LU87059A1 - METHOD FOR THE MASS PRODUCTION OF VIROID AND VIRUS-FREE POTATO PROPELLING MATERIAL - Google Patents

Description

L-3294

Ο 7 GRAND-DUCHÉ DE LUXEMBOURG

»Ir-T _’ i. t * · v / V ^ ^ $ Monsieur ie Ministre ül - décembre x ^ Sl de! 'Économie et des Classes Mienne-

Service de 3a Propriété Intellectuelle ntic üclivre 1

________ _ LUXEMBOURG

Demande de Brevet d’invention I. Requête

Novotrade RT, 24-26 Fürst Sandor utca, H-1136 Budapest,:: représentée par Monsieur Jean Waxweiler ,, 55 rue des Bruyères, Howald, agissant en qualité de mandataire dépose! nti ce premier décembre mil neuf cent quatre-vingt-sept, 4 · à ^ ... heures, au Ministère de l'Économie et des Classes Moyennes, à Luxembourg: 1. la présente requête pour l'obtention d'un brevet d 'invention concernant:

Procedure for. the mass production of ... viroid- ..and virus-. . 5> Free potato propagation smaterial .............................

2. la description en langue allemande ........... de l’invention entrais exemplaires:?. /. planches de dessin, en trois exemplaires; 4. la quittance des taxes versées au Bureau de l’Enregistrement à Luxembourg, le 01.12.1987 5. la délégation de pouvoir, datée de le .........

6. ie document d’ayant cause (authorization): déclare! Nt) en assumant la responsabilité de cette déclaration, que l ’(es) inventeur (s) est (sont): i o.

Ferenc dr. FÖGELEIN, 83-85 Patakhegyi ut, H-1028 Budapest; Istvan dr. SUM, 27 Gerecse u., H-1028 Budapest; Gyula OLEÄR, 45, Haman K. uPilisvörösvax 2085, Hongrie; Miklos MAGYAR, 216-226 Biitibo ut, H-1026 Budapest; Annamaria MES2AROS, 41 Benko utca, H-1147 Budapest; Miklos SZEGEDI, 83-85 Patakhegvi ut, H-1028 Budapest revendiquent I pour la susdite demande de brevet la priorité d'une (des) demande (s) de (7 · / ..... ... déposée (s) en (8) ..... / le (9> / ........... ...............

sous le N ° (10) / au nom de (11) .... Z ..... ...... ...................

élit (elisent) domicile pour lui (elle) et, si désigné, pour son mandataire, à Luxembourg 55 rue des Bruyères, Howald ............... d2t sollicitent) la délivrance d'un brevet d'invention pour l'objet décrit et représenté dans les annexes susmentionnées, avec ajournement de cette délivrance à AJ mois 1 - '

Le déposai®: / mandataire: (U

'II. Procès-verbal de Dépôt

La susdite demande de brevet d’invention a été déposée au Ministère de l’Économie et des Classes Moyennes. Service de la Propriété Intellectuelle à Luxembourg, en date du- 01.12.1987 -i 'r * .... % ’'’ · Y Pt. le Ministre de l’Économie et des Classes Moyennes s - a 15.00 heures i γ .'- 'ψΐ V ’* p-d.

• * -ÏÏy \ ç ·: Le chef du service de la propriété intellectuelle.

-. 'a ^ · * - ____ U Λ-l IJ ....... 4T1VFS AIFORM:: AÏRF DE * nFP' '/ t f L-3204 *

DESCRIPTION

FOR A PATENT APPLICATION IN

GRAND DUCHY OF LUXEMBOURG

NOVOTRADE RT

24-26, Prince Sandor utca 1136 Budapest

Hungary

Process for the mass production of viroid and virus-free potato propagation material f - 17 098 55 I 1 R · \ f t m *>

Process for the mass production of viroid and virus-free Ivar to ff eivermehr ungsmat eria 1

Field of application of the invention; '

The invention relates to a process for the mass production of viroid and virus-free potato consumption material from plant tissue cultures * / .¾ - 2 -

Characteristic of the known state of the art

The potato (Solanum tuberosum) is a major food rider, so great experimental and practical efforts are being made to improve its fertility and resistance to disease and to develop a good adaptability to the environment. Despite all efforts, no cultivars have so far been cultivated drawn * that are resistant to all major diseases and at the same time have other appropriate properties.

Most potato cultivars are classified under the subspecies Solanum tuberosum tetraploid ssp «tuberosum (Howard, H, f ·, Genetics of the potato (Solanum tuberosum) (genetics of the potato). * Logos Press, London (1970) 126 p *), although dihaploid and monohaploid potato lines can also be produced (Houglas, B «W., Ploquin, 3rd j ,, Gabert, A, C«, Bffect of seed parent and pollinator on frequency of haploids in Solanum tuberosum (effect from seed strain and pollinator to the frequency of Haploldén in Solanum tuberosum), Crop, Sei, ^ (1964), pp. 593-595) # 3s, it is generally assumed that the potato must be largely heterozygous to maintain maximum fertility and growth rate . By reducing the inter-express heterozygous character through endogamy, the growth rate of the potato seedlings is also significantly reduced.

The ecological conditions of different countries do not allow different cultivars of the potato to be kept free of disease for a long time, and it was therefore the aim of the improvement to cultivate cultivars, * 3

5 I

which are resistant to environmental conditions and various pathogens, for which wild «resistant - species have been used as hybrid-disruptive material ·. . However, the improvement in the survival rate was only modest

Disasters, since the so-called "Binsehritt method" was not applicable to the potato (this method produces the new * desired properties while maintaining other advantageous merlanals) *

There were different ones. Methods for the vegetative in vitro yerage cultivation of the potato developed (Hoch «W * M *, Esponoza * H * 0 *, Roes, M # R * .f Bryan * J« B * e A tisane culture raethod for the rapid propagation of potatoes (Bine tissue tower method for the rapid multiplication of potatoes) »Am Pots» to J * ||, 69-1 - 701 (t $ 7S>? Goodwin * P * 3rd, Kim, Y «C ** Adi- sarwsnto, 2 ·, Propagation of potato by shoot-tip culture (propagation of the potato by Iriebspitzeakultur) * Potato Res * 23 * 9 - 23 (1980)} Rosst * S * r Bohelmann * G * S · * IsT vitro adventitious bud techniques for vegetative propagation and mutation breeding of potato (Solanum tuberosum X) * I. Vegetative propagation in vitro through adventitious shoot formation (Premdhnaq) en-in vitro techniques for vegetative propagation and mutation breeding of potatoes * I * Vegetative propagation in vitro through vine shoot formation ) * Potato Res * 22, 16? - 131 (1930)} Huasey * ß., Staeey * I * J «, In vitro propagation of potato (Solanum tuberosum X) (in vitro propagation of the Potato) * Am. Bot * 43 * 787-796 (1981); Hsnaz & y, X. E ** Rnyingi * K **. 3zabS, I *, îissue culture technology »nology for lodgtarm siorage and propagation of potato (Solanum tuberosum L *) (tissue culture technology for long-term storage and propagation of the potato) seedlings (Plant Cell * • 4 * 3 ».

Culture la Crop # Improvament S «K * S en * K« ailes ad «, Plénum Press, Bew York, London (1983)» pp. 9 - 18) «

These methods are especially recommended for the propagation of virus-free potato lines. "Live selection there is another way of generating virus-free lines, ie," the isolation of the meristem cells of the virus-infected plants and their cultivation in vitro (Morel, G " , Müller, J "P", La culture in vifco du m & rist & rae apicale de la pomme de terre, Compt "Hend", 258, 5250 - 5252 (1964); Mellon, H "C", Stace-Smith, K * * Virus -free potatoes by tissue culture, "Applied and fundamental Aspects of Plant Cell, Pissue and Organ Culture" (Applied and fundamental aspects of the plant to the urine, tissue and organ culture), Edited by Heinert, J «and Bajaä, Y.B * S«, Springer-Verlag, Berlin - Hew York (1977) 615 - 637; Paociolo, G «, Eubies-Autonell, G«, PVX and PXY distribution in potato aeristem-tips and their éradication by the use of thermotherapy and meriatea-tip culture (PVY and PVY distribution tnKartoffel-Meristemspitzen and their eradication by the use of thermal therapy and the meristem tip culture, Phytopath Z 103, 66-76 (1982). The reason for this is that the meristem cells of the plants are free of viruses as a result of certain biochemical processes * who have only partially found an explanation so far *;

Virus liberation and vegetative in vitro propagation are common methods for the conservation of potato species, both of which are based on meristem cultivation. «A well-known method is the in viiro production of small, so-called mini tubers of the potato (American Potato Journal 33 - 37 (1982)) «- 5 - *. The further cultivation of »the potato plants that were produced in vitro» takes place in greenhouses * Sinus sphincisms are also frequently used due to the multiplication of the virus-free mother plants (Simarjono, H *, Krisna-wati, Y., Potato seed multiplication by stem cutting * X « The influence of the mother plant age on the growth and yield of the cutting (propagation of the potato sarane by stem cut · I * The influence of the age of the mother plant on the growth and the yield of the cut} · Bull «Penel, Hort« 8 (2 ) 39 - 47 (1980); Goodwin, P * B *, Brown * G «, Pield performance of potato shoot-tips proliferated in culture (Peld behavior of potato shoot tips that were proliferated during the culture) · Potato Ses * 23 «449-452 (1980}; Goodwin * P. 3 ·, Rapid propagation of potato by single node cuttings (rapid multiplication of potatoes by means of single knot cuts), Pield Oops Res. 165 - 173 (1981).

Despite all efforts made to win the potato propagation material by cell culture. new * it has so far not been possible to produce propagation materials in large quantities and economically by tissue culture * although there are various partial results, there is not yet a complex system that has been successfully used in which a suitable propagation material of the potato · on a large scale to create, and that could be a solution that meets the requirements of the production of different propagation material, d. h *, root cuts ^ mini tubers uhd / or small tubers, adjusts *

Purpose of the invention?

It is an object of the invention to provide viroid-free (and virus-free) "- β -

Propagation material of the potato - d * h *, root balls, mini-tubers and / or small tubers - to be produced in large quantities by tissue culture.

State of the Invention}

According to the invention, germinated cartilage cells (final tissue of the ïriabes) are isolated * the fractions isolated from the virus and non-virus are separated and up to 6 to 8 buds are grown in vitro in a nutrient * later. that they are rooted in a nutrient or mini tubers are brought to training on the grains? the in vitro-rooted plants are transplanted into greenhouses, where they are drawn separately as meristem lines to give mother plants, or mini-tubers are again formed on the in-vitro-rooted plants and mother plants are drawn from these mini-tubers * and out of them Mother plants can be seedlings or small tubers obtained as reproductive material, or the mini tubers can be used as reproductive material and the formation of seedlings or tubers can be controlled by influencing the patatin synthesis of the mother plants.

The ancestor embodied in the invention can be used profitably for the economic production of potato propagation material in large quantities and is suitable for the constant supply of consumable material throughout the year *

The isolation of the meristem cells, the cultivation and multiplication of the iriabe, the rooting and the formation of the mini tubers are carried out under laboratory conditions, the mother plants are grown from the rooted plants in glass houses, and the seedlings are grown or small. Tubers, from the mother plants. is carried out in greenhouses or in fields * under a vector-free bypass ·

The first step according to the present invention is to select the potato lines free of viruses and viruses.

On potato tubers of the same cultivars, the formation of shoots is induced, for example, by gibberic acid, the tubers are germinated and placed in a climatic chamber without lighting. After about 3 weeks, 0.2 - 0.3 mra large frieze tips are removed from the 5 - 10 cm long experiences under aseptic conditions · The isolated strain tips are individually placed in a sterile nutrient · Each meristem is treated and multiplied as a separate line ·

The maintenance of each multiplied line of Tirai and 71Λ roiden is controlled by viralogue test methods, i. h ·, the Bnzyraimraun analysis (BIA) (J. gen. Tirol # 32 »165 to 167 (1976)} Revue Suisse Agrîc.ij., 253 - 265 (1979)} J. gen. Virol · 24 * 475 - 483 ( 1977); NSv & nyvêdelein 15 (8), 354 to 358 (1979); Phytopath »Z * $ 0, 364 - 368 (1977); Phytopath. 61, 145 - 150 (1977)).

Only the meristem lines were used for further propagation. which are free of all potato viruses and viroids · After the in vitro propagation has started, the freedom from viruses of the selected lines is checked several times during the process ·

The appropriately controlled and selected meristems are stored in Wassermann tubes, in a nutrient, up to 6 "* Q - ** t.

8β 8 buds pulled. The agitator has the following composition - »3etaung {M3 macro elements 1/2 degree of concentration MS micro element e 1

Inozit '100 mg / 1

Thiamine HCl 0.5 mg / 1

Pyridoxine HCl 0.5 mg / 1

Kicotinic acid 5 ^ 0 ffig / i

Glycine 2 * 0 rag / 1 '

Polyacid 0.5 mg / 1

Biotin 0.05 mg / 1

Hydrolyzed casein 500.0 mg / 1

Ca panthotenate 2.0 mg / 1

Haphihylacetic acid 0.00t mg / 1

Sucrose t, 5%

Agar 0.8%

For the MaktfÖ * and microelements see the publication by Hurashige and Skoog, Physiol. Plant 1j |, 473 - 497 (1962) «

The developed meristem plants with 6 - 8 buds are placed horizontally in a solid multi 'which is similar to the one listed above (the macro elements have the original concentration, sucrose 3 $) t they are easily pressed into the medium' by this method it becomes possible that all buds can sprout. In this phase, the plants are propagated in vitro.

The same nutrient used for propagation as for propagation but without agar is used. The nutrients given in the Hungarian patent specifications 183 978 and t87 396, t - 9 - • can also be used for the formation of mini-tubers. "In fluid, the buds of the shoots float quickly and form, root tissue" become 10 - 12 days the branching roots are torn off and taken out at once «There are very few leaves on the balance sheets and it is very advantageous to plan them in greenhouses. They are then influenced in the known manner, 3® after whether root shoots or mini tubers are to be obtained (Annals öf Botany 53 »565 * 578 0984) 5.

The plants rooted in vitro are grown on solid ground in the greenhouse.

The rooted plants are placed in bundles on the surface of the soil. "To keep the moisture level at the correct level, the plants are covered with polish and the light must be controlled." The plants under polish must be aerated every day, and in the second week, the plants are gradually uncovered in order to adapt the plants to the conditions in the greenhouse.

Three weeks after the plants are used, they grow uncovered and it is possible to prune them.

The plant growth is not so uniform that seedlings can be obtained directly from the plants, so these plants are used as mother plants "from which shoots are obtained." To produce a uniform seedling material, tip cuts (with 3 to 4 buds) are rooted in trays .

The cuts are made on young shoots with 1 - 2 cm long • i, '0 - 10 -

Leaves carried out because the older, overgrown tubers develop and are therefore not suitable for growing rooted seedlings ε / us. The seedlings are rooted under cover and they are grown on the dish for two to five weeks. The seedlings can be planted in greenhouses or in the field in a vector-free environment.

\ '·

The plant obtained from the mini tubers induced during the shoot propagation phase can also be used as a mother plant.

, t

The seedlings that were grown in greenhouses should be transplanted to a vector-free field in such a way that. just the ./ upper leaves of the seedlings are above the ground level. Later, shoots and tubers develop from all underground buds of the horizontally placed plants.

It is advisable to plant the plants separately according to meristem lines in the vector-free environment so that the purity of the variety and the fertility can be checked.

In this way it is possible to separate out bad lines and to select the advantageous properties.

This propagation material can be propagated further in the field using conventional agricultural methods «f

As mentioned above, the cuts obtained from the starlings are trasped in order to obtain tubers. "If no tubers are to be obtained, the goal is to obtain more staram plants from which cuts can be obtained U.N-

Ti i • 11 * become blind in order to increase the päigliglitite »to form shingles * After the tuber formation has considered, the cuts can no longer be rooted effectively *

It was found that the initiation of the formation of the potato tubers is closely related to the beginning of the patina synthesis of the potato. The patatin synthesis that begins in the leaves * is the Yorankiîndigimg dW initiation of the tubers formation · The patatin synthesis and thus the induction or suppression of tuber formation can be followed by an enzyme-linked 'Patatln antiserum analysis *

Based on the control of the patatin synthesis, the tuber formation can be influenced by changing the environmental conditions or by chemical treatment · The patatin synthesis can be done by cooling or by removing the inorganic nitrogen (ammonia, nitrate) or by replacing it with organic nitrogen ^ elle (carbamide) are induced ·

According to one of the exemplary embodiments of the method «which is described in the present invention, the carbon image of the laboratory shoots is induced, so that in vitro mini-tubers with a diameter of a few millimeters are obtained * These mini tubers are very suitable as propagation material In contrast to "seasonal shoots", the mini tubers can be produced continuously all year round, and they can be stored in small quantities in the refrigerator * \

The laughing part of the mini tubers, however, is that they have a very loose consistency, that they vibrate very easily * and that they can be easily damaged * These disadvantages can be eliminated if the in vitro produ- i - 12 - «· * Covered tubers with a protective layer to protect them from shrinkage and mechanical damage * The covered tubers can be spread with a seeder * 3s is worked according to the following procedure% laohdem the mini tubers have been removed from the bottles, the residues of the Stalks are removed, the tubers are gread-. loaned to wash and dry * The (drying can be done on sieve bottom, in one or two layers, at a (temperature of 20 ° C and a humidity of 40%) over about 2 weeks * the (drying must be continued for so long until the original volume of the tubers is reduced by a third «Then the tubers are coated by granulation in the known manner (0.5-0.6 mm) · The granulation can be carried out according to the conventional methods used for the Coating Seeds to be applied * The coated tubers must be dried and stored at a (temperature of around 2 ° - 5 ° 0). No spontaneous genetic changes were found in the course of practicing this procedure.

Design examples;

The following examples illustrate the invention without restricting it.

example 1

Production of virus-free breeding material and in vitro maintenance of cultivars p

Ten pieces of potato tubers, Somogy gyöngye "are washed with a solution of 10% Katrium hypochlorite and 0.1% wetting agent k» - 13 - Ά Λ and, with a hand brush, and they are soaked in the same solution for 30 minutes * After sterilization wash the tubers three times with sterile, distilled water! * and then dry # In order to promote germination, the dry tubers will be soaked in a GA-fj solution (oil-fluoric acid solution) of 10 rag / 1 for 30 minutes » they germinated in a climate box at a temperature of 5 ° 0, a relative humidity of 70-80% and without lighting for 18-22 days until 4-6 cm long shoots were formed after they had been dried beforehand.% The cultures are au mark and register according to tubers and shoots # <

The cut shoots must be sterilized in a solution of 4 tiger sodium hypochlorite * then washed three times with sterile, distilled water, and the tri-tip must be cut under a microscope with a sharp lancet and, as explained above, placed on a nutrient # A shoot tip meristem is placed in a test tube and begins to grow in 3 - 4 weeks at a temperature of 20 ° - 22 ° 0 and a light intensity of 500 lux. # Over the growing crops, the light intensity is increased to 2000 lux, which causes the plantlets develop 5-7 leaves within 6-8 weeks #

The plants must be removed from the tea tubes and placed in a nutrient similar to that mentioned above, but the saecharaose content should be increased to 3% #

A standardized container (400 ml) with a screw cap containing 40 ml of the nutrient (1 plant / container) is suitable for this purpose. At 20 - 22 ° 0 and a light intensity of 3000 lux, all buds form in 6 - 8 weeks , and 4 - 8 cm long shoots develop in the vessel # At this - 4 - H - aom must be checked for the first time, 0¾ the plants are free of viruses *

The virus-infected pine trees are excluded from further cultivation, and the satisfactory shoots are cut into pieces with one or two buds, placed in other grow boxes and pulled in the manner described above. At least three further checks must be carried out in each of these cycles , then the virus-free pine trees can be multiplied * Using the procedure outlined above, unlimited amounts of manzas can be obtained from each pine tree *

Example 2

Production of mini tubers

The plants that were cultivated according to Example 1 are further cultivated in the above-mentioned (liquid) nutrient that does not contain agar * (22 ° C., 3000 dux). The vessels are braided through the plants, and then - after about three weeks - the feeding solution has to be replaced by another one that favors patatin synthesis * This solution differs from the previous one, it contains 10 mg / 1 benzyl adenine, 10 mg / 1 coumarin and 8% sucrose, but only 10 % of the original amount of the inorganic nitrogen source. The cultivation takes place at a temperature of 17 ° 0, 1000 pus over a period of 10 to 12 weeks * At this time, 40 to 60 mini-tubers (0.3 - 0.6 cm in diameter) have developed * The vessel must tip over the plants must be dried, the tubers must be abnormal and can then rest for a week at room temperature (suberification), later they are stored at + 2 ° to 4 ° C in a dry and dark place * - 15 · «* ' «

Example 3

Production of stem plants and seedlings

The plants that were produced according to Example 1 are planted in loose bundles in the greenhouse in a soil that contains a mixture of peat and per lit (500 - 750 plants per m2). The floor must be kept at a temperature of 20 ° C, the air at 24 ° C, and to ensure moisture, the table must be covered with film. The culture must be adapted to the conditions in the greenhouse in 2 to 4 weeks 7 / er den: the plants become stronger, and 7 to 8 leaves develop. This is the parent plant, which prevents the formation of tubers by preventing the patatin -Synthesis is prevented, for which the plants are sprayed with a solution of artificial fertilizer at $ 0.2 micramid «

From this parent plant can in turn top shoots. 4 to 5 leaves can be obtained, and these can be cultivated after rooting, or they can be put on the market as potato seedlings * 80 to 100 seedlings can be obtained from one culture vessel.

Example 4

Production of small tubers

Small tubers can be pulled from the mini tubers according to Example 2 or from the seedlings according to Example 3 in greenhouses or in isolation systems. * Laying or planting takes place in a mixture of peat and pearlite, which contains 0.5 1¾ per carbamide artificial fertilizer (400 Plants / m2) *

The plants must be watered according to their needs at natural temperature and lighting. The supply of sewing materials can take place through the leaves. Hach 4> »- η - {« real weeks the foliage development must be prevented by the use of Qhlor-Ch $ j | in ~ Chlaridf a growth regulator, the irrigation must be stopped so that 85 to SD "-

Hunt after laying 3-4 small tubers (0.5 - 2 # 0 om diameter) Mhinen * i, \ \ • · \ * / / / r

Claims (4)

3. The method according to item 1 or 2, characterized in that the mini-tuber propagation material obtained is coated with a protective layer. 4 · Procedure according to point 1 for the production of potato seedlings as propagation material, characterized in that the grades obtained by in vitro propagation are rooted in vitro in a nutrient, then transplanted into a greenhouse and from stem plants and that setaling propagation material is obtained from these parent plants, while the patatin synthesis of the StamrapAlause is prevented · \ 5 # method according to point t · or 4, characterized * in that the patatin synthesis by changing the environmental conditions or by using a chemical treatment is affected * 6 · Process for the production of small potato tubers "as a nutritional material according to item 1, characterized in that tuber formation is stimulated on the starara plant, which is cultivated from the plants rooted in vitro, or on the mini tubers ·· 7 · Method according to item t or 4, characterized * in that other shoots are cultivated * from the shoots * obtained from the mother plants *. 8 * Method according to one of the items' 1 to 7, characterized in that the potato feeding material may be free of viruses and viroids. are generated. "I 9 *