EP0288460A1 - Process for mass production of potato's propagation material free from viroids and viruses - Google Patents

Abstract

The method described involves the cultivation of plant tissue, in such a way that the cells isolated from the tissue of the tip of the potato shoot are cultivated in vitro in a nutritive agent, then they are rooted or else one provokes the formation of mini-tubers. Rooted plants are bred to become brooders, or new mini-tubers are induced from which other brooders are bred; from these we obtain young planes or small tubers which serve as multiplication substances, or else the mini-tubers serve as multiplication substances.

Description

PROCESS FOR MASS PRODUCTION OF POTATO'S PROPAGATION MATERIAL FREE FROM VIROIDS AND VIRUSES

The invention relates to a process for the mass production of potato's propagation material, free from viroids and viruses, by plant tissues culture.

The potato (Solanum tuberosum) is an essential food, therefore great experimertal and practical efforts are made in order to improve its fertility and its resistance against diseases and to develop a good adaptability to the environment. In spite of all efforts, no cultivars have been produced so far, resisting all the important diseases and at the same time owing other appropriate characteristics.

Most cultivars of potato are classed under subspecies tetraploid Solanum tuberosum ssp. tuberosum (Howard, H.W.: Genetics of the potato (Solanum tuberosum), Logos Press, London (1970) 126. p.), 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. Sci. 4. 593-595. (1964)). It is generally believed that in crder to maintain the maximal fertility and the growth rate, the potato must be heterozygote, the greatest possible. However, by reducing the interallelic heterozygote character by endogamy, also the growth rate of the potato seedlings will be significantly reduced.

The oecological conditions of several countries do not allow the long-term maintenance of different potato cultivars free from diseases, and so the aim of the improvement was to produce cultivars resisting the environmental conditions and the different pathogens by using wild - resisting - species, as hybridisation material.

However, the resistance improvement had only moderate results, since in case of the potato the so-called "single step" traditional method did not work (by this method the new wanted characteristics are produced by preserving the other favourable features.)

Several methods have been developed for the in vitro vegetative propagation 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 3. 55. 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 (Slanum 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., Szabd 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 recommended primarily for the propagation of virus-free potato lines. Besides the selection there is another possibility for producing the virus-free lines i.e. by isolating the meristem cells of the virus-infected plants and by raising them in vitro (Morel, G., Mϋller, J.F.: La culture in vitro du meristéme 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", edit.: Reinert J. and Bajai Y.P.S., Springer-Verlag, Berlin- New York, (1977) 615-637; Faccioli, G.; Rubies-Autonell, C: PVX and FVY distribution in potato meristem-tips and their eradication by the use of thermotherapy and meristemtip culture, Phytopath. Z. 103, 66-76 (1982)) . The reason of it is, that the meristem cells of the plants are free of viruses, as a result of certain biochemical processes, being explained partly so far.

To exempt from viruses and the in vitro vegetative propagation, both based on meristem cultivation, are widely popular methods for preserving the potato species. The in vitro production of the small, so called minitubers of the potato, is also a known method (American Potato Journal 59. 33-37. (1982)) . The further cultivation of the potato plantlets, produced in vitro, will be carried out in glasshouses. The singlebud cuttings are also widely used with the multiplication of the virus-free mother plants (Sunarjono, H., Krisnawati, Y.: Potato seed multiplication 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. 23, 449-452 (1980); Goodwin, P.B.: Rapid propagation of potato by single node cuttings, Field Crops, Res. 4, 165-173 (1981).

In spite of all efforts, made in order to produce the propagation material of the potato by tissue culture, it is not yet resolved to produce the propagation material in great quantities and economically. Although several partial results are available, no complex system is available so far, which could be succesfully used for producing an appropriate propagation material of potato on large scale, and which could effer a solution, adaptingitself to the demands for the production of different propagation materisl, i.e. rooted cuttings, minitubers and/or small tubers.

It has been found - and it is the subject of our invention - that the potato's propagation material free from viroids (and viruses) - i.e. rooted cuttings, minitubers and/or small tubers - can be produced in great quantities by tissue culture as follows: the germinated potato meristem cells (terminal tissue of the shoot) will be isolated, the isolated fractions free from viroids and viruses will be separated and raised in nutritive agent in vitro up to 6-8 buds, and later on it will be rooted in nutritive agent, or minitubers will be induced on the shoots; the in vitro rooted plants will be transplanted into glasshouses where separated as meristem lines they will be raised to become mother plants, or minitubers will be induced again on the in vitro rooted plants, and mother plants will be raised from the minitubers and from these mother plants seedlings or small tubers may be obtained as propagation materials, or the minitubers will be used as propagation materials, and the formation of seedlings or tubers can be directed by influencing the patatin-synthesis of the mother-plants.

The process detailed in this invention, can be yieldingly applied for the economical production of the potato's propagation material in great quantities, and it is suitable for the steady supply of propagation material all the year rourd.

The isolation of the meristem cells, the raising and the multiplication of the shoots, the rooting and the formation of minitubers are carried out under laboratory conditions, the raising of the mother plants from the rooted plants is carried out in glasshouses, and the raising of the seedlings or small tubers from the mother plants will be performed in greenhouses or on the fields, under vector-free environment.

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

On the potato tubers of the same cultivars, shoot formation is induced, e.g. by gibberellic acid, then the tubers will be germinated and forced in a climate-chamber without lighting. After about 3 weeks, 0.2-0.3 mm shoot apex will be isolated from the top of 5-10 cm shoots, under aspectic conditions. The isolated shoot apex will be individually set in sterile nutritive agent. Each meristem will be treated and multipled as separate lines. The exemption of each multiplied line from viruses and viroids will be controlled by virological test methods i.e. Enzyme immuno assay (EIA). (J. gen. Virol. 33. 165-167 (1976); Revue Suisse Agric. 11. 253-265 (1979); J. gen. Virol. 34. 475-483 (1977); Novenyvedelem 15(8), 354-358 (1979); Phytopath. Z. 90. 364-368 (1977); Phytopath. 67. 145-150 (1977)). For longer propagation only those meristem lines have been used, which were free from all potato-viruses and -viroids. After starting the in vitro propagation, the exemption of the selected virus-free lines are several times controlled in the course of the process.

The meristems satisfactorily controlled and selected, are raised in Wassermann-tubes, in nutritive agents, up to 6-8 buds. The composition ofthe nutritive agent is as follows: MS macroelements 1/2 degree of concentration MS microelements

Inozit 100 mg/L

Thiamine HCl 0.5 mg/L Pyridoxine HCl 0.5 mg/L

Nicotinic acid 5 mg/L

Glycine 2 mg/L

Folic acid 0.5 mg/L

Biotin 0.05 mg/L Hydrolysed caseine 500 mg/L

Ca-panthotenat 2 mg/L

Naphtylacetic acid 0.001 mg/L Saccharose 1.5 %

Agar 0.8 %

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

The developed meristem-plants with 6-8 buds will be set horisontally in solid nutritive agent, similar to the above-mentioned (the macroelements have the original concentration, 3 % saccharose), slightly squeesed into the medium. By this method it will be rendered possible that all buds should start up. In this stage the plants are propagated in vitro.

For the rooting the same nutritive agent will be used as for the propagation, however without agar. For the formation of minitubers the nutritive agents, given in the Hungarian patent specification Nos. 183.978 and 187.396 can be used, too. In liquids, the buds of the shoot-pieces are quickly shooting and are generating root-tissues. After

10-12 days the ramifying roots will be bundled up and can be taken out at once. There will be only very little leaves on the plants, and it is very advantageous as regards the planting of them in the greenhouses. It will be influenced in the known manner, whether rooted shoots or minitubers should be obtained (Annals of Botany 53. 565-578 (1984)).

The plants, rooted in vitro, will be raised in greenhouses, on solid soil. The rooted plants are taken in bounds on lhe surface of the soil. In order to maintain the degree of humidity at the appropriate level, the plants will be covered with foil and the light must be controlled. The plants under the foils must be aerated each day, and the second week the plants will be uncovered step by step, so adapting the plants to the environment conditions in the greenhouse. 3 weeks after the plants having been transplanted, the plants are growing without covering, and they are suitable for getting cuttings from them.

The growth of the plant is not uniform enough to be suitable for producing seedlings directly from them, and so these plants are used as mother-plants, giving, shoots.

For producing a uniform seedling material, top-cuttings (with 3-4 buds) will be rooted on trays. The cuttings are made of young shoots with

1-2 cm long leaves, because the older shoots will develop tubers and so they are not suitable for raising rooted seedlings of them. The seedlings are rooted uider coverage and they will be raised on the tray for two weeks, up to 5-6 leaves. The seedlings may be transplanted into greenhouses or on the field in a vector-free environment.

The plant, gained from minitubers induced in the shoot propagation stage, can also be used as mother-plant.

The seedlings, cultivated in greenhouses, are to be transplanted in a vector-free field in a way, that just the upper leaves of the seedlings should get over the ground level. From all the under- ground buds of the horisontally set plants, shoot and later tuber will develop.

It is advisable to set the plants separated according to the meristem lines in the environment free from vectors, in order that the trueness to variety and the fertility could be controlled. In that way it is possible to remove the bad lines and to select the favourable characteristics.

The further propagation of this propagation material on the field may be carried out by traditional agricultural methods.

As mentioned above, the cuttings, got from the parent plants, will be transplanted in order to obtain tubers. In case, if we don't want to gain tubers, but our aim is to have further parent plants, giving cuttings, the tuber-formation must be inhibited, in order to increase the ability of its cutting-formation. After the tuber-formation has started, the cuttings cannot be efficiently rooted.

It has been found that the induction of the potato's tuber formation is in close connection with the beginning of the potato's patatin-synthesis. The patatin-synthesis, starting in the leaves, is a prediction of the induction of tuber-formation. The patatin-synthesis and so the induction or inhibition of the tuber-formation may be followed up by an enzymelinked patatin antiserum assay. On the basis of the control of the patatin-

-synthesis, the tuber-formation may be influenced by changing the environmental conditions or by chemical treatment. The patatin-synthesis may be induced by cooling or by withdrawing the inorganic nitrogen (ammonia, nitrate), resp. by replacing it with organic nitrogen source carbamide) .

According to one of the embodiments of the process, disclosed in the present invention, the tuber-formation of the laboratory shoots will be induced, so in vitro minitubers will be obtained, being of some mm diameter. These minitubers are very convenient as propagation materials. In contradiction to the seasonal shoot production, the minitubers can be steadily produced all the year round, and they can be stored in small volumes in refrigerators.

However, the disadvantages of the minitubers are, that they are of loose consistence, they wither very easily and they may be damaged very easily. These disadvantages may be eliminated by coating the tubers, produced in vitro, with a protective layer, so protecting them from withering and from the mechanical damages. The coated tubers are suitable for sowing by seading machine.

The process is as follows: after having taken the minitubers from the flasks, the residues of the stem must be removed, the tubers must be thoroughly washed and dried. The drying can be carried out on trayers, in 1-2 layers, under 20 ºC temperature, 40 % humidity, for appr. 2 weeks. The drying must be kept on until the original volume of the tubers will be reduced by the third part of it. Then the tubers will be coated in the known manner

(0.5-0.6 mm) by granulation. The granulation may be carried out by the usual methods used for coating the seeds. The coated tubers are to be dried, and stored under about 2-5 ºC temperature. In the course of the practical realization of this process, no spontaneous genetic changes have been found.

The following examples illustrate the invention without limiting it. Example 1

Production of virus-free breeding stock and in vitro reservation of cultivars 10 pcs "Somogy gyöngye" potato tubers are washed with the solution of 10 % sodium hypochlorite and 0.1 % wetting agent and with hand-scrub, and in a same solution they are steeped for 30 min. After the sterilization, the tubers are to be flushed 3 times with sterile distilled water, and then dried. In order to promote the germination, the dry tubers are to be steeped in GA-3 (gibberellic acid) solution of 10 mg/litre for 30 min., and then dried and they will be germinated in a climate box under 5 ºC temperature, 70-80 % relative humidity, without lighting, for 18-22 days, up to 4-6 cm shoots. The cultures are to be signed and recorded by tubers and shoots. The cutted shoots are to be sterilized in a solution of 4 % sodium-hypochlorite, then flushed 3 times with sterile distilled water, and the shoot apex must be cut with a sharp lancet, under microscope, and put on a nutritive agent as detailed above. One shoot apex meristem is put in a test-tube, and it begins growing in 3-4 weeks, under 20-22 ºC temperature, and 500 Lux light intensity. Above the growing cultures the intensity of light will be increased on 2000 Lux, whereby plantlets with 5-7 leaves will develop during 6-8 weeks.

The plants must be taken off the test- -tubes, and put in a nutritive agent, similar to the above mentioned one, except the saccharose content, which must be increased up to 3 % . A standardised screw-capped jar (400 ml), containing 40 ml nutritive agent, is convenient for this purpose (1 plaht/jar). Under 20-22 °C and 3000 Lux light intensity, all buds will start up in 6-8 weeks, and 4-8 cm shoots will develop in the jar. At this stage the first control of the exemption from viruses must be carried out.

The virus-infected lines are eliminated from the further cultivation, and the satisfactory shoots are cut into pieces with one-two buds, put in other raising jars, and they will be raised in the above mentioned manner. In each grafting cycle further controls must be carried out at least 3 times, and then the virus-free lines can be multipled. Using the process detailed above, unlimited quantities of plants can be produced from each lines. Example 2

Producing minitubers The plants, produced according to example 1 will be further cultivated in the above mentioned nutritive (liquid) agent, containing no agar (22 ºC, 3000 Lux). The jars will be woven by the plants, and then - about after 3 weeks - the nutritive agent must be changed by another one, promoting the patatin-xynthesis. This agent differs from the other one: it contains 10 mg/litre benzyl-adenine, 10 mg/litre coumarin and 8 % saccharose, but only 10 % inorganic nitrogen source of the original quantity. The cultivation is carried out under 17 °C temperature, 1000 Lux, for 10-12 weeks. By this time 40-60 pcs minitubers (0.3-0.6 cm diam.) have been developed.

The jar must be turned out, the plants are to be dried, the tubers must be taken off, let them rest for a week at room temperature (suberification), and later on they are to be stored at +2 -4 °C- temperature, in a dry and dark place. Example 3

Production of parent plants and seedlings The plants, produced according to example 1 are to be transplanted into greenhouses, in loose bounds, in a soil containing the mixture of peat and perlite (500-750 plants/m²). The soil must be kept on 20 °C and the air on 24 ºCtemperature, and in order to assure the humidity, the table is to be covered with foil. The culture may be adapted in 2-4 weeks to the conditions of 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 hindering the patatin-synthesis, by spraying them with artifical fertilizer solution of 0.2 % Mikramid. From this parent plant top-end shoots with 4-5 leaves can be gained again, and these may be cultivated after having been rooted, or they may be put on the market as potato seedlings. 80-100 pcs seedlings can be gained from one culture-jar. Example 4

Production of small tubers From the minitubers according to example 2 or from the seedlings according to example 3 small tubers may be produced in greenhouses or in isolators. The sowing or the planting will be carried out in a mixture of peat and pearlite, containing 0.5 kg/m³ carbamide artificial fertilizer (400 plants/ /m²).

The plants must be watered under natural temperature and lighting, meeting their requirements, resp. The supply of the nutritives can be carried out through the leaves. After 8 weeks the development of the foliage must be hindered by using chlor-choline-chloride, a growth regulator, the watering must be stopped, so in 85-90 days after the sowing, 3-4 small tubers (stock) (0.5-2.0 cm diam.) may be gained.

Claims

What we claim is:

1. A process for the mass production of potatoes' propagation material free from viruses and viroids by using tissue culture, characterized in that germinated cells of potato's shoot apex tissue are isolated, the cells are cultivated in vitro in nutritive agent, then they are rrultipled in vitro in nutritive agent, the obtained shoots are rooted in nutritive agent or tuber formation is induced on the shoots, the plants, rooted in vitro, are transplanted into greenhouses, and raised to become parent plants, or, on the plants, rooted in vitro, tuber formation is induced, if required from the minitubers mother plants are cultivated, and from the mother plants propagation material of seedling or small tuber are gained, or the obtained minitubers are used as propagation materials, and the formation of seedling, resp. tubers. is suitably directed by influencing the patatin-synthesis of the parent plants.

2. A process as claimed in claim 1 for the production of minitubers, as propagation material, characterized in that on the shoots, gained by in vitro propagation, after direct or in vitro rooting, the formation of tuber is induced.

3. A process as claimed in claim 1 or 2, characterized in that the obtained minituber propagation material is coated with a protective layer.

4. A process as claimed in claim 1 for the production of potato seedling as propagation material, characterized in that the shoots, gained by in vitro propagation, are rooted in vitro in nutritive agent, then transplanted into greenhouses, and raised to become parent plants, and from these parent plants seedling propagation material is obtained, while the patatin-synthesis of the parent plant is hindered.

5. A process as claimed in claims 1 or 4 characterized in that the patatin-synthesis is influenced by changing the environmental conditions, or by using chemical treatment.

6. A process as claimed in claim 1 for the production of potato's small tubers as propagation material, characterized in that on the parent plant cultivated from the in vitro rooted plants, or on the minitubers, tuber formation is induced.

7. A process as claimed in claim 1 or 4, characterized in that from the shoots, obtained from the mother plants, further mother plants are cultivated.

8. A process as claimed in any one of claims 1 to 7, characterized in that the potato's propagation material is produced under circumstances free from viruses and viroids.