KR101066011B1 - Embryonic Callus Induction and Plant Regeneration Methods from Mature Seed of Miscanthus sinensis - Google Patents

Abstract

The present invention relates to a method of inducing embryogenic callus and regeneration of plant from mature seeds, more specifically 25-35 g / L sucrose, 700-800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L Method for inducing embryogenic callus from pampas grass mature seeds by using germ-derived callus induction medium consisting of gelrite and MS medium containing 2 to 4 mg / L of 2,4-D and 25 to 35 g / L sucrose, 700-800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L gelrite, 1-3 mg / L 2,4-D and 1.5-2.5 mg / L The present invention relates to a method for regenerating a plant from embryogenic callus using a medium for plant regeneration using a combination of BA or MS medium containing 1 to 3 mg / L kinetin alone.

Pampas grass, callus, regeneration

Description

Embryo Callus Induction and Plant Regeneration Methods from Mature Seed of Miscanthus sinensis

The present invention relates to a method for inducing embryogenic callus and regenerating plants from pampas grass mature seeds, and more specifically, to inducing embryogenic callus from pampas grass mature seeds by using a medium containing a plant growth hormone specified by the present invention at a specified concentration. And a method for regenerating a plant from this callus.

Miscanthus sinensis is a perennial herb widely distributed all over Korea and shows the maximum photosynthetic efficiency at 30 ~ 35 ℃ and the fastest growth rate between July and August. Pampas grass is one of the representative plants that make up secondary vegetation by developing well in the place where existing vegetation is destroyed by natural and artificial disturbance such as storm, forest fire, felling, and cultivation because of its strong living power. Moreover, it has not only been widely used as an important forage for cattle or horses since ancient times, but also has been widely used as a cover plant to prevent the loss of mountain soils because of its ability to grow well under strong roots and dry environmental conditions.

Recently, due to the global craze for bio energy development, research on bio ethanol raw material crops is being actively conducted. Among them, Korean pampas grasses growing in Korea have a lot of production and grow very well in various environments around the country. It is one of the bioethanol raw crops. Pampas grass is very efficient in terms of photosynthesis, moisture and nutrient use, so the yield is 40 tons per hectare per year. In addition, unlike corn and sugar cane, since it is a non-edible crop, the potential is greater in terms of avoiding the ethical problem of developing food or feed as a raw material when it is developed as a bioenergy raw material crop.

In manufacturing bioethanol from cellulosic raw material crops such as silver grass, the most important thing is to secure raw material crops with a large quantity per unit area and to develop an efficient saccharification and fermentation process. Therefore, it is very important to develop crops of high yield and raw material crops having high saccharification and fermentation efficiency.

Among the components of the raw material crops, cellulose and hemicellulose are available as bioethanol. Therefore, research should be conducted to develop new varieties that significantly reduce the content of lignin, an inhibitor that cannot be glycosylated and inhibits the saccharification reaction of cellulose or hemicellulose.

Traditional breeding and molecular breeding methods can be used to develop new breeds of pigs with reduced lignin content. Breeding new varieties by traditional breeding methods requires a lot of time, effort and space, and has several disadvantages such as limitations on the genetic traits that can be introduced. Therefore, the development of new breeds by molecular breeding methods such as transformation can secure competitiveness in a short time. For new breed molecular breeding, an efficient tissue culture and transformation system must be established. Moreover, Agrobacterium transforming technique is most frequently used to introduce useful genes most economically and efficiently. To this end, a high efficiency regeneration system must be established.

Therefore, in the present invention, in order to develop new varieties of silver grasses widely distributed throughout Korea, the Agrobacterium transformation technique, first, a system for inducing high embryogenic callus from mature seeds of silver grasses and regenerating plants from them To establish.

We have found that embryonic callus induction from mature pigs was 3 mg / L in MS medium containing 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O, and 2 g / L gelrite. The medium with 2,4-D added was the most efficient, and the plant regeneration from the embryogenic callus was 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O, and 2 g / L gelrite. It was confirmed that the medium in which 1 mg / L of 2,4-D and 2 mg / L of BA were added to the contained MS medium was the most efficient, and the present invention was completed.

The present invention, in one aspect, 25-35 g / L sucrose, 700-800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L gelrite and 2 ~ 4 mg The present invention relates to a method for inducing embryonic callus from mature seeds by culturing in a medium for induction of embryogenic callus composed of MS medium containing 2,4-D of / L.

MS medium in the embryogenic callus induction medium may be obtained commercially, or may be prepared by referring to components and concentrations known in the art. Preference is given to using MS medium which does not contain sucrose. MS medium in the present invention is preferably composed of a composition as shown in Table 1 below.

Preferred Composition of MS Medium Macro elements ingredient Content (mg / L) Concentration (mM) CaCl ₂ 332.02 2.99 KH ₂ PO ₄ 170.00 1.25 KNO ₃ 1900.00 18.79 MgSO ₄ 180.54 1.50 NH ₄ NO ₃ 1650.00 20.61 Micro elements ingredient Content (mg / L) Concentration (μM) CoCl ₂ .6H ₂ O 0.025 0.11 CuSO ₄ .5H ₂ O 0.025 0.10 FeNaEDTA 36.70 100.00 H ₃ BO ₃ 6.20 100.27 KI 0.83 5.00 MnSO ₄ .H ₂ O 16.90 100.00 Na ₂ MoO ₄ .2H ₂ O 0.25 1.03 ZnSO ₄ .7H ₂ O 8.60 29.91 Vitamins ingredient Content (mg / L) Concentration (μM) Glycine 2.00 26.64 myo-Inositol 100.00  554.94 Nicotinic acid 0.50 4.06 Pyridoxine HCl 0.50 2.43 Thiamine HCl 0.10 0.30

In addition, MS medium which is a basal medium in the embryogenic callus induction medium can be replaced with N6 medium. In addition, sucrose, which is a carbon source, can also be replaced with glucose.

The embryogenic callus induction medium contains 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O and 2 g / L gelrite, and 3 mg / L of 2,4-D More preferably, it consists of MS medium.

In addition, in incubating the pampas grass mature seeds in the embryogenic callus induction medium to induce embryogenic callus, the temperature is 22 ~ 26 ℃, light conditions 40 ~ 60 μE / m ² · s, preferably 50 μE / m ² · s, the culture period is set for 3 to 5 weeks, preferably 4 weeks.

Furthermore, the present invention relates to the above-mentioned callus induction medium used to induce mature silver seed into embryogenic callus. Here, the MS medium which is the basic medium is preferably composed of the composition shown in Table 1 above, and may be replaced with N6 medium. In addition, sucrose, which is a carbon source, can also be replaced with glucose.

In another aspect, the present invention, 25 ~ 35 g / L sucrose, 700 ~ 800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L gelrite The present invention relates to a method for redifferentiating from embryogenic callus to a plant by culturing in a medium for plant regeneration comprising 1 to 3 mg / L 2,4-D and MS medium containing 1.5 to 2.5 mg / L BA. Instead of 1-3 mg / L 2,4-D and 1.5-2.5 mg / L BA in the medium, 1-3 mg / L kinetin may be used.

In addition, MS medium which is a basal medium in the plant regeneration medium is preferably made of the composition shown in Table 1, it can be replaced with N6 medium. In addition, sucrose, which is a carbon source, can also be replaced with glucose.

The medium for plant regeneration is 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O and 2 g / L gelrite, 1 mg / L 2,4-D and 2 mg / L BA It is more preferable that it consists of MS medium containing. 2 mg / L kinetin alone may be used instead of 1 mg / L 2,4-D and 2 mg / L BA in the medium.

In addition, when the embryogenic callus is cultured in the plant regeneration medium and redivided into plants, the temperature is 22 to 26 ° C., and the light conditions are 14 to 16 hours in light / 8 to 10 hours in dark conditions. Preferably, 16 hours day / 8 hours night conditions, the culture period is set to 3 to 5 weeks, preferably 4 weeks.

Furthermore, the present invention relates to the above-described vegetation regeneration medium used to regenerate embryogenic callus into plants. Here, MS medium that is a basic medium is preferably made of the composition shown in Table 1, it can be replaced with N6 medium. In addition, sucrose, which is a carbon source, can also be replaced with glucose.

As biofuels grow in importance, silver grass is a biomass with great potential to meet these demands. Therefore, new species such as biomass-rich varieties, varieties with reduced content of lignin, a glycosylation inhibitor, and the like should be developed. To this end, a technique must first be established to induce callus from pampas grass and to regenerate plants from the callus.

According to the present invention, embryonic callus can be induced from mature soybean seeds with high efficiency by cultivating mature soybean seeds in a callus induction medium.

In addition, by culturing the embryogenic callus derived from the mature seeds of the Pampas grass in the plant regeneration medium of the present invention can regenerate the plant from the embryogenic callus with high efficiency.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Plant Material and Seed Sterilization

As a plant material, mature seeds of domestic pampas grass were used. After removing the seed, mature seeds were surface sterilized in 70% ethanol for 1 minute, washed three times with sterile water, and then surface sterilized with 30% sodium hypochlorite solution and stirred for 1 hour. The sterilized seeds were washed 5 times or more with sterile water and then transferred to sterilized filter paper to remove water and then wound into callus induction medium.

Example 2 Embryonic Callus Induction

Basic callus induction medium to induce callus from mature seeds contains 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O, 2 g / L gelrite and various concentrations of plant growth hormone MS medium was used. In order to investigate the induction efficiency of embryogenic callus according to the type and concentration of plant growth hormone in induction of callus, auxin (2,4-D, dicamba, NAA) and cytokinin (BA, kinetin) were used alone in the induction medium. Or mixed medium was used. After sterilizing the seed sterilized in the medium, and incubated for 4 weeks in a light condition (50μE / m ² · s) in a growth room at 24 ± 2 ℃, and compared with the callus induction efficiency.

Seeds were incubated in callus-derived medium supplemented with 2,4-D, dicamba, and NAA at concentrations of 0, 3, 5, 7, and 10 mg / L, respectively. 2,4-D was the highest at 76% and 5 mg / L of dicamba was the second highest at 54%. In the case of NAA, the highest induction efficiency of 10 mg / L was 5.3%, indicating that NAA was not suitable for induction of pneumoniae callus (Table 2).

Effect of Auxin on Callus Induction from Mature Seeds Growth regulators Concentration (mg / L) Callus induction rate (%) 2,4-D


3 76.0 5 70.6 7 67.3 10 56.0 dicamba

3 50.6 5 54.0 7 50.6 10 51.3 NAA


3 2.0 5 1.3 7 2.6 10 5.3

On the other hand, when a combination of auxin (2,4-D, dicamba) and cytokinin (BA, kinetin) was incubated by adding 0.1 mg / L of kinetin to 3 mg / L of 2,4-D The highest was 28% (Table 3).

Effect of Auxin and Cytokine on Callus Induction from Mature Seeds Growth regulators and concentrations (mg / L) Callus Induction Efficiency (%) 2,4-D BA 3 0.1 20 3 0.3 22 2,4-D kinetin 3 0.1 28 3 0.3 10 Dicamba BA 5 0.1 15 5 0.3 13 Dicamba kinetin 5 0.1 20 5 0.3 13

Therefore, the optimal conditions for inducing embryogenic callus from mature seeds were 3 mg / L in MS medium containing 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O, 2 g / L gelrite. It was proved to be a medium to which 2,4-D was added.

Example 3: Plant Regeneration from Callus

Regeneration media for regeneration of callus derived from mature seeds into plants include 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O, 2 g / L gelrite, and various concentrations of plant growth. MS medium containing hormones was used. Specifically, four-week-old embryogenic callus was used alone or mixed with auxin (2,4-D, dicamba) and cytokinin (BA, kinetin) to investigate the type and concentration of plant growth hormone appropriate for plant regeneration. Transferred to the regeneration medium was incubated for 4 weeks at 24 ± 2 ℃, 16 hours day / 8 hours night conditions and shoots grown to 1 cm or more formed in each treatment was irradiated with the re-differentiated individuals.

As a result of incubating embryogenic callus formed for 4 weeks in a callus-induced medium added with 3 mg / L 2,4-D in a regeneration medium added with kinetin and BA at a concentration of 0, 2 and 4 mg / L, respectively, 2 mg / L kinetin showed the highest regeneration efficiency of 41% and 2 mg / L BA showed 38% regeneration efficiency (Table 4). Therefore, there was no significant difference between kinetin and BA, and both plant growth hormones showed a marked decrease in plant regeneration efficiency at 4 mg / L (Table 4).

Effect of Growth Regulators on Plant Regeneration Growth regulators Concentration (mg / L) Plant Regeneration (%)
kinetin
0 14.0 2 41.0 4 19.0
BA
0 13.0 2 38.0 4 22.0

In addition, when a combination of auxin (2,4-D, dicamba) and cytokinin (BA, kinetin) was incubated by adding 2 mg / L BA to 1 mg / L 2,4-D The highest was 45% (Table 5). That is, in the case of plant regeneration from embryogenic callus, the mixed treatment of 2,4-D and BA showed higher efficiency than the single treatment.

Effect of Auxin and Cytokine on Plant Regeneration Growth regulators and concentrations (mg / L) Plant regeneration rate (%) 2,4-D BA One 2 45 3 2 33 2,4-D kinetin One 2 23 3 2 28 Dicamba BA One 2 18 3 2 36 Dicamba kinetin One 2 11 3 2 23

Therefore, in a subsequent experiment, a medium in which 3 mg / L of 2,4-D was added for induction of embryogenic callus, and a medium in which 1 mg / L of 2,4-D and 2 mg / L of BA were added for plant regeneration Was used. When cultured under these conditions, embryogenic callus was formed at 70% or more after 4 weeks of culture in callus induction medium, and embryogenic callus-derived shoots were formed at 40% or more after 4 weeks of culture in regeneration medium. Re-differentiated shoots were cultured for 2 weeks in a rooting medium consisting of 1/2 MS, differentiated into complete plants, and then transplanted into pots (FIG. 1E and F).

Figure 1 shows the plant regeneration process from callus seed-derived callus (A: mature seeds of callus placed on callus-inducing medium, B: callus development from mature seed-derived callus, C: seed cultured for 4 weeks in callus-inducing medium). Callus, D: plant regeneration from embryogenic callus cultured for 4 weeks in regeneration medium, E: newsletter cultured in rooting medium, F: whole plant grown in pollen under greenhouse).

Claims (14)

Pampas grass mature seeds were 25-35 g / L sucrose, 700-800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L gelite, and 2-4 mg / L of 2,4-D. A method of increasing embryogenic callus induction comprising culturing in a medium for embryogenic callus induction comprising MS medium. The method of claim 1, The embryogenic callus induction medium comprises glucose in place of sucrose or N6 medium in place of the MS medium. The method of claim 1, The embryogenic callus induction medium is MS medium containing 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O and 2 g / L gelite, 3 mg / L of 2,4-D Characterized in that the method. The method according to any one of claims 1 to 3, The method characterized by culturing for 3 to 5 weeks at a light condition of 40-60 μE / m ² · s at a temperature of 22 to 26 ° C. With MS medium containing 25-35 g / L sucrose, 700-800 mg / L MgCl ₂ · 6H ₂ O and 1-3 g / L gelite, and 2-4 mg / L 2,4-D A medium for induction of embryonic callus from mature seeds of made pampas grass. The method of claim 5, A medium comprising glucose in place of the sucrose or N6 medium in place of the MS medium. The method of claim 5, A medium characterized by consisting of MS medium comprising 30 g / L sucrose, 750 mg / L MgCl ₂ · 6H ₂ O and 2 g / L zeolite and 3 mg / L of 2,4-D. delete delete delete delete delete delete delete

Images