KR20220045625A - Promoting methods for rooting of in vitro shoots of Diospyros kaki and mass propagation of plants through these techniques - Google Patents

Abstract

Disclosed in the present invention are a method for promoting rooting of shoots of Diospyros kaki and a mass production method for tissue-cultured Diospyros kaki seedlings using the same. According to the present invention, the rooting rate of Diospyros kaki shoots is significantly increased. In addition, the shoot induction rate, shoot growth, and shoot rooting rate are significantly increased from growth point culture of Diospyros kaki seedlings, so disease-free tissue-cultured Diospyros kaki seedlings can be mass-produced in vitro. In addition, the tissue-cultured Diospyros kaki seedling has the same genetic traits as those of a mother plant having excellent genetic traits, thereby being useful for being distributed to fruit farms. The mass production method for tissue-cultured Diospyros kaki seedlings comprises the following steps: cutting growth points; growing shoots; carbonizing cut surfaces of the grown shoots; and inducing rooting.

Description

Method for promoting rooting of astringent persimmon tree shoots and mass production of tissue culture seedlings using the same

The present invention relates to a method for promoting the rooting of new shoots of astringent persimmon tree and a method for mass production of tissue culture seedlings using the same, and more particularly, to a method for promoting the rooting of shoots of astringent persimmon tree using the carbonizing method, and a method using the same It relates to a method for mass production of astringent persimmon trees through tissue culture.

The persimmon tree is a special species in the temperate zone of East Asia, and is a fruit tree widely cultivated in northern central China, Japan, and south central Korea. There are sweet persimmons and astringent persimmons. Most of the native varieties of persimmon are astringent persimmons, and all sweet persimmons currently cultivated are varieties introduced from Japan.

In general, persimmon trees are propagated by sexual propagation through seed sowing, or by asexual propagation using the grafting method of cutting branches and attaching the folds to the Goyam tree, so there is a limit to mass propagation.

Recently, attempts have been made to apply tissue (organ) culture technology using growth points or axonal explants to develop an efficient asexual reproduction method in fruit trees. In particular, among the organ differentiation methods through tissue culture, in-flight plant propagation using growth points has the advantage of producing virus-free seedlings free of high-risk viruses that can cause fruit quality deterioration and death damage (Registration Patent 10) -1453903).

However, there was a difficulty in mass production through tissue culture because the rooting efficiency of new shoots was low when culturing the growth point of the astringent persimmon tree.

Therefore, there is an urgent need to develop a mass production method through tissue culture by promoting the rooting of astringent persimmon tree shoots to improve the redifferentiation efficiency.

Accordingly, as a result of continuous research to meet the needs of the prior art, the present inventors confirmed that, when the cut surface of the shoot of astringent persimmon was carbonized, the rooting of the shoot was significantly improved, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a method for promoting the rooting of astringent persimmon shoots.

Another object of the present invention is to provide a method for mass production of tissue culture astringent persimmon tree using the above method.

In order to achieve the above object of the present invention, the present invention

It provides a method for promoting the rooting of astringent persimmon shoots, comprising the step of carbonizing the cut surface of the astringent persimmon shoots.

In the present invention, varieties of astringent persimmons may include Sangjudongsi, Gapjubaekmok, Cheongdobansi, Sagoksi, Goseongsi, Danseongsi, Jangdungi, Wolhashi and Bongok, and most preferably Sangjudongsi.

In the present invention, 'shoot' refers to a newly grown branch, and a branch that grows from the in-flight culture of the tissue, a branch that grows from the lateral bud when the tip of the stem is wetted, the hidden eye or upper part of the branch, or on the back Includes strong branches, such as branches growing out of located eyes.

In the present invention, 'in-flight culture' refers to a method of cultivating an organism using an artificial incubator.

In the present invention, 'rooting' refers to rooting, and includes differentiation (formation) of roots in plant organs.

In the present invention, 'carbonization treatment' means carbonizing (burning) with a flame, preferably, carbonization treatment of the cut surface of the shoot can be carried out by treatment with an alcohol lamp or a torch, and most preferably an alcohol lamp flame It can be carried out by processing for 40 to 60 seconds.

Carbonizing method is commonly used as one of the cut flower preservation methods used to maintain the lifespan of cut flowers longer. . However, prior to the present invention, there have been no reports of cases in which the carbonization method is applied to promote rooting of shoots in the field of tissue culture.

According to the rooting promotion method according to the present invention, the rooting rate of astringent persimmon tree shoots is significantly improved (FIG. 5).

In order to achieve another object of the present invention, the present invention

i) cutting off the growth point containing 4 leaf primordia from the axillary persimmon tree;

ii) inducing and growing shoots by culturing the explants at the growth point in-flight;

iii) carbonizing the cut surface of the grown shoots; and

iv) transplanting the carbonized shoots into a culture medium and culturing to induce rooting.

If necessary, the method of the present invention may further include, after step iv), v) acclimatizing the rooting-induced plant by transplanting it into the top soil.

Step i) Cutting the growth point of the astringent persimmon tree

The growth point containing 4 leaf primordia is cut from the astringent persimmon tree.

In the present invention, 'axillary (腋芽)' is also called axillary eyes, which are eyes that run on the leaf axils. In general, in some cases, it is a flower bud, and in others it is an eye ready to release a new stem from a branch when the stem is damaged.

In the present invention, 'leaf primordium' refers to a leaf that is in an embryo state at an early stage of development.

As in the present invention, when growth point explants containing 4 leaf primordial stages of astringent persimmon tree were used, the shoot induction rate was excellent. (Fig. 1).

Step ii) shoot induction and growth

Growth point explants are transplanted into a culture medium and cultured in-flight to induce and grow shoots.

In this step, the culture medium is a solid MS (Murashige and Skoog) medium containing 2.0 ~ 5.0 mg/L zeatin, 2% ~ 3% sucrose, and 0.2% ~ 0.4% gelite, and the most Preferably, an MS medium supplemented with 3.0 mg/L zeatin, 3% sucrose and 0.3% gelite is used.

When a hormone other than zeatin is used as the plant growth hormone, shoot growth is not efficient, and when the concentration of zeatin is less than 2.0 mg/L, the shoot induction rate is low and shoot growth is not performed properly, and the concentration of zeatin is 5.0 When it exceeds mg/L, shoot induction rate and shoot growth are lowered again.

In this step, the culture is performed at 25° C. to 30° C. for 15 to 17 hours a day, 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained, preferably at 30° C.

In this step, the culture period for induction of shoots is preferably 4 to 6 weeks, the culture period including up to growth of shoots is preferably 6 to 8 weeks, and most preferably 6 weeks and 8 weeks, respectively.

When using a culture medium containing zeatin at a specific concentration as described above and culturing under specific conditions, the shoot induction rate of Japanese persimmon tree is enhanced and the shoot length is remarkably increased (Table 1, FIG. 2, FIG. 3a, 3b).

Step iii) Carbonization of shoots

The shoot cuts from step ii) are carbonized.

In this step, shoot cutting is cut to include the stem of the shoot.

Carbonization treatment is performed by carbonizing (burning) the cut surface of the shoot with a flame, as defined above, and most preferably, it can be performed by treating the cut surface of the shoot with an alcohol lamp flame for 40 to 60 seconds (Fig. 4).

The rooting rate of the carbonized astringent persimmon tree shoots is significantly improved (FIG. 5).

Step iv) induction of rooting

Rooting is induced by transplanting the carbonized shoots into a culture medium and culturing them.

At this stage, the culture medium for rooting is 1/ with 2.0~4.0 mg/L indole-3-butyric acid (IBA; indole-3-butyric acid), 2~4% sucrose and 0.2~0.4% gelite. 2 MS medium (a medium in which the amount of basal salt is reduced by half) is used, and most preferably, 1/2 MS medium with 3 mg/L IBA, 3% sucrose and 0.25% gelite is used.

After inducing rooting by culturing in the culture medium for 2-3 weeks, it is transferred to 1/2 MS medium from which growth hormone (IBA) has been removed, and cultured for 2-3 weeks to grow roots.

The culture environment is maintained at a temperature of 25±1°C with illumination (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) for 16 hours a day.

Rooting rate is improved about 1.5 to 5 times in the carbonized shoots compared to the non-carbonized shoots (FIG. 5).

v) purification

The rooting-derived plants from step iv) are transplanted into top soil and acclimatized.

At this stage, acclimatization involves transplanting the plant into artificial soil in an acclimatization container where humidity of 80 to 90% is maintained to obtain an acclimated young plant.

In this step, the acclimatization container can be used without particular limitation as long as it can maintain a humidity of 80 to 90%.

In this step, artificial soil may be a mixture of vermiculite, peat moss and perlite, and preferably vermiculite: peat moss: perlite mixed in a ratio of 1:1: 1 (v:v:v) is used.

In this stage, the proliferation is carried out under the condition that the illumination is maintained at 25±1°C for 15-17 hours a day, and 50-70 μmol·m ^-2 ·s ^-1 light.

The period of the proliferation is preferably 3 to 5 weeks, and most preferably 4 weeks.

According to the method for promoting the rooting of shoots of astringent persimmon tree according to the present invention, the rooting rate of shoots of astringent persimmon tree is significantly improved.

In addition, according to the mass production method of the tissue-cultured astringent persimmon tree of the present invention, the shoot induction rate and shoot growth are remarkably improved from the growth point culture of the astringent persimmon tree, and the shoot rooting rate is also significantly improved, so that disease-free tissue-cultured astringent persimmon trees are mass-produced in-flight. can produce

In addition, since the tissue-cultivated astringent persimmon tree according to the present invention has the same genetic traits as the model having excellent genetic traits, it is useful for supplying fruit trees.

1 is a photograph showing the result of induction of shoots according to the growth point of astringent persimmon tree and the type of plant growth hormone.
2 is a graph showing the effect of inducing shoots according to the concentration of plant growth hormone and the culture temperature.
Figure 3a is a graph showing the effect of promoting shoot growth according to the concentration and culture temperature of the plant growth hormone.
Figure 3b is a photograph showing the results of shoot growth according to the concentration and culture temperature of the plant growth hormone.
4 is a photograph showing the process of carbonizing the cut surface of the shoot.
5 is a graph showing the effect of promoting the rooting of shoots according to the carbonization treatment.
6 is a photograph of an astringent persimmon tree re-differentiated by inducing rooting after carbonizing the cut surface of the shoot.

Hereinafter, the configuration and effect of the present invention will be described in more detail with reference to specific examples in order to help the understanding of the present invention. However, the following examples are merely illustrative in order to understand the present invention more clearly, and the scope of the present invention is not limited by the following examples.

Example 1: Induction of shoots through growth point cultivation of astringent persimmon tree

In the present invention, as an astringent persimmon tree, Sangju-dongsi was used as a test material.

In order to find a growth point suitable for induction of shoots during growth point culture, growth points containing 2 or 4 leaf primordia were cut off from the axillary tip of the upper spout and prepared (FIG. 1).

In addition, in order to find out the type and concentration of plant growth hormone suitable for induction of shoots, 6-benzylaminopurine (BA) or zeatin, 3% sucrose and 0.3% gelite at the concentrations shown in Table 1 were added. A solid phase MS (Murashige and Skoog) culture medium was prepared and used for each group.

After transplanting 30 growth points in each group into the culture medium of each group, they were cultured for 6 weeks to induce shoots. The culture environment was maintained at 25° C. for 15-17 hours a day, and 50-70 μmol·m ^-2 ·s ^-1 lighting was maintained.

A photograph of the culture result was shown in FIG. 1 , and the shoot induction rate was calculated by averaging those from which shoots were induced, and the results are shown in Table 1.

section type growth hormone
(mg/L) new induction rate
(%) Shincho length
(mm) Growing point with 2 leaf genitalia Control (0.0) 0 0 BA 0.5 0 0 BA 1.0 0 0 BA 2.0 0 0 zeatin 0.5 0 0 zeatin 1.0 0 0 zeatin 2.0 0 0 Growth point with 4 leaf genitalia Control(0.0) 0 0 BA 0.5 37.4 1.56 BA 1.0 64.5 2.06 BA 2.0 68.2 1.94 zeatin 0.5 12.4 1.96 zeatin 1.0 18.6 2.83 zeatin 2.0 54.7 4.53

1 is a photograph showing the results of induction of shoots according to the growth point culture of Sangjudongsi, an astringent persimmon tree. As shown in Figure 1, when the growth point including 4 leaf primordia was used, shoots were well induced (photo below), but when the growth point including 2 leaf stages was used, it was confirmed that shoots were not induced at all. Yes (pictured above).

Table 1 shows the effect of inducing shoots according to the type of growth point and the type of plant growth hormone (BA or zeatin) at the time of sangsouti. As shown in Table 1, the shoot-inducing effect showed a large difference according to the type of growth point and the type of growth hormone. Shoots were induced from the growth point with 4 leaf primordial stages, and shoot formation was not performed at the growth point with 2 leaf primordia. As a result of culturing the growth points containing 4 leaf primordia in a medium supplemented with 1.0-2.0 mg/L BA, the shoot induction rate was as high as 64% or more, but it was confirmed that the shoot induction was not performed properly due to the short shoot length. . On the other hand, in the medium to which 2 mg/L of zeatin was added, the shoot induction rate was as high as 54% or more, and the shoot length was as high as 4.53 mm.

Therefore, it can be seen that it is efficient to use a growth point containing 4 leaf progenitors as an explant for culturing for shoot induction when culturing the growth point at the periosteum, and to use a medium containing 2 mg/L or more of zeatin for induction of shoots. there is.

Example 2: Analysis of concentration and culture temperature of zeatin optimal for induction of shoots

Solid phase to which 0, 1.0, 2.0, 3.0, or 5.0 mg/L zeatin, 3% sucrose and 0.3% gelite were added to determine the optimal concentration and culture temperature of zeatin during induction of shoots by culturing the growth point at the time of stagnation. of MS culture medium was prepared and used for each group, and the growth point including 4 leaf stage prepared as in Example 1 was 30 for each group, except that the culture temperature was divided into groups at 25 ° C or 30 ° C. After transplanting each in a culture medium, they were cultured for 6 weeks to induce shoots. The culture environment was maintained for 15-17 hours per day, and 50-70 μmol·m ^-2 ·s ^-1 lighting.

As a result of culturing, the shoot induction rate was calculated by averaging those from which shoots were induced, and the results are shown in FIG. 2 .

As shown in FIG. 2 , the shoot induction rate was high in the group containing zeatin at a concentration of 2 to 5 mg/L, and in particular, in the group containing zeatin at a concentration of 3.0 mg/L, the shoot induction rate was 87% or more. it was As for the incubation temperature, the rate of shoot induction was high at 30°C when zeatin was at a concentration of 1 to 3 mg/L and at 25°C when zeatin was at a concentration of 5 mg/L. At the culture temperature of 30°C and 3.0 mg/L zeatin treatment, the shoot induction rate was the highest at 91.7%.

Example 3: Analysis of concentration and culture temperature of zeatin optimal for the growth of induced shoots

In order to determine the suitable plant growth hormone concentration and culture temperature for promoting growth of the induced shoots, the shoots induced in Example 2 were additionally cultured under the same conditions (medium and culture temperature, culture environment) for 2 weeks to grow the induced shoots. and the average length of the grown shoots was calculated and the results are shown in FIG. 3a. Also, pictures of the grown shoots are shown in Figure 3b.

As shown in Figure 3a, the growth of shoots is promoted in the group containing zeatin at a concentration of 2 to 5 mg/L, and it has been shown that the growth of shoots is promoted when cultured at a culture temperature of 30 ° C. When the culture temperature was 30° C. and 3.0 mg/L zeatin treatment, the shoot length was 17.75 mm and the best growth was achieved.

Also by the bar shown in FIG. 3b, it can be confirmed that the shoot growth promoting effect is excellent at a culture temperature of 30° C., and it can be confirmed that the shoot length is the longest when 3.0 mg/L zeatin is treated.

Example 4: Cutting surface carbonization treatment and rooting induction of shoots

After cutting the shoots grown in Example 3 including the stem, the cut surface was prepared by carbonizing treatment for about 50 seconds in an alcohol lamp flame as shown in FIG. 4, and for comparison, a shoot without carbonization treatment was prepared.

1/ with 0, 1, 3, 4 or 5 mg/L indole-3-butyric acid (IBA, indole-3-butyric acid), 3% sucrose and 0.25 gelite added to 30 each of the shoots prepared as above 2 It was transplanted into MS medium and cultured for 3 weeks to induce rooting, then transferred to the same medium in which only IBA was removed and cultured for 2 weeks. The culture environment was maintained at a temperature of 25 °C with illumination (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) for 16 hours a day. The rooting rate of shoots of each group was averaged and calculated, and the results are shown in FIG. 5 . Figure 6 shows a photograph of a redifferentiated plant (Sangjudongsi) that was rooted and re-differentiated after carbonization.

As shown in Figure 5, the rooting rate of shoots (control) not treated with carbonization were all 40.7% or less. On the other hand, the rooting rate of shoots carbonized according to the present invention was improved by 1.5 to 5 times in each group.

The rooting rate of carbonized shoots was higher than 70% in the group added with 2.0 to 4.0 mg/L IBA, and was highest at 77.8% in the medium supplemented with 3.0 mg/L IBA. At the high concentration of IBA (6.0 mg/L), the rooting rate of shoots was reduced to 55.6%.

As shown in FIG. 6 , it can be confirmed that the roots are well formed in the shoots when rooting is induced after the carbonization treatment according to the present invention.

Claims (11)

A method for promoting rooting of astringent persimmon shoots comprising the step of carbonizing the cut surface of the astringent persimmon shoots.
The method of claim 1, wherein the carbonization treatment is to carbonize with a flame.
The method according to claim 1, wherein the carbonization treatment is performed by treatment with an alcohol lamp flame for 40 to 60 seconds.
The method of claim 1, wherein the astringent persimmon tree is sangjudongsi.
i) cutting off the growth point containing 4 leaf primordia from the axillary persimmon tree;
ii) inducing and growing shoots by culturing the growth point explants in-flight;
iii) carbonizing the cut surface of the grown shoots; and
iv) inducing rooting by transplanting the carbonized shoots into a culture medium and culturing;
A method for mass production of tissue-cultured astringent persimmon trees, including
The method of claim 5, wherein the culture medium in step ii) is a solid MS (Murashige and Skoog) medium to which 2.0 to 5.0 mg/L zeatin, 2% to 3% sucrose, and 0.2% to 0.4% gelite are added. A method for mass production of tissue-cultured astringent persimmon trees.
The method according to claim 5, wherein in step ii), the culture is carried out at 25° C. to 30° C. for 15 to 17 hours a day, 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained, and for 6 to 8 weeks. A method for mass production of tissue-cultured astringent persimmon trees by culturing and inducing and growing shoots.
The method of claim 5, wherein the carbonization treatment in step iii) is carbonization with a flame.
The method of claim 5, wherein the carbonization treatment in step iii) is performed by treatment with an alcohol lamp flame for 40 to 60 seconds.
[Claim 6] The method of claim 5, wherein the astringent persimmon tree is sangjudongsi.
The method according to claim 5, wherein the culture medium in step iv) is 1/2 MS medium to which 2.0-4.0 mg/L indole-3-butyric acid, 2-4% sucrose and 0.2-0.4% gelite are added. A method for mass production of tissue-cultured astringent persimmon trees.

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