KR100620799B1 - In vitro regeneration and acclimatization of oleaceae plant - Google Patents

Abstract

The present invention provides a method for effectively re-differentiating the ash tree and the plant from the cotyledons and embryonic sections of the plant germinated in the cabin by extracting the zygote embryo of the ash tree of the ash family using tissue culture technology and the method of re-differentiated ash tree It is about soil purifying and transplanting. In other words, the present invention, after extracting the zygomatic embryo from the aspergillus of the dung beetle seeds and healed in a medium that does not contain hormones, after cutting the cotyledons and hypocotyls to make the explants and different types and concentrations of hormones Induce callus and shoot on one plant solid medium, elongate shoots in medium to which benzylamino purine (BAP) is added, and then shoot shoots on medium to completely redifferentiated ash It relates to a method for mass production of trees and plants.

Regeneration of the ash plant.

Description

In vitro regeneration and acclimatization of oleaceae plant}

1 Seedlings of ash tree germinated in the conjugate used in the present invention

Figure 2 high frequency induced malaria in the hypocotyl explants of the seedlings of the ash family according to the present invention (culture 2 weeks)

Fig. 3 (3 weeks in culture) elongated from the hypocotyl explants of the seedlings of the ash family according to the present invention

Figure 4 Elongation of stems derived from malaria in accordance with the present invention to the stem

Figure 5 elongated shoots showing the development of normal shoots before rooting induction according to the present invention

Figure 6 of the newly developed shoots and roots in the aircraft according to the present invention

Fig. 7 Primate plant of the ash tree purified and transplanted into soil according to the present invention.

The present invention provides a method for effectively re-differentiating ash tree and plant from cotyledon and hypocotyl fragments of plants germinated in the cabin by extracting the zygote of ash and rodent tree using tissue culture technology and purifying soil of re-planted ash family As described in more detail, as described in more detail, after extracting the zygote from aseptically treated ash and rodent seed, healed in a medium that does not contain hormones, cut cotyledons and hypocotyls to make excisions, Callus and shoots were induced on solid media of different types and concentrations, the shoots were stretched in a medium to which benzylamino purine (BAP) was added, and the shoots obtained here were rooted on the medium. Re-differentiated and re-differentiated ash that produces large quantities of fully re-differentiated ash family The present invention relates to a method for purifying soil of the Lepidaceae family.

Generally, the ash tree of the ash family is Ligustrum obtusifolium S. et Z., and it is a deciduous shrub whose flowers form a gunshot or abdominal inflorescence, 2 ~ 3cm long at the end of the branch, with fine hairs, and blooms in May ~ June. . Fruit is berry, ovate round, 7 ~ 8mm long, mature in purple black in October. (See Coloring Book of Korean Trees, Tae-Wook Kim, published.)

Induction of somatic embryos of woody plants is a step forward in coniferous species, and several hundred thousand improved seedlings have been commercially produced in the pine family spruce (1997 IUFRO Conference, Aug. 12-16, Quebec, Canada). In the pines such as Teda pine, research is being actively promoted as an applicable field for industrialization in recent years. In hardwood species, there are many researches on the regeneration of plants using somatic embryos from Moktulips, locusts, oaks, and conifers (Merkle, SA. 1995. Plant Tissue Culture and Biotechnology Vol. 1 (3)).

Korean Registered Patent Publication No. 10-302206 describes a method for mass production and forge transplant of Mindum elm,

Korean Patent Publication No. 10-257991 describes a method of breeding prickly pears using somatic embryos.

Conventionally, no mass breeding technique has been reported by inducing somatic embryos of selected individuals of ash family.

In order to solve the above problems, the present invention provides a method for effectively regenerating a complete plant from cotyledon and hypocotyl explants of the ash of the ash tree using tissue culture technology, and to purify and transplant the regenerated ash tree. It is about. The present invention provides a step of inducing callus and shoots from a slice of the ash of the ash tree on aseptic medium, elongating the induced shoots, rooting the shoots from the shoots, and purifying the rooted ash tree to the soil to complete ash. It is a technical object of the present invention to provide a method for regeneration of soil and regeneration of re-differentiated ash trees and plants comprising the steps of producing trees and plants.

In order to achieve the above object, the present invention is a method for effectively re-differentiating the complete ash tree and the plant from the cotyledon and hypocotyl explants of the ash tree Asteraceae using tissue culture technology and soil purification, transplantation of the re-differentiated ash tree It is about. The present invention is to induce the callus and shoot bud from the explants of the ash tree on the sterile medium, to elongate the induced shoots, to root from the shoots, to purify the roots of the ash ash plant to the soil to produce a complete ash tree The present invention relates to a soil regeneration method of redifferentiated and redifferentiated ash tree plants composed of steps.

1. Callus and Shoot Induction Steps

Seeds of sterile ash and pellets, which have been treated in a known manner, are sown in plant solid medium and germinated. After seeding, cotyledons, hypocotyls, and roots are cut from germinated seedlings to form sections. The sections were prepared to have a size of 1-5 mm, preferably 1-2 mm, and the prepared sections were then 0.002-2 mg / L thidiazuron, 0.1-1 mg / L isadichlorophenoxy. It is wound on plant solid medium mixed with acetic acid (2,4-dichlorophenoxyacetic acid, 2,4-D) or naphthaleneacetic acid (NAA). After about 2-3 weeks, callus and shoots are induced, and enough to differentiate into the next step, the shoot extension phase. It is important to note that the regeneration rate of shoots is greatly influenced by the cutting time and size of the sections, and thus the sections should be manufactured with the correct timing and size.

2. Shoot Extension

After incubation for about 2-3 weeks in the callus and shoot shoot induction medium, the plant stem pieces (Figs. 2 and 3) are transferred to a solid medium to which benzylaminopurine is added to stretch shoots. The shoots are stretched from the induced shoots by transfer to a solid medium to which about 1.0 mg / L benzylaminopurine is added (FIG. 4). At this time, after 4 weeks of culture, the concentration of benzylaminopurine was lowered and passaged to a solid medium to which about 0.2 mg / L benzylaminopurine was added, thereby promoting elongation of shoots (FIG. 5). Phenolic compounds and the like are secreted from the fragments during the cultivation, and immediately transfer to a fresh medium to prevent browning of the fragments.

3. Root Induction Stage

In the culture condition, shoots grown sufficiently are cut at the bottom of shoots and transplanted into a solid medium without hormones to induce rooting (FIG. 6). Alternatively, the same medium to which 0.02 g / L activated carbon is added is used. In the rooting induction step, it is important to incubate for 2 weeks in a weak or dark condition and then move to a weak light condition to induce rooting, and remove the area whenever the lower part of the shoot is browned and callused. Every 15 days or as soon as the medium browns, it is replaced with a fresh medium of the same composition.

4. Soil Purifying Stage of Plant

Rooted well-developed in-flight ash trees and plants were washed with running water to remove the medium attached to the roots, and then transplanted into soil mixed with vermiculite, peat moss, and tops in a ratio of 1: 1: 1 and grown for about one month. And stems grow into healthy seedlings (FIG. 7).

Hereinafter, the present invention will be described in detail with reference to the following Examples.

Example

First step (sowing of seeds)

Seed from the ash of the Asteraceae plants in 1-2% of 70-100% ethanol for 1 minute, then discard the ethanol and add 1 or 2 drops of tween20 to 1-30% hypochlorous acid solution. After sterilization by shaking for 10 to 20 minutes, wash each time 3-5 times with sterile water for 5 to 10 minutes, and then remove the germ pears from the seed with a sharp knife so that they do not contain hormones. 0.002-2 mg / L thidiazuron, 0.1-1 mg / L isadichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) of about 5.8 10-20 individuals were mixed in the solid medium containing) and then the germination of the spliced spliced embryos and the culture conditions of the whole tissue culture process were fixed at a constant temperature of about 25-27 ° C. and a working condition of 16 hours. ,

2nd process (callus and shoot induction)

Explants cut 1-2 mm of cotyledons, hypocotyls, and roots from germinated plants (Fig. 1) at 10-14 after zygote placement to determine effective plant growth regulators and optimal concentrations for inducing callus and shoots Was mixed with 0.002 to 2 mg / L tidiazuron alone and 0.001 to 1 mg / L tidiazuron with 0.1, 0.5 mg / L isadiide or naphthalinacetic acid, and contained 20 to 30 g / L sugar. After incubation on the plant solid medium with acidity of 5.8 for about 2-3 weeks,

Third process (extension of shoot)

Sections were cultured in the callus and shoot shoot induction medium for 2-3 weeks, and then transferred to plant solid medium containing 0.001-1 mg / L benzylaminopurine. The culture period was about 3 weeks.

(The shoots grown during the incubation period were cut about 1 cm and transferred to a 1/2 medium plant solid medium without hormones to remove the hormones.)

On the other hand, after 3 weeks of cultivation, it was transferred to a plant solid medium to which 0.1-0.5 mg / L benzylaminopurine was added and cultured by lowering the concentration of hormones, which was passaged every 2 to 3 weeks, promoting continuous induction and extension of shoots,

4th process (induction of rooting from normal shoots)

In the third step, cultured for about 8-10 weeks, the development of normal shoots is visible, and when the node between shoots and shoots is sufficiently elongated (Fig. 5), the lower part of the re-differentiated shoots is cut and 20 ~ 30g / L sugar, 3 ~ Roots were induced by transplanting them into a test tube containing 10 g / L agar containing 1/2 of the hormone-free plant solid medium (pH 5.8) (FIG. 6).

(In this period, when the cut surface of the lower shoot is changed brown or callus is formed, the area is cut by a thin blade (less than 1mm in thickness) and removed.)

After the main roots are generated, they are passaged (redifferentiated) with a medium having a low hardness of 4.5 g / L on the same medium to promote the production of the roots.

5th process (Plant soil purification step)

Plants rooted to about 3-5 cm in size are removed from the test tube, washed with a running water, and the roots are immediately transplanted into soil mixed with vermiculite, peat moss, and soil in a 1: 1: 1 ratio. It was.

For 3 ~ 4 weeks after transplanting, the seedlings were covered with vinyl so as not to wither, and then sprayed with a sprayer to grow the leaves so that they did not dry out. 7).

Experimental Example

Seed from the ash of the Asteraceae plants in 1-2% of 70-100% ethanol for 1 minute, then discard the ethanol and add 1 or 2 drops of tween20 to 1-30% hypochlorous acid solution. Sterilize by shaking for 10-20 minutes. After disinfection, rinse each 3-5 times for 5 to 10 minutes using sterile water, and then remove the seed germ pear from the sharp knife so as not to be injured, and then place it in a plant solid medium having a pH of about 5.8 without hormones. -20 individuals were wounded. The germination of the injured spliced embryos and the culture conditions of the whole tissue culture process were fixed at a constant temperature of about 25 ~ 27 ℃ and sun conditions of 16 hours.

To determine the plant growth regulators and optimal concentrations effective for inducing callus and shoots, the fragments cut from cotyledons, hypocotyls and roots 1-2 mm from the germinated plants (Fig. 1) at 10 days after the splicing were placed. , 0.002, 0.02, 0.2, 1, 2 mg / L tidiazuron alone and mixed with 1.0 mg / L tidiazuron and 0.1, 0.5 mg / L isadiide or naphthalinacetic acid, 30 g / L sugar It was inoculated on a plant solid medium having an acidity of 5.8 and cultured for about 3 weeks (FIG. 2). This experiment was repeated three times and the results are described in Table 1. Shoot differentiation rate was different depending on the explant site and plant growth regulator. Regeneration rate according to the explant site was effective in cotyledon and hypocotyl explants, but no shoots were formed in root explants. Depending on the plant growth regulators, the redifferentiation efficiency was high in the tidiazuron alone treatment, and the hypocotyl at the 1.0 mg / L tidiazuron concentration was the highest in the cotyledon section at the 2.0 mg / L tidiazuron concentration. On the other hand, in the mixed treatment of 1.0 mg / L tidiazuron, isadi, and naphthalinacetic acid, mainly callus tended to be formed, and browning tended to change as the culture continued. The shoot regeneration according to the size of the sections showed a higher shoot regeneration rate than the sections of 3-5 mm when the section size was 1-2 mm (Table 2).

Table 1. Effect of explant site and plant growth regulator on ash regeneration of ash tree of Ash family

Growth regulator (mg / L) Callus Formation Rate 2,4-D NAA TDZ Intercept cotyledon Axle Root 0 0 0 0 0 0 0.1 0.5 0 0 0 0 0 0 0 0 0 0 0.1 0.5 0 0.002 0.02 0.2 1 2 1 1 1 1   0.0 (0.0) 27.3 (3.0) 72.7 (3.0) 96.4 (3.6) 100 (11.4) 100 (35.6) 96.2 (0.0) 100 (0.0) 80.0 (0.0) 95.5 (4.6) 0.0 (0.0) 34.4 (3.1) 100 (4.2) 88.9 (14.8) 100 (32.9) 97.0 (18.2) 100 (11.8) 100 (0.0) 100 (17.4) 100 (15.0) 0.0 (0.0) 0.0 (0.0) 62.7 (0.0) 66.7 (0.0) 60.9 (0.0) 90.9 (0.0) 100 (0.0) 100 (0.0) 80.0 (0.0) 100 (0.0)

Table 2. Effect of Explant Size and Plant Growth Regulators on the Regeneration of Primrose Root from Ash Trees

Growth regulator (mg / L) Intercept size 1-2 mm 3-5 mm TDZ cotyledon Axle Root cotyledon Axle Root 0 0.002 0.02 0.2 1 2 0.0 3.0 3.0 3.6 11.4 35.6 0.0 3.1 0.0 14.8 32.9 18.2 0.0 0.0 0.0 0.0 0.0 0.0 0 0 0 0 6.3 18.5 0 0 9.1 16.7 21.4 9.1 0 0 0 0 0 0

The present invention prepared as described above is capable of producing seedlings of a large amount of ash trees and plants in a short time by a simple method, and as a result of cultivating seedlings, roots and stems are cultivated soundly to promote forest greening in the mountains and the mountains of the country, furthermore It has the effect of contributing to the forest economy.

Claims (5)

In the soil recycling transplantation method of re-differentiated and re-differentiated ash tree, First step (sowing of seeds) Seed from the ash of the Asteraceae plants in 1-2% of 70-100% ethanol for 1 minute, then discard the ethanol and add 1 or 2 drops of tween20 to 1-30% hypochlorous acid solution. After sterilization by shaking for 10 to 20 minutes, wash each time 3-5 times with sterile water for 5 to 10 minutes, and then remove the germ pears from the seed with a sharp knife so that they do not contain hormones. 5.8 0.002-2 mg / L thidiazuron, 0.1-1 mg / L isadichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid, 2,4-D) or naphthaleneacetic acid (NAA) After 10-20 individuals were flocculated into the mixed-added plant solid medium, the germination of the injured spliced embryos and the culture conditions of the whole tissue culture process were fixed at a constant temperature of 25-27 ° C. and a working condition of 16 hours. 2nd process (callus and shoot induction) Explants cut 1-2 mm of cotyledons, hypocotyls, and roots from germinated plants (Fig. 1) at 10-14 after zygote placement to determine effective plant growth regulators and optimal concentrations for inducing callus and shoots Was mixed with 0.002 to 2 mg / L tidiazuron alone and 0.001 to 1 mg / L tidiazuron with 0.1, 0.5 mg / L isadiide or naphthalinacetic acid, and contained 20 to 30 g / L sugar. After incubation for 2 to 3 weeks on the plant solid medium with a pH of 5.8, Third process (extension of shoot) Sections were cultured in the callus and shoot shoot induction medium for 2-3 weeks, and then transferred to plant solid medium containing 0.001-1 mg / L benzylaminopurine. The culture period was about 3 weeks. (The shoots grown during the incubation period were cut about 1 cm and transferred to a 1/2 medium plant solid medium without hormones to remove the hormones.) On the other hand, after 3 weeks of cultivation, it was transferred to a plant solid medium to which 0.1-0.5 mg / L benzylaminopurine was added and cultured by lowering the concentration of hormones. 4th process (induction of rooting from normal shoots) In the third process, normal shoot development was observed after 8-10 weeks, and when the node between shoots and shoots was sufficiently elongated, the lower part of the subdivided shoots was cut and 20 ~ 30g / L sugar, 3 ~ 10g / L agar Rooting was induced by transplantation into a test tube containing 1/2 of the hormone-free plant solid medium (pH 5.8). (In this period, when the cut surface of the lower shoot is changed brown or callus is formed, the area is cut by a thin blade (less than 1mm in thickness) and removed.) After the main roots are generated, they are passaged (redifferentiated) with a medium having a low hardness of 4.5 g / L on the same medium to promote the production of the roots. 5th process (Plant soil purification step) Plants grown to around 3-5 cm in size were removed from the test tube and washed with a running water to wash the medium attached to the root, and immediately transplanted into vermiculite, peat moss, and soil mixed in a 1: 1: 1 ratio. After that, the seedlings are covered with vinyl for 3 to 4 weeks, and then sprayed with a sprayer to grow the leaves so that the leaves do not dry out. delete delete delete delete

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