concentration of MS medium was less than ½ times and the sucrose concentration was 15 g / L and 7.5 g / L, 1/4 MS medium containing 15 g / L sucrose showed the best rooting rate. In all cases, 1.0 mg / L IBA was added.

Sucrose concentration MS salt concentration: 1 times MS salt concentration: 1/2 times MS salt concentration: 1/4 times 30 g / L - - + 15 g / L - ++ +++ 7.5 g / L + ++ ++

* Rooting status

-: Very bad, +: Bad, ++: Moderate, +++: Good

Table 9 below shows the effect of IBA on the rooting of apple rootstock M9 shoots after 6 weeks of cultivation, and it was confirmed that rooting rate and root number were the best when IBA was 1.0 mg / L.

process
(mg / L) Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment IBA 0.0 0 1.0 0 0 137 0.5 46.7 1.1 2.4 4.7 231 1.0 57.8 1.2 7.3 5.6 249 2.0 43.5 1.0 4.2 6.4 253 3.0 37.3 1.0 3.4 4.3 267 5.0 23.4 1.2 1.8 4.7 289

* Use medium with 1/2 MS + sucrose 30 g / L

Table 10 below compares the effect of sucrose on the rooting of apple rootstock M9 shoots after 6 weeks of cultivation, and it was confirmed that the rooting rate, root number and root length were the best when sucrose was 15.0 g / L.

Sucrose
(g / L) Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment 7.5 58.7 1.0 6.4 5.7 265 15.0 72.3 1.1 7.6 7.6 245 30.0 33.5 1.1 6.2 7.3 232

* Use media with 1/2 MS + IBA 1.0 mg / L added

Table 11 below compares the effect of MS medium salt concentration on the rooting of apple rootstock M9 shoots after 6 weeks of cultivation, and it can be confirmed that the rooting rate is the highest, such as the root number is highest when rooting in 1/4 medium.

MS salt Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment 1 x 63.6 1.0 5.4 4.9 278 1/2 x 67.7 1.0 6.3 5.4 263 1/4 x 85.2 1.0 5.6 5.7 224

* The medium used was medium supplemented with Sucrose 15 g / L + IBA 1.0 mg / L

Table 12 below shows the rooting rate of shoots of "Bae Yeon 3" shoots of the abalone trees. When the salt concentration of the MS medium was less than ½ times and the sucrose concentrations were 15 g / L and 7.5 g / L, Especially in 1/4 MS medium containing 15 g / L sucrose. In all cases, 2.0 mg / L IBA was added.

Sucrose concentration MS salt concentration
1x MS salt concentration
1/2 times MS salt concentration
1/4 times 30 g / L - - + 15 g / L - ++ +++ 7.5 g / L + ++ ++

* Rooting status

-: Very bad, +: Bad, ++: Moderate, +++: Good

The following Table 13 shows the effect of IBA concentration on the rooting of the shoots of the "No 3" shoot after 6 weeks of cultivation. When the root was rooted at 2.0 mg / L, the root yield was the highest and the root length was the longest. Of the respondents.

process
(mg / L) Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment IBA 0.0 0 1.0 0 0 89  0.5 7.3 1.2 0.7 4.9 245 1.0 15.8 1.0 1.8 5.2 269 2.0 53.6 1.0 3.4 5.6 293 3.0 45.0 1.0 2.2 3.5 284 5.0 36.7 1.1 1.9 3.4 207

* Use medium with 1/2 MS + sucrose 30 g / L

The following Table 14 shows the effect of sucrose concentration on the rooting of the "Bae Yeon-no 3" shoot of the pine tree after 6 weeks of cultivation. When rooting at 15 g / L, the root number is the highest and the root length is the longest. Of the respondents.

Sucrose
(g / L) Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment 7.5 65.3 1.0 3.6 5.5 246 15.0 75.6 1.0 4.7 6.3 294 30.0 50.1 1.0 3.2 6.1 224

* The medium used was medium supplemented with 1/2 MS + IBA 2.0 mg / L

Table 15 below compares the effect of MS medium salt concentration on the rooting of the "Bacillus no. 3" shoots after 6 weeks of incubation. It shows the highest rooting rate, I could confirm.

MS salt Rooting rate
(%) Number of shoots
(dog) Number of roots
(dog) Root length
(cm) Fresh weight (mg)
/ Fragment 1 x 60.7 1.0 3.4 5.8 267 1/2 x 73.4 1.0 5.6 5.3 236 1/4 x 87.6 1.0 5.2 5.8 207

 * The medium used was medium supplemented with Sucrose 15 g / L + IBA 2.0 mg / L

Claims (15)

A method of producing an in-flight plant for harvesting point growth for cultivating a growth point, comprising the steps of cutting and disinfecting the plant's fertilization in vitro and tissue culture of the sterilized fertilizer in-flight. The method of claim 1 wherein the length of the well is 5-10 mm. The method according to claim 1, wherein when the plant is planted benzyl adenine (BA) 1-5 mg / L, indole-2-butyric acid (IBA) 0.2-1 mg / L and sucrose (15-30 g / L) Production method, characterized in that the tissue culture in MS medium. The method of claim 1, wherein when the plant is an apple tree, production is characterized in that the tissue culture in MS medium containing BA 0.5-2 mg / L, IBA 0.2-1 mg / L and sucrose (15-30 g / L) Way. Obtaining a growth point in the in-flight plant produced according to the production method of claim 1, and the virus-free disease plant production method comprising the step of culturing the obtained growth point in-flight without disinfection process. 6. The method according to claim 5, further comprising the step of removing the virus infection through a virus assay. 6. The method according to claim 5, wherein the plant is an apple or a pear tree. The method according to claim 5, wherein when the plant is an apple tree, it is cultured in an MS medium containing 0.5 to 2 mg / L of BA, 0.2 to 1 mg / L of IBA and 15 to 30 g / L of sucrose, &Lt; / RTI &gt; [5] The method according to claim 5, wherein the plant is an apple tree. The method includes rooting in 1/4 to 1/2 MS medium containing 0.5 to 5 mg / L of IBA and 7.5 to 30 g / L of sucrose . 6. The method according to claim 5, wherein the plant is cultured in an MS medium containing 1-5 mg / L of BA, 0.2-1 mg / L of IBA and 15-30 g / L of sucrose, &Lt; / RTI &gt; [7] The method according to claim 5, wherein when the plant is a donepezil, it comprises rooting in 1/4 -1/2 MS medium containing 1-5 mg / L of IBA and 7.5-30 g / L of sucrose . Proliferation medium for virus-free diseased apple tree culture by growth point culture, characterized in that it contains BA 0.5-2 mg / L, IBA 0.2-1 mg / L and sucrose 15-30 g / L in MS medium. . Rooting medium for viral disease-free apple tree cultivation by growth point culture, characterized in that it comprises 0.5-5 mg / L of IBA and 7.5-30 g / L of sucrose in 1 / 4-1 / 2 MS medium. A growth medium for virus-free pathogen culture by growth point culture, comprising BA 1-5 mg / L, IBA 0.2-1 mg / L and sucrose 15-30 g / L in MS medium. Rooting medium for virus free bottle culture by growth point culture, characterized in that it comprises IBA 1-5 mg / L and sucrose 7.5-30 g / L in 1/4 -1/2 MS medium.