KR20100026018A - Method to derive callus from seed or tissue of leymus chinensis trin and culture medium thereof - Google Patents

Abstract

PURPOSE: A method and medium for inducing callus from seed or tissue of Leymus chinensis Trin. is provided to enhance inducing rate of callus and produce callus having excellent plant regeneration ability. CONSTITUTION: A callus is induced by culturing seed or tissue of Leymus chinensis Trin. in a MS medium containing 2,4-D. The MS medium additionally contains 10mg/L of L-glutamic acid. The MS medium additionally contains 50 g/L of sucrose and 10 g/L of gelrite. The tissue of Leymus chinensis Trin. is leaves of Leymus chinensis Trin.

Description

Method and derive callus from seed or tissue of Leymus chinensis Trin and culture medium

The present invention relates to a method and a medium for inducing callus from seed or tissue of a candle, and more particularly, to a method and a medium for increasing the induction rate of callus.

The destruction of the global scale ecosystem due to rapid industrialization and population growth has emerged as the biggest social problem of human survival in the 21st century. In particular, China's desertification due to unreasonable reclamation and grazing of livestock is developing at a rapid pace, and it is urgently required for various countermeasures against it, which causes enormous industrial damage to Korea.

Candles ( Leymus chinensis Trin.) is a perennial plant and plant native to northeast Asia's dry and alkaline soils (pH8.5-11.5), including China, Siberia and Mongolia. It is an important feed crop that can be used for high-quality hay containing high protein as well as suppressing soil breeding and yellow dust (Jia 1987).

On the other hand, the distribution of candles is greatly reduced due to large-scale clearing and livestock grazing in areas where desertification is advanced (Liu and Wang 1999). This is so severe that the soil is extremely devastated. Therefore, it is important to identify the excellent environmental adaptability of candles that adapt well to high alkaline soils and dry areas at the molecular level, and to develop candles that are more resistant to environmental stresses.

On the other hand, Jin (2004) recently isolated about 1,500 ESTs from high alkali treatments on candles to identify the function of individual genes using transformation technology. In order to directly identify the function of the environmental resistance gene of a candle, it is important to establish the transfection system of the candle, but there are only one research report about the transfection of the candle. In order to establish the transformation system of candles, it is necessary to establish an efficient regeneration system. The study of the tissue culture of candles is carried out by plant regeneration through immature embryos (Liu et al. 2002), callus formation and immature differentiation using immature inflorescence. Plant regeneration (Liu et al. 2004). Plant regeneration using mature embryos (Kim Myung-deok et el. 2005) was created in 2005, but the callus induction rate was low.

Accordingly, the present invention is to identify the environmental adaptability of the candle and to develop a transgenic candle having complex environmental stress resistance, in the effective plant regeneration system by somatic embryogenesis, the callus having good plant regeneration ability while increasing the induction rate of callus It is an object of the present invention to provide a method and a medium for inducing callus from the seed or tissue of an inducing candle.

In order to achieve the above object, the present invention provides a candle (MS) in 2,4-D-containing MS medium ( Leymus) Induction of callus by culturing sections of seeds or tissues of chinensis Trin.), L-glutamic acid is additionally contained in MS medium containing 2,4-D. Provided are methods for inducing callus from seeds or tissues.

In addition, the present invention provides a medium for inducing callus from the seed or tissue of the candle, characterized in that L-glutamic acid is further contained in MS medium containing 2,4-D.

Hereinafter will be described in detail with respect to the problem solving means of the present invention.

Candles are an important feed crop that is relatively well adapted to harsh environments and can be used for high quality, high quality hay containing high protein, as well as inhibiting soil breeding and yellow dust in areas where desertification is advancing. Inadequate adaptation to dry and dry areas. To address these challenges, genetic transformation has resulted in the development of new plant varieties with new agronomically important features such as growth rates, increased resistance to biological and abiotic stresses, increased yields and flexible adaptation to environmental stresses. Essential conditions for such plant gene engineering are effective systems for tissue culture and plant regeneration and viable gene delivery devices. The latter is genetically technically well developed and the gene of interest can be transferred to any plant species of interest, but not all plant species are readily accessible to genetic transformation, and some agricultural plants are genetically engineered. It is known to be sensitive to. Moreover, the experimental information on certain systems for tissue culture of candles is very limited.

Accordingly, the present invention provides the optimal conditions for effectively inducing undifferentiated plant tissues capable of regenerating plants, particularly callus, and the candle ( Lymus) in MS medium containing 2,4-D Induction of callus by culturing sections of seeds or tissues of chinensis Trin.), L-glutamic acid is additionally contained in MS medium containing 2,4-D. It is a method of inducing callus from seed or tissue. The present invention was confirmed in Experiment 1 that can increase the induction rate of callus up to 75% due to the addition of L- glutamic acid, can induce a callus excellent in plant regeneration ability.

On the other hand, the content of L-glutamic acid in the medium is not limited to a specific range, but preferably contains at most 10 mg / L in the medium, because when it exceeds 10 mg / L efficiency is lower than the material content.

On the other hand, the content of 2,4-D in the medium is not limited to a specific range, but preferably contains at most 2.5 mg / L in the medium.

On the other hand, the MS medium used in the method of inducing callus from the seed or tissue of the candle of the present invention is not limited to a specific kind, but preferably further contains sucrose and gelrite, wherein sucrose And zeolite are most preferably contained at most 50 g / L and 10 g / L, respectively, in the medium.

On the other hand, the tissue is not limited to a specific one, but is preferably a leaf because the induction rate of callus is high.

On the other hand, the present invention provides a medium for inducing callus from the seed or tissue of the candle, characterized in that L-glutamic acid is further contained in MS medium containing 2,4-D.

The present invention provides a medium characterized in that L-glutamic acid is additionally contained in MS medium containing 2,4-D to increase the rate of inducing callus from seeds or tissues of candles. In this case, L-glutamic acid is preferably contained at most 10 mg / L in the medium.

On the other hand, the content of 2,4-D in the medium is not limited to a specific range, but preferably contains at most 2.5 mg / L in the medium.

On the other hand, the MS medium is not limited to a specific kind, but preferably further contains sucrose and gelrite, wherein sucrose and gelite are most preferably in the medium, at most 50 g / L, 10 g / L each is good.

As described above, the method and the medium for inducing callus from the seed or tissue of the candle of the present invention increase the induction rate of callus by incorporating L-glutamic acid in the medium, and exhibit the effect of producing a callus having good plant reproducibility. do.

In addition, the present invention can increase the induction rate of callus and produce a callus having good plant reproducibility, it is possible to establish a regeneration system to effectively build a transformation system of candles.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Example  1: L- Glutamic acid  In the containing medium From candle seeds Callus  Judo

Step 1: Plant Materials and Seed Sterilization

Candles ( Leymus chinensis Trin. Surface sterilization was performed with chlorite (sodium hypochlorite) solution for 20 minutes with stirring. The sterilized seeds were washed 3 to 5 times with sterile water and then transferred to sterilized filter paper to remove water and induce callus from the seeds.

Step 2: Callus  Judo

The medium for inducing callus from the seed of the candle was 5 mg / L L-glutamic acid, 30 g / L sucrose, 4 g / L gelrite and various concentrations in MS basal medium. Of 2,4-D, each named: M 0 (5 mg / L L-glutamic acid + 0 mg / L 2,4-D), M 1 (5 mg / L L-glutamic acid + 1.0 mg / L 2,4-D), M 2 (5 mg / L L-glutamic acid + 2.0 mg / L 2,4-D), M 2.5 (5 mg / L L-glutamic acid + 2.5 mg / L 2,4-D), M 4 (5 mg / L L-glutamic acid + 4.0 mg / L 2,4-D)

The medium was adjusted to pH 5.8 and autoclaved at 121 ° C. for 20 minutes, and then M 0, M 1, M 2, M 2.5, and M 4 were each 0.2 μm 'filter-sterilized' and then added to each medium.

The sterilized seeds were incubated for 10 to 4 weeks at 28 ° C dark conditions for each medium and cultured once for 2 weeks. When seeds germinated after 5-7 days, the small shoots and roots were completely excised.

Example  2: L- Glutamic acid  From candle leaves in the containing medium Callus  Judo

Step 1: Plant Materials

Fifteen seeds sterilized in Example 1 were soaked in a pot (100 × 40 mm) containing 35-40 ml of MS basal medium and sterilely sprouted after about one week. The leaves were cut into 1 cm sections and toothed in Petri dishes ((90 X 20 mm) to induce callus from the leaves.

Step 2: Callus  Judo

The medium for inducing callus from the leaves of candles was 5 mg / L L-glutamic acid, 30 g / L sucrose, 4 g / L gelrite and various concentrations in MS basal medium. Of 2,4-D, each named: M 0 (5 mg / L L-glutamic acid + 0 mg / L 2,4-D), M 1 (5 mg / L L-glutamic acid + 1.0 mg / L 2,4-D), M 2 (5 mg / L L-glutamic acid + 2.0 mg / L 2,4-D), M 2.5 (5 mg / L L-glutamic acid + 2.5 mg / L 2,4-D), M 4 (5 mg / L L-glutamic acid + 4.0 mg / L 2,4-D).

The medium was adjusted to pH 5.8 and autoclaved at 121 ° C. for 20 minutes, and then M 0, M 1, M 2, M 2.5, and M 4 were each 0.2 μm 'filter-sterilized' and then added to each medium.

Leaf sections were cultured for 4-5 weeks in three replicates and passaged once for 2 weeks.

Comparative example  One: L- Glutamic acid  From candle seeds in medium without Callus  Judo

Step 1: Plant Materials

As the plant material of Comparative Example 1, seeds sterilized in the same manner as in Example 1 were used.

Step 2: Callus  Judo

The medium for inducing callus from candle seeds contains only 30 g / L sucrose in MS basal medium, 4 g / L gelrite and 2,4-D in varying concentrations and no L-glutamic acid. Did: MSD 0 (0 mg / L 2,4-D), MSD 1 (1.0 mg / L 2,4-D), MSD 2 (2.0 mg / L 2,4-D), MSD 2.5 (2.5 mg / L 2,4-D), MSD 4 (4.0 mg / L 2,4-D).

The medium was adjusted to pH 5.8 and autoclaved at 121 ° C. for 20 minutes, followed by 0.2 μm 'filter-sterilized' of MSD 0, MSD 1, MSD 2, MSD 2.5, and MSD 4, respectively, and then added to each medium.

Each sterilized seed was incubated for 10 to 4 weeks at 28 ° C. in a dark condition, and then passaged once for 2 weeks. When seeds germinated after 5-7 days, the small shoots and roots were completely excised.

Comparative example  2: L- Glutamic acid  From candle leaves in medium free Callus  Judo

Step 1: Plant Materials

Fifteen seeds sterilized in Example 1 were soaked in a pot (100 × 40 mm) containing 35-40 ml of MS basal medium and sterilely sprouted after about one week. The leaves were cut into 1 cm sections and toothed in Petri dishes ((90 X 20 mm) to induce callus from the leaves.

Step 2: Callus  Judo

The medium for inducing callus from the leaves of candles is MS basal medium containing 30 g / L sucrose in MS basal medium and containing only 4 g / L gelrite and 2,4-D in various concentrations and L No glutamic acid was contained: MSD 0 (0 mg / L 2,4-D), MSD 1 (1.0 mg / L 2,4-D), MSD 2 (2.0 mg / L 2,4-D), MSD 2.5 (2.5 mg / L 2,4-D), MSD 4 (4.0 mg / L 2,4-D).

The medium was adjusted to pH 5.8 and autoclaved at 121 ° C. for 20 minutes, followed by 0.2 μm 'filter-sterilized' of MSD 0, MSD 1, MSD 2, MSD 2.5, and MSD 4, respectively, and then added to each medium.

Leaf sections were cultured for 4-5 weeks in three replicates and passaged once for 2 weeks.

Experimental Example  One: Callus  Induction rate comparison

Callus induction ratios of Example 1, Example 2, and Comparative Example 1 and Comparative Example 2 were compared.

Induction rates of Examples 1 and 2, Comparative Example 1 and Comparative Example 2 callus are shown in Table 1.

MS medium with 2,4-D (Comparative Example 1, Comparative Example 2) MS medium with 2,4-D and L-glutamic acid (Example 1, Example 2) 2,4-D of different concentrations (mg / L) callus formation (%) 2,4-D of different concentrations (mg / L) callus formation (%) Seed Leaf Seed Leaf MSD 0 20 ± 1.2 8 ± 0.4 M 0 20 ± 1.1 11 ± 0.9 MSD 1 55 ± 1.0 19.3 ± 0.8 M 1 55 ± 0.4 24 ± 0.5 MSD 2 52 ± 0.6 24.4 ± 0.6 M 2 75 ± 0.8 30 ± 0.3 MSD 2.5 50.2 ± 0.8 22.3 ± 1.0 M 2.5 40 ± 1.0 18 ± 0.5 MSD 4 30 ± 0.8 10 ± 1.2 M 4 20.2 ± 0.2 15.6 ± 0.8

Comparison of callus induction rate of candles showed that callus induction was higher in MS medium added with 2,4-D and L-glutamic acid than in MS medium containing only 2,4-D, especially in M 2 medium. Induction rate was shown.

In addition, the callus condition induced in the medium to which L-glutamic acid was added was good, but the callus type which is effectively regenerated into plants has a dense and rigid appearance, while the callus which does not exhibit any regeneration is soft and weak. As a result of observation based on these morphological cues, callus of Comparative Example 1 (FIGS. 1 to 3) and Calli of Comparative Example 2 (FIGS. 4 and 5) derived from a medium containing no L-glutamic acid contained moisture. Examples 1 (FIGS. 1 to 3) and 2 (FIGS. 4 and 5) derived from a medium containing L-glutamic acid, while being high in content and not only hard but exhibiting a lot of white color, but also small in size ) Callus has a light yellow color, low moisture content, hard grains, large size and fast growth.

From the above results, the present invention was confirmed that the induction rate of the callus of the candle can be confirmed, it was confirmed that the callus excellent in plant regeneration effect can be induced.

1 is a diagram comparing the state of callus derived from candle seeds (Comparative Example 1) in a medium containing L-glutamic acid and the callus derived from candle seeds (Example 1) in a medium containing L-glutamic acid. .

2 is a diagram comparing the state of callus derived from candle seeds (Comparative Example 1) in a medium containing L-glutamic acid and callus derived from candle seeds in a medium containing L-glutamic acid (Example 1). .

FIG. 3 is a diagram comparing the state of callus derived from candle seeds (Comparative Example 1) in a medium containing L-glutamic acid and callus derived from candle seeds in a medium containing L-glutamic acid (Example 1). ..

4 is a diagram comparing the state of callus derived from the candle leaf (Comparative Example 2) in the medium containing no L-glutamic acid and callus derived from the candle leaf (Example 2) in the medium containing L-glutamic acid. .

5 is a diagram comparing the state of callus derived from the candle leaf (Comparative Example 2) in the medium containing no L-glutamic acid and the callus derived from the candle leaf (Example 2) in the medium containing L-glutamic acid. .

Claims (11)

The 2,4-D-containing MS medium candles (Leymus Induction of callus by culturing sections of seeds or tissues of chinensis Trin. Method to induce callus from seed or tissue of candle, characterized in that L-glutamic acid is additionally contained in MS medium containing 2,4-D The method of claim 1, L-glutamic acid, Method of inducing callus from seed or tissue of a candle, characterized in that it contains up to 10 mg / L in the medium The method of claim 1, 2,4-D, Method of inducing callus from seed or tissue of a candle, characterized in that it contains up to 2.5 mg / L in the medium The method of claim 1, MS badge, A method of inducing callus from seed or tissue of a candle, further comprising sucrose and gelrite The method of claim 4, wherein Sucrose and gelite, Method for inducing callus from seeds or tissue of a candle, characterized in that it contains up to 50 g / L, 10 g / L, respectively, in the medium The method of claim 1, Organization, A method of inducing callus from seeds or tissue of a candle characterized in that it is a leaf Medium for inducing callus from seed or tissue of a candle, characterized in that L-glutamic acid is additionally contained in MS medium containing 2,4-D. The method of claim 7, wherein L-glutamic acid, Medium for inducing callus from seed or tissue of a candle, characterized in that it contains up to 10 mg / L in the medium The method of claim 7, wherein 2,4-D, Medium for inducing callus from seed or tissue of a candle, characterized in that it contains up to 2.5 mg / L in the medium The method of claim 7, wherein MS badge, Medium for inducing callus from seed or tissue of a candle, characterized in that it further contains sucrose and gelrite The method of claim 10, Sucrose and gelite, Medium for inducing callus from seed or tissue of a candle, characterized in that it contains up to 50 g / L and 10 g / L, respectively, in the medium

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