WO2010076954A2 - Mass production of potato microtubers by tissue culture and method for producing seed potatoes - Google Patents

Abstract

The present invention relates to a method for producing seed potatoes free of virus through the massive propagation of potato microtubers by growing sterile potato stems in a bio-reactor culture device containing MS liquid culture medium reduced to 1/2 to 1/4 of the amount thereof, increasing the used MS liquid culture medium by 1-2 times, and culturing the potato stem in a dark place. Between 7 and 20 potato microtubers having stems with a length of 3-5 cm produced germ-free by tissue-culture and sprouts grown to 2-5cm are sown per 10 ㎠ on a seedbed filled with bed-soil at a depth of 3-6cm. The small seed potatoes grow under the bed-soil, and are cultivated. The potatoes of 5g or more are used as the seed potatoes for farmhouses, and the potatoes of less than 5g are sown on the seedbed to produce the seed potatoes. The small seed potatoes produced by the present invention are capable of convenient storage, can reduce labor forces since the potatoes do not need to be cut when being sown on the open field, and have the advantage of supplying farmhouses with virus-free seed potatoes.

Description

Mass Production and Seed Potato Production Method of Tissue Cultured Potato Seeds by Bioreactor Incubator

The present invention relates to a method for producing a large amount of seed potatoes by cultivating them in rice seedlings in large quantities by seed production from potato stems aseptically cultured in a bioreactor incubator containing a liquid medium.

The basic plants of the virus-free seed potato were grown in a Petri dish culture container containing agar containing agar (10 cm in diameter, 3 cm thick, and 100 mL medium), and then seeded in the same culture container. The Korean Journal of Plant Tissue Culture, Vol. 19, No. 67-73; The Korean Society for Horticultural Science, Vol. 19, No. 36, 46-49; This conventional cultivation method has a lot of production costs, the seed size is small, there are a number of problems in cultivating potato seeds in house or field to obtain seed potatoes.

Bioreactor incubator is an incubator that can cultivate liquid media from several liters to hundreds of liters, and is advantageous for producing inexpensively large quantities of cultures. However, this culture method is not used well because of the disadvantages of complex culture technology and expensive facilities.

In the case of potatoes, when cultivating potato stems in a bioreactor liquid medium, the stems are first grown and grown, and the medium is exchanged so that a seed is formed on the stems, and there is a difficulty in transferring to a dark and low temperature environment. In addition, when growing potato stems, they are divided into several pieces of one or two pieces and cultured in a liquid medium containing inorganic nutrients and a carbon source (mainly sugar). There is this.

Plant tissue culture should be cultured while keeping the culture vessel and medium completely sterilized, and if the medium is used or grown in an unsterilized container, the culture will die immediately. The bioreactor sterilizes a large amount (more than 10 liters) of the liquid medium at high temperature and high pressure (121 ° C., 1.2 atm), so that the sterilization takes a long time and the pH and the media components are changed during sterilization, thereby preventing the culture from growing.

Existing seed potato production and supply system is to produce small seed potatoes (basic species) by planting potato stems and seeds (sterile artificial seed potatoes) in the house by top soil or hydroponic cultivation. It is farmed twice a year and supplied to farms. Such a system has a disadvantage in that it takes about 5 to 6 years to produce a seed seed potato supplied to a farmhouse from a tissue-cultivated potato stem or seed. The reason for the long time is that it takes a lot of cost and time to produce the first tissue cultured potato seed, and the multiplication process is needed because the system for mass production is not established yet.

An object of the present invention is to provide a method for culturing potato stems with a bioreactor to produce a large amount of potato stems or seeds and transfer them to seedlings for cultivation to produce a small, uniform seed potato in large quantities. In other words, the present invention is a method for producing potato stems and seeds without virus by culturing potato stems in a bioreactor, which is a spatially limited sterile culture vessel, and cultivating them in multi-stage rice seedlings at a house in small seed potatoes. To produce in large quantities to provide to the potato farming farmers.

The present invention for solving the above problems, (A) 1 to 5% by weight of the carbon source (MS) in which the content of NH ₄ NO ₃ and KNO ₃ is reduced to 1/2 to 1/4 (optimum 1/3) ( Optimum 3%) inoculated sterilized potato stems into a sterile liquid medium of pH 8 ~ 4 added, potato stem growth step of incubating for 2 to 4 weeks at 2000 ~ 4000Lux, 20 ~ 27 ℃ condition; (B) A sterile liquid medium having a pH of 8 to 4 with 3 to 7% by weight (5% by weight) of carbon source added to MS medium having increased contents of NH ₄ NO ₃ and KNO ₃ by 1 to 2 times (optimum 1.5 times). Inoculating the propagated potato stems in the dark, sprouting potato seeds after harvesting by growing potato seed cultured for 4 to 6 weeks at 12 to 20 ° C .; (C) filling the seedling plate with 3-6 cm of topsoil, and planting 7-20 seeds per 100 cm 2 of the seedlings of potato stems or shoots grown about 2-5 mm; (D) Feeding the medicinal solution (nutrient solution diluted to 1/20 to 1/10 of MS medium component of Table 1) at 1 to 3 days intervals at 15 to 25 ° C and 60 to 90% humidity in the nursery house. Cultivating seed potatoes by cultivating in normal light and dark conditions, the weight of the seed potato is more than 5g to use as a seed cultivation for farmhouse, less than the step of sowing to seedlings in step (C) to utilize as potato seed; It relates to a sterile seed potato mass production method comprising a.

In the present invention, when cultivating small potato seed in the (C) and (D) in the present invention, by placing a seedling plate on a shelf consisting of 2 to 8 stages and cultivating in multiple layers in the same space to efficiently a large amount in a narrow space Can produce.

In addition, after washing the produced seed potatoes with water and dry with air-dried and stored in a dark room at 5 ~ 15 ℃, sowing seedlings after seeding 30 ~ 40 days before transfer to 15 ~ 25 ℃ bright condition It relates to a mass production method of excellent seedless potatoes.

The present invention, as described through the above embodiment in the bioreactor incubator to grow a large number of potato stems and cultured to produce a seed from them to produce a large amount of potato seeds and then cultivate them in rice seedlings to obtain a large amount of seed potatoes It is about a method.

In this way, potato seeds and seed potatoes can be produced more robustly and cheaply in large quantities. In other words, we solved the problems of quality and economics in tissue culture, the first step in the production of virus-free seed potatoes, and developed a very efficient method of growing them in seed plants by first growing them in house.

Since the seed seed potato produced by cultivating the seed seed potato (seed potato obtained by cultivating tissue cultured stem or seed-first generation) or the original seed seed potato (seed potato obtained from the basic species-second generation) by seedling plate is all small according to the present invention, There is no need to cut seed potatoes during sowing, which can prevent viral infection and save labor costs. In addition, it is possible to shorten the period of production and dissemination of seed potatoes for farmhouse supply from one to two years from the existing five to six years.

This will enable the production of high quality, bottleless seed potatoes at low cost in a short period of time, laying the foundation for harvesting high quality potatoes and further contributing to the increase of farm income.

1 is a photograph comparing the size of the seed cultured in the solid medium of the present invention and the cultured in the bioreactor.

Figure 2 is a photograph showing the process of growing by culturing potato stem in the vial reactor of the liquid medium of the present invention.

Figure 3 is a photograph showing the results of culturing large amounts of disease-free potato seed from potato stem in the bioreactor incubator of the present invention.

Figure 4 is a photo of the potato seed produced in the bioreactor of the present invention and sprouting them grown on top soil.

Figure 5 is a photograph showing the appearance of tissue culture potato stem (top) and seed (bottom) used to plant in the seedling plate of the present invention.

Figure 6 is a photo showing planting potato seedlings (top) in tissue seedlings, and stacking a plurality of seedlings in multiple layers (bottom).

Fig. 7 shows the seedlings arranged in multiple stages on the seedling table (left side). Picture showing growing potatoes (right) with frequent fertilization.

Figure 8 is a photograph showing that a small seed potato is produced in the lower part of the top soil of the seedlings when cultivated for about 1 to 2 months after sowing potato seeds.

Figure 9 is a photo showing a potato seed sowing seedlings on the seedling plate (left) and enough to harvest the potato seed (right) that can be harvested.

10 is a photograph showing washing the seed potatoes harvested from the nursery board (left) and placing them in a box so that water drains well (right).

Figure 11 is a photograph showing the dried seed potatoes to be weighed by the size of less than 5g (left) and more than 5g (right).

Figure 12 is a photograph showing the sprouting (left) of the seed potatoes weighing less than 5g (left) and the sprouting of the seed potatoes weighing more than 5g (right).

Figure 13 is a photograph showing the results of confirming the virus-free seed virus according to the present invention (top) and planted in the farmer's field (bottom).

The present invention comprises the steps of: (1) preparing a liquid medium in the bioreactor suitable for potato stem and seed culture and sterilization so that the medium components do not change when sterilized; (3) Tissue Culture Potato stems and seeds are grown on rice seedlings to produce small seed potatoes.

In the present invention, the components of the liquid medium used for potato stem and seed culture are the same as the MS medium of the existing solid medium, but the amount of NH ₄ NO ₃ and KNO _{3 in} the inorganic components is 1/2 to 1/4 (optimal 1/3). ) Or medium (1.5 MS medium) increased by 1-2 times (optimal 1.5 times) was used (Table 1).

Table 1

The two components account for an extremely large amount in the MS medium, which solves two problems by reducing their amount. The first is to lower the concentration of the liquid medium to prevent injury to the cells of the cut potato stem. In liquid medium, when the concentration of minerals is high, such as agar medium, liquid medium enters the inside of the cut potato stem, which injures cells and affects growth. Secondly, when sterilizing more than 5 liters of MS liquid medium sterilization time is short, sterilization is not properly, if the sterilization time is long, the pH of the medium is lowered to less than 5, if the cultivation of potato stem under this condition hardly grow. However, 1/2 ~ 1/4 MS medium that reduced the amount of NH ₄ NO ₃ and KNO ₃ to 1/2 ~ 1/4 does not have any problem in cultivating potato stems because the pH does not drop below 5 even if sterilized for more than 60 minutes. .

Potato stem culture in the present invention was used a bioreactor incubator containing more than 5 liters of 1/2 ~ 1/4 MS liquid medium containing 3% of sugar, the density of potato stem per liter of the medium was 10. In addition, no plant growth regulator was added to the medium for potato culture.

Stem and seed cultures of conventional potatoes were grown in a solid medium (25 ℃ under fluorescent light for 4-5 weeks in MS medium containing 1-5% sugar) and then 6-9% sugar was added. Transferred to a solid MS solid medium and cultured in a dark and 16 ℃ culture conditions potato potato seed is generated at the bottom of the potato stem (Fig. 1). In solid media with small culture vessels, potato stems are inherently thin and weak, so the number of seeds is small and the size is small.

However, according to the present invention, since the cultivation vessel is large and the potato stem grows thick and large in the bioreactor using the liquid medium, the potato seed produced therefrom was large in size and large in number (Fig. 1, Fig. 3). Therefore, potato seeds grown in bioreactor incubators are more robust than those produced in solid medium, and when grown in a house or a field, they are resistant to drought and pests, thereby obtaining a large amount of seed potatoes.

The growth of potato stems from bioreactors to produce potato seeds from them is divided into two steps. The first step is to grow only stems by culturing potato stems, and the second step is to produce seeds from the grown stems. First, potato stem propagation is carried out in a bioreactor containing a liquid medium (1-5% (optimum 3%) sugar added 1 / 2-1 / 4 MS medium) prepared by the above method, about 10 potato stems per liter Put 3,000Lux, 23 weeks incubation at 23 ℃ culture room temperature conditions potato stems approximately 5 times proliferation (Fig. 2). By cutting the proliferated potato stem and dividing it into a bioreactor under the same culture conditions, it can grow about 5 times potato stem every 3 weeks. Thus, it can be said that the present invention is the first to complete a system for growing potato stems in a bioreactor liquid medium of 5 liters or more. This is possible by minimizing the medium (reducing the concentration by 1/2 to 1/4) to minimize the damage of the cut cell of the cut potato stem and by the sterilization time when sterilizing liquid medium of more than 5 liters. This is because the medium is prepared so that it does not go below 5.

In order for the seed to form in the grown potato stem, the medium should be replaced with the seed medium and the culture environment should be changed. Pour the potato stem medium incubated for 3 weeks, exchange it with a seeding medium (liquid medium added with 3-7% (optimum 5%) sugar in 1-2 MS medium (optimal 1.5 MS medium)), and 18 ° C. Incubation at dark temperatures and in dark conditions produces seed.

Under the above conditions, potato seeds started to be produced from the first week of culture, and the production was almost completed after about 3 weeks, after which the seed was enlarged and growth was almost completed after about 5 weeks (Fig. 3). Potato stems are cultivated in bioreactor incubators using large liquid media of more than 5 liters in size to change medium exchange (enhancing medium) and culture environment (cancer culture under low temperature and light) The present invention is also the first to produce a system.

Tissue cultured potato stems to be grown in seedlings are 3 ~ 5cm in size, and the roots and leaves are used as shown in the above picture of Fig. 5, and the seeds are about 2 ~ 5 mm long and green as shown in the picture below of Fig. 5. Use the one with. Seedlings can utilize existing plant seedlings that can be placed thinly, including rice seedling seedlings. As shown in the upper side of Figure 6, the planting seedlings filled with the manufacturing soil is planted in the tissue culture potato stem and potato seed 7 to 20/100 cm ² each.

When grown under the condition of 15 ~ 20 ℃ temperature and 60 ~ 90% humidity, feeding medicinal solution (nutrient solution diluted to 1/20 of MS medium component of Table 1), shoots emerge from the top and bottom In the portion, a small seed potato is generated as shown in FIG. When the diameter of the seed potato reaches 2-4cm, the seed potato is harvested, and the seed potato having a weight of 5g or more (right side of FIG. 11) is used as the seed potato for farming, and the lower one (left side of FIG. 11) is used as a seedling cultivation potato seed. use. Since the harvested seed potatoes are smaller than the seed potatoes harvested in general greenhouses or fields, there is no need to cut and use labor when sowing, thereby reducing labor costs.

When the small seed potatoes according to the present invention were planted and grown in a farmer's field, the growth was superior to that of conventional seed potatoes, and the pest damage was less. This may be because the primary seed (first generation) seed or the original seed (second generation) seed potato obtained by cultivating tissue culture stems or seeds on seedlings is much higher than the existing seed seed (5-6 generation).

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and embodiments. However, these drawings and embodiments are only examples for easily explaining the contents and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

Example 1 Potato Stem Culture and Growth in a Bioreactor

The first potato stems cultured in a bioreactor incubator are grown in a conventional solid medium. The modified 1/3 MS liquid medium containing 3% of sugar was transferred to the bioreactor aseptically from the solid medium. 1) were cultured under the conditions. The injection of air into the bioreactor is arranged to continue to be pumped under the vessel using a compressor or aquarium air pump. Air was allowed to pass through the Midisart 2000 (pore size 0.2 μm) for injection of sterile air. The culture environment was set at 3,000 Lux or higher in a bright place with a window or fluorescent light to the south with direct sunlight at a temperature of 23 ± 1 ° C.

Potato stems have a solution in each node between the leaves, and when cut and incubated, one stem emerges from each solution. Therefore, stems sufficiently grown have five or more nodes so that they can be cut and cultured in fresh medium to produce five times more stems (FIG. 2).

In a solid medium, potato stems are cut and put on stems in MS medium, which is generally used in plant tissue culture, and stems grow well. In the case of liquid medium, however, no growth was achieved when cultured under the same conditions as solid medium. As a result of examining the cause, the medium medium does not enter the cut part in the solid medium, so the medium grows well because the medium does not enter the cut part of the potato stem even if the medium with high mineral concentration is used. They found that because the components were introduced through the stem sections, the cells were injured and difficult to culture. Therefore, it is possible to culture only by preparing a liquid medium of a suitable concentration harmless to stem growth in the present invention was successful in culturing potato stem slices by lowering the medium concentration.

Potato stem propagation is carried out in a bioreactor containing a medium prepared by the above method (1/3 MS medium added with 3% sugar) and incubated at about 3,000Lux, 23 ° C incubation temperature at 10 liters per potato per liter. The leaves also grow large with the growth of the potato stem (Fig. 2). Larger potato stems and leaves grow larger when the seed is made from them, so it is desirable to grow potato stems as large as possible. Potato stems can be cut and divided into bioreactors under the same culture conditions, and cultured up to 5 times more potato stems every three weeks. These are used to make potato seeds.

Example 2 Mass Production of Potato Seeds from Potato Stem

When potato stems and leaves are sufficiently grown in the bioreactor, the medium in the bioreactor should be replaced with a medium for seeding (liquid medium added with 5% sugar in 1.5 MS medium) and the culture environment should be changed to generate the seed.

First, the medium exchange is carefully followed by the medium of potato stem cultured in the bioreactor for 3 weeks and exchanged with the medium for seed formation. All work must be done aseptically, sterilely, so take special care to avoid contamination when pouring large amounts of media. The newly changed bioreactor transfers the culture chamber to a dark dark room and the temperature is about 18 ° C. Under these conditions, potato seed is produced at each node from the lower part of the stem from 1 week of culture (FIG. 3). Potato seeds can be produced for up to three weeks, after which the seeds will be enlarged and after five weeks they will be hard and large enough to be harvested. If potato seeds are incubated in bright light for 4-5 days before being removed from the bioreactor, the seeds become green and harder.

Most potato seeds produced in solid medium were less than 0.3 g, but most of them produced more than 0.5 g and more than 1.0 g in liquid medium. As such, a system for producing a large amount of potato seed in a bioreactor incubator using liquid medium is much more efficient than a system for producing seed in a conventional solid medium.

Example 3 Storage and Shooting of Potato Seeds

Potato seeds obtained in the same manner as in Example 2 were washed clean and dried in a constant temperature room at 20 ℃, and then stored at an appropriate temperature until the top soil cultivation in the house.

Potatoes dormant for 2-3 months at 25 ° C room temperature. Potatoes dormant longer when stored at low temperatures. Cultured seeds can be produced year-round, so dormant periods are adjusted so that the shoots are sprouted and grown on topsoil.

The dormant periods of potato seedlings were 2 days old seedlings and grounds at 20-25 ℃ for about 60 days and 15 ℃ for 90 days (Fig. 4). The first varieties, Sumi and Daedae, took 90 days at 20-25 ° C and 110 days at 15 ° C. The purple potato Jasim was more dormant than those. If all potato seeds are stored at low temperature of 5-10 ℃, it can be stored for a long period of time without sprouting for more than 6 months. Therefore, potato seeds produced in the year-round culture room has a convenient point that can be sown sowing sprouts dormant break in accordance with the planting period after storing at low temperature (Fig. 4).

Example 4 cultivation of seedlings of tissue stem potato stem and seed

Potato stems produced aseptically by tissue culture are 3 to 5 cm in size, and roots and leaves are used as shown in the upper side of Fig. 5, and seedlings sprout about 2 to 5 mm and green as shown in the lower side of Fig. 5. It is preferable to use one.

Seedlings can utilize existing plant seedlings that can be placed thinly, including rice seedling seedlings. Soil used for seedlings was manufactured by mixing clean sand: ocher: horticultural clay in a ratio of 1: 1: 3 but mixed with appropriate amount of compost to commercial horticultural clay or commercial soil. It is irrelevant to use. The use of ocher and sand in gardening topsoil prevents the topsoil from blowing out of dry seedlings or spilling out of the seedlings when watering, and also prevents the roots of plants from being easily pulled out.

After filling the seedling plate with 3 ~ 6cm of top soil, it was squeezed and pressed slightly so that the top soil did not flow out of the seedling plate when watering. Tissue cultured potato stalks and potato seedlings were planted in 7 ~ 20/100 / 100cm2 each, and the water was chopped while planting the seedlings.The stems were fixed so that the stems could not be easily moved or the roots were inadvertently removed. The upper side of Figure 6 shows a planting potato seed on the seedling plate.

Potato seedlings sown on the seedlings seedlings are preferably stacked in a layered layer of 20-30 stages as shown in the lower side of Figure 6 in order to maximize the efficiency of the space before the shoots emerge on the top soil. In the winter or early spring, greenhouse temperatures are low, so stacking seedlings in double layers and covering them with a warm cloth can prevent cold damage, as well as sprouting evenly from potato seeds. The seedlings can be arranged in 2-8 stages in the seedlings as shown in the left side of FIG. Cultivation conditions are 15 ~ 20 ℃ temperature, 60 ~ 90% humidity, and leaves and stems are well lit by feeding nutrient solution (nutrient solution diluted to 1/20 of MS medium component of Table 1) every 1 ~ 3 days. The seedlings were arranged to receive. At this time, the nutrient solution was supplied so that the soil could be soaked in the range which does not flow out of the seedling plate.

Tissue cultures in seedlings grow more than 10cm or 3 weeks after sowing seedlings. When watering every 1 ~ 3 days, give 1/10 of diluted MS medium (Table 1), and leaves and stems Arrange the nursery boards to receive them well. Seedling cultivation environment is about 1 month after sowing, the temperature condition of 25 ℃ after 20 ℃ 1 month, the humidity is preferably 60 to 80% and the normal light and dark conditions.

When potato stems grow in the nursery board, the sunscreen spreads up and down or changes the position of the nursery board, and the sunlight is evenly distributed to the inner nursery board. Seedlings on the seedlings are thin, so the moisture is easily dried, and nutrients are easy to dry, so as the potato stem grows, the potato seed is often cultivated to grow the potatoes, even in the little seedlings as shown in the right side of FIG. 7 and the left side of FIG. You can grow enough potato stems to make small seed potatoes.

When grown in seedlings for about 1 to 2 months, small seed potatoes are formed in the upper part of the seedlings as shown in FIG. 8. When the diameter of the seed potato reaches 2 to 4 cm as shown in the right side of Fig. 9, the seed potato is harvested, and the seed potato having a weight of 5 g or more (right side of Fig. 11) is sprouted (Fig. 12 right) and used as a seed for cultivating farmhouse, and less than that (Left of FIG. 11) is used to produce a seed seed potato by replanting the seedlings after sprouting (left of FIG. 12). Harvested seed potatoes, as shown on the left side of Figure 10, the surface is smooth and close to a spherical shape, since the size is smaller than the seed potatoes harvested in a general greenhouse or field, there is no need to cut and use when sowing.

The harvested seed potatoes are washed with water (left side of FIG. 10) and firstly drained from a well-drained box (right side of FIG. 10), followed by air drying in a constant temperature room of about 20 ± 5 ° C.

The dried seed potatoes are stored in the dark room, and the storage temperature is selected in consideration of the seed potato varieties and sowing time, preferably 5 to 15 ° C. If the seed potato is stored at a low temperature of about 5 ℃, it will not sprout for more than 6 months, so it can be stored for a long time. Therefore, seed potatoes, which have been produced about three times a year by growing seedlings in greenhouses, are stored at low temperature and dormantly broken so that they can be sown so that they can be sown.

The dried seed potatoes stored in the dark room are transferred to the light condition of 15 ~ 25 ℃ before sowing and sprouted. Seed potatoes are dormant for 2 to 4 months at 20 ℃ depending on the variety, but they are longer when stored at lower temperatures. The period of dormancy of seed potatoes is 2 days of early harvest, Chubaek and land, which are about 60 days at 20 ° C, and 1 seasoned variety, Sumi and Daedae, takes about 90 days, and purple potato Jasim is longer than these.

Seeds produced by the present invention were identified as virus-free when tested for viruses, and they were planted and grown in farm fields (FIG. 13). The small seed potato according to the present invention has better growth and less pest damage than when the conventional seed potato is grown. This is because the primary seed (first generation) seed or the original seed (second generation) seed potato obtained by cultivating the stem or seed of tissue culture on seedling plate is much higher than the existing seed seed potato (5-6 generation). do.

The present invention has improved the method of preparing a liquid medium and sterilizing medium which is a problem in the process of culturing potato stems with bioreactor to make potato seed from them. In addition, we have developed a method for growing stems in bioreactors and a new method for generating large amounts of seeds from them. Tissue cultured potato stems and seeds produced by this technology were planted in rice seedling seedlings to develop a breakthrough method for mass production of small virus-free seed potatoes. By this method, small sized bottle-free seed potatoes with uniform size can be mass-produced at low cost, and seed potatoes can be supplied to farmers within 1 to 2 years from tissue-cultivated potato stems or seeds, which is economical in industrialization.

Claims (5)

1. (A) Inoculated with sterilized potato stems in sterile liquid medium at pH 8-4 with 1-5% by weight of carbon source added to MS medium whose NH4NO3 and KNO3 contents were reduced to 1 / 2-1 / 4, and then 2000 Potato stem growth step incubated for 2 ~ 4 weeks at ~ 4000Lux, 20 ~ 27 ℃ conditions; And

   (B) after inoculating the grown potato stem in a sterilized liquid medium having a pH of 8 to 4 added with a carbon source of 3 to 7% by weight in MS medium 1 to 2 times the content of NH4NO3 and KNO3, dark room, 12-20 Potato seed growth stage incubated for 4-6 weeks at ℃ condition; Artificial seed potato production method comprising a.
2. The method of claim 1,

   2 to 7 days before harvesting the potato seed in the step (B) artificial seed potato production method, characterized in that the seeding by ripening the recording by irradiation of 2000 ~ 4000Lux.
3. (A) After filling the seedling plate with 3 to 6 cm of topsoil, planting 7 to 20 potato seeds per 100 cm 2 of seedlings with 3 to 5 cm sized potato stems or shoots grown 2 to 5 mm aseptically by tissue culture. ;

   (B) Normal light and dark conditions in the nursery house at 15-25 ° C. and 60-90% humidity at 1 ~ 3 day intervals, supplying the elixir solution (diluted solution of MS medium in Table 1 by 1/20). Initial seedling step;

   (C) When the stem grows about 10cm, supplying the medicinal solution (nutrient solution diluted 1/10 of MS medium component of Table 1) at intervals of 1 ~ 3 days at 20 ~ 30 ℃ and 50 ~ 80% humidity condition. Late seeding under normal light and dark conditions;

   (D) harvesting seed potatoes when the seed potato reaches about 2 to 4 cm in diameter under the topsoil, and having a weight of 7 to 12 g or more is used as a seed potato for farmhouse cultivation, and a less than one is used as potato seed in step (A); Sterile seed potato mass production method characterized in that it comprises a.
4. The method of claim 1,

   Sterile seed potato mass production method characterized in that the seedlings are arranged in 2 to 8 stages in the step (B), (C).
5. After washing the seed potatoes produced according to claim 1 or 2 with water, remove the water with air-dry and store it in a dark room at 5 to 15 ° C, and transfer to light conditions of 15 to 25 ° C 30-30 days before sowing to make sprouts. Aseptic seed potato mass production method characterized in that the seeding.

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