KR20160023034A - Method for tissue culture of Aster scaber - Google Patents

Abstract

The present invention relates to a method for mass-producing Aster scaber seedlings using callus derived from Aster scaber plant tissues which includes: (1) inoculating tissue fractions of Aster scaber in a MS solid medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA) and culturing the same to induce and propagate callus; (2) redifferentiating shoot from the propagated callus in a redifferentiation medium; and (3) inducing growth of the root of the redifferentiated shoot in a medium for rooting to differentiate the same to a plant, and Aster scaber seedlings produced by the method.

Description

[0001] The present invention relates to a method for culturing australia,

More particularly, the present invention relates to a method of culturing a tissue of an aesthetic, and more particularly, to a method of culturing a tissue of an azalea, comprising the steps of: (1) Aze scaber tissue fragment is dipped and cultured in MS solid medium supplemented with NAA (naphthaleneacetic acid) Inducing and propagating callus; (2) regenerating a shoot from the propagated callus in regeneration medium; (3) inducing roots of the regenerated shoots in a medium for rooting to differentiate into plants, and a method for mass production of colony plants using a callus derived from a marine plant tissue and a method for producing ≪ / RTI >

As the national income increases, the interest and popularity of health food and non - food is gradually increasing among the people. Among them, anger is also the most representative alive.

Aster scaber is a dicotyledonous plant belonging to the Asteraceae family. It is known to be native to Korea, Japan, and China. It has traditionally been used as a therapeutic agent for bruises, detoxification, headache, and dizziness. Triterpene glycoside and volatile compound have been isolated and identified in anthrax, and in recent research, various pharmacologically active substances showing anti-Alzheimer's, antioxidant and antiviral (HIV-1) And has been attracting much attention as a development material for pharmacologically active substances.

Recently, various studies ranging from seed germination experiments to storage, sorting and chemical properties have been carried out. The anchovy is sown in the autumn to direct the seed, or to give up two or three abandonments in autumn or early spring. However, the period from seed production to seedling production is too long. Even if germination occurs, it may be necessary to break the dormancy or make a second dormancy, so that the germination is uneven and the germination rate is not high. Therefore, we sought to find out the possibility of breeding method using in - flight culture, which is more productive than the seeds or abandonment that has been used up to now,

Plant tissue culture is a valuable technology for various bioengineering applications such as mass production of pharmacologically active substances and breeding through genetic engineering. However, up to now, it has been known that tissue culture system for anthrax, especially tissue culture system via somatic cell allocation Has not been established.

Korean Patent Registration No. 1078283 discloses 'a method of culturing a marine antelope using an organic medium containing a waste mushroom medium', Korean Patent Publication No. 2007-0003653 discloses a 'method of tissue culture of a mushroom' , There is no known 'method for tissue culture of an avocado' as in the present invention.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-described needs. The present inventors have found that the concentration of NAA and BA, which are growth-regulating substances, are differently added in order to establish optimal conditions in which callus induction and proliferation are taken into consideration As a result, it was confirmed that when 5 μM NAA and 0.05 μM BA were added, induction and proliferation of callus with high efficiency were achieved. In addition, shoot regeneration was most successful in the MS solid medium supplemented with 25 μM NAA and 25 μM BA at high concentrations, and rooting was found to be best in the absence of growth regulators.

Through this, the present inventors have completed the present invention by establishing a mass production system of persimmon seedlings through tissue culture of an asteroid.

In order to solve the above problems,

(1) inducing and proliferating callus by dipping a tissue fragment of an aster scaber into an MS solid medium supplemented with NAA (naphthaleneacetic acid) and BA (benzyladenine) and culturing;

(2) regenerating a shoot from the propagated callus in regeneration medium;

(3) inducing roots of the regenerated shoots in a medium for rooting to differentiate into plants, which method comprises mass-producing the plants of the present invention using callus derived from the rootstock plant tissue.

In addition, the present invention provides a metamorphosis plant produced by the above method.

The present invention also provides a culture medium for inducing and proliferating callus from a tissue fragment of an aquatic animal comprising MS solid medium supplemented with 3 to 10 μM NAA (naphthaleneacetic acid) and 0.01 to 0.2 μM BA (benzyladenine) as an active ingredient to provide.

In addition, the present invention provides a medium composition for regeneration of shoots from azalea callus containing MS solid medium supplemented with 20-30 μM NAA (naphthaleneacetic acid) and 20-30 μM BA (benzyladenine) as an active ingredient.

Through the present invention, mass production system of persimmon seedlings can be established through the tissue culture of the anthrax, so that it can be very usefully used in the food industry and the medicine industry using the anthrax, and can contribute to the related field.

FIG. 1 shows the results of investigation of callus induction efficiency and proliferation efficiency from three different tissue sections (leaves, petiole, roots) of an asterisk in MS base medium in which various concentrations of NAA and BA were singly or mixed. A: callus induction efficiency, B: callus propagation efficiency.
Figure 2 shows representative five forms of callus derived from three different tissue sections (leaves, petiole, roots) of an asteroid. A: green and hard, B: white, but also roots, soft, C: white and soft. D: brown and soft. E: white and hard. B and C callus is a callus that differentiates into embryogenic callus.
FIG. 3 is a graph showing the plants growing in the soil through the whole process and the purification process in which the plant is regenerated through the somatic cell allocation process from the callus of the anemone. A: callus with differentiation ability, B: shoot through somatic cell differentiation process in embryogenic callus, C: shoot differentiated from callus, D: complete plant derived from plant without addition of growth regulator Result: E: Plant before being transferred to soil after purification process, F: Plant that is growing in soil after purification process.
Figure 4 shows the overall process and titration conditions for deriving complete plants via somatic cell sorting from leaf slices.

In order to achieve the object of the present invention,

(1) inducing and proliferating callus by dipping a tissue fragment of an aster scaber into an MS solid medium supplemented with NAA (naphthaleneacetic acid) and BA (benzyladenine) and culturing;

(2) regenerating a shoot from the propagated callus in regeneration medium;

(3) inducing roots of the regenerated shoots in a medium for rooting to differentiate into plants, which method comprises mass-producing the plants of the present invention using callus derived from the rootstock plant tissue.

In the method according to one embodiment of the present invention, the NAA and BA added to the MS solid medium of step (1) above may be between 3 and 10 μM NAA and between 0.01 and 0.2 μM BA, preferably between 5 μM NAA and 0.05 μM BA But is not limited thereto.

In the present invention, callus induction and proliferation are inhibited by callus induction induced by petioles or roots when the concentration of NAA is too low (0.5 μM) (25 μM), and in the case of callus derived from leaves, the concentration of NAA is low (0.5 μM) proliferation was inhibited.

In addition, when the BA concentration was high and the NAA concentration was low, the callus tended to be hard, and when the NAA concentration was too high, the callus induced from the leaf and the callus induced from the root tended to browse. In conclusion, white and soft calli (including roots) with well-regenerated shoots were best formed on medium supplemented with NAA 5.0 μM + BA 0 ~ 0.5 μM.

In the method according to an embodiment of the present invention, the regeneration medium of step (2) may be MS solid medium to which 20-30 μM NAA and 20-30 μM BA are added, preferably 25 μM NAA and 25 μM BA added MS solid medium, but are not limited thereto.

In the method according to an embodiment of the present invention, the medium for rooting in the step (3) may be MS medium to which the growth regulator is not added, but is not limited thereto.

In the present invention, induction of roots of regenerated shoots is not significantly different from the case of using MS base medium in which various concentrations of NAA are used and in which growth regulators are not added. Rather, new callus induction and delayed shoot growth , And it is preferable to use an MS base medium to which no growth regulator is added.

In the method according to one embodiment of the present invention, the odoriferous tissue fragment may be, but is not limited to, an odoriferous leaf tissue fragment.

The method according to an embodiment of the present invention

(1) inducing and propagating callus by denting and culturing a leaf tissue fragment of an aster scaber on an MS solid medium supplemented with 3 to 10 μM NAA and 0.01 to 0.2 μM BA;

(2) regenerating a shoot from the proliferated callus in MS solid medium supplemented with 20-30 μM NAA and 20-30 μM BA;

(3) inducing roots of regenerated shoots for 8 to 10 weeks in MS medium without growth regulators to differentiate into plants, the callus derived from the leaves of persimmon leaves is used for mass production , ≪ / RTI >

Preferably,

(1) inducing and proliferating callus by streaking and culturing a leaf tissue fragment of aster scaber in an MS solid medium supplemented with 5 [mu] M NAA and 0.05 [mu] M BA;

(2) regenerating a shoot from the proliferated callus in MS solid medium supplemented with 25 [mu] M NAA and 25 [mu] M BA;

(3) inducing roots of regenerated shoots for 8 to 10 weeks in MS medium without growth regulators to differentiate into plants, the callus derived from the leaves of persimmon leaves is used for mass production But the present invention is not limited thereto.

In addition, the present invention provides a metamorphosis plant produced by the above method.

The present invention also provides a culture medium for inducing and proliferating callus from a tissue fragment of an aquatic animal comprising MS solid medium supplemented with 3 to 10 μM NAA (naphthaleneacetic acid) and 0.01 to 0.2 μM BA (benzyladenine) as an active ingredient to provide. The composition comprises MS solid medium with 3 to 10 μM NAA (naphthaleneacetic acid) and 0.01 to 0.2 μM BA (benzyladenine) as an active ingredient, and 3 to 10 μM NAA (naphthaleneacetic acid) and 0.01 to 0.2 μM BA ) Can be induced and proliferated by culturing a tissue fragment of an asterisk in an MS solid medium supplemented with a callus.

In addition, the present invention provides a medium composition for regeneration of shoots from azalea callus containing MS solid medium supplemented with 20-30 μM NAA (naphthaleneacetic acid) and 20-30 μM BA (benzyladenine) as an active ingredient. The composition includes MS solid medium to which 20-30 μM NAA (naphthaleneacetic acid) and 20-30 μM BA (benzyladenine) are added as an active ingredient, and 20-30 μM NAA (naphthaleneacetic acid) and 20-30 μM benzyladenine The seeds can be regenerated by culturing the callus in a solid medium supplemented with MS.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples

Materials and methods

Plant material

The plant used for tissue culture was anchovy seedlings germinated in the cabin. The production process of germination anchovy seedlings is as follows. The seaweed seeds were immersed in a 70% ethanol solution for 1 minute and immersed in a sterilization solution (5% of commercial lactose + 1% Tween 20 solution) for 5 hours. After transferring to an aseptic incubator, it was immersed in 70% ethanol for 2 minutes, washed three times with sterilized water, transferred onto filter paper to remove water, and 30 g / L maleic acid and 8 g / L agar , And the pH was adjusted to 6.0 using 1N KOH. Then, the cells were sterilized by high pressure steam sterilization (121 ° C, 20 minutes), and were placed in Petri dishes. It was cultured in dark condition for 2 days and transferred to plant growth chamber (26 ° C / photoperiod or 24 ° C / cancer cycle for 12 hours).

Callus  Judo

The callus induction condition was determined by three kinds of tissue slices (0.5 ~ 1.0 cm size pieces) of leaf, petiole, and root of seedlings 5 to 6 weeks after aseptic germination. Single or mixed media was used. The MS basal medium was the same as that used for the production of sterile germinated plants, and the concentrations of NAA and BA added to the medium were as shown in Table 1. The callus induction efficiency was calculated by counting the number of fragments forming the callus after culturing for 5 weeks. Culture conditions for 5 weeks were 26 ° C / photoperiod or 24 ° C / dark cycle for 12 hours. The proliferation efficiency of callus was calculated by weighing callus formed for 5 weeks.

Plant regeneration

In order to investigate the regeneration efficiency of shoots, the callus induced for 5 weeks was subcultured in each medium and propagated for about 4 weeks. After mixing all the white and soft calli, they were mixed and the medium for examination of callus induction condition (Table 1) Respectively. In order to examine the regeneration efficiency of shoots, nine callus clusters per medium were used, and these were repeated three times, and the number of clumps forming callus was calculated. However, even if several shoots are formed in the same callus mass, it is counted as one, and since the shoot formation rate is not so high, it is difficult to calculate the average and deviation for the repetition. Respectively. The use of various concentrations of NAA was used to induce the rooting of regenerated shoots, but there was no significant difference from the use of MS basal medium without growth regulators, and new callus induction and delayed shoot growth MS base medium without growth regulator was used.

Plant regeneration and soil transplantation

The plant regenerated in the cabin was first cultivated for 3 days (1 hour on day 1, 2 hours on day 2, and 3 hours on day 3) by gradually opening and incubating the cap of the culture vessel in an aseptic culture zone. After 3 days, the plants in the culture container were taken out, washed thoroughly with flowing water, and transferred to sterilized soil moistened with MS inorganic nutrients. The plants were covered with transparent vinyl packs to maintain the humidity well In a plant growth chamber (26 [deg.] C / photoperiod for 12 hours or 24 [deg.] C / cancer cycle). The transparent vinyl pack which was put in order to maintain the humidity was opened sequentially one week later and completely removed after 2 weeks. Water was supplied twice a day for one week and once a day thereafter. After 3 to 4 weeks of purity in the plant growth chamber, the growth was transferred to a suitable natural condition.

Example  One. Callus  Induction and propagation conditions

The callus was induced after 1 to 2 weeks after induction on 25 calli induction mediums. The medium with induction efficiency of over 65% in all tissue sections was M14 To M25 medium (Fig. 1A). This result shows that auxin (NAA) plays a more important role in inducing callus than cytokinin (BA). When NAA is 5.0 ~ 25.0μM, callus induction occurs vigorously regardless of BA concentration . BA also affected the callus induction when the NAA concentration was low. The callus induction rate in the M11 ~ M15 medium was found to increase with increasing BA concentration at the same NAA concentration (0.5 μM). However, BA had little effect on callus induction when NAA concentration was high, and when NAA concentration was low, there was almost no callus (M1 ~ M10) even when BA concentration was high. In conclusion, it was confirmed that high concentration of NAA (5.0 ~ 25.0μM) is very important for callus induction.

The callus proliferation efficiency was found to be superior to the NAA 5.0 mu M with or without the addition of BA (M16 ~ M18 medium), and it was confirmed that about 300 mg of callus was induced / proliferated for 5 weeks (Fig. 1B). The specificity is that the concentration of NAA is too low (0.5 μM) (25 μM) to inhibit callus induction from petioles or roots. In the case of callus derived from leaves, if the concentration of NAA is low (0.5 μM) (25 μM) proliferation was not significantly suppressed while proliferation was suppressed. These results show that the concentration of NAA is also important for callus proliferation. In conclusion, when both callus induction and proliferation were considered, it was confirmed that the most optimal medium was M17 (NAA 5.0 μM + BA 0.05 μM) medium.

Example  2. Callus  shape

The culture medium induced and grown in M14 ~ M25 medium showed 5 different forms. Five forms were green and hard, white, but also roots, soft, white and soft, brown and soft, white and hard (Fig. 2). Among the five types, embryogenic calli showing good seed regeneration were white, with roots, soft, white and soft. These embryogenic calli were well formed when M16 ~ M18 medium was used in all tissue sections of leaf, petiole, and root. When the BA concentration was high and the NAA concentration was low (M14 ~ M15, M19 ~ M20, M25 medium), the callus tended to be hard and the NAA concentration was too high. (M21 ~ M25) induced tendency to browse. In conclusion, white and smooth callus (including rooting) which regenerated shoots were best formed in M16 ~ M18 medium supplemented with NAA 5.0 μM + BA 0 ~ 0.5 μM.

Example  3. Shinshu  subdivision

After induction / proliferation of white and soft calli (including rooting) were all standardized, a certain amount of lumps (9X3) were plucked into each medium and cultured. As a result, shoots were formed after about 8 weeks (Fig. 3B and Fig. 3C). The shoots were confirmed to be formed from this callus after the white, soft callus was differentiated into light yellow, embryogenic callus (FIG. 3B).

It can be seen that the formed shoots show the initial shape of heart shape and cotyledon, which is one of the typical shapes of somatic cell distribution (Fig. 3B), and shoot regeneration was judged to be via somatic cell division. The optimal medium for shoot regeneration was determined to be different depending on the callus-induced tissue sections. Leaf - derived calli were found to have higher regeneration efficiency than 10% in media with high NAA and BA concentrations (NAA 25.0 μM + BA 5.0 ~ 25.0 μM) like M23 ~ M25, Regeneration efficiency was higher than 10% in NAA and BA medium (NAA 25.0 μM + BA 5.0 μM) as in medium. In contrast to the callus induced from leaves or roots, the callus derived from petioles showed the highest shoot regeneration efficiency (11.1%) in M15 medium containing NAA 0.5 μM + BA 25.0 μM, indicating that high NAA is not required Respectively. Overall, the regeneration efficiency of shoots was determined by the high BA concentration in general, although the callus induction was different depending on which tissue the callus was derived from. The callus derived from the petiole was found to have a low NAA concentration and a high BA concentration of M15 (NAA 0.5 μM + BA 25 μM), and the M25 (NAA 25 μM + BA 25 μM) or M24 ) Medium was found to be suitable.

Example  4. Root induction

It has been known that auxin plays an important role in root induction. The root induction efficiency was compared between the medium supplemented with various concentrations of NAA and the regenerated shoots in the medium not treated with the growth regulator, The results showed that the medium supplemented with NAA (0.05 μM) induces new calli and inhibits shoot growth. In conclusion, it was confirmed that a medium without growth regulator was more suitable for root induction of regenerated shoots (Fig. 3D).

Example  5. Regeneration Plant purification

Purification of regenerated plants was accomplished by the same method as mentioned in Materials and Methods for 3 to 4 weeks and then grown to the full plant by growing under appropriate natural conditions (FIG. 3E and 3F).

Claims (9)

(1) inducing and proliferating callus by dipping a tissue fragment of an aster scaber into an MS solid medium supplemented with NAA (naphthaleneacetic acid) and BA (benzyladenine) and culturing;
(2) regenerating a shoot from the propagated callus in regeneration medium;
(3) inducing roots of the regenerated shoots in a medium for rooting to differentiate into plants, and using the callus derived from the rootstock plant tissue to mass produce the plants. 2. The method of claim 1, wherein the NAA and BA added to the MS solid medium of step (1) are between 3 and 10 [mu] M NAA and between 0.01 and 0.2 [mu] M BA. The method according to claim 1, wherein the regeneration medium of step (2) is MS solid medium to which 20-30 μM NAA and 20-30 μM BA are added. [3] The method according to claim 1, wherein the medium for rooting in step (3) is an MS medium to which no growth regulator is added. 2. The method of claim 1, wherein the odoriferous tissue fragment is an odoriferous leaf tissue fragment. The method according to claim 1,
(1) inducing and propagating callus by denting and culturing a leaf tissue fragment of an aster scaber on an MS solid medium supplemented with 3 to 10 μM NAA and 0.01 to 0.2 μM BA;
(2) regenerating a shoot from the proliferated callus in MS solid medium supplemented with 20-30 μM NAA and 20-30 μM BA;
(3) inducing roots of regenerated shoots for 8 to 10 weeks in MS medium without growth regulators to differentiate into plants, the callus derived from the leaves of persimmon leaves is used for mass production . 10. A petroglyphs plant produced by the method of any one of claims 1 to 6.  A medium composition for inducing and proliferating callus from a tissue fragment of an azalea containing MS solid medium supplemented with 3 to 10 μM NAA (naphthaleneacetic acid) and 0.01 to 0.2 μM BA (benzyladenine) as an active ingredient. A medium composition for regeneration of shoots from azalea callus containing MS solid medium supplemented with 20-30 μM NAA (naphthaleneacetic acid) and 20-30 μM BA (benzyladenine) as an active ingredient.

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