KR101934775B1 - Multiple propagation methods of Moringa Oleifera in vitro plantlets using callus culture - Google Patents

Abstract

The present invention relates to a method of inducing callus formation and repetitive induction in a medium, regeneration of the formed seedling plant, growing the regenerated seedling plant, and purifying the seedling plant, And more particularly,
The step of growing the seedling plant comprises growing the seedlings planted in the step of forming the seedling plant and regeneration of the plant by 30 g to 40 g / L of sucrose, 30 g / L of D-sorbitol, Wherein the growth medium is grown in a medium supplemented with 0.1 g / L of inositol and 8 to 10 g / L of agar in the basic medium. It is advantageous to increase the number of planted plants by increasing the physiological activity without declining leaves, and it is possible to shorten the formation period of the whole seedling plant to 3 to 4 months, so that a large amount of seedling seedling can be supplied to the farmers, Since the leaves, stalks and roots can be harvested from the farmhouse even in the short domestic climate environment from January to October, it is possible to increase the income of farm households and import substitution effect of Moringa.

Description

[Background Art] [0002] Multiple propagation methods of Moringa Oleifera in vitro plantlets using callus culture,

The present invention relates to a method for mass propagation of a moringa plant through callus culture, and more particularly, to a method for inducing and culturing callus in an improved medium composition and culture condition in Moringa plant tissue, The present invention relates to a method of forming an in-cabbage plant of Moringa improved to form a plant.

Moringa Oleifera is a perennial soybean plant in the Moringaceae family, which is distributed in tropical and subtropical climatic regions. It is an important arboreous tree 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 nutrient in the world, containing more than 90 nutrients needed for the human body, "Miracle Tree" was also reported.

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 It has been recognized as a beneficial value, such as functioning, digestion, antioxidation, immune system enhancement, anti-inflammatory action, and blood sugar coagulation, and its application to functional health supplements and functional cosmetics And demand is also increasing.

These moring herbs have been cultivated in India, Vietnam, the United States, and the Philippines and processed and sold as oil, dried leaves and powder. Recently, however, growing areas such as Moringa have been growing in Japan and China .

Moringa began to be introduced in Korea about four or five years ago. It is mainly imported and processed by importing direct products and raw materials of foreign products, and the import amount of Moringa is rapidly increasing. Domestic cultivation is minimal. Moringa is a perennial plant that grows in 20 ~ 30 years. It grows well in the tropics, but it can not survive the cold winter in Korea. Therefore, it is the biggest problem in Korea that it can grow only from early May to late October.

In Korea, Moringa can be harvested for about 5 months in the open field to obtain the desired leaves, stems, roots, etc. However, it is impossible to harvest the seeds because the cultivation period is short. Therefore, the interest of farmers has increased with the increase of demand, and some farmers are importing seeds from abroad and cultivating them through seedling. Currently, most seeds distributed in Korea are supplied from Thailand, Vietnam, India, These imported seeds have a problem of extremely low germination rate of 10 ~ 15% due to mixing of immature seeds, old seeds, and impurities (see Figure 1).

Therefore, it is necessary to secure the domestic production technology of the tissue culture seedlings which are able to supply the moringa seedlings in accordance with the time when the virus is not infected and are free from disease, the root growth is fast, the initial growth rate is fast and the mass supply is possible.

Regarding the technology of producing such tissue culture seedlings, Korean Patent Laid-Open No. 10-2006-0078100 discloses a method for producing embryogenic seedlings by inducing embryogenic cells and calli aseptically from petioles of thorny stem plants and culturing somatic embryos from embryogenic calli A step of inducing and growing a mammary plant through a process of inducing somatic embryogenesis, a step of inducing and proliferating a second embryogenic callus using the cultured in-flight plant, Inducing a large amount of secondary somatic embryo from a callus through a liquid and solid medium; producing a large amount of a flowering plant from the secondary somatic embryo; and purifying the infant plant grown on the flowering plant to soil A method for mass production of seedlings is disclosed.

As another example, Korean Patent Laid-Open No. 10-2010-0026019 discloses a method for inducing regeneration from callus of a candle, wherein casamino acid is added to MS medium containing kinetin and NAA .

Although culturing methods inducing shoot formation and plant regeneration through callus (or callus (union tissue, wound tissue, oily tissue), which refers to the indefinite amorphous cell mass, are known, It is very short, from May to October, and the environment such as temperature and sunshine conditions are different from tropical regions, and because of falling leaves in the initial culture, they fail to enter into independent nutrition by promoting photosynthesis. We have completed the optimal Moringa in vitro plant cultivation technology using callus cultivation which can supply a large amount of Moringa seeds.

Korean Patent Laid-Open No. 10-2006-0078100 (published on July 05, 2006) Korean Patent Laid-Open No. 10-2010-0026019 (published on Mar. 10, 2010)

The object of the present invention is to directly utilize any one selected from the stem, root and leaf of a Moringa plant cultivated in the cabin according to the domestic situation in which the mass production of Moringa must be carried out through tissue culture inevitably according to the domestic climate environment In order to optimize the callus culturing conditions to enable mass proliferation through callus culturing and to determine the optimal culture conditions for inducing callus formation ability or shoot differentiation ability at the initial stage of culturing in the process of forming shoots after callus formation To provide a method for forming an in-flight plant of Moringa.

In order to achieve the above object, Moringa's method for forming an in-cabbage plant of the present invention comprises the steps of forming callus and inducing hyperglycaemia in a culture medium, regenerating the formed osseous plant, growing the regenerated osseous plant Wherein the step of inducing callus formation and hyperosinosis comprises culturing a small plant tissue collected from the stem, roots and leaves of an in-vitro cultured mammal plant with 0.1 g / L of casamino acid, 2.4-dichloro phenoxy acetic acid (20 mg / L), sucrose (30 g / L) and phytagel (2 to 3 g / L) to the basal medium.

In the step of regenerating the plant and regenerating the plant, the plant cultured in the step of inducing callus formation and hyperosinosis is differentiated to produce 20 mg / L of kinetin, 1-naphthaleneacetic acid (1-naphthaleneacetic acid; (NAA) 2 mg / L, Sucrose 30 g to 40 g / L, D-sorbitol 20 g / L and Pytagel 2 to 3 g / L to the basic medium desirable.

The step of growing the seedling plant comprises growing the seedlings planted in the step of forming the seedling plant and regeneration of the plant by 30 g to 40 g / L of sucrose, 30 g / L of D-sorbitol, It is preferable to grow in a medium in which 0.1 g / L of inositol and 8 to 10 g / L of agar are further added to the basic medium.

The medium in the step of inducing callus formation and hyperosin was KNO3 1,900mg / L, NH4NO3 1,650mg / L, CaCl2H2O 440mg / L, MgSO47H2O 370mg / L, KH2PO4 170mg / L, MnSO44H2O 22.3mg / L, ZnSO47H2O 8.6mg / L, H3BO3 6.2mg / L, KI 0.83mg / L, CuSO45H2O 0.025mg / L, Na2MoO42H2O 0.25mg / L, CoCL26H2O 0.025mg / L, FeSO47H2O 27.8mg / L, Na2EDTA2H2O 37.3mg / L, myo-inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine HCl 0.5 mg / L, thiamine HCl 0.1 mg / L, sucrose 3% and casamino acid 100 mg / L.

The medium in the step of forming the seedling plant and regenerating the plant was a medium containing 1,900 mg / L of KNO3, 1,650 mg / L of NH4NO3, 440 mg / L of CaCl2H2O, 370 mg / L of MgSO47H2O, 170 mg / L of KH2PO4, 22.3 mg / L of MnSO44H2O, L of inorganic salts of Na2EDTA2H2O and 37.3 mg / L of Na2EDTA2H2O were added to the reaction mixture at a concentration of 0.1 mg / L, 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, CuSO45H2O 0.025 mg / L, Na2MoO42H2O 0.25 mg / L, CoCL26H2O 0.025 mg / , glycine 2 mg / L, myo-inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine HCl 0.5 mg / L, thiamine HCl 0.1 mg / L, sucrose 3% and sorbitol 2%.

The medium in the step of growing the seedlings was a medium containing 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 moringa planting method according to the present invention directly induces callus induction and plant regeneration in plant tissues collected from stem, roots and leaves to directly utilize Moringa's tissue under cultivation It is advantageous to increase the number of flowering plants in the proliferating culture process in which the culture scale is enlarged, and since the formation period of the whole flowering plants can be mass produced in 6 months, And it is possible to harvest the leaves, stalks and roots in the farmhouse even in the domestic climate environment where the cultivation period is short from May to October, so that the farm income increase and the import substitution effect of Moringa can be obtained .

Fig. 1 is a photograph showing Moringa sowing and poor germination state.
Fig. 2 is a photograph showing the seed teeth and adventitious bud formation for callus cultivation of Moringa.
FIG. 3 is a photograph showing step-by-step plant formation processes using callus cultures of Moringa 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 study on the production technology of Moringa tissue culture using the callus culture carried out in the present invention was carried out in order to induce the callus induction in Moringa plant tissue for the mass production of Moringa seedlings and to optimize the culture conditions such as the medium composition necessary for plant formation .

In other words, since the shoot formation rate and differentiation rate of moringa vary depending on the kind and concentration of additives including growth regulators, when the tissue culture method is used, it is possible to increase the planting rate of Moringa, It is necessary to optimize the composition of the medium such as the kind and concentration of the medium supplement including the growth regulator.

The present invention uses stems, roots and leaves of Moringa seedlings of the seedless seedlings as a test material in the cabin. The above-mentioned Moringa seeds were selected by selecting a large and robust seed, immersed in 70% ethanol for 10 seconds as a pretreatment, washed 3 to 4 times with sterilized distilled water, immersed in 1% sodium hypochlorite solution for 15 to 20 minutes , And further washed with sterilized water three to four times. After removing the moisture of the seed using sterilized filter paper, it is cultured on a medium of agar or phytagel. The viability of the seedlings grown in the medium A good one is selected and used as a tissue culture material (see Fig. 2).

The present invention includes an initial callus formation and multi-shoot induction step, a plant formation step, and a root formation (rooting) step for culturing. This step transition is shown in Fig.

The method of Moringa plant formation using callus cultivation according to the present invention comprises the following steps:

(a) Small plant tissues collected from stems, roots and leaves of in-flight cultivated plants were treated with 0.1 g / L of casamino acid, 20 mg / L of 2.4-dichloro phenoxy acetic acid, 2.4- 30 g / L of sucrose and 2 to 3 g / L of phytagel to induce early callus formation and hyperosinosis;

(b) 20 mg / L of Kinetin, 2 mg / L of 1-naphthaleneacetic acid (NAA), 30 g to 40 g / L of Sucrose, 20 g / L of D-sorbitol, Forming and plant regeneration in a medium supplemented with 5 kinds of Pytagel (2 to 3 g / L); And

(c) a medium supplemented with 4 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 / The growing stage of metaphoric plants.

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.

Kinetin, a growth regulator, is a hormone that promotes cell division in plants. It induces the formation of buds, promotes leaf growth, and delays aging. 1-naphthaleneacetic acid (NAA), one of the natural auxins, plays a role in promoting the elongation and rooting of the stem and apical root, rooting and rooting.

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>

Moringa callus culture method

&Lt; 1 > Moringa callus culture medium

Tissues were harvested from leaves of moringa cultured in vitro and plated on the callus formation medium.

Early callus induction and multi-shoot media were prepared by adding 0.1 g / L casamino acid, 2.4-D (2.4-dichloro phenoxy acetic acid) to 4 g / L MS medium (Murashige & Skoog) / L, 30 g / L of sucrose and 2 to 3 g / L of phytagel as an additive and adjusting the pH to 5.8. The composition of the medium is shown in Table 1 below.

Formation of callus and morphogenesis in Moringa division Ingredients Content (mg / L) Inorganic salts
(Heavy element) KNO ₃ 1,900 NH4NO ₃ 1,650 CaCl ₂ 2H ₂ O   440 MgSO ₄ 7H ₂ O   370 KH ₂ PO ₄   170 Inorganic salts
(Trace element) MnSO ₄ 4H ₂ O      22.3 ZnSO ₄ 7H ₂ O      8.6 H ₃ BO ₃      6.2 KI       0.83 CuSO ₄ 5H ₂ O       0.025 Na ₂ MoO ₄ 2H ₂ O       0.25 CoCL ₂ 6H ₂ O       0.025 FeSO ₄ 7H ₂ O    27.8 Na ₂ EDTA ₂ H ₂ O    37.3 Organic substance glycine   2 myo-inositol 100 nicotinic acid     0.5 pyridoxineHCl     0.5 thiamine HCl     0.1 sucrose 3% casamino acid 100

Each of the medium components was mixed and prepared, and then 100 to 150 mL of the solution was dispensed into a culture bottle (outer diameter: 81 mm, height: 132 mm) at a temperature of 121 ° C for 15 minutes. Ten plant tissues were streaked on the culture medium containing the medium and cultured for 8 weeks in a culture room maintained at 26 ± 1 ° C. As shown in Table 2, the initial callus and shoot differentiation characteristics of the 8th week of culture were very good, and the state of 8th week of culture was the same as the first picture on the left side of FIG.

8th Callus formation and shoot differentiation characteristics Callus formation rate (%) Differentiation rate of shoots (%) Number of shoots / (plantlet) 52.7 ± 5.2 15.7 ± 1.5 11.2 ± 1.1

<2> Growth culture for plant and roots of 'Moringa'

After inducing callus formation and hyperhidrosine induction culture, the cells were treated with 20 mg / L of kinetin, 1 mg / L of 1-naphthaleneacetic acid (NAA), 4 mg / Sucrose 30 g to 40 g / L, D-sorbitol 20 g / L and Pytagel 2 to 3 g / L were added to form a medium. 1, planting and plant regeneration were induced for about 4 to 6 weeks in the culture bottle of the second photograph, and then grown to the same height as in the third photograph of FIG. 1. The characteristics of the 12-week and 20-day old regeneration cultivated plants were as shown in Table 4 below.

Moringa regeneration medium composition division Ingredients Content (mg / L) Inorganic salts
(Heavy element) KNO ₃ 1,900 NH ₄ NO ₃ 1,650 CaCl ₂ 2H ₂ O   440 MgSO ₄ 7H ₂ O   370 KH ₂ PO ₄   170 Inorganic salts
(Trace element) MnSO ₄ 4H ₂ O     22.3 ZnSO ₄ 7H ₂ O      8.6 H ₃ BO ₃      6.2 KI       0.83 CuSO45H2O        0.025 Na2MoO42H2O       0.25 CoCL26H2O        0.025 FeSO47H2O    27.8 Na2EDTA2H2O    37.3 Organic substance glycine   2 myo-inositol 100 nicotinic acid     0.5 pyridoxineHCl     0.5 thiamine HCl     0.1 sucrose 3% sorbitol 2%

12, 20 callus formation and shoot differentiation characteristics division Differentiation rate of shoots (%) Number of plantlets New plant length (mm) 12 parking 31.7 ± 3.1 27.5 ± 2.7 25.2 ± 2.5 20 Parking 52.7 ± 5.2 45.7 ± 4.5 42.8 ± 4.2

The rate of differentiation of shoots, number of shoots and change of length from 12th to 20th weeks were significantly increased.

&Lt; 3 & Moringa's  Culture for growth induction

In addition, 30 g / L of sorbitol (D-sorbitol) and 0.1 g / L of inositol were added to 6 g / L of MS medium (Murashige & Skoog) The ingredients are shown in Table 5. The characteristics of the seedlings grown from the previous stage medium to the 24th week were as shown in Table 6, and the growth state of the fourth photograph of FIG. 1 was obtained.

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%

20th Plant Growth Characteristics Plant Formation Rate (%) Number of plantlets Leaf Length (mm) 52.5 ± 5.2 3.7 ± 3 75.4 ± 7.5 94 ± 9.4

&Lt; 4 > Moringa Purification process for the formation of tissue cultured seedlings

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, 50gu) containing general peat moss for gardening, shielding 30 ~ 40% of sunlight and covering transparent vinyl in greenhouse keeping maximum 25 ℃ and minimum 18 ℃, . 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 that has not been attempted due to the inability of the domestic climate to grow due to the tissue culture of the seedling plant. It is a mass production of Moringa seeds containing various amino acids and nutrients .

Claims (6)

A method for forming an in-cabbage plant of Moringa comprising the steps of forming callus and inducing hyperglycaemia in a medium, regenerating a formed seedling plant, and growing a regenerated seedling plant,
In the step of inducing callus formation and hyperosinosis, small plant tissues collected from the stem, root and leaf of the in-vitro cultured plant were treated with 0.1 g / L of casamino acid, 2.4-dichloro phenoxy acetic acid ), 20 mg / L of Sucrose, 30 g / L of Sucrose and 2 to 3 g / L of phytagel were added to the basal medium,
In the step of regenerating the plant and regenerating the plant, the plant cultured in the step of inducing callus formation and hyperosinosis is differentiated to produce 20 mg / L of kinetin, 1-naphthaleneacetic acid (1-naphthaleneacetic acid; NAA) 2 mg / L, Sucrose 30 g to 40 g / L, D-sorbitol 20 g / L and Pytagel 2 to 3 g / L to the basal medium,
The step of growing the seedling plant comprises growing the seedlings planted in the step of forming the seedling plant and regeneration of the plant by 30 to 40 g / L of sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and 8 to 10 g / L of agar is further added to the basic medium.
delete delete The method according to claim 1,
The medium in the step of inducing callus formation and hyperosin was KNO3 1,900mg / L, NH4NO3 1,650mg / L, CaCl2H2O 440mg / L, MgSO47H2O 370mg / L, KH2PO4 170mg / L, MnSO44H2O 22.3mg / L, ZnSO47H2O 8.6mg / L, H3BO3 6.2mg / L, KI 0.83mg / L, CuSO45H2O 0.025mg / L, Na2MoO42H2O 0.25mg / L, CoCL26H2O 0.025mg / L, FeSO47H2O 27.8mg / L, Na2EDTA2H2O 37.3mg / Wherein the composition is comprised of an organic substance of 2 mg / L, myo-inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine HCl 0.5 mg / L, thiamine HCl 0.1 mg / L, sucrose 3%, and casamino acid 100 mg / Moringa's method for forming an infant feeder plant.
The method according to claim 1,
The medium in the step of forming the seedling plant and regenerating the plant was a medium containing 1,900 mg / L of KNO3, 1,650 mg / L of NH4NO3, 440 mg / L of CaCl2H2O, 370 mg / L of MgSO47H2O, 170 mg / L of KH2PO4, 22.3 mg / L of MnSO44H2O, L of inorganic salts of Na2EDTA2H2O and 37.3 mg / L of Na2EDTA2H2O were added to the reaction mixture at a concentration of 0.1 mg / L, 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, CuSO45H2O 0.025 mg / L, Na2MoO42H2O 0.25 mg / L, CoCL26H2O 0.025 mg / , glycine 2 mg / L, myo-inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine HCl 0.5 mg / L, thiamine HCl 0.1 mg / L, sucrose 3%, sorbitol 2% Moringa's method for forming an infant feeder plant.
The method according to claim 1,
The medium in the step of growing the seedlings was a medium containing 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 / , Inorganic salts of 9.1 mg / L H3BO3, 1.2 mg / L KI, 0.037 mg / L CuSO45H2O, 0.37 mg / L Na2MoO42H2O, 0.037 mg / L CoCl26H2O, 41.7 mg / L FeSO47H2O, 55.9 mg / L Na2EDTA2H2O, glycine 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% Method of forming a seedling plant.

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