KR102612091B1 - Promoting methods for rooting of in vitro shoots of Diospyros kaki and mass propagation of plants through these techniques - Google Patents

Abstract

In the present invention, a method for enhancing the rooting of astringent persimmon shoots and a method for mass production of astringent persimmon trees using tissue culture are disclosed.
According to the method for promoting the rooting of astringent persimmon tree shoots according to the present invention, the rooting rate of astringent persimmon tree shoots is significantly improved. In addition, according to the method for mass production of tissue-cultured astringent persimmon trees of the present invention, the shoot induction rate and shoot growth are significantly improved from the growing point culture of the astringent persimmon tree, and the shoot rooting rate is also significantly improved, allowing mass production of disease-free tissue-cultured astringent persimmon trees in-flight. can be produced In addition, the tissue cultured astringent persimmon tree according to the present invention has the same genetic characteristics as the mother plant with excellent genetic characteristics, so it is useful for dissemination to orchard farmers.

Description

Method for promoting rooting of astringent persimmon tree shoots and mass production of tissue cultured seedlings using the same {Promoting methods for rooting of in vitro shoots of Diospyros kaki and mass propagation of plants through these techniques}

The present invention relates to a method for enhancing the rooting of shoots of an astringent persimmon tree and a method for mass producing tissue culture seedlings using the same. More specifically, a method for promoting the rooting of shoots of an astringent persimmon tree using the carbonizing method and a method for promoting the rooting of shoots of an astringent persimmon tree using the same in an airplane. This relates to a method of mass producing astringent persimmon trees through tissue culture.

The persimmon tree is a fruit tree native to the temperate zone of East Asia and widely cultivated in north-central China, Japan, and central and southern Korea. There are two types of persimmons: sweet persimmons and astringent persimmons. Most of the native varieties of Korea are astringent persimmons, and the sweet persimmons currently cultivated are all varieties introduced from Japan.

In general, persimmon trees are propagated either sexually by sowing seeds or asexually by cutting branches and grafting them onto a persimmon tree, which limits mass reproduction.

Recently, attempts have been made to apply tissue (organ) culture technology using growing points or sap explants to develop efficient asexual propagation methods in fruit trees. In particular, among the organ differentiation methods through tissue culture, in-flight plant propagation using growing points has the advantage of producing virus-free seedlings free from high-risk viruses that can reduce fruit quality and cause death damage (registered patent 10). -1453903).

However, when cultivating the growing point of astringent persimmon trees, the rooting efficiency of shoots was low, making mass production through tissue culture difficult.

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

Accordingly, as a result of continuous research to meet the needs of the prior art, the present inventors confirmed that carbonization of the cut surface of the shoot of an astringent persimmon tree significantly improves the rooting of the shoot, and thus completed the present invention.

Therefore, the purpose of the present invention is to provide a method for promoting rooting of astringent persimmon tree shoots.

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

In order to achieve the purpose of the present invention, the present invention

A method for promoting rooting of astringent persimmon tree shoots is provided, which includes the step of carbonizing the cut surface of the astringent persimmon tree shoots.

In the present invention, varieties of astringent persimmon trees may include Sangjudungsi, Gapju Baekmok, Cheongdo Bansi, Sagoksi, Goseongsi, Danseongsi, Jangdungi, Wolhasi and Bongok, and the most preferred is Sangjudungsi.

In the present invention, 'shoot' refers to a newly grown branch, such as a branch newly grown by in-plant culture of the tissue, a branch growing from a lateral bud when the tip of the stem is wetted, a hidden bud, the upper part of a branch, or the dorsum. Includes strong branches such as those growing from located buds.

In the present invention, ‘in-flight culture’ refers to a method of growing organisms using an artificial culture medium.

In the present invention, ‘rooting’ means rooting and includes the differentiation (formation) of roots in the organs of the plant.

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

The carbonizing method is commonly used as one of the cut flower preservation methods used to maintain the lifespan of cut flowers longer. It burns the area around the cut surface of the stem to prevent rotting of the cut surface, absorbs water well, and prevents air bubbles. . However, until the present invention, there has been no report of a case in which the carbonization method was applied to promote rooting of shoots in the field of tissue culture.

According to the rooting enhancement 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 a growing point containing four leaf primordia from a bud of an astringent persimmon tree;

ii) culturing the growing point explant in vitro to induce shoot growth;

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

iv) transplanting carbonized shoots into a culture medium and culturing them to induce rooting; providing a method for mass production of tissue cultured astringent persimmon trees, including the step of inducing rooting.

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

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

The growing point containing four leaf primordia is cut from the sap of the astringent persimmon tree.

In the present invention, ‘apillary buds’ are so-called axillary buds, which are buds that grow in the axils of leaves. In some cases, they are generally flower buds, and in other cases, they are buds prepared to produce new stems from branches when the stem is damaged.

In the present invention, ‘leaf primordium’ refers to a leaf in an embryonic state at the beginning of development.

When using growing point explants containing 4 leaf primordia of astringent persimmon trees as in the present invention, the shoot induction rate was excellent. In contrast, when growing points containing less than 4 leaf primordia were used, no shoots were induced at all. (Figure 1).

Step ii) Shoot induction and growth

The growing point explant is transplanted into the culture medium and cultured in vitro to induce and grow shoots.

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

When hormones other than zeatin are used as plant growth hormones, shoot growth is not efficient, and if the concentration of zeatin is less than 2.0 mg/L, the shoot induction rate is low and shoot growth does not occur properly, and if the concentration of zeatin is less than 5.0 mg/L, shoot growth is not achieved properly. If mg/L is exceeded, the shoot induction rate and shoot growth decrease again.

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

In this step, the culture period for shoot induction is preferably 4 to 6 weeks, and the culture period including shoot growth is preferably 6 to 8 weeks, 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 Astringent persimmon trees is improved and the shoot length is significantly increased (Table 1, Figure 2, Figure 3a, Figure 3b).

Step iii) Carbonization treatment of shoots

The shoot cut surface from step ii) is carbonized.

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

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

The rooting rate of carbonized astringent persimmon shoots is significantly improved (Figure 5).

Step iv) Rooting induction

The carbonized shoots are transplanted into a culture medium and cultured to induce rooting.

The culture medium for rooting in this step is 1/ with the addition of 2.0 to 4.0 mg/L indole-3-butyric acid (IBA), 2 to 4% sucrose, and 0.2 to 0.4% gelite. 2 MS medium (medium with the amount of basic salts reduced by half) is used, most preferably 1/2 MS medium supplemented with 3 mg/L IBA, 3% sucrose and 0.25% Gelite.

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 lighting (cool white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) for 16 hours a day.

The rooting rate of carbonized shoots is increased by about 1.5 to 5 times compared to non-carbonized shoots (Figure 5).

v) Purification

The rooting-induced plant from step iv) is transplanted into the top soil and acclimatized.

At this stage, acclimatized plants are transplanted into artificial soil in an acclimatization container where humidity is maintained at 80-90% to obtain acclimatized young plants.

In this step, the purification container can be used without particular restrictions as long as the humidity of 80-90% can be maintained.

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

In this step, proliferation is performed under conditions where 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained at 25 ± 1°C for 15 to 17 hours a day.

The growth period is preferably 3 to 5 weeks, and most preferably 4 weeks.

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

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

In addition, the tissue cultured astringent persimmon tree according to the present invention has the same genetic characteristics as the mother plant with excellent genetic characteristics, so it is useful for dissemination to orchard farmers.

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

Hereinafter, in order to facilitate understanding of the present invention, the configuration and effects of the present invention will be described in more detail through specific examples. However, the following examples are merely illustrative for a clearer understanding of the present invention, and the scope of the present invention is not limited by the following examples.

Example 1: Shoot induction through growing point culture of astringent persimmon tree

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

In order to identify growing points suitable for shoot induction during growing point culture, growing points containing 2 or 4 leaf primordia were cut from the tip of the shoots of the rostral stems and prepared (Figure 1).

In addition, in order to determine the type and concentration of plant growth hormone suitable for shoot induction, 6-benzylaminopurine (BA) or zeatin, 3% sucrose, and 0.3% gelite were added at the same concentration as Table 1. Solid-phase MS (Murashige and Skoog) culture medium was prepared and used for each group.

30 growing points from each group were transplanted into each group's culture medium and cultured for 6 weeks to induce shoots. The culture environment was maintained at 25°C for 15 to 17 hours a day with 50 to 70 μmol·m ^-2 ·s ^-1 lighting.

A photo of the culture results is shown in Figure 1, and the shoot induction rate was calculated by averaging the shoots that were induced, and the results are shown in Table 1.

Explant type growth hormone
(mg/L) Shoot induction rate
(%) Shoot length
(mm) Growing point containing 2 leaf primordia Control (0.0) 0 0 B.A. 0.5 0 0 B.A. 1.0 0 0 B.A. 2.0 0 0 zeatin 0.5 0 0 zeatin 1.0 0 0 zeatin 2.0 0 0 Growing point containing 4 leaf primordia Control(0.0) 0 0 B.A. 0.5 37.4 1.56 B.A. 1.0 64.5 2.06 B.A. 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

Figure 1 is a photograph showing the results of shoot induction according to growing point culture of Sangjudungsi, an astringent persimmon tree. As shown in Figure 1, when a growing point containing 4 leaf primordia was used, shoots were induced well (photo below), but when a growing point containing 2 leaf primordia was used, shoots were not induced at all. (top photo).

Table 1 shows the shoot induction effect according to the type of growth point and type of plant growth hormone (BA or zeatin) of Sangjudungsi. As shown in Table 1, the shoot induction effect showed significant differences depending on the type of growing point and type of growth hormone. Shoots were induced from growing points containing four leaf primordia, but shoot formation did not occur at all from growing points containing two leaf primordia. When growing points containing 4 leaf primordia were cultured in medium containing 1.0-2.0 mg/L BA, the shoot induction rate was high at over 64%, but it was confirmed that shoot induction was not performed properly due to the short shoot length. . On the other hand, in the medium containing 2 mg/L zeatin, the shoot induction rate was high at over 54% and the shoot length was high at 4.53 mm.

Therefore, it can be seen that it is efficient to use a growing point containing 4 leaf primordia as a culture explant for shoot induction when cultivating a growing point at a crown, and to use a medium containing more than 2 mg/L of zeatin for shoot induction. there is.

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

In order to determine the optimal concentration and culture temperature of zeatin when inducing shoots by culturing the growing point of the crown, 0, 1.0, 2.0, 3.0 or 5.0 mg/L zeatin, 3% sucrose and 0.3% Gelite were added to the solid phase. MS culture medium was prepared and used for each group, and each group had 30 growth points, including 4 leaf primordia, prepared as in Example 1, except that the groups were cultured at a culture temperature of 25°C or 30°C. Each seed was transplanted into culture medium and cultured for 6 weeks to induce shoots. The culture environment was maintained at 50 to 70 μmol·m ^-2 ·s ^-1 lighting for 15 to 17 hours per day.

The shoot induction rate was calculated by averaging the shoots induced as a result of the culture, and the results are shown in Figure 2.

As shown in Figure 2, the shoot induction rate was high in the group containing zeatin at a concentration of 2 to 5 mg/L, and in particular, the shoot induction rate was over 87% in the group containing zeatin at a concentration of 3.0 mg/L. It was. The shoot induction rate was high at a culture temperature of 30°C at a zeatin concentration of 1 to 3 mg/L and at 25°C at a zeatin concentration of 5 mg/L. The shoot induction rate was highest at 91.7% when cultured at 30°C and treated with 3.0 mg/L zeatin.

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

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

As shown in Figure 3a, shoot growth was promoted in the group containing zeatin at a concentration of 2 to 5 mg/L, and shoot growth was found to be promoted when cultured at a culture temperature of 30°C. When cultured at a temperature of 30°C and treated with 3.0 mg/L zeatin, the shoots grew best with a length of 17.75 mm.

As shown in Figure 3b, it can be confirmed that the shoot growth promotion effect is excellent at a culture temperature of 30°C, and that the shoot length is the longest when treated with 3.0 mg/L zeatin.

Example 4: Carbonization treatment of cut surfaces of shoots and induction of rooting

After cutting the shoots grown in Example 3, including the stem, the cut surface was prepared by carbonizing it in an alcohol lamp flame for about 50 seconds as shown in Figure 4. For comparison, a shoot whose cut surface was not carbonized was prepared.

30 shoots prepared as above were mixed with 0, 1, 3, 4, or 5 mg/L indole-3-butyric acid (IBA), 3% sucrose, and 0.25 gelite. 2 After transplanting to MS medium and culturing for 3 weeks to induce rooting, they were transferred to the same medium from which only IBA was removed and cultured for 2 weeks. The culture environment was maintained at a temperature of 25°C with lighting (cool white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) for 16 hours a day. The rooting rate of shoots in each group was averaged and calculated, and the results are shown in Figure 5. A photograph of a plant (Sangjudungsi) that was induced to root and redifferentiated after carbonization treatment is shown in Figure 6.

As shown in Figure 5, the rooting rate of shoots (control) that were not carbonized were all 40.7% or less. In comparison, the rooting rate of shoots carbonized according to the present invention was increased by 1.5 to 5 times in each group.

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

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

Claims (11)

delete delete delete delete i) cutting a growing point containing four leaf primordia from a bud of an astringent persimmon tree;
ii) culturing the growing point explant in vitro to induce shoot growth;
iii) performing a carbonization treatment of carbonizing the cut surface of the grown shoot with a flame; and
iv) Transplant the carbonized shoots into a culture medium of 1/2 MS medium supplemented with 2.0-4.0 mg/L indole-3-butyric acid, 2-4% sucrose, and 0.2-0.4% gelite and root by culturing. Inducing a step;
Mass production method of tissue cultured astringent persimmon trees comprising.
The method of claim 5, wherein the culture medium in step ii) is solid MS (Murashige and Skoog) medium supplemented with 2.0 to 5.0 mg/L zeatin, 2% to 3% sucrose, and 0.2% to 0.4% gelite. Mass production method of tissue cultured astringent persimmon trees.
The method of claim 5, wherein in step ii), the culture is carried out at 25°C to 30°C for 15 to 17 hours per day, with 50 to 70 μmol·m ^-2 ·s ^-1 lighting maintained, and for 6 to 8 weeks. A mass production method of tissue cultured astringent persimmon trees, which involves culturing to induce and grow shoots.
delete The method for mass production of tissue cultured astringent persimmon trees according to claim 5, wherein the carbonization treatment in step iii) is performed by treatment with an alcohol lamp flame for 40 to 60 seconds.
The method for mass production of tissue cultured astringent persimmon trees according to claim 5, wherein the astringent persimmon trees are Sangjudungsi.
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