NO882418L - PROCEDURE FOR MASS PRODUCTION OF POTATO PRODUCTS FREE OF VIROIDS AND VIRUSES. - Google Patents

Description

The present invention relates to a method for the mass production of potato crop material which is free of viroids and viruses, using plant tissue culture.

The potato (Solanum tuberosum) is an essential food, and therefore great experimental and practical efforts have been made to improve its fertility and its resistance to diseases and to develop a good adaptation to the environment. Despite all efforts, no cultivars have yet been produced which resist all the serious diseases and at the same time have other suitable characteristics.

Most cultivars of the potato are classified under the subspecies tetraploid Solanum tuberosum ssp. tuberosum (Howard, H.W.: Genetics of the potato (Solanum tuberosum), Logos Press, London (1970) p. 126), although dihaploid and monohaploid potato lines can also be produced (Houglas, R.W., Ploquin, S.J., Gabert A.C.: Effect of seed parent and pollinator on frequency of haploids in Solanum tuberosum. Crop. Sei. 4, 593-595 (1964)).

It is generally believed that to maintain maximum fertility and growth rate the potato must be heterozygous,

and largest possible. By reducing the interallelic heterozygous character in endogamy, the growth rate of the potato seedlings will also be significantly reduced.

The ecological conditions in many countries allow

not long-term retention of different potato cultivars that are free from diseases, and thus the aim of the improvement was to produce cultivars that resist the environmental conditions and the various pathogens by using wild, resistant species as hybridization material.

However, the resistance improvement had only moderate results as the traditional so-called "single-step" method in the case of potatoes did not work (in this method, the new, desired characteristics are produced while retaining the other advantageous traits).

Many methods have been developed for the in vitro vegetative breeding of the potato (Roca, W.M., Esponoza, N.O., Roca, M.R., Bryan, J.E.: A tissue culture method for -the rapid propagation of potatoes; Am. Potato J. 5_5, 691- 701 (1978); Goodwin, P.B., Kim, Y.C. Adisarwanto, T.: Propagation of potato by shoot-tip culture, Potato Res. 23, 9-23 (1 80); Roest, S., Bokelmann, G.S.: In vitro adventitious bud techniques for vegetative propagation and mutation breeding of potato (Solanum tuberosum L.) I. Vegetative propagation in vitro through adventitious shoot formation. Potato Res. 23, 167-181 (1980); Hussey, G., Stacey, N.J.; In vitro propagation of potato (Solanum tuberosum L.) Ann. Bot. 48, 787-796 (1981); Henszky, L.E., Enyingi, K., Szabo I: Tissue culture technology for longterm storage and propagation of potato (Solanum tuberosum L. ) germplants (Plant cell Culture in Crop. Improvement, S.K. Sen, K.L. Giles ad., Plenum Press, New York, London, (1983), 9-18).

These methods are primarily recommended for breeding virus-free potato lines. Next to selection, there is another possibility of producing virus-free lines, i.e. by isolating the meristem cells of the virus-infected plants and cultivating them in vitro (Morel, G., Muller, J.F.: La culture in vitro du méristéme apicale de la pomme de terre; Compt. Rend. 258, 5250-5252 (1964); Mellor, R.C., Stace-Smith, R.: Virus-free potatoes by tissue culture, "Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture", ed.: Reinert J. and Bajaj Y.P.S., Springer-Verlag, Berlin-New York, (1977), 615-637;

Faccioli, G.; Rubies-Autonell, C.: PVX and PVY distribution in potato meristem-tips and their eradication by the use of thermotherapy and meristem-tip culture, Phytopath. Z. 103, 66-76 (1982)). The reason for this is that the meristem cells in the plants are free of viruses as a result of certain bio-chemical processes which have so far been partially explained.

Protecting from viruses and the in vitro vegetative cultivation, both based on meristem cultivation, are very popular methods for the protection of potato species. The in vitro production of the small, so-called mini tubers of the potato is also a known method (American Potato Journal _59 , 33-37 (1982)).

The further cultivation of potato plants, produced in vitro, will be carried out in greenhouses. Separated single buds are also widely used for multiplication of virus-free mother plants (Sunarjono, H., Krisnawati, Y.: Potato seed multi-plication by stem cutting I. The influence of the mother plant age on the growth and yield of the cutting. Bull. Penel, Hort, 8(2) 39-47 (1980); Goodwin, P.B., Brown, G.: Field performance of potato shoot-tips proliferated in culture. Potato Res. 2J3 , 449-452 (1980); Goodwin ,P.B.: Rapid propagation of potato by single node cuttings, Field Crops, Res. 4_, 165-173 (1981).

Despite all the efforts made to produce the crop material from the potato by tissue culture, it has not yet been achieved to produce the crop material in large quantities and economically. Although several partial results are available, no complex system is available so far, which can be successfully applied for the production of a suitable crop material of potato on a large scale, and which could provide a solution that adapts to the requirements for the production of different crop materials, i.e. .rooted cuttings, mini tubers and/or small tubers.

It has been found, and it is the purpose of the present invention, that the potato crop material which is free of viroids (and viruses), i.e. rooted cuttings, mini tubers and/or small tubers, can be produced in large quantities by tissue culture as follows: the germinated potato meristem cells (the endwood of the shoot) will be isolated, the isolated fractions that are free of viroids and viruses will be separated and grown in nutrients in vitro up to 6-8 buds, and later they will root in nutrient, or mini-tubers will be induced on the shoots. The plants rooted in vitro will be transferred to greenhouses where they will be separated as meristem lines and grown to become mother plants, or mini tubers will again be induced on the in vitro rooted plants, and mother plants will be grown from the mini tubers, and from these mother plants, seedlings can be obtained or small tubers as crop materials, or the mini tubers will be used as crop materials, and the formation of seedlings or tubers can be directed by influencing patatin synthesis in the mother plants.

The method set out in detail in the present invention can be used with benefit for the economic production of the potato's crop material in large quantities,

and it is suitable for a stable supply of crop material all year round.

The isolation of the meristem cells, the cultivation and multiplication of the shoots, the rooting and the formation of mini-tubers are carried out under laboratory conditions, the cultivation of the mother plants from the rooted plants is carried out in a greenhouse, and the cultivation of the seedlings or small tubers from the mother plants will be carried out in the greenhouse or in the field, under vector-free environments .

The first step according to the present invention is to select potato lines that are free from viruses and viroids.

On potato tubers of the same cultivars, shoot formation is induced, e.g. with gibberellic acid, then the tubers will germinate and be forced in a climate chamber without lighting. After approx. After 3 weeks, 0.2-0.3 mm shoot tip will be isolated from the top of 5-10 cm shoot, under aseptic conditions. The isolated shoot tip will be placed individually in sterile nutrient medium. Each meristem will be treated and multiplied as separate lines. The protection of each multiplied line from viruses and viroids will be checked using virological test methods, i.e. enzyme immunoassay (EIA). (J. gen. Virol. 33, 165-167 (1976); Revue Suisse Agric. 11, 253-265 (1979);

J. gen. Virol. 3_4 , 475-483 (1977); Novényvédélem 15(8), 354-358 (1979); Phytopath. Z. 90, 364-368 (1977); Phytopath. 67, 145-150 (1977)).

For longer cultivation, only those meristem lines that are free from all potato viruses and viroids are used. After starting the in vitro crop, the preservation of the selected virus-free lines is checked several times during the process.

The meristems which have been satisfactorily controlled and selected are grown in Wasserman tubes, in nutrients, up to 6-8 buds. The composition of the nutrient is as follows:

Regarding macro- and micro-elements, see the publication of Hurashige and Skoog: Physiol. Plant 15, 473-497 (1962).

The developed meristem plants with 6-8 buds are placed horizontally in solid nutrient, similar to that mentioned above (the macroelements have the original concentration, 3% sucrose), which is slightly squeezed into the medium. Using this method, it will be possible for all the buds to begin to grow. At this stage, the plants are grown in vitro.

For rooting, the same nutrient will be used as for cultivation, but without agar. For the formation of mini tubers, the nutrients specified in the Hungarian patents 183,978 and 187,396 can also be used. In liquids, the tubers shoot off the shoot parts quickly and generate root tissue. After 10-12 days, the branched roots will be tied up and can be taken out straight away. There will be very few leaves on the plants, and this is very advantageous when it comes to planting them in greenhouses. They will be affected in a known manner so that either rooted shoots or mini-tubers are obtained (Annals of Botany, _5_3, 565-578 (1984)).

The plants that are rooted in vitro will be grown in greenhouses on solid soil.

The rooted plants are taken in bands on the surface of the soil. To maintain the degree of humidity at an appropriate level, the plants are covered with foil, and the light must be regulated. The plants under the foils must be aired every day, and in the second week the plants will be uncovered step by step to adapt the plants to the environmental conditions in the greenhouse. 3 weeks after the plants are transplanted, the plants grow without a cover and cuttings can be taken from them.

The growth of the plant is not uniform enough to be suitable for direct production of seedlings from them, and thus these plants are used as mother plants to produce shoots.

For the production of a uniform seedling material, top cuttings (with 3-4 buds) will be rooted on trays.

The cuttings are formed from young shoots with 1-2 cm long leaves because the older shoots will develop tubers, and they are thus not suitable for growing rooted seedlings. The seedlings are rooted under cover, and they will be grown on trays for two weeks, up to 5-6 leaves. The seedlings can be transferred to a greenhouse or in the field in a vector-free environment.

Plants obtained from mini tubers induced in the shoot growth stage can also be used as a mother plant.

The seedlings grown in greenhouses must be transferred to a vector-free field in such a way that only the upper leaves of the seedlings are above ground level. From all the underground buds in the horizontally placed plants, shoots and later tubers will develop.

It is recommended to place the plants separated according to the meristem lines in an environment that is free from vectors so that the correctness of the variety and fertility can be checked. In this way it is possible to remove the bad lines and to select the advantageous characteristics.

The further harvesting of this crop material on the field can be carried out using traditional agricultural methods.

As mentioned above, the cuttings obtained from the parent plants will be transferred to obtain tubers. In cases where it is not desirable to obtain tubers, but the goal is to obtain additional parent plants that give cuttings, tuber formation must be inhibited in order to increase its ability to form cuttings. After tuber formation has started, the cuttings cannot be rooted effectively.

It has been found that the induction of potato tuber formation is closely related to the onset of potato patatin synthesis. The patatin synthesis that starts in the leaves is a predictor of the induction of tuber formation. Patatin synthesis and thus the induction or inhibition of tuber formation can be followed up with an enzyme-linked patatin antiserum test.

On the basis of the control of patatin synthesis, tuber formation can be influenced by changing environmental conditions or by chemical treatment. Patatin synthesis can be induced by cooling or by removing inorganic nitrogen (ammonia, nitrate), respectively. by replacing it with an organic nitrogen source (carbamide).

According to one of the embodiments of the method described in the present invention, the tuber formation of the laboratory shoots will be induced so that mini-tubers with a diameter of a few mm will be obtained in vitro. These mini tubers are very suitable as crop materials. In contrast to the seasonal shoot production, the mini tubers can be produced evenly all year round, and they can be stored in small volumes in a refrigerator.

However, the disadvantage of the mini tubers is that they have a loose consistency, they wilt very easily and they can be destroyed very easily. These disadvantages can be eliminated by coating the tubers produced in vitro with a protective layer to protect them from exposure and mechanical damage. The coated tubers are suitable for sowing with seed drills.

The procedure is as follows: after taking the mini tubers from the kolhen, the remains of the stem must be removed, the tubers must be thoroughly washed and dried. The drying can be carried out on trays, in 1-2 layers, at 20°C temperature, 40% humidity,

for about. Two weeks. Drying must be continued until the original tuber volume has been reduced by a third. The tubers are then coated in a known manner (0.5-0.6 mm) by granulation. The granulation can be carried out using the methods that are common for coating seeds. The coated tubers must be dried and stored at a temperature of approx. 2-5°C. During the practical implementation of this method, no spontaneous genetic changes have been found.

The following examples illustrate the invention without limiting it.

Example 1

Production of virus-free crop material and in vitro reservation of cultivars 10 pcs. "Somogy gyongye" potato tubers are washed with a solution of 10% sodium hypochlorite and 0.1% wetting agent and with a hand scrub, and they are soaked in the same solution for 30 minutes. After sterilization, the tubers are rinsed 3 times with sterile, distilled water and then dried. To promote germination, the dry tubers are soaked in GA-3 (gibberellic acid) solution of 10 mg/liter for 30 minutes and then dried, and they will germinate in a climate box at a temperature of 5°C, 70-80% relative humidity, without light, for 18-22 days, up to 4-6 cm shoots. The Kul-tours must be marked and recorded with tubers and shoots.

The cut shoots must be sterilized in a solution of 4% sodium hypochlorite, then rinsed 3 times with sterile, distilled water, and the shoot tip must be cut under a microscope with a sharp lancet and placed on a nutrient medium

as detailed above. A shoot tip meristem is placed in a test tube and it begins to grow within 3-4 weeks, at a temperature of 20-22°C and a light intensity of 500 Lux. Above the growing cultures, the light intensity will

is increased to 2000 Lux, whereby small plants with 5-7 leaves will develop within 6-8 weeks.

The plants must be taken out of the test tubes and placed in a nutrient similar to that mentioned above, except for the sucrose content, which must be increased to 3%. A standard container with a screw cap (400 ml), containing 40 ml of nutrient, is suitable for this purpose (1 plant/container). At 20-22°C and a light intensity of 3000 Lux, all the buds will start to grow within 6-8 weeks, and

4-8 cm shoots will develop in the container. At this stage

the first virus exclusion check must be performed.

The virus-infected lines are eliminated from further cultivation, and the satisfactory shoots are divided into pieces of 1 to 2 tubers, put into other culture containers, and they will be cultivated in the above-mentioned manner. In each inoculation cycle, further checks must be carried out at least 3 times, and then the virus-free lines can be multiplied. Using the method detailed above, unlimited quantities of plants can be produced from each line.

Example 2

Production of mini tubers

The plants produced according to example 1 will be grown further in the above-mentioned nutrient (liquid) which does not contain agar (22°C, 3000 Lux). The containers will be woven through by the plants, and then, after approx. 3 weeks,

the nutrient must be replaced with another that promotes patatin synthesis. This substance differs from the other: it contains 10 mg/liter benzyl-adenine, 10 mg/liter coumarin and 8% sucrose, but only 10% inorganic nitrogen source of the original amount. Cultivation is carried out at a temperature of 17°C, 1000 Lux, for 10-12 weeks. At this point, 40-60 pieces have been developed. mini tubers (0.3-0.6 cm diameter). The container must be emptied, the plants dried, the tubers removed, they are left for a week at room temperature (corking), and then they are stored at +2 - 4°C in a dry and dark place.

Example 3 Production of parent plants and seedlings

The plants produced according to example 1 are transferred to a greenhouse, in loose bundles, in a soil containing a mixture of peat and perlite (500-750 plants/m 2 ). The soil must be kept at 20°C and the air at 24°C, and to ensure humidity the table must be covered with foil. The culture can be adapted for 2-4 weeks to the conditions in the greenhouse: the plants become stronger and 7-8 leaves will develop. This is the parent plant where the formation of tubers will be inhibited by preventing patatin synthesis, by spraying them with artificial fertilizer solution with 0.2% Micramide. From this parent plant, top-end shoots with 4-5 leaves left can be obtained and these can be grown after being rooted, or they can be marketed as potato seedlings. 80-100 pcs. seedlings can be obtained from a growing container.

Example 4

Production of small tubers

From the mini tubers according to example 2 or from the seed plants according to example 3, small tubers can be produced in greenhouses or in insulators. Sowing or planting is carried out in a mixture of peat and perlite, containing 0.5 kg/m 3 of the artificial fertilizer carbamide

2

(400 plants/m ).

The plants must be watered at a natural temperature and light that satisfies their requirements. The supply of nutrients can take place through the leaves. After 8 weeks, the development of the foliage must be prevented by using chlorine-choline chloride, a growth regulator, watering must be stopped, so that 85-90 days after sowing, 3-4 small tubers (stock material) can be obtained (0.5- 2.0 cm diameter).

Claims (8)

1. Method for the mass production of potato crop material that is free from viruses and viroids using tissue culture, characterized in that germinated cells from tissue of potato shoot tops are isolated, the cells are grown in vitro in nutrient, they are then multiplied in vitro in nutrient, the obtained shoots are rooted in nutrient or tuber formation is induced on the shoots, the plants that are rooted in vitro are transferred to a greenhouse and grown to become parent plants; if tuber formation is induced on the plants that are rooted in vitro, if desired, mother plants are grown from the mini tubers, and from the mother plants it is obtained crop material in the form of seedlings or small tubers, or the obtained mini-tubers are used as crop material, and the formation of seedlings, respectively. tubers, is directed in a suitable way by affecting patatin synthesis in the parent plants. 2. Method according to claim 1 for the production of mini tubers, as crop material, characterized in that the formation of tubers is induced on the shoots, which have been obtained by harvesting in vitro, after direct or in vitro root attachment. 3. Method according to claim 1 or 2, characterized in that the obtained mini tuber crop material is coated with a protective layer. 4. Method according to claim 1 for the production of potato seedlings as crop material, characterized in that the shoots obtained by cropping in vitro are rooted in vitro in nutrient, then transferred to a greenhouse, and cultivated to become parent plants, and from these parent plants seedling crop material is obtained, while patatin synthesis in the parent plant is prevented. 5. Method according to claim 1 or 4, characterized in that patatin synthesis is affected by changing the environmental conditions, or by using chemical treatment. 6. Method according to claim 1 for the production of small potato tubers as crop material, characterized in that tuber formation is induced on the parent plant grown from plants that have been rooted in vitro, or on the mini tubers. 7. Method according to claim 1 or 4, characterized in that additional mother plants are grown from the shoots obtained from the mother plants. 8. Method according to any one of claims 1 to 7, characterized by the fact that the potato crop material is produced in environments that are free from viruses and viroids.