KR20150093517A - Method for mass propagation of callus and method for mass division of Populus euramericana utilizing stem segments of Populus euramericana - Google Patents

Abstract

The present invention relates to a method for mass propagation of callus of Populus euramericana and a method for mass division of Populus euramericana. According to the present invention, a method for mass propagation of Populus euramericana is capable of mass-producing outstanding Populus euramericana by redividing a plant in a short period of time with a method for cutting the stem with the thickness of 1-2 mm and redividing a plurality of shoots from a cambium inside the stem, not with existing node culture.

Description

[0001] The present invention relates to a method for mass propagation of callus and populer populations of Populus euramericana using an Italian poplar stem segment,

The present invention relates to a method for mass proliferation of Italian poplar callus and a method for mass proliferation of Italian poplar.

FAO estimates that demand for wood will increase by 25% between 1996 and 2010, while actual forests are currently declining by 9.4 million ha per year. In addition, due to climate change, Korea's temperature is also warming more than twice as fast as the global average, which is expected to greatly affect water quality and wood quality.

In Korea, the demand for the use of wood biomass is increasing as the low carbon green growth policy is promoted. In particular, the demand for wood energy materials is expected to increase due to the introduction of the greenhouse gas target management system. to be.

In order to produce and supply domestic woody biomass, it is necessary to develop large-scale forestation and production technology. To this end, Populus euramericana , which produces the best biomass for a short period of time and grows well in a variety of soil, is considered to be a suitable target species and a large amount of seedlings should be secured in a short period of time in order to form a large area of wood energy forest.

In the case of actual seedlings using seeds, poplar seeds such as poplar from Italy are not only difficult to obtain seeds because of their reduced vitality in a short period of time, but also can not distinguish cancerous trees from seedlings in actual seedlings. There are many side effects such as seeds with spring-loaded hairs. Therefore, it is necessary to mass-propagate clone seedlings in a short period of time for a good clone having excellent growth characteristics and good wood characteristics in a flowering tree which does not fly on the flowering period.

However, existing breeding methods such as cutting have been limited in the production of seedlings unless large - scale watering can be provided.

Therefore, it is a time to develop a mass production system of Italian poplar seedlings, which has good growth, high adaptability in barnyard soil, and has good wood quality for later use as woody energy.

Korean Patent No. 0522437 Korean Patent No. 1114578

It is an object of the present invention to provide a method for mass proliferation of calli of Italian poplar using an Italian poplar stem slice in order to solve the above problems.

It is also an object of the present invention to provide a method for massively differentiating Italian poplar using an Italian poplar stem slice.

To achieve these and other advantages and in accordance with the purpose of the present invention,

(a) Italian poplar ( Populus euramericana ) to a size of 5-7 cm and sterilizing the surface; (b) cutting the surface sterilized stems into pieces of 1 to 2 mm thickness from the aseptic stand; And (c) cut by the cutting surface in contact with the culture medium comprising: a callus proliferation; comprising the, Italy poplar (Populus euramericana ) callus. < / RTI >

In an embodiment of the present invention, the step of sterilizing in step (a) is carried out by immersing in tap water flowing in a stem cut to a size of 5 to 7 cm for 30 minutes, and then sterilized in 70% ethanol for 10 seconds to 1 minute, It can be surface-sterilized with 0.2% HgCl ₂ for 20-30 minutes and with 2% NaClO for 10-20 minutes and washed 3-5 times with sterile water.

In one embodiment of the present invention, the step (c) is performed in a medium supplemented with 0.2 mg / L of BA and 0.1 mg / L of zeatin in a 1 / 2MS (Murashige and Skoog) It can be cultured for weeks.

In one embodiment of the present invention, the pH of the medium is 5 to 6, and the autoclave may be autoclaved at 110 to 130 ° C for 10 to 20 minutes.

In one embodiment of the present invention, the culturing in step (c) may be carried out in an aseptic culture room maintained at 23 to 25 ° C. for 14 to 18 hours.

In addition,

(a) Italian poplar ( Populus euramericana ) to a size of 5-7 cm and sterilizing the surface; (b) cutting the surface sterilized stems into pieces of 1 to 2 mm thickness from the aseptic stand; And (c) culturing in contact with the cut surface cut into the medium inducing the roots; comprising the, Italy poplar (Populus euramericana ).

In an embodiment of the present invention, the step of sterilizing in step (a) is carried out by immersing in tap water flowing in a stem cut to a size of 5 to 7 cm for 30 minutes, and then sterilized in 70% ethanol for 10 seconds to 1 minute, It can be surface-sterilized with 0.2% HgCl ₂ for 20-30 minutes and with 2% NaClO for 10-20 minutes and washed 3-5 times with sterile water.

In one embodiment of the present invention, the step (c) is performed in a medium supplemented with 0.2 mg / L of BA and 0.1 mg / L of zeatin in a 1 / 2MS (Murashige and Skoog) It can be cultured for weeks.

In one embodiment of the present invention, the pH of the medium is 5 to 6, and the autoclave may be autoclaved at 110 to 130 ° C for 10 to 20 minutes.

In one embodiment of the present invention, the culturing in step (c) may be carried out in an aseptic culture room maintained at 23 to 25 ° C. for 14 to 18 hours.

The method of mass proliferation of Italian poplar according to the present invention is a method of regenerating a large number of shoots from a cambium inside a stem by cutting a tree stem into a piece having a thickness of 1 to 2 mm instead of a conventional rod culture to regenerate the plant in a short time, It has the effect of mass production of poplar.

Fig. 1 is a photograph showing a plant regeneration pattern diagram by a flaky culture method (above: a conventional method of culturing a nodule, and the following: a flaky cultivation method of the present invention).
FIG. 2 is a graph showing the number of pieces that can be harvested from a 30 cm long stem. FIG.
Fig. 3 is a graph showing the number of shoots regenerated in shoots of 30 cm long stems per culture method.
FIG. 4 is a graph showing the total number of plants regenerated in a 30 cm long stem according to the culture method.
FIG. 5 shows the results of comparing the average plant regeneration numbers according to the culture method.
FIG. 6 shows the results of photographs of the production of Italian poplar superior clone seedlings by the interlaminar flaky culture method.

The present invention relates to a method for the detection of < RTI ID = 0.0 & euramericana ) callus. < / RTI >

In the present invention, " Populus euramericana ) is a deciduous arboreous tree of the dicotyledonous willow tree. It grows well on fertile ground below the foot of the mountain, about 30 meters high and 80 meters wide. The bark is silvery white, and the branches are round and have no hairs. Leaves are triangles, young leaves are reddish and green, petiole is flat and red, about 3/4 of the leaf length. It is the origin of Italy, and it is a selection among the hybrid of the US native cottonwood and the European native cotton cane, and is seemingly similar to a cottonwood. They are planted with trees and trees, and they are used for boils and hemostats, burns, and fever in the private sector.

In addition, with the recent low carbon green growth policy, demand for utilization of wood biomass is increasing, especially demand for wood energy material is expected to increase due to the introduction of greenhouse gas target management system. Therefore, it is necessary to secure a stable supply source of wood- to be. To this end, the Italian poplar, which produces the greatest amount of biomass for a short period of time and grows well in a variety of soil, has received a lot of attention recently as a suitable target species.

Therefore, the inventors of the present invention have conducted various studies to mass produce Italian poplar which is difficult to propagate in comparison with other poplar species, which is superior in properties for wood energy. In the case of the Italian poplar, And it was confirmed that large amount of Italian poplar could be propagated when cultivated by the sliced flaky culture method.

Specifically, the method according to the present invention

(a) Italian poplar ( Populus euramericana ) to a size of 5-7 cm and sterilizing the surface; (b) cutting the surface sterilized stems into pieces of 1 to 2 mm thickness from the aseptic stand; And (c) culturing the cut section so as to contact with the medium to thereby proliferate the callus.

The method for mass propagation of the Italian poplar according to the present invention will now be described in more detail in stages.

Stage 1: Stems of Italian poplar 5-7 cm And then sterilizing the surface

First, in the mass proliferation method of Italian poplar according to the present invention, a new shoot stalk having leaves removed for surface sterilization is cut into 5 to 7 cm so as to include one or two nodes, and then immersed in flowing tap water for 20 to 40 minutes Then sterilized with 70% ethanol for 10 seconds to 1 minute, with 0.2% HgCl ₂ for 20-30 minutes and with 2% sodium hypochlorite (NaClO) for 10-20 minutes in a clean bench and sterilized for 3 - It is preferable to wash five times.

Step 2: Remove the sterilized stem In aseptic band  1-2 mm  Step of cutting into thick slices

The surface-sterilized stems can be cut into aseptic strips of stem or slices of Italian poplar to a certain length, preferably about 1 to 2 mm in length.

Step 3: Cut  The sections were incubated in contact with the medium Callus  The step of proliferation

The slices cut with 1 ~ 2mm thick flakes can be cultured with the cut surface contacting the medium. The composition of the medium is 0.2mg / L of BA and 0.2mg / L of zeatin 0.1 / 2MS (Murashige and Skoog) mg / L may be added.

Also, the medium can be cultured in the medium for 2 to 6 weeks. At this time, pH of the medium is adjusted to 5 to 6, and sterilized at 110 to 130 ° C for 10 to 20 minutes. Cultivation can be carried out in an aseptic incubator maintained at a temperature of 23 to 25 ° C for 14 to 18 hours.

Meanwhile, in the present invention, the 'callus' is a tissue that is formed when a plant is injured, and is also referred to as an oily tissue, a fusion tissue, or a callus. Callus is an undifferentiated irregular cell mass that is typical of tumor tissues formed by cleavage tissue around the wound when the plant is injured. Plants are composed of dividing tissues that are largely dividing cells and permanent tissues. When cells of the dividing tissues are first put into the nutrient medium, callus is formed and then the somatic embryo is formed and differentiated into plants. This callus is often called 'plant stem cells'.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Material preparation

The present inventors used five clones of Italian poplar, which were selected by the National Forestry Academy as having excellent growth and superior wood physicochemical properties. The clones used in the present invention are 1) Dorskamp, 2) ECO-28, 3) I-476, 4) Venziano, and 5) Ay-48. The stem of the harvested Italian poplar was cut into a length of 30 cm and used in the present invention.

< Example  2>

Surface sterilization

The present inventors cut new cut shoots with 5 ~ 7 cm so as to include one or two nodes, and then immersed in running tap water for 30 minutes and then washed with 70% ethanol For 30 seconds, 0.2% HgCl ₂ for 25 minutes, 2% NaClO for 15 minutes and sterilized water for 3 to 5 times. Surface sterilized stems were transferred aseptically for this experiment.

< Example  3>

Culture method

The surface-sterilized stem of the method of Example 2 was divided into a conventional muddy culture method and a stem-flaky culture method of the present invention. At this time, the existing culture method (nodule culture method) was cut into 3 ~ 5 cm lengths so that one axillary bud was included, and one third of the stem was cultured so as to be embedded in the medium.

Stem slice culture was carried out by cutting the surface sterilized stem to a clean bench with a slice of 1 to 2 mm in thickness and cultivating the slice to contact the medium to induce shoot regeneration from the surrounding formation layer of the slice (see FIG. 1).

The slices (nodules, flakes) cut in the conventional nodule culture method and the stem flake culture method of the present invention were prepared by adding 0.2 mg / L of BA and 0.1 mg / L of zeatin to 1 / 2MS (Murashige and Skoog) And cultured in medium (see Table 1) for 4 weeks. At this time, the pH of the medium was adjusted to 5.8 and sterilized by high pressure steam at 121 ° C for 15 minutes. The cultivation was carried out in an aseptic incubator maintained at a temperature of 24 ± 1 ° C and a light period of 16 hours.

< Example  4>

Measurement of proliferation efficiency by culture method

In order to accurately compare the efficiency of the conventional round cultivation method and the stem flaky culture method of the present invention, the present inventors measured the number of interspersed sections obtained from the stem of 30 cm in length, And total number of shoots obtained from the stem of 30 cm length after 4 weeks of culture were compared by Italian poplar clones. And finally, the proliferation efficiency of the two methods was compared by presenting the average value of the plants obtained when cultivated by both culture methods.

As a result, in the conventional culture method, 3.6 cm (Venziano) ~ 6.4 (Ay-48) pieces were obtained from 30 cm stems, whereas in the case of using the stem flake culture method, 36.7 ~ 64.2 pieces of the culture medium were obtained to obtain a large amount of culture samples (see FIG. 2).

 In addition, the culture efficiency of Ay-48, which is the clone with the lowest regeneration rate, was slightly different in each clone's ability to regenerate, and the number of the subdivided slices was 2.8 times higher than that of the nodule culture method and the Venziano clone In the case of interspecific flaky cultivation, the regeneration was performed in 16 times larger sections than in the field culture, and when all five clones tested in the present invention were compared, the intermittent flaky culture method was 7.9 times more It was confirmed that shoots were produced from the sectioned bodies (see Fig. 3).

The present inventors investigated the number of regrown shoots in each clone-specific fragment after 4 weeks of culture. As a result, it was found that Ay-48, which is a clone with low regeneration rate, was able to obtain 6.4 shoots when cultivated in the nodule, but 59 shoots were obtained when cultivated in slice culture to yield 9.2 times more shoots there was. In addition, the best regenerated Venziano clones were found to produce 1,138 shoots when the slices were cultured using a flake culture method at a length of 30 cm, and 316 times more shoots could be produced than 3.6 of the seed culture method (See FIG. 4).

As a result of comparing the average plant regeneration numbers according to the cultivation method, it was found that the average number of 492.3 pieces of the interleaf flaky culturing method of the present invention was regenerated, and that 4.8 pieces were regenerated on average in the node culture method (see FIG. 5).

Therefore, it was found that the stem flake culture method of the present invention is a very efficient method for the rapid proliferation of new shoots or a small amount of material for rapid proliferation, and that clones having high regenerative ability are more efficient. The method of the present invention regenerates the shoots 102 times more than the conventional node cultures, and these results may be applied to the aborted relatives although there may be some differences between species or clones .

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

(a) Italian poplar ( Populus euramericana ) to a size of 5-7 cm and sterilizing the surface;
(b) cutting the surface sterilized stems into pieces of 1 to 2 mm thickness from the aseptic stand; And
(c) cut by the cutting surface in contact with the culture medium comprising: a callus proliferation; comprising the, Italy poplar (Populus euramericana) methods of mass proliferation of callus. The method according to claim 1,
The step of sterilizing in step (a) is carried out by immersing in tap water flowing in a stalk cut to a size of 5 to 7 cm for 30 minutes, followed by incubation with 70% ethanol for 10 seconds to 1 minute, 0.2% HgCl ₂ for 20 to 30 minutes And 2% NaClO for 10 to 20 minutes, and washed 3 to 5 times with sterilized water. The method of mass proliferation of Populus euramericana callus. The method according to claim 1,
Wherein step (c) comprises culturing in medium supplemented with 0.2 mg / L of BA and 0.1 mg / L of zeatin in 1 / 2MS (Murashige and Skoog) medium for 3 to 5 weeks. Populus euramericana) methods of mass proliferation of callus. The method according to claim 1 or 3,
The pH of the culture medium is 5-6, and, characterized in that the eseo 110 ~ 130 ℃ high-pressure steam sterilization 10 ~ 20 minutes, Italy poplar (Populus euramericana) methods of mass proliferation of callus. The method according to claim 1,
Wherein the culturing in step (c) is performed in an aseptic culture room maintained at a temperature of 23 to 25 DEG C for 14 to 18 hours in a photoperiod, and a method for mass proliferation of Populus euramericana callus. (a) Italian poplar ( Populus euramericana ) to a size of 5-7 cm and sterilizing the surface;
(b) cutting the surface sterilized stems into pieces of 1 to 2 mm thickness from the aseptic stand; And
(c) culturing in contact with the cut surface cut into the medium inducing the roots; comprising the, Italy poplar (Populus euramericana ). The method according to claim 6,
The step of sterilizing in step (a) is carried out by immersing in tap water flowing in a stalk cut to a size of 5 to 7 cm for 30 minutes, followed by incubation with 70% ethanol for 10 seconds to 1 minute, 0.2% HgCl ₂ for 20 to 30 minutes and 2% of 10-20 minutes surface sterilization and sterilized with NaClO in 3-5 times, characterized in that for cleaning, Italy poplar (Populus euramericana ). The method according to claim 6,
Wherein the step (c) is carried out for 4 weeks in a medium supplemented with 0.2 mg / L of BA and 0.1 mg / L of zeatin in 1 / 2MS (Murashige and Skoog) medium. Populus euramericana ). 9. The method according to claim 6 or 8,
The pH of the culture medium is 5-6, and, characterized in that the eseo 110 ~ 130 ℃ high-pressure steam sterilization 10 ~ 20 minutes, Italy poplar (Populus euramericana ). The method according to claim 6,
(C) the bulk of the differentiation method is a step in culture which comprises culturing in a sterile growth chamber are at a temperature of 23 ~ 25 ℃ groups of 14-18 hours Gwangju oil, Italian poplar (Populus euramericana).

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