KR20180027118A - Methods of cultivating plantlets in vitro derived from stem node of Moringa - Google Patents

Abstract

The present invention relates to a method of forming plantlets of moringa in vitro. According to the present invention, the method comprises the steps of: forming a callus and inducing multiple shoots in a medium, and redifferentiating formed plantlets; growing the redifferentiated plantlets; and acclimatizing the plantlets. The step of redifferentiating plantlets comprises: (a) a step of excising a fixed size of tissues of a stem node region including growing points, from a stem of moringa being cultured in vitro; and (b) a step of growing the excised tissues of the redifferentiated plantlet in a growth medium prepared by adding, to a basal medium, 30-40 g/L of sucrose, 30 g/L of D-sorbitol, 0.1 g/L of inositol, and 8-10 g/L of agar. According to the present invention, by enabling an early supply of a large quantity of moringa to farms, the method allows the farms to harvest leaves, stems, and roots of moringa even in limited climatic conditions in Korea, and thus enables the production of functional food materials containing moringa as the main ingredient. In addition, the method makes it possible to culture moringa in a convenient and efficient manner by directly using the tissues of moringa being cultured in vitro in improved culture conditions, and thus can shorten the moringa plantlet formation period to 8 weeks, and can enable mass propagation culture. In addition, the method can avoid losses due to poor germination rates in seedlings grown from imported seeds in conventional farms, and thus can contribute to increased economic gain in the farms, replacement of imported moringa, and expansion of moringa growing farms.

Description

[0001] The present invention relates to a method for cultivating stem cells of Moringa,

The present invention relates to a method for forming an in-flight plant through moringa stem cultivation, and more particularly, to a method for producing plant seedlings by cutting plant tissues from the moringa stem nodal region and cultivating the seedlings under an improved culture condition, The present invention relates to a method for forming an in-cabbage plant of Moringa, which is improved so that it can be supplied in large quantities.

Moringa Oleifera is a perennial leguminous plant belonging to the Moringaceae family, which is distributed in tropical and subtropical climatic regions. It is a shrub ranging from 5 to 12 meters in length, It is native to the northwestern part of India.

Mouring contains more than 90 nutrients including 16 vitamins, 17 minerals, 20 essential amino acids, 15 fatty acids, and 3, 6, and 9 omega. Is the most abundant plant nutrient. It contains more than 90 nutrients needed for the human body and it is reported as "Miracle Tree".

It is known that Moringa has many effects such as prevention of cholesterol, anti-inflammation, improvement of eyesight, hypertension, immunity enhancement, antioxidation, anti-aging, tumor prevention and diabetes. In particular, Moringa leaf is known to have various effects such as protein, calcium, iron, Vitamin A and Vitamin E), Vitamin C and Vitamin E are very abundant, and it is possible to increase the natural defenses of the body by various active ingredients, provide nutrition to eyes and brain, promote metabolism, improve cell structure, improve wrinkles, normalize liver and kidney Its functional value, digestive function, antioxidant effect, immune system enhancement, anti-inflammatory action and blood sugar co-action.

These moring herbs have been cultivated in India, Vietnam, the United States, and the Philippines and have been processed and sold as oil, dried leaves and powder. Recently, however, Moringa has been cultivated in Japan and China. Accordingly, the utility of functional health supplements and functional cosmetics is expanding, and demand is also increasing.

As a result of the widespread nutritional overgrowth such as obesity, adult disease, gout, and metabolic syndrome which are common in developed countries due to the economic growth of Korea, the demand for health functional foods has been greatly increased due to increased interest in health, , There is an increasing effort to utilize the functional ingredients obtained from the existing plants as food materials, and the public is increasingly interested in the Moringa introduced through the media.

As a result, Moringa was introduced to Korea about 4 ~ 5 years ago. In Korea, imports, processing, and sales of foreign direct products and raw materials are mainly imported, and the import amount of Moringa is rapidly increasing. However, , Which is estimated to be in the range of 50 ~ 60, is leading to cultivation in Cheorwon, the jurisdiction of the present applicant.

Moringa is a perennial plant that grows in 20 ~ 30 years. It grows well in the tropics but can not grow in the domestic cold winter season. Domestically, it can be cultivated from early May to late October. to be.

In Korea, it is necessary to harvest the desired leaves, stems, roots, etc. for about 5 months, so that it is not possible to collect the seeds because the cultivation period is short. However, since the interest of the farmers is increased, However, since most of the seeds currently distributed in Korea are supplied from Thailand, Vietnam, India, and the Philippines, and these imported seeds are used for edible and processing purposes, such as seeds, Is 10 to 15%, which is extremely low.

Therefore, it is very necessary to secure domestic production technology of tissue culture seedlings which can be used as a disease-free disease free from viruses, root growth is fast, initial growth rate is high,

The culture method that induces shoot formation and plant regeneration through callus (callus, or union tissue, wound tissue, oily tissue), which refers to an indefinite amorphous cell mass, is a method that utilizes the totipotency of a plant Refers to the ability of whole plants to regenerate from a single cell or part of plant tissue. All cells have this typical performance, but the results expressed by the degree of differentiation of the cell tissue, the harvesting site, the composition of the medium, the culture environment, etc. are significantly different, and the cultivation period is very short from May to October In order to supply a large quantity of Moringa seedlings in accordance with the domestic situation, Moringa's technology for forming in-flight herbaceous plants is required.

Therefore, the domestic production of the cultured seedlings using the stem of the stem node, which is well-differentiated, is able to supply the Moringa seedlings in accordance with the time when the virus is not infected, the root growth is strong, the initial growth rate is fast, Technological securing is very necessary.

It is an object of the present invention to provide a method for producing a moringa seedlings which is inevitably produced through tissue culture by the domestic climate environment, And to provide a method of forming a Moringa inflorescence plant that cultivates healthy Moringa seedlings under improved culture conditions.

In order to accomplish the above object, the present invention provides a method for forming an in-

(a) cutting a stem node tissue including a growth point in a Moringa stem to a predetermined size,

(b) 30 g to 40 g / L of Sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and And growing the medium in a growth medium supplemented with 8 to 10 g / L of agar to the basic medium.

The growth medium for growing the cut tissue of the seedlings was KNO3 2,850 mg / L, NH4NO3 2,470 mg / L, CaCl2H2O 660 mg / L, MgSO47H2O 550 mg / L, KH2PO4 255 mg / L, MnSO44H2O 33.1 mg / L, ZnSO47H2O 12.3 mg / L, H3BO3 9.1 mg / L, KI 1.2 mg / L, CuSO45H2O 0.037 mg / L, Na2MoO42H2O 0.37 mg / L, CoCL26H2O 0.037 mg / L, FeSO47H2O 41.7 mg / L, Na2EDTA2H2O 55.9 mg / L, 3,000 mg / L of myo-inositol, 0.75 mg / L of nicotinic acid, 0.75 mg / L of pyridoxine HCl, 0.15 mg / L of thiamine HCl, 3% of sucrose and 3% of sorbitol.

The base medium is preferably one selected from the group consisting of MS (Murashige & Skoog) and WPM (Lloyd & McCown Woody Plant Basal Salt Medium).

According to the present invention, stem cells including the growth point are cut off from stem of Moringa plant which is cultured in vitro, and cultured and proliferated rapidly and in large quantity under improved culture conditions, It is advantageous to increase the number of flowering plants in the proliferation culture process and to shorten the formation period of the whole flowering plants by 5 to 8 weeks so that a large quantity of seedling can be supplied to the farmhouse, Leaves, stem and roots can be harvested under limited climatic conditions from the end of May to the end of October, enabling the provision of functional food ingredients based on Moringa, thereby increasing farm income, replacing imported Moringa, A diffusion effect can be obtained.

FIG. 1 is a photograph showing morphological processes of forming Moringa plant in the stomach by cutting and culturing stem nodes including the growth point of Moringa stem according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

The method of forming Moringa inflorescences according to the present invention is to obtain a healthy tissue of Moringa and to optimize the culture conditions so that the moringa plant can grow healthily and quickly.

For this purpose, in the present invention, in order to obtain a healthy tissue of Moringa, a stem node region including a growth point is cut and used in the stem of Moringa seedlings planted in vitro or moringae plant growing outside, For the mass production of successful tissue culture seedlings, the composition of the medium including growth regulator and the type and concentration of the medium additives was optimized for tissue culture seedlings growth.

As a preliminary step for obtaining a healthy tissue required for Moringa's method for forming in-flight herbaceous plants according to the present invention, large and robust seeds were selected and immersed in 70% ethanol for 10 seconds as a pretreatment, washed three to four times with sterilized distilled water Then, it is immersed in a 1% sodium hypochlorite solution for 15 to 20 minutes, washed again with sterilized water 3 to 4 times, the moisture of the seed is removed using sterilized paper, and then agar or pita gel cultured on a phytagel medium, and selected as a viable medium among the seedlings grown in the medium and used as a moringa tissue culture material.

The method of forming Moringa in-flight herbaceous plants according to the present invention is characterized in that the Moringa early callus formation and multi-shoot formation cultivated in the preceding process for Moringa tissue culture necessary for formation of in- , Regenerating the formed seedlings to grow them, and purifying the plants from the cabin (culture room) to the outside (greenhouse).

According to the method of the present invention,

(a) cutting a stem node region (including a growth point) produced in a Moringa stem that is cultured in a cabin in a regeneration process of the seedling plant to a predetermined length size;

(b) Four kinds of sucrose 30 g to 40 g / L, D-sorbitol 30 g / L, inositol 0.1 g / L and agar 8 to 10 g / And further comprising a step of growing a seedling plant in which the plant tissue cut out from above is dipped in a medium to which it is added to grow into a seedling plant.

Tissue culture medium is an inorganic salt of tissue culture medium excluding oxygen, carbon and hydrogen, including inorganic salts, vitamins, growth regulators, carbon and energy sources, other organic substances and other additives (agar, Elements required for cell and tissue culture include trace elements such as nitrogen, phosphorus, calcium, calcium, magnesium and sulfur and trace elements required in relatively small amounts such as calcium, sodium, manganese, molybdenum, copper, zinc, boron, cobalt and iron do.

These elements play an important role in the development of not only plants but also cells and tissues. Particularly, P is a component of nucleic acid and is essential for cell division and growth. Ca is a constituent of cell wall and it binds to pectin in middle lamella, It plays an important role in cell division and growth. Iron (Fe) in trace elements plays a role as a cofactor to complement and assist in the function of enzymes that regulate various metabolic processes occurring in plants. It also plays an important role in catalyzing photosynthesis, such as the transfer of electrons through redox action, and is also a component of respiratory enzymes.

In the present invention, MS (Murashige & Skoog Medium, Basal Salt Mixture, NH4NO3 Free) was used as a basic medium and only essential substances necessary for plant cultivation were included. In addition, the pH of the basic medium was used in the range of 5.8 to 6.0.

In addition, the present invention can be used not only in the stem of Moringa cultured in the cabinet but also at the stem node (including the growth point) of the Moringa plant growing outside, as a moringa tissue culture material. In this case, a conventional sterilization process Use coarse tissue. In this case, the cut stems (nodules) were washed well with running water, and the washed tissues were taken in a clean bench, immersed in 70% ethanol for several seconds (10 to 20 seconds), and then sterilized in distilled water Sterilized), rinsed 3 to 4 times, then immersed in 1% sodium hypochlorite solution for 15 to 20 minutes, rinsed 3 to 4 times with sterilized water, and then cut to a predetermined size centered on a sterilized scalpel Appear on the badge.

The method of the present invention was carried out as follows. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Examples>

How to cultivate the axillary region of Moringa stem

<1> Arrangement of stem nodes including Moringa stem growth

Stem nodes including the growth point were cut into a predetermined length, for example, about 30 mm, on the stem of the Moringa cultured in the cabin, and the stem was placed on the growth medium.

<2> Cultivation for growth induction of Moringa

For the growth of the stem node tissue of the moringa stem cut from the previous stage, 30 g / L of sorbitol (D-sorbitol) and 0.1 g / L of inositol were added to the basic medium of 6 g / L of MS (Murashige & Skoog) , And used as a growth medium. Each of the media components was mixed and prepared, and then 100 to 150 mL of the culture solution was dispensed into a culture bottle (for example, outer diameter: 81 mm, height: 132 mm) and sterilized at 121 ° C for 15 minutes. Ten plant tissues were streaked in a culture bottle containing the medium and cultured for 8 weeks in a culture room maintained at 26 ± 1 ° C.

The culture medium components of the above growth medium are shown in Table 1 below. As shown in the first photograph on the left side of FIG. 1, the result of culturing for 1 week in the growth medium described above shows that the stem nodal region (including the growth point) As shown in Table 2, the growth characteristics of the stem node tissue including the moringa growth point were confirmed, and the leaf number was greatly increased and the plant length was also elongated as shown in Table 2 below .

Growing medium composition of Moringa division Ingredients Content (mg / L) Inorganic salts
(Heavy element) KNO3 2,850 NH4NO3 2,470 CaCl22H2O   660 MgSO47H2O   550 KH2PO4   255 Inorganic salts
(Trace element) MnSO44H2O      33.1 ZnSO47H2O      12.3 H3BO3       9.1 KI       1.2 CuSO45H2O         0.037 Na2MoO42H2O        0.37 CoCL26H2O         0.037 FeSO47H2O      41.7 Na2EDTA2H2O      55.9 Organic substance glycine     3 myo-inositol   250 nicotinic acid        0.75 pyridoxineHCl        0.75 thiamine HCl        0.15 sucrose 3% sorbitol 3%

8th Plant Growth Characteristics Plant Formation Rate (%) Number of plantlets Leaf Length (mm) 72.5 ± 7.2 4.5 ± 4 72.5 ± 7.2 135.5 ± 13.5

<3> Purification process for morphogenesis cultivation of Moringa

Tissue cultures cultured in the growth medium become independent complete plants only through an external purification process. During fertilization, it forms shoots, anterior and lateral roots, and continues nutrition growth. It is planted in a tray (27x53x12.5cm, 50 cm) containing general peat moss for gardening, covering 30 ~ 40% of sunlight and covering transparent vinyl in a greenhouse maintained at 25 ℃ maximum and 18 ℃ minimum. . When new leaves were formed, they gradually increased the light intensity and lowered the humidity. Then, while checking the condition of the plants, the vinyl was gradually removed and cured by fully exposed to the environment of the outside air.

Although MS (Murashige & Skoog) is used as a basic medium in the description of the above embodiments, it is not limited thereto and other medium such as WPM (Lloyd & McCown Woody Plant Basal Salt Medium) may be used as a basic medium .

The present invention relates to a perennial soybean plant distributed in a subtropical climate region, which is a tropical plant which has not been attempted due to the inability of the domestic climate to grow due to the tissue culture of the seedling plant. The seedling of Moringa, which contains various amino acids and nutrients, And can be used for mass production.

Claims (3)

In Moringa's method for forming infant plants,
(a) cutting a stem node tissue including a growth point in a Moringa stem to a predetermined size,
(b) 30 g to 40 g / L of Sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and (Agar) in a growth medium supplemented with 8 to 10 g / L of the agar in the basic medium.
The method according to claim 1,
The growth medium for growing the cut tissue of the seedlings was KNO3 2,850 mg / L, NH4NO3 2,470 mg / L, CaCl2H2O 660 mg / L, MgSO47H2O 550 mg / L, KH2PO4 255 mg / L, MnSO44H2O 33.1 mg / L, ZnSO47H2O 12.3 mg / L, H3BO3 9.1 mg / L, KI 1.2 mg / L, CuSO45H2O 0.037 mg / L, Na2MoO42H2O 0.37 mg / L, CoCL26H2O 0.037 mg / L, FeSO47H2O 41.7 mg / L, Na2EDTA2H2O 55.9 mg / Wherein the composition is comprised of organic matter of 3 mg / L, myo-inositol 250 mg / L, nicotinic acid 0.75 mg / L, pyridoxine HCl 0.75 mg / L, thiamine HCl 0.15 mg / L, sucrose 3%, sorbitol 3% Of the plant.
The method according to claim 1,
Wherein said basic medium is any one selected from the group consisting of MS (Murashige & Skoog) and WPM (Lloyd & McCown Woody Plant Basal Salt Medium) medium.

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