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DEVICES, COMPOSITIONS AND METHODS OF INCREASING VICINITY AND CROP PRODUCTIVITY OF VEGETABLE CROPS
INVENTION
Patent of the Russian Federation RU2075289

METHOD OF MASS RECOVERY OF MICRO-CLASS POTATOES

METHOD OF MASS RECOVERY OF MICRO-CLASS POTATOES

The name of the inventor: Hiyuk Yong [KR]; Yang-Riol Liu [KR]; Yu-Bong Hong [KR]; Seung-Guyong Young [KR]; Heng-Sung Lee [KR]; Yad Heung John [KR]; Yeung-Sook Ku [KR]
The name of the patent holder: Korea Institute of Science and Technology (KR)
Address for correspondence:
The effective date of the patent: 1990.03.07

Use: agriculture and biotechnology. SUMMARY OF THE INVENTION: The mass multiplication of microtubers involves the induction of microcubber-free germ-free germs, their low temperature treatment, and cultivation on a modified nutrient medium by placing the shoots in flat culture vessels, kept on a solid nutrient medium for a week at light at 30 ° C and for the next Weeks in the dark at 10 ° C, and then with a 6-hour photoperiod with an air temperature of 20 ° C during the day and 12 ° C at night. The process is carried out with the indicated compositions of nutrient media.

DESCRIPTION OF THE INVENTION

This invention relates to a novel method for the mass production of artificial seed potatoes (microtubers of potatoes) free of pathogens (especially viruses) using the technique of plant tissue culture.

Potato is a kind of plant that belongs to the family of Solanaceae and is characterized by vegetative reproduction by means of tubers. One of the most serious problems, usually found in most technical crops of vegetative reproduction, is a decrease in the level caused by a viral infection, and in the case of potatoes, the damage caused by it is particularly serious (Manzer FE Merriam DC and Helper PR 1978. Am Potato Jour. 55: 601 609. Schultz EC and Bonde R. 1944. Am., Potato J. 21: 278 283. Wright NS 1977. Am., Potato J. 54: 147 149. Korean seed potato program: organization, impact, results. International Potato Center, p. 19-24). Therefore, ensuring the supply of virus-free seed potatoes plays a decisive role in solving the yield of the technical potato crop every year.

It is well known that most viral infections are caused by plant aphids that can easily carry the virus through their mouth. Therefore, in order to produce a seed-free seed potato, the production area should be located in a high mountain area where the population of plant aphids carrying viruses is very low. However, using this traditional method, the production efficiency of a virus-free seed potato is so low that it is very difficult to produce an enormous amount of good quality seed potatoes that is needed annually.

Recently, thanks to the rapid development of the technique of cultivation of plant tissue, it became possible to mass-breed many varieties of plants using in vitro technique. In the case of potatoes, a system for rapid dilution of virus-free plants using plant growth point culturing techniques is well established (Goodwin, PB Kim YC and Adisarwanto, T. 1980. Potato Res., 23 9 23. Hussey, G. and Stacey, NJ 1981. Ann Bot. 48: 787,796. Roset S. and Bokelmann GS 1976. Potato Res 19: 173,178) and is already used commercially to some extent. However, based on production efficiency, the above system has several serious drawbacks, one of which is that the process of in vitro transplantation into soil is a process requiring such time and labor that intense attention is always needed and thus during this process Many in vitro sprouted tender potato sprouts can not withstand a sudden change in the environment.

Since 1970, there have been several reports of the formation of potato microtubers in vitro, but the production efficiency has been so low that the researchers used the phenomenon of microtuber formation only as an experimental tool for studying the physiology of tuber formation of potatoes (Garcia-Torres L. and Gomez-Campo C. 1973. Potato Res 16: 732 79. Abbott AJ and Belcher AR 1986. Jnplant Tissue Culture and its Agricultural application wortus Butter, pp. 113 122. Hussey, G. and Stacey, NJ 1984. Ann., 53: 565-578).

Recently, several attempts have been made to obtain in large quantities a seed-free potato free from a virus of good quality by growing potato microtubers produced in vitro using a cultivating method in a liquid nutrient medium (Wang PJ and HU, CY 1982. Amer Potato Jour 59: 33 39. International Patent application Number: PCT / HU 86/00053, 1986). However, the production efficiency of microtubers using the above methods is still too low to replace natural seed potatoes. Moreover, the microtubers that were obtained by the aforementioned cultivation methods in a liquid nutrient medium have several decisive disadvantages, such as light drying during storage and frequent glazing of microtubers, rendering artificial seed potatoes unsuitable. Despite these problems, the potato microtubers that are formed during the cultivation of the plant tissue of potato sprouts may seem to be used as one of the ways to replace potato growth points, because potato microtubers are significantly less gentle and easier to manipulate in stage Transplantation than with the plants grown in the cultivation of plant tissue. In the end, due to the characteristic feature of the vegetative reproduction of potatoes, the amount of seed potatoes required annually is so huge that even if microtubers need to prove their practical importance, the importance will be reduced to zero if one does not develop any way of mass production of a huge number of microtubers on a small And thus provide them with farmers at a low price, sufficient to replace natural seed potatoes.

Therefore, in this invention, we intend to develop a new way of mass production of potato microtubers at a price low enough to provide them directly to farmers as real substitutes for natural seed potatoes.

Accordingly, some of the objects and advantages of our invention are to develop a new method for cultivating plant tissue for mass production of an artificial seed potato (microtuber of potato) free from a pathogen that will be able to replace natural seed potatoes or be used as a substitute for seed potatoes directly Preceding the production of natural seed potatoes. As a result of this invention, it is now possible to mass produce artificial seed potatoes, at least more than 30 times more efficient than the already known method of producing microtubers. Thus, thanks to this invention, it is possible to produce artificial seed potatoes at a significantly cheaper price, sufficient to provide farmers as a substitute for natural seed potatoes. Other objects and advantages of our invention will become apparent from the consideration of the Figures and the Detailed Description of the Invention.

FIG. 1. Sprouts that give microtubers of the "Superior" variety, which quickly sprouted on artificial media for cultivation in a Petri dish.

FIG. 2. Microtubers of the potato "Superior", which quickly formed on artificial media for cultivation in a petri dish.

FIG. 3. Microtubers of the potato of the "Superior" type, which were stored sterile in a Petri dish at low temperature for a long period of storage.

FIG. 4. Potatoes immediately before harvest, obtained by germination of microtubers.

FIG. 5. The average yield of potatoes harvested from a plant obtained from a single microclub of the "Superior" variety.

The whole method of mass production of artificial seed potatoes of our invention is several stages. The following experiments will illustrate in more detail what the invention really is, but it does not follow that this invention is limited to them.

Experimental Example 1 . The basis of the method for induction, care and mass germination of germs, giving microtubers virus-free potatoes.

The experimental material is a virus-free potato of the "Superior" variety, which is obtained from the Horticultural Experiment Station of the Rural Development Administration. It is cleaned by rinsing in tap water from a tap, soaked in 70% ethyl alcohol for 3 minutes, the surface is sterilized with 20 Clorox (industrial) for 10 minutes and finally sown in square pots containing autoclaved soil (1: 1 pearlite vermiculite) . After about a week, the germination of nodules begins, which is observed in the growth chamber in a 16-hour light-day mode at a constant cultivation temperature (25 ° C).

After two weeks, when the shoots grow to an average length of 5 10 cm, the growth points (1 2 cm in length) are cut off and used as a base material for culturing growth points. The cut sprouts are rinsed with sterilized distilled water 3 times, soaked in 70 ethyl alcohol for 30 seconds, the surface is sterilized with 10 Clorox for 10 minutes and then sown ultimately on liquid or solid media of a specific formula (see Tables 1, 2) for induction and Growth sprouts, giving microtubers.

The environmental conditions of the chambers for growth were at the same time identical to those of the mother tuber. A week after sowing under the same external conditions of cultivation, axillary shoots begin to appear and in most cases in 3-4 weeks they grow as quickly as required for the subculture. At the same time, the technique of multiplication by layers is used to stimulate as much as possible the induction of axillary shoots, but in the case of vessels or test tubes, the technique requires so much skill that in this experiment the shoots are cultivated in flat Petri dishes (diameter 10 cm, height 1, 5 cm), thus obtaining in vitro reproduction automatically, resulting in a significant increase in the number of axillary sprouts (see Table 3). In general, the growth rate of sprouts in a liquid nutrient medium for cultivation exceeded the rate of growth of germs in a solid nutrient medium. But when subcultures remained for a long period in a liquid nutrient medium, degeneration or vitrification of the shoots often resulted from excessive moisture absorption, preventing the normal growth of potato germs in vitro. As a result, only liquid nutrient media are used at the initial stage and then mainly solid nutrient media are used.

It is noted that the point is that not all sprouts multiplied on artificial media for cultivation are able to form microtubers when transferred to the next stage to form microtubers, but only sprouts with unique features, in other words, with slightly elongated interstices with stems that do not have stem , With strong roots and especially buds with buds at the ends of the shoots (see Figure 1), are able to form microtubers in large numbers (see Table 5). Therefore, we named sprouts with such specific features as "microtuber sprouts" and they were registered and deposited in Korean Type Culture Sollection as patented plant cell lines ("Superior", patented cell line N 8445 p). Such sprouts giving microtubers of potato are formed exclusively on induction media to form microtubers of a specific composition and, once formed, they retain their characteristics even after at least 24 consecutive years of cultivation per year.

Experimental Example 2 . Method of mass production of potato microtubers.

The microtubule germs in the rapid multiplication stage illustrated in Experimental Example 1 were first transferred to a high temperature growth vessel (30 ° C) (other culture conditions identical to the above microprocessor germs) for a week and then transferred to a low temperature (10 ° C) C) the growth chamber into total darkness for another week. After a low temperature treatment, the microtuber sprouts were seeded on induction media to form microtubers (see Tables 2 and 4), densely paraffin waxed, and cultured in a growth chamber in which the day temperature was maintained at 20 ° C and the night temperature maintained at 12 ° C , While the photoperiod was 6 hours of light and 18 hours of darkness. The intensity of light was about 500 lux. In order to use the entire space of the growth chamber, we stacked the Petri dishes as much as possible. In most cases, after about 10 days, they were transferred to such conditions for induction of microtubers, that the microtubers of potatoes began to form and after 40 to 50 days of the cultivation period microtubers were formed, small as soybeans, in the amount of more than 10 pieces, on an average per cup Petri. (See Figure 2).

With growth, a growth inhibitor such as phosphon D, Amo 1618, B-905 (N-dimethylamino-amylamide of succinic acid), and Chlorine Choline Chloride (XXX) are added to culture media at a concentration of 50 ppm. The production efficiency of microtubers is significantly increased.

In the case of the "Superior" variety, a comparative study was made of the production efficiency per unit of the culture space with a single vessel, which showed a difference in results between the traditional cultivation method in a vessel with a liquid nutrient medium using non-microtuber sprouts and the cultivation method on a solid nutrient medium in a Petri dish using Microtubers of germs developed in this invention, including all other treatments to increase the rate of induction of microtubers. And the results are presented in Table 6 and Table 7.

Experimental Example 3 . A method for the long-term storage of potato microtubers and methods for inhibiting and stimulating germination during storage.

Microcliners of the "Superior" variety, obtained quantitatively in Petri dishes, are sterilely collected, washed with sterilized distilled water 3 and 4 times, thus purifying from the culture media remaining on the surface. Then they are laid out and dried inside a clean rack until the moisture completely disappears from the surface. After drying, the microtubers are placed in empty sterile Petri dishes and tightly covered with three layers of paraffined film and kept in a refrigerator at a low temperature of 4 ° C (see Figure 3). After about 2 months of soaking in the refrigerator, rest is disturbed and then germinated with ease, leaving at room temperature or for two weeks or so as needed (see Table 8). When it is desired to preserve them for a long time without germination, they are pretreated with 5 mg / l abscisic acid solution for 3 hours before final storage at low temperature. In this way it is possible to keep microtubers in healthy conditions for more than a year until it is confirmed that the ability to germinate or harm is lost (see Table 9). When it is required to obtain germination shortly after harvest, light germination is a possible way of disturbing the rest by treatment with gibberellic acid or along with treatment in a warm water bath at 38 ° C before treatment with gibberellic acid (see Table 8). This type of treatment with gibberellin and high temperature was and was used to shorten the period required for germination in the case of microtubers whose rest is already disturbed (see Table 10).

Experimental Example 4 . A test for the yield of potato microtubers of the grade "Superior".

We conducted a control experiment to compare the yield grown with the help of natural replaceable potatoes with the crop grown with the help of potato microtubers. When the length of the shoots of germinating microtubers reached 2 to 3 mm in length after treatment for germination, as described in Experimental Example 3, microtubers were planted directly into the soil. At an early stage, the growth of microtubers was rather weak compared to the growth of natural replaceable potatoes, but after the middle stage they showed very rapid growth and by the time of harvest, 3 months after planting, microtubers over the soil grew two-thirds higher than natural seed potatoes . The final yield on the plant and shows about the same ratio as the growth rate over the soil. A plant derived from microtubers produces approximately 507 grams of potatoes per plant, while natural seed potatoes are approximately 812 grams per plant (see Table 11 in Figures 4 and 5). In other words, the average yield of potato microtubers reached about 60 70% of natural seed potatoes if they were planted in the same way as natural seed potatoes. However, since the plants obtained from microtubers were much smaller than those obtained from natural seed potatoes, it seems that a more dense planting is possible in order to increase the yield per acre.

CLAIM

1. A method for mass production of potato microtubers, comprising: induction of virus-free microtuberogenic shoots of the type "Suppior" on a medium for induction of microtubers, treatment of microtuberigenic sprouts at low temperature, and cultivation of treated germs on a modified medium for induction of microbes containing an additional growth inhibitor to produce microbes , Characterized in that induction of microtuberogenic germs is carried out and microtubers are prepared on a solid medium contained in suitably arranged vessels of flat Petri dishes, the shoots are allowed to stand for 1 week in the light at 30 ° C and for the next week in the dark at about 10 ° C and Microtubers were prepared by culturing said aged shoots for 6 hours at 20 ° C in light and for 18 hours at 12 ° C in the dark.

2. A method according to claim 1, characterized in that microtuberogenic germs are induced on a solid medium having the following composition, mg / l:

  • Ammonium nitrate 2000
  • Potassium nitrate 2500
  • Calcium chloride, dichloride 440
  • Magnesium sulphate seven-water 370
  • Potassium Phosphate 170
  • Sodium EDTA 37.25
  • Iron sulfate, seven-fold 27,85
  • Manganese sulphate single-water 16.9
  • Boric acid 6.2
  • Zinc sulfate seven-fold 8,6
  • Potassium iodide 0.83
  • Sodium molybdate double-water 0.25
  • Copper sulphate five-water 0.025
  • Cobalt chloride, hexahedral 0.025
  • Myoinositol 100
  • Ascorbic acid 50
  • Gibberellic acid 0.1
  • Zeatin ribozide 0.1
  • Sucrose 20000
  • Agar 10000
  • Cyanocobalamin 1,5
  • Folic acid 0.5
  • Riboflavin 0.5
  • Biotin 1
  • Choline Chloride 1
  • Calcium pantothenate 1
  • Thiamine hydrochloride salt 1
  • Nicotinamide 2
  • Pyridoxine HCl 2
  • Paraaminobenzoic acid 0.5

3. The method of claim 1, wherein the microtubers are prepared on a solid medium having the following composition, mg / l:

  • Ammonium nitrate 1000
  • Potassium nitrate 1500
  • Calcium chloride, dichloride 440
  • Magnesium sulphate seven-water 370
  • Potassium phosphate 500
  • Na 2 EDTA 37.25
  • Ferrous sulphate of semivalent 27,85
  • Manganese sulphate single-water 16.9
  • Boric acid 6.2
  • Zinc sulfate seven-fold 8,6
  • Potassium iodide 0.83
  • Sodium molybdate dichloride 0.25
  • Copper sulphate five-water 0.025
  • Cobalt chloride, hexahedral 0.025
  • Myoinositol 100
  • Zeatin ribozide 0.1
  • Ascorbic acid 50
  • Chlorocholine chloride, or phosphon D, or Amo-1618, or B-905 100
  • Sucrose 90
  • Agar 10
  • Cyanocobalamin 1,5
  • Folic acid 0.5
  • Riboflavin 0.5
  • Biotin 1
  • Choline Choline 1
  • Calcium pantothenate 1
  • Thiamine hydrochloride salt 1
  • Nicotinamide 2
  • Pyridoxine HCl 2
  • Paraaminobenzoic acid 0.5 °

print version
Date of publication 06.03.2007гг