KR102403394B1 - Method of producing high quality potato microtuber by optimizing dark culture - Google Patents

Abstract

A method for increasing the productivity of direct-sown microtuber potato seeds is presented. According to the method of the present invention, based on the composition of the MS medium, the ammonium nitrate content is 200 to 800 mg/L, the potassium nitrate content is adjusted to 2,500 to 3,000 mg/L, and 2 to 4 ppm of ClO ₂ A cancer culture medium comprising ClO 2 By culturing under shaking conditions, it is possible to finally produce a large amount of high-quality microtuber potato seeds with a large size.

Description

{Method of producing high quality potato microtuber by optimizing dark culture}

The present invention relates to a method for producing potato microtubers, and more particularly, to a method for producing large-sized microtube potato seeds through optimization of a dark culture environment during tissue culture.

Potatoes are one of the world's four major food crops, after rice, wheat, and maize, because of their short growing period, relatively high production per unit area, and strong environmental adaptability. Potatoes are used as a staple food in each country or mainly used to make various processed products, but consumption of potatoes in Korea is on the rise.

Potatoes can be propagated by true seeds or vegetative propagules, but most of them are prone to malformations, and it is difficult to produce uniform potatoes.

Potato production by vegetative propagation increases the yield by sowing and cultivating disease-free and excellent seed potatoes, but in reality, the production and supply of disease-free and excellent seed potatoes is not sufficient, and the supply rate of disease-free seed potatoes in Korea is only 25 to 30%.

Disease-free seed potatoes are usually grown in house soil or hydroponically to produce small-sized seed potatoes, which are then grown twice in the open field and then grown and supplied to farms. As such, it takes a long time to supply and demand disease-free seed potatoes, because it takes a lot of time to produce the first tissue cultured seed, and a system for mass production is not established, so several propagation processes are required. In particular, since the risk of exposure to diseases, pests or viruses increases during the propagation period, shortening the seed potato propagation period is very important in the supply system of disease-free and high-quality seed potatoes.

On the other hand, the mass production method of disease-free seed potatoes using a bioreactor incubator has an advantage in mass production of a culture by using a liquid medium of several hundred liters. However, this culture method is complicated in culture technology and expensive to install, and an additional planting process is required to secure tubers of mini-tube size.

The seed potato production method through in-flight microtuber culture without using a bioreactor has the advantage of being able to produce disease-free seed potatoes year-round even in a small space at any time. However, this method has not yet been industrialized like the method of producing seed potatoes through nutrient solution small tubers or thinly shoveled tubers, because the size of the microtubers produced in the plane is too small or the quality is poor, so direct seeding like commercialized seed potatoes is impossible. it was In addition, even if a rather large-sized microtuber is produced, there is a problem in that it is difficult to mass-produce it due to a low yield.

The present invention has been devised to solve the above problems, and by optimizing the dark culture conditions during tissue culture of potato plants, it is confirmed that it is possible to ultimately produce a large amount of disease-free and excellent seed potatoes of the present invention completed.

It is an object of the present invention to provide a method capable of direct seeding through optimization of the dark culture environment during tissue culture and increasing the productivity of large-sized microtuber potato seeds.

In order to achieve the above task,

According to one aspect of the present invention,

(a) based on MS medium, ammonium nitrate content of 800 to 1,200 mg / L and potassium nitrate content of 3,000 to 4,000 mg / L, and denting sterile potato stems in a medium adjusted to 2 to 4% carbon source Culturing the proliferation stem by culturing for 10 to 30 days while irradiating a light source with a light amount of 70 to 110 umol/m ² /sec; and

(b) Based on MS medium, ammonium nitrate content is 200 to 800 mg/L, potassium nitrate content is 2,500 to 3,000 mg/L, and carbon sources are 7 to 10 %, it is possible to present a method for producing microtuber potato seeds, including the step of culturing a shake culture for 70 to 100 days under dark conditions in a medium adjusted to %.

In the method of the present invention, the shaking culture of (b) may be performed by operating the shaking culture apparatus in which the culture vessel is located at a speed of 20 to 40 rpm.

In addition, the medium for the cancer culture of (b) may contain 2 to 4 ppm ClO ₂ .

The method for producing a potato microtuber of the present invention is applicable to potato varieties such as Sumi, Haryeong, Early Fry, Baron, Geumseon and Saebong, and is preferably applied to Sumi varieties.

In addition, according to another aspect of the present invention, it is possible to present the microtuber potato seeds produced by the above method.

우량의 보급용 씨감자를 생산하는데 걸리는 시간과 비용을 단축할 수 있다. According to the method for producing microtuber potato seeds according to the present invention, it is possible to produce a large amount of disease-free microtube potato seeds that can be directly sown through optimization of the cancer culture environment during tissue culture. As a result, disease free

It is possible to reduce the time and cost required to produce high-quality seed potatoes for supply.

1 is a schematic diagram briefly showing the shaking culture apparatus adopted in the present invention. In the shaking culture apparatus, the culture plate performs seesaw motion according to the driving of the motor, and the movement of the culture plate is indicated by a dotted line.

Hereinafter, the configuration and operation of the present invention will be described in more detail through preferred embodiments of the present invention. However, this is presented as a preferred example of the present invention and cannot be construed as limiting the present invention for any reason.

Content not described herein will be omitted because it can be technically inferred sufficiently by a person skilled in the art to which the present invention pertains.

The present invention provides a method for producing microtuber potato seeds,

(a) based on MS medium, ammonium nitrate content of 800 to 1,200 mg/L, potassium nitrate content of 3,000 to 4,000 mg/L, and sterile potato stems in a medium adjusted to 2 to 4% of carbon source Culturing the proliferation stem by culturing for 10 to 30 days while irradiating the light amount of the light source at 70 to 110 umol/m ² /sec; and

(b) Based on the MS medium, the 1-3 nodes of the lower part remaining after cutting the growth stem have an ammonium nitrate content of 200 to 800 mg/L, a potassium nitrate content of 2,500 to 3,000 mg/L, and a carbon source of 7 to 10 A method for producing microtuber potato seeds, including the step of culturing with shaking for 70 to 100 days under dark conditions in a medium adjusted to %, can be provided.

Shake culture of (b) in the method of the present invention can be performed by placing the culture vessel containing the plant and medium on the shaking culture apparatus and operating the apparatus at 20 to 40 rpm. The shaking culture apparatus includes a culture plate in which the culture vessel is located. The culture plate may be a rectangular plate or shelf on which several to tens of culture vessels can be placed, and may be provided with a column or side plate for preventing the culture vessel from being tilted.

According to the rotation of the motor of the culture apparatus, the culture plate is tilted in the east-west or north-south direction based on the center of the culture plate, and the seesaw motion is performed (FIG. 1).

The inclination of the seesaw motion may be about 30° up and down (± 30°) based on the inclination of the culture plate when the inclination of the culture plate is 0°.

According to the seesaw movement of the culture plate, the liquid medium in the culture vessel flows, thereby preventing the stem and roots of the plant from being continuously submerged in the medium.

In this process, air circulation efficiency increases and the supply of nutrients required for tuber production becomes active, which can affect the size of the tuber. In addition, as the continuous submergence of the stem is prevented, it is possible to reduce the hypertrophy of the cortex and increase the quality of the stalk.

The microtuber potato seed of the present invention is based on the tissue culture method, by collecting and culturing the growth point of a potato plant to obtain a basic stem, cutting/culturing the upper part of the stem to obtain a proliferation stem, and cutting the proliferation stem It is produced by a series of processes of cancer-culturing the remaining lower nodes to form wells and tubers.

MS medium (Murashige & Skoog, 1962) was originally developed for tissue culture of tobacco plants, but is a plant medium widely used for culturing other plants. MS medium is the most commonly used for plant cell/tissue culture, demonstrating effective growth in both dicot and monocot plants.

As a result of many years of efforts by the present applicant to find a medium composition suitable for potato microtuber production, significant results were obtained in the composition change of compounds containing nitrogen (N) and potassium (K) based on MS medium.

The optimal medium composition required for each plant is different, and in particular, the contents of nitrogen and potassium, which are large amounts of elements, are important. Nitrogen is divided into nitrate nitrogen and ammonia nitrogen when absorbed by plants. Since the form of nitrogen absorbed well by plants is different, it is better to increase the content of nitrogen compounds with high absorption rate.

According to the study of the present applicant, potato plants showed a better absorption rate of nitrate nitrogen than ammonia nitrogen. In addition, the content of potassium was also shown to have an effect on potato tubers.

That is, the present applicant based on the MS medium, nitrogen increased the content of nitrate nitrogen (NO ₃ ), reduced the content of ammonia nitrogen (NH ₄ ), increased the absorption of nitrogen, and increased the content of potassium to produce tuber raised the

The medium of the light culture in the method of the present invention, based on the MS medium, ammonium nitrate (NH ₄ NO ₃ ) 800 to 1,200 mg/L, preferably 900 to 1,100 mg/L, including potassium nitrate (KNO ₃ ) It may contain 3,000 to 4,000 mg/L, preferably 3,200 to 3,800 mg/L. The carbon source may include 2 to 4% sucrose.

When brightly cultured in a medium containing an excess of ammonium nitrate and potassium nitrate than the above range, the stem is weakened, and there is a risk of necrosis when subcultured continuously. When cultured in a medium containing a trace amount than the above range, the stem There was a problem in that the number and area of the leaves became smaller as they became thinner.

On the other hand, the cancer culture medium for forming welfare and inducing and growing microtubers is 200 to 800 mg/L, preferably 400 to 600 mg/L, of ammonium nitrate, and 2,500 to 3,000 mg/L of potassium nitrate. L, preferably 2,600 to 3,000 mg/L, may contain 7 to 10% of sucrose as a carbon source. In the case of containing ammonium nitrate and potassium nitrate in the above range, the wells and microtubers were well induced and grown.

When the content of ammonium nitrate and potassium nitrate in the dark culture medium is higher or lower than the above-mentioned range, 30% or more of the total microtubers produced in the final production have a particle diameter of 7.9mm or less, and less than 35% of 9mm or more. This was similar. On the other hand, when the content of ammonium nitrate and potassium nitrate satisfies the above-mentioned ranges, 22% of those having a diameter of 7.9 mm or less and 49% of those having a size of 9 mm or more were produced, indicating that high-quality microtubers with large sizes were produced in large quantities.

The medium for the cancer culture may contain 2 to 4 ppm of ClO ₂ for disinfection, sterilization and induction of tuber formation.

When the content of ammonium nitrate and potassium nitrate in the dark culture medium is within the above-mentioned range, if ClO ₂ is contained in excess of 2 to 4 ppm, ClO ₂ adversely affects the plant and slows down growth, the quantity of tubers may decrease, and if severe, It is also dismissed

The tissue culture medium used in the present invention does not contain artificial growth regulators or hormones.

On the other hand, the amount of light required for plant growth may also vary depending on the plant, and the present applicant has built a light condition suitable for the characteristics of the variety by optimizing the amount of light in the bright culture stage of the potato plant.

In the method for producing a potato microtube of the present invention, a light quantity of 70 to 110 umol/m ² /sec, preferably 80 to 100 umol/m ² /sec, is maintained by using an LED or a fluorescent lamp as a light source for bright culture. it's good Here, the photoperiod can be adjusted in the range of 12 to 18 hours: 12 to 6 hours (L:D).

In the present invention, in particular, it was attempted to produce high-quality potato microtubers through optimization of the dark culture environment.

In the method of the present invention, shaking culture was adopted from dark culture, unlike before. To this end, the present inventors place a culture vessel replaced with a dark culture medium on a locker after light culture of potato plant stems, and 20 to 40 By operating the apparatus at rpm, the liquid medium in the culture vessel was allowed to flow according to the inclination of the culture plate.

When all other conditions were the same, microtubers produced in the case of shaking culture and stationary culture, respectively, during dark culture, the production rate of microtubers larger than 11.2 mm in static culture was the lowest. In the case of shaking culture with an rpm value lower than the above range, the ratio of tubers having a size of 11.2 mm or more was higher than that of stationary culture, but it was lower than the case of shaking culture at the rpm of 20 to 40 rpm, and 7.9 mm or less. The production rate of small tubers was more than 15%. In addition, when it was excessively higher than the above rpm range, it was found that the average production amount per container was smaller than that of stationary culture (Table 1).

Therefore, when the shaking culture is performed at an appropriate rate, it can be expected that the plant body and the medium components and the supply of oxygen are good, so that the proliferation is uniform and the growth is improved.

In the method for producing potato microtubers of the present invention, light culture can be cultured for 10 to 30 days at 24-28 ° C. to grow stems and leaves, and dark culture is cultured for 70 to 100 days at 18 to 22 ° C. You can grow microtubers. The temperature and the number of incubation days can be adjusted within the above range in consideration of the growth rate, the size of the container, the efficiency of the operation, and the like.

The present applicant has been researching a method for increasing the productivity of microtuber seeds for several varieties of potatoes for many years. As one aspect, efforts have been made to produce large-sized microtuber seeds, and the appearance rate of large-sized microtubers when sown directly into the field (the ratio of sprouts appearing on the soil surface 20 days after sowing compared to sown seeds) This is because there was a correlation that the production quantity per week and production weight also increased.

According to the method of the present invention, it is possible to ultimately produce large-sized and high-quality potato microtubers through optimization of the cancer culture environment during tissue culture. Since the microtuber produced in this way produces potato seeds that can be directly planted in the field, the time it takes to produce the conventional sterile seed potatoes for supply can be dramatically shortened, and the production cost can be lowered to help farmers.

Hereinafter, the present invention will be described in more detail through preferred embodiments of the present invention. This is for illustration of the present invention and cannot be construed as limiting the present invention.

Optimization of medium composition for cancer culture

Experimental Example 1

After culturing the growth point of potato plants (Sumi variety), virus-free individuals were selected through virus assay. 30 stems were placed in a 20*30*15 cm culture vessel containing a culture support plate (non-woven fabric, PP 30g sm, Noja Co., Ltd.) divided into each compartment to obtain a proliferated stem by densely culturing the obtained basic stem. did

The medium for culturing the proliferation stem was adjusted to the nitrogen and potassium composition of the MS medium composition (NH ₄ NO ₃ 1,000 mg/L, KNO ₃ 3,600 mg/L), and a liquid medium containing 3% (w/v) sucrose (w/v) 200 mL per container). The culture temperature was 26°C and the photoperiod was 16h/8h for 14 days. At this time, the amount of light was maintained at 90 umol/m ² /s using a white LED light source.

After cutting the propagation stem, the remaining 1~2 nodes of the lower part were replaced with microtuber production medium (300ml per 20 * 30 * 10 cm container). The medium for this cancer culture is a liquid medium containing 9% sucrose and 3ppm of ClO ₂ adjusted to the nitrogen and potassium contents in the MS medium composition (NH ₄ NO ₃ 500 mg/L, KNO ₃ 2,800 mg/L). This was cultured in the dark for 90 days at 18°C in dark conditions.

Comparative Experimental Example 1

Microtuber by culturing under the same conditions as the method of Experimental Example 1, except that the medium for cancer culture contained 250 mg/L and 1,400 mg/L of NH ₄ NO ₃ and KNO ₃ , respectively, and did not contain ClO ₂ was produced.

Comparative Experimental Example 2

Microtuber by culturing under the same conditions as the method of Experimental Example 1, except that the medium for cancer culture contained 1,000 mg/L and 5,600 mg/L of NH ₄ NO ₃ and KNO ₃ , respectively, and did not contain ClO ₂ was produced.

Comparative Experimental Example 3

Microtubers were produced by culturing under the same conditions as in Experimental Example 1, except that the medium for cancer culture contained 6 ppm ClO ₂ .

Comparative Experimental Example 4

Microtubers were produced by culturing under the same conditions as in Experimental Example 1, except that the medium for cancer culture contained 9 ppm ClO ₂ .

Microtuber productivity evaluation 1

The potato microtubers produced in Experimental Example 1 and Comparative Experimental Examples 1 to 4 were harvested from each 100 containers and the size was analyzed (Table 1).

The average production per container is the total production divided by 100 containers, and the average production per stem is the number produced by one container divided by 30 (the number of stems placed in the container).

[Table 1]

As a result, as shown in Table 1, in Comparative Experimental Examples 3 and 4 containing ClO ₂ at a high concentration, it can be confirmed that the average production amount of microtubers is lowered. When the content of potassium nitrate and ammonium nitrate is too small (Comparative Example 1) or too large (Comparative Example 2), there is no significant difference in the overall production (average production), but looking at the size of the produced tuber, the It can be seen that the proportions are similar. In particular, in Comparative Experimental Examples 1 and 2, it can be confirmed that the seedable size of 8 mm or more does not exceed 70%.

Therefore, from the above results, the concentrations of ammonium nitrate and potassium nitrate in the cancer culture medium were 500 mg/L and 2,800 mg, respectively, and the concentration of ClO ₂ was established as 3 ppm and applied to subsequent experiments.

Example

After culturing the growth point of potato plants (Sumi variety), virus-free individuals were selected through virus assay. 30 stems were placed in a 20*30*15 cm culture vessel containing a culture support plate (non-woven fabric, PP 30g sm, Noja Co., Ltd.) divided into each compartment to obtain a proliferated stem by densely culturing the obtained basic stem. did

The medium for culturing the proliferation stem was adjusted to the nitrogen and potassium composition of the MS medium composition (NH ₄ NO ₃ 1,000 mg/L, KNO ₃ 3,600 mg/L), and a liquid medium containing 3% (w/v) sucrose (w/v) 200 mL per container). The culture temperature was 26°C and the photoperiod was 16h/8h for 14 days. At this time, the amount of light was maintained at 90 umol/m ² /s using a white LED light source.

After cutting the propagation stem, the remaining 1~2 nodes of the lower part were replaced with microtuber production medium (300ml per 20*30*10 cm container). The medium for this cancer culture adjusts the nitrogen and potassium contents based on the MS medium composition (NH ₄ NO ₃ 500 mg/L, KNO ₃ 2,800 mg/L), and contains sucrose 9% (w/v), ClO ₂ 3ppm It is a liquid medium that The culture vessel was placed on a rocker (self-made, culture plate size 1200 mm * 600 mm), which is a shaking culture device, under dark conditions at 18 ° C., and darkly cultured for 90 days while operating at 30 rpm.

Comparative Example 1

Microtubers were produced in the same manner as in Example 1, except that the culture was stationary without a shaking culture device during dark culture.

Comparative Example 2

Microtubers were produced in the same manner as in Example 1, except that the rpm of the shaking culture apparatus was set to 10 during dark culture.

Comparative Example 3

Microtubers were produced in the same manner as in Example 1, except that the rpm of the shaking culture apparatus was set to 90 during dark culture.

Microtuber productivity evaluation 2

The potato microtubers produced in Examples and Comparative Examples 1 to 3 were harvested from each 100 containers and the size was analyzed.

The average production per container is the total production divided by 100 containers, and the average production per stem is the number produced by one container divided by 30 (the number of stems placed in the container).

[Table 2]

As a result, as shown in Table 2, in the case of stationary culture (Comparative Example 1) during dark culture, less than 10% of tubes of 11.2 mm or more were produced. When cultured with shaking at a low speed of 10 rpm (Comparative Example 2) or cultured with shaking at a high speed of 90 rpm (Comparative Example 3), the average yield per container was smaller than that of stationary culture. The rate of seedable tubers of 8 mm or more was confirmed to be 89% in Example, 78% in Comparative Example 1, 83% in Comparative Example 2, and 74% in Comparative Example 3. Therefore, it is confirmed that the size of the microtuber can be increased by circulating the air in the vessel through shaking culture at an appropriate speed.

In the past research conducted by the present applicant, microtubers of less than 8 mm are easy to store, but because of their small size, they often dry out or freeze. In addition, the germination rate decreased after the dormant period and the emergence rate was not good due to the sensitivity to the external environment after sowing. If the size of the microtuber is 8mm or more, the tuber has high fidelity and robustness, can be stored for a long time at low temperatures, and has a good germination rate.

Therefore, by the method of the present application applying specific medium conditions and dark culture environment, it is possible to effectively produce a large amount of microtuber seeds having a size of 8 mm or more, as well as high average production, ultimately, high-quality potato seeds with high productivity can be obtained. .

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but may be implemented in a variety of different forms, and those of ordinary skill in the art to which the present invention pertains will appreciate the technical spirit of the present invention. However, it will be understood that the invention may be embodied in other specific forms without changing essential features. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (5)

(a) based on MS medium, ammonium nitrate content of 800 to 1,200 mg / L and potassium nitrate content of 3,000 to 4,000 mg / L, and denting sterile potato stems in a medium adjusted to 2 to 4% carbon source Culturing the proliferation stem by culturing for 10 to 30 days while irradiating a light source with a light amount of 70 to 110 umol/m ² /sec; and
(b) Based on the MS medium, the lower 1-3 nodes remaining after cutting the growth stem have an ammonium nitrate content of 400 to 600 mg/L, a potassium nitrate content of 2,500 to 3,000 mg/L, and a carbon source of 7 to 10 A method for producing microtuber potato seeds, comprising the step of culturing with shaking for 70 to 100 days under dark conditions in a medium adjusted to %.
The method of claim 1, wherein the shaking culture of (b) is performed by placing the culture vessel on a shaking culture device and operating at 20 to 40 rpm.
The method of claim 1, wherein the medium for cancer culture of (b) contains 2 to 4 ppm of ClO ₂ , the method for producing microtuber potato seeds.
The method according to claim 1, wherein the potato variety is selected from the group consisting of Sumi, Haryeong, Early Fry, Baron, Geumseon and Saebong.
A microtuber potato seed produced by the method of claim 1.

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