KR100533120B1 - Method of plant cell culture from cambium - Google Patents

Abstract

The present invention relates to a method for plant cell culture using a cambium, which is a dividing tissue of a plant, comprising: extracting the cambium after sterilizing the stem of the plant; Callus inducing step from the forming layer in callus induction medium; And cell proliferation step through suspension culture of the callus in cell proliferation medium.

Conventionally, seed culture was carried out when plant cells were cultured. However, the present invention enables rapid mass production during cell culture by culturing the formation layer which is a dividing tissue, and supplies raw materials without destroying the natural ecosystem and environment in producing useful secondary metabolites. This will make a significant contribution in environmental, economic and industrial terms.

Description

Plant cell culture method using the cambium {METHOD OF PLANT CELL CULTURE FROM CAMBIUM}

The present invention relates to a plant culture method for rapidly producing a large amount of plant cells, and more particularly, to a plant cell culture method of inducing callus through a cambium culture rather than a general seed culture.

Plant cell culture is aseptically placed on artificial media (cells, tissues, organs, etc.) in artificially produced medium (solidified liquid or liquid supplying nutrients, such as agar), and adjusted to the appropriate temperature by artificial light. It is common to maintain and culture.

In 1904, Hanning began to cultivate immature embryos under sterile conditions and obtained plantlets. After 1920, egg seed germination, callus culture, and organ culture were performed. After 1945, these various forms were tied together and called plant tissue culture. (Barz, W., Reinhard, E. & Zeuk, M.H., 1977).

Plant tissue culture has evolved into a wide range of fields out of the early stages of technology and has become an essential technology for completing genetic engineering techniques in higher plants, especially in combination with genetic engineering techniques.

Recently, with the development of molecular biology and the development of research equipment, it is possible to study the mechanisms related to tissue culture at the genetic level. It is believed that when these studies are conducted continuously, plant tissue culture will have a ripple effect across the field of biotechnology unlike now, and will occupy a firm place in plant-based foundations and applications.

However, plant cell culture often encounters several problems such as low content of necessary ingredients and instability of productivity.

As a plant transformation method employing genetic engineering methods to improve the commercial value by improving the shortcomings of varieties, but the secondary problem of transformation may include genetic damage, this also is a complete alternative It cannot be.

Therefore, in particular, when extracting the active ingredient from the plant is a situation that requires a rapid mass production method of plant cells without genetic damage.

The present invention is invented to solve the above problems,

An object of the present invention is to provide a method for culturing plant cells for mass production of cells.

Another object of the present invention to provide a raw material supply method for extracting a large amount of the active ingredient of plant cells.

In order to achieve the above object, the present invention consists of the following steps.

Formation layer collection step after the sterilization process of the plant stem;

Callus inducing step from the forming layer in callus induction medium; And

Cell proliferation step through suspension culture of the callus in cell proliferation medium.

Conventionally, seed culture was performed during plant cell culture, but the present inventors found that mass production is possible within a short time by culturing the cambium, which is a meristem, and thus, the present invention has been completed. In the present invention, in particular, the callus induction rate was improved by using the cambium collected from the stem of the plant.

The cambium is also called a tissue, and is a tissue that allows a plant to grow in volume. This tissue is present between the woody and phloem of the stem or root and actively divides to thicken the stem or root. Cell division is the most active meristem, and when used as explants of plant tissue culture, high-speed and large-scale production of cells is enabled.

In addition, callus (CALLUS) is when the wound or grafted to the plant when the de-differentiation occurs in the cut surface to form an irregular cell mass, which is also referred to as a non-differentiated cell tissue in tissue culture. (Pierik, R.L.M., 1987).

The present invention is intended to mass-produce cells by harvesting the cambium and inducing callus (CALLUS) by suspending the callus.

Through the following examples will be described in more detail the present invention.

Example 1

Preparation of plant material

Seeds of pears (pears), needles (leader tissues), and stems (forming layers) were collected seasonally (winter, spring, summer). Surface sterilization was carried out in different ways due to the morphological and physiological characteristics of the materials.

① Seed (pear): surface sterilization in 70% ethanol for 1 minute, and then immersed in 1% sodium hypochlorite solution for 48 hours, washed 3-4 times with sterile water to extract the conjugate embryo from the seed was used as an experimental material. .

② Needle leaf and stem: immersed in 1% benomil + 1% streptomycin solution for 24 hours, washed with flowing water for 30 minutes, surface sterilization in 70% ethanol for 1 minute, and then immersed in 1% sodium hypochlorite solution for 30 minutes 3-4 times as a test material was used. The stems were cut to a size of 0.05 cm and made into discs so that the cross section of the forming layer was in contact with the medium surface.

Example 2

Effect of Explants on Callus Induction

In Example 1, the different plant growth regulators {2,4-D (2,4-Dichlorophenoxyacetic acid), MS with α-naphtalene acetic acid (NAA), Indole-3-acetic acid (IAA), 3-Indolebutyric acid (IABA), Dicamba, Picloram} and 0.4% gelrite, 3% sucrose (Murashige, T. and Skoog F., 1962), B5 (Gamborg et al., 1968), modified B5, and SH (Schenk, RU and Hildebrandt, AC, 1972) were added to the medium to prepare the medium.

The medium was adjusted to pH 5.8 before gelrite addition and autoclaved at 121 ° C. and 1.2 atm for 15 minutes. In addition, gibberellic acid (GA ₃ ), which is required for experimental purposes, was added to the sterilized medium aseptically.

The sterilized medium was dispensed into petridish by about 25 mL to dentify each explant that had undergone surface sterilization. Cultivation of the test material was carried out under dark conditions of 25 ± 1 ℃.

B5 medium is one of the main mediums used in plant culture. It is a combination of inorganic salts and organics designed by Gamborg et al. In 1968 for the culture of soybean tissues. (Gamborg et al., 1968, Nutrient requirement of suspensions cultures of soybean root cells.Exp. Cell.Res., 50, 151). This is called modified B5 medium. In this experiment, modified B5 medium was modified by doubling the amount of KNO ₃ in the original B5 medium composition and doubling the amount of vitamins, which are organic substances, in order to induce maximum callus.

The composition of the B5 medium and the modified B5 medium used in the present invention are shown in Table 1 below.

Furtherance B5 badge Modified B5 Medium Macronutrients (mg / L) KNO ₃ 2500 5000 CaCl ₂ · 2H ₂ O 150 150 MgSO _{4 7} H ₂ O 250 250 NaH ₂ PO ₄ · H ₂ O 150 150 (NH ₄ ) ₂ SO ₄ 134 134 Micronutrients (mg / L) MnSO ₄ 4H ₂ O 13.2 13.2 ZnSO ₄ · 7H2O 2 2 H ₃ BO ₃ 3 3 KI 0.75 0.75 CuSO ₄ · 5H ₂ O 0.025 0.025 Na ₂ MoO ₄ 2H ₂ O 0.25 0.25 CoCl ₂ · 6H ₂ O 0.025 0.025 Vitamin (mg / L) Inositol 100 200 Thiamine HCl 10 20 Nicotinic acid One 2 Pyridoxine One 2

The result of the above experiment is as follows.

As shown in FIG. 1, the callus of light yellow began to be induced in the cambium from the 10th day of culture, and a significant amount of callus was formed on the 20th day of culture. On the other hand, callus formation was not observed in 20 days of culture, and callus induction was observed in the lobules of needles, but the amount was very low. The fresh weight of callus derived from the cambium of stem showed about 10 times higher proliferation rate than callus derived from the lobules of needles. As such, callus induction from each explant was most effective in forming layers of stems.

FIG. 2 is a diagram illustrating a culturing layer formed from foliar tissue and stems collected from a leaf, and illustrates a process of inducing callus by initiating cell division while inflating.

Unlike the cambium collected from the stem at the time of co-cultivation, the foliar tissue collected from the leaf can be seen that the cell expansion step for cell division only.

In addition, the highest induction rate of callus was 78.56mg per explant from the formation layers of the stems collected seasonally, especially in winter, and the callus induction rate decreased as the temperature of the collection time increased. In order. Thus, callus induction rate was significantly different depending on the collection time and the type of explants, so the best callus induction rate was obtained from the cambium in winter. This is the same as FIG. 3.

Example 3

Identification of Auxins for Callus Induction of Formation Layers Extracted from Stems

To investigate the effective auxin in callus induction from the cambium of stem, 2,4-D (2,4-Dichlorophenoxyacetic acid), NAA (α-naphtalene acetic acid), IAA (Indole-3-acetic acid), IBA (3- Indolebutyric acid), Dicamba, and Picloram were added to modified B5 medium.

To determine the appropriate concentration of plant growth regulators, low concentration groups of 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 8 (mg / L) and high concentrations of 10, 20, 30, 40, 50 (mg / L) As a result of dividing into groups, the callus induction rate was lower in the low concentration group than in the high concentration group, and some of them were necrotic during the subculture.

Callus induction was not observed in NAA, α-naphtalene acetic acid (NAA), Indole-3-acetic acid (IAA), and 3-Indolebutyric acid (IABA), but induction was incomplete in dicamba. In addition, some of the callus was also induced in 2,4-D (2,4-Dichlorophenoxyacetic acid) treatment, but browned and necrotic in culture. Therefore, callus induction of the auxin-based picloram was effective, as shown in Figure 4, it can be seen that the optimal concentration is 30 mg / L picloram.

 Example 4

Effect of Gibberellic Acid (GA ₃ ) on Callus Induction

Gibberellic acid (GA ₃ ), which is known to be involved in the formation of the cambium in tissue development, was added to the medium to obtain a positive effect on callus induction.

The addition of 0.5 mg / L gibberellic acid (GA ₃ ) resulted in double callus induction compared to the control that was not added.

This is as shown in FIG.

Example 5

Selection of medium for callus induction

Each medium was used to determine the induction of callus according to the salt composition and the organic composition of the medium, and there were many differences. The callus induction was different from the salts and organics of the modified B5 medium compared to B5 medium, MS and SH medium. It was the most effective in the composition and the lowest in the MS medium. This is as shown in FIG.

As a result of the experiment in the embodiment it was confirmed that the optimum composition of the callus induction medium from the formation layer of the yew tree is shown in Table 2.

Callus Induction Optimum Badge ^※ B5 major salts2X B5 vitamins Activated charcoalGA ₃ SucrosePiclorampHGelrite 2X KNO ₃ 0.01% 0.5 mg / L3% 30 mg / L5.80.4%

^※ Gamborg B5 medium: Gamborg et al., Exp. Cell Res., (1968)

Example 6

Selection of Additives for Cell Proliferation

 After incubation for about 20 days in a callus-induced medium, a good cell line was selected from callus formed from explants. Cell lines were cultured at 110 rpm in shaker culture using modified B5 medium in which picloram and NAA (α-naphtalene acetic acid) alone and in combination. Cell proliferation was better under combined conditions than single treatment and was most effective in modified B5 medium supplemented with 5 mg / L picloram and 2 mg / L NAA (α-naphtalene acetic acid). Regardless of the concentration, 2,4-D (2,4-Dichlorophenoxyacetic acid) and NAA (α-naphtalene acetic acid) alone showed lower cell proliferation rate than picloram supplemented medium. The most effective auxin in cell proliferation is picloram, and the addition of NAA (α-naphtalene acetic acid) induces a synergistic effect. This is as shown in FIG.

In the drawings the symbols are as follows:

P2.5N1 was added to 2.5 mg / L picloram and 1 mg / L α-naphtalene acetic acid (NAA) in modified B5 medium;

P5N2: addition of 5 mg / L picloram and 2 mg / L NAA (α-naphtalene acetic acid);

P10N4: addition of 10 mg / L picloram and 4 mg / L NAA (α-naphtalene acetic acid);

P20N8: addition of 20 mg / L picloram and 8 mg / L NAA (α-naphtalene acetic acid);

P40N16: Added medium of 40 mg / L picloram and 16 mg / L NAA (α-naphtalene acetic acid).

In the above example, the optimal composition of the cell proliferation medium derived from callus was confirmed as shown in Table 3.

Cell proliferation optimal medium ^※ B5 major salts2X B5 vitamins GA ₃ Sucrose PicloramNAA (α-naphtalene acetic acid) pH 2X KNO ₃ 0.5 mg / L3% 5 mg / L2 mg / L5.8

^※ Gamborg B5 medium: Gamborg et al., Exp. Cell Res., (1968)

As described above, the formation of culturing layer, in particular, the stem formation layer culture in modified B5 medium has the effect of maximizing the growth and proliferation of cells that produce useful secondary metabolites in a large amount in a short time.

In particular, the medium effective for callus induction was modified B5 medium with picloram 30 mg / L, and cell proliferation was modified B5 medium with 5 mg / L picloram and 2 mg / L NAA (α-naphtalene acetic acid). You can see that it works best.

The cultivation method of the present invention will be applied to various wood plants having an effective ingredient that is difficult to cultivate, and will greatly contribute in terms of environmental, economic and industrial derivatives derived from them as well as raw materials that do not destroy the environment in the production of useful secondary metabolites.

1 is a photograph showing the results of culturing embryos (embryo) isolated from seeds, mesophylls collected from needles, and cambiums collected from stems simultaneously. Results for the formation layer.

Figure 2 is a photograph showing the results of the culturing layer of leaf foliar tissue and stem, the upper right picture is a photograph of foliar tissue, the remaining three is that the formation layer taken from the stem cells to form a callus It is a picture showing.

3 is a graph showing the callus induction rate according to the type of explants collected seasonally.

4 is a graph showing the effect of picloram, an additive suitable for callus induction.

FIG. 5 is a graph illustrating the effect of gibberellic acid (GA ₃ ), an additive suitable for inducing callus.

6 is a graph comparing callus induction rate in culture in various media.

7 is a graph comparing cell proliferation with additives.

Claims (9)

Plant cell culture method for mass production of plant cells, comprising the following steps: Formation layer collection step after the sterilization process of the plant stem; Callus inducing step from the forming layer in callus induction medium; And Cell proliferation step through suspension culture of the callus in cell proliferation medium. The method of claim 1, wherein the stem is harvested in winter plant cell culture method for mass production of plant cells.  The method of claim 1 or 2, wherein the plant cell culture method for mass production of plant cells, characterized in that the addition of picloram (caloram) to the callus induction medium. The plant cell culture method of claim 1 or 2, wherein gibberellic acid (GA ₃ ), which is involved in cambium division, is added to the callus-inducing medium. The method of claim 1 or 2, wherein the callus induction medium is a modified B5 medium in which BNO medium doubles KNO ₃ and vitamins. The method of claim 3, wherein the concentration of picloram (picloram) is a plant cell culture method for mass production of plant cells, characterized in that 30mg / L. The method of claim 4, wherein the concentration of gibberellic acid (GA ₃ ) is a plant cell culture method for mass production of plant cells, characterized in that 0.5mg / L. The method for mass production of plant cells according to claim 1 or 2, wherein a modified B5 medium to which picloram and NAA (α-naphtalene acetic acid) are added is used as a cell proliferation medium. Plant cell culture method. 9. The method of claim 8, wherein the concentration of picloram and NAA (α-naphtalene acetic acid) is 5 mg / L and 2 mg / L, respectively.