HU192837B - Method for large-scale production of seed-potato poor in virus - Google Patents

Abstract

In the method according to the invention, in a manner known per se, a meristemic tissue containing a culture medium, e.g. Put in a vial. The medium is sterilized during preparation at about 120 ° C and 1.5-1.8 bar. Breeding vessels containing meristems isolated on the medium, e.g. vials are placed at about 20-25 ° C in a periodically darkened room of 1500-2000 lux and held there until the meristems have 4-6 leaf shoots. Plants of the inertic origin are preferably cut into petal segments in a petri dish and grown on a culture medium containing plant hormones for propagation in a culture room, preferably in 16/8 hour illumination / darkening periods, with 6-8 leaf shoots. They are chopped up again, and the split segment isolates are formed by chopping. The process is repeated until the required number of plants is reached, and the plants in the in vitro phase are shifted and roots are placed in a pellet in an intermediate phase for 14-21 days in insulated microclimate conditions. Thereafter, 8-10 leaves in deciduous state are prepared in soil prepared under torsors, which is enriched with fertilizers supplemented with micro- and macro-elements. In the future, we continue to make up, in proportion to the development of the plants, until we develop tubers suitable for propagation. 10 15 20 25 192837 -1-

Description

The present invention relates to a process for the large-scale production of virus-poor seed potatoes, in which the propagation material is produced from plant seedlings by cell culture.

Potatoes are known to respond sensitively to viral contamination with reduced yield. Due to the contamination of the environment and the large number of aphids that make up the viral vector, viral degradation of the propagation pairs cannot be prevented by the conventional field method.

It is known that the concentration of virus towards plant germinal suppositories, even in the case of virus-infected plants, is greatly reduced. No virus is detected in the meristematic cells or tissues themselves.

The rapid advancement of tissue culture techniques has allowed the isolation of this tissue of the plant and its in vitro cultivation to produce a complete virus-free plant.

It is an object of the present invention to provide a propagation cycle and system that allows large scale production of virus-free or virus-free potato tubers based on tissue culture.

SUMMARY OF THE INVENTION It is an object of the present invention to utilize the results of meristems cultivation and to further develop them on the basis of large-scale potato growing technology, and to provide a virus-free or virus-free potato tuber commercially available and a virus-free variety.

The invention thus provides a method for the large scale production of virus-poor seed potatoes, which comprises the selection of virus-free or virus-free tubers of the variety, based on bioassay serological assays, and cultivation of internodium shoots at room temperature without illumination. About 3 to 6 bud-bud shoots are detached from the tubers, and each tip shoot and bend starter is used to isolate a meristoma by placing the meristem tissue in a culture medium, e.g. vial. The medium was prepared at about 120 ° C and

Sterilize at 1.5-1.8 bar. Breeding vessels containing meristem isolated on the medium, e.g. vials, about

It is placed at 20-25 'C, 1500-2000 lux illuminated, periodically darkened, and kept there until the meristem has 4-6 leaf shoots. Thereafter, the merlemae-derived plants are suitably sliced into nodal segments in the Petri dish and grown on growth media containing plant hormones in a culture room, preferably for 16/8 hour illumination / darkening periods.

6-8 leaf shoots are grown. They are again sliced and sliced to form new nodal segment isolates. This process is repeated until sufficient plant slag has been reached and then the plants formed in the physiological in vitro phase are planted in rooting soil in an intermediate phase for 14 to 21 days in an isolated phase suitable for microclimate formation. This is followed by the transplanting of the plants from the propagation beakers with soil ball, in soil prepared under an isolation tent with 8 to 10 foliage leaves - enriched with fertilizers containing micro and muco elements. In the following, we continue to charge fc commensurately with the development of the plants until we develop tubers suitable for propagation.

In a preferred embodiment, the tubers obtained from plants of in vitro origin are further propagated in two cycles, in isolator tent, in viral vectors, and virologically inspected, followed by desiccation and seeding of the tuber when seedlings develop.

The present invention will now be described in more detail without limiting the scope of the invention to the embodiment.

The tubers of the plants, which were grown under vector-free conditions, were virtually virus-free, visualized by bioassay and serological (ELISA) and were treated with Rindilievel, which stimulates the germination potential.

The treated tubers are kept at room temperature without illumination at 80-90% relative humidity, while the tubers develop long internodiumoB cell shoots of 6-10 cm, with at least

3-6 bud buds are observed. Shoots in this condition are separated from the tubers and washed thoroughly in running tap water for 2-5 minutes.

After washing, shoots are placed in aseptic glass jar, filled with 70% ethyl alcohol, and kept under shaking for 1-1 1/2 minutes.

Following the treatment with ethyl alcohol, the alcohol was poured and a 0.1% HgCl solution was added to the meristomas remaining in the glass jar. Depending on the size and maturity of the shoots, HgCl treatment is applied for 1-2 minutes with constant shaking. The sterilization solution is then discarded and the shoots are rinsed three times with sterile distilled water. During each rinse, with sterile shaking, keep sterile distilled water on the shoots for 2-3 cartridges.

Surface sterilization of shoots should be performed in aseptic laminar boxes from the start of treatment with 70% ethyl alcohol.

Hereinafter, the * meristene is isolated. The surface-sterilized shoots were kept in a sterile container under a luminous box until isolation began.

Λζ we use the propulsion chute of some shoots now, using the shuttle noduBzou located underneath, to isolate the meristem.

The sterile microscope is used to gradually remove the meristematic leaflets under the stereoscopic microscope. The thus released, 0.3 to 0.5 mm diameter, single-stem meristem tissue with a very rudimentary leaf primordium is removed from the surrounding tissue by sharp cutting perpendicular to the drive axis and placed in a culture vessel (vial) prepared as described below. Each meristem comes in a separate vial. The whole isolation operation is carried out in a laminar box.

The medium is a modified Murashige-Skoog medium and is prepared as follows:

Trace element trace solution: mg / 100 ml HjBOj 620.0 MnSO 4 4 H 2 O 2230.0 ZnSCU 2H ₂ O 860.0 NaaMoO * · 2H ₂ O 25.0 CUSOt · 5HxO 2.5 CoClt · GthO 2.5 Ee ₂ Na ₂ EDTA lotion solution: mg / 100 ml EeSO <7H ₂ O 278.0 NI EDTA 373.0 KJ Torso Solution: mg / 100 ml KJ 75.0 Vitamin body page: mg / 100 ml Nikolinsav 10.0 Thiamin HCl 100.0 Pyridoxine HCl 10.0 Moro-inositol. 1,000,0 CaCb stock solution: mg / 100 ml CaCl ₂ · 2H ₂ O 15000.0 BA (benzioladenine) stock solution: mg / 100 ml BA 100.0 CA pantothenate bulk solution: mg / 100 ml CA-pantothenate 100.0 The following trace elements are added to 150 ml of Dissolve in water: NiUNOj mg / 100 ml 1600

1900

MgSÜ «· TiiiO 370

KibPO. 170

Add the following amounts of stock solutions:

Trace element solution ι _n .i

CaCl 2,9 ml

KJ '1.1 ml

Fej-Naj EDTA ,. 10.0 ml

Vitamin 10.0 ml

Ca-Pantotc torso solution 2 ml / (

BA 0.5 ml / l

GA:> "0.5 ml / l

The modifiers thus prepared are dissolved in 30 g of sucrose and then dissolved. Make up to 100 ml.

The pH of the solution was adjusted to 5.8 by addition of 0.1 π HCl or 1 N NaOH as needed.

Dissolve g of agar-agar (Difco) in 100 ml of water by heating.

After complete dissolution of the agar-agar, it is added to the pre-prepared medium and made up to 1000 cm 'by addition of bi-distilled water. Stirring ensures perfect homogeneity of the solution.

After mixing, dispense from the medium into sterile vials 9 cm high and 25 mm in diameter, pouring into 10 ml-L. The meadow vial is sealed with an aluminum foil cap.

The culture medium vials are sterilized in an oval oven at 120 ° C for 1.5-20 min at a pressure of 1.5-1.8 atm.

After aeration, the vials are kept at room temperature until the medium is completely solidified and used.

The vials containing the meristomas isolated on the culture medium are placed in a culture room at 20-25 ° C and 1500-200 lux illumination. The length of the light period is 16 hours and the dark period is 8 hours per day.

The flavor should be maintained under these conditions until 4-5 of the inert systems develop into shoot shoots. If the duration of the enr.ek is greater than 0 weeks, then, after 4 weeks of merestenii, n is sprayed onto fresh medium of the composition described above. If necessary, the passage should be repeated every 3-4 weeks.

This is followed by micro-propagation of plants of meristema origin.

The medium for microsuphorization was prepared in the same manner as described for the preparation of Lapululus for the isolation of the meristem. Λ The only difference is that the plant hormones (U / és and G-í) are added to the culture medium so that its final concentration is as follows:

BA (benzyladenine) 0.5 mg / L

GAj (gibberellin) 0.2 mg / L

The culture medium is autoclaved and dosed as described above.

In the following, micro-reproduction is carried out as follows;

The 4- to 8-leaf shoots developed from the meristemis isolates are separated by sterile means under a laminar box (providing aseptic conditions), removed from the vial and placed in a sterile Petri dish.

The shoots are also cut with sterile cleavage lance to form so-called nodal segments.

The shoots are cut in such a way that the shoots are cut above and below each leaf row - the shorter part of the stem above the leaf floor, the longer one below - cut perpendicular to the axis of the shoot. Leaves on the resulting drive segment should not be removed.

The entire drive shaft is cut as described. The Bzegment pieces are individually or more conveniently placed in a vial containing the medium for microsporation. The shoot segments are placed in the medium so that the basal shoot underneath leaves reaches the level of the leaf origin.

After inserting the isolate, cover the vial with an aluminum foil cap.

Naturally, the shoots must be removed and cut under aseptic conditions (laminar box, sterile cabin, etc.), and the sterility requirement must be carefully observed.

The vials containing the isolated shoot segments are maintained at 20-25 ° C with a minimum light intensity of 2000 lux with 16/8 hours light / dark period in the culture room. The isolates are cultured under these conditions until an 8-10 cm long, 6-8-leaf shoot is formed from the dorsal fin.

The shoots of such development are again cut according to the method described above and made into isolated segment isolates.

The process is repeated - the nodal segment-plant-kennal segment plant - until the required number of plants is reached.

Plants of nodal germ cell cultures, in vitro shoots and roots, should be kept under controlled environmental conditions for 14 to 21 days (this is called the intermediate phase).

Materials and devices used in the intermediate phase:

(a) an insulated glasshouse with a sluice-gate housing:

- evaporator with automatic regulator

- microclimate equipment

- radiators with regulating valves

(b) propagation table

(c) propagation glasses

d) crazy perlite

(e) garden land

(f) a fungicide with the active ingredient mancozeb and bonomyl.

The commercial garden seedling is filled into a perforated propagation cup at the bottom, watered with a solution containing 0.16% mancozeb and 0.05% benomyl, and the soil is kept moist until planting.

The roots of the plants extracted from the vials are washed with tap water and the plants are planted in the prepared soil in a propagation glass so that no more than 3-4 foliage leaves are placed above the soil surface. The planted plants are e.g. Watered with Hoagland's medium.

The plants, together with the propagation cup, are embedded in moistened perlite on the tables.

Plants are particularly sensitive to changes in environmental conditions during the intermediate phase, so constant temperature, humidity, and illumination time should be provided for the following values:

temperature: 22/17 ° C, 16/8 day period illumination: 15,000 lux, 16 hour continuous illumination relative humidity for plants

95-100% for 4-5 days.

From now on, a reduction value of 60% should be maintained until planting.

The soil of the plants and the perlite should be kept moist during the intermediate phase. The plants are treated, e.g. Water with Hoagland solution.

Plants during the planting period a

They must reach a level of 8 to 10 leaves.

Following the intermediate stage, the plants must be grown under isolated conditions to obtain virus-free propagation material of appropriate quality. For this purpose we use an insulating spool.

The frame, with a span of about 7.5 m, is mounted on a concrete base parallel to the longitudinal axis of the tent. At the beginning of the isolation ridge, a sluice-gate of at least 3.0 lengths shall be provided, bounded on one side by a door opening to the potato and on the other. The substrate of the sluice-gate must be covered with cement or concrete for cleaning.

Doors and frames enclosing the sluice-gate section shall be insulated in such a way as to prevent any gap between the apertures and other insects.

The frame thus prepared is covered with a 0.3 mm mesh insulator screen.

The 3.0 mm wide insulator mesh is sewn from four parts to 12 m wide to cover the entire circumference of the frame and to be grounded. The core of the insulating mesh can be covered with plastic foil to prevent premature wear.

An irrigation system consisting of PVC tubing and impact nozzles shall be mounted on the frame on both sides so that the two irrigation wings supply the entire surface of the tent with irrigation water. If necessary, a drip or other irrigation may be used instead of irrigation irrigation.

At the bottom of the isolation tent, a 1: 3 mixture of peat and sand is laid in a 35 cm thick layer.

Ai / onto the soil uniformly spread over the mortar dazomet insecticidal active substance is incorporated into 50 g of ^one dose. After application, the soil is thoroughly watered and covered with airtight plastic foil.

After two weeks, remove the mulch foil and ventilate the soil by rotating it several times for another two weeks. Then use bioassay methods (cerebral digestion of lettuce or white mustard) to ensure that the soil is free of harmful residues.

Commercially available Volldünger fertilizer is applied at 0.1 t / ha to ensure adequate nutrient supply to the plants.

In addition to Volldünger fertilizers, ie fertilizers with macro and micro elements, phosphorus and potassium fertilizers must be added. Potassium should only be administered in the form of potassium phosphate.

From Bzaporito glasses, the potato plants, together with the ground ball, are planted in the soil of the isolation tent so that the upper 3-4 leaves of the plants are placed above the soil surface. The row spacing of the herd should be set to 70 cm and the spacing to 22 cm.

After planting, the herd is watered. During planting, the plants are separated by line and subsequently kept open.

As the plants evolve, the rows are continuously filled until a ridge of 23-27 cm in height and 45-50 cm is reached. ·

Unlike tuber propagation, the stock must be watered continuously from the time of planting. It is preferable not to dispense more water artificially at one time, but to keep the soil in a constant wet state.

The critical period for irrigation is the period after planting, ranging from tubing to the beginning of the lesson.

The needs of the pond water for the construction of the irrigation system should be designed so that if necessary, 1500 m ^{3 of} water per hectare can be sprinkled during the growing season.

The stock shall be chemically protected from the appearance of aphid aphids, from the inception of aphids to loin-sealed aphids, from the first viral vector captured in Moerich's yellow foxes.

From the 10 cm state of the plants, each plant unit should be visually inspected every two weeks. All plants showing abnormal lesions and phenotypically different plants should be selected for visual examination. Material selection must also be removed during selection.

During the 4th week after planting the plants should be sampled line by line and assayed by ELISA (Enzyme-linked Immunoassay Assay). If the virus infection of the line is confirmed by the above method, the line shall be selected.

The tubers obtained by growing in vitro plants are propagated in two cycles, one spring and one summer. Autumn oil harvesting, organic and fertilization shall be carried out on the entire land necessary for propagation as described in large-scale technology. Beginning in the spring season, half of the area is cultivated as black fallow till summer planting and the other half is prepared for spring planting according to the previous technology.

Before planting, tubers are seeded with 3000 g / t Chinoin Fundazol or Topsin-methyl.

Planting is done with 4 rows of Cramer SH-3001, Hassia GLB-42, or 4-SABPD planters in a 75 cm row, with 8 rows per row. A band of at least 3 m in width is omitted between the tracks. The transplanting machines make the so-called coins in the same coins as planting. primary rabbits. The final rake formation is done shortly after planting with the Van Rumpt SFZ-5 type milling machine. Following sedimentation of rushes, a pre-emergent herbicide spray is performed with a field sprayer (which may be Novor-1005) using known single and kernel combinations.

In order to prevent the emergence of emerged plants and viral vectors, 6 m wide lanes of 8 m should be covered with an isolation tent between planting and emergence. For this purpose, the skeleton described above must be mounted above the strips. Λ strips drill holes on both sides, corresponding to the wingspan spacing, at a width of 7,5 m and a distance of 1,5 m from each other, with holes of 80 cm depth corresponding to the frame arches.

-51 I ground. On the erected and assembled frame, the irrigation equipment consisting of the PVC pipe and the impact nozzle described above is mounted. The frame shall be designed and erected in such a way as to secure the two-door, insulated sluice-gate space.

All parts of the insulator set are sprayed with contact insecticide after the insulation has been completed.

The herd is continuously protected against phytophthora from the time of Myzus perzicae caught in the isolation house area from the time of the foliage closure.

From the start of tuber formation, the soil is kept moist by irrigation.

Negative selection based on visual inspection of the plants shall be carried out continuously, but at least three times, starting from the 10 cm state of the plants. During this time, all plants showing symptoms of virus infection should be removed from the herd, together with the materialum. At week 3 post-treatment, flocks should be randomly sampled per line and examined by a reliable virological method (ELISA bioassay). In case of a positive result, remove the plant and, if necessary, the entire line.

The test should include a phenotypic examination of the herd. If phenotypic (different developmental) plants are present in the herd, they should be selected.

Once the tubers have reached the optimum ratio of tuber size under the roots in the isolation barrier, we begin to dismantle the isolation tents, which are completed by crushing the potato shoots immediately with a mechanical shredder or chemically. Following this crushing, we carry out chemical decontamination with Ms S. EC.

Following stalk crushing 10-14. On the day when the potato tubers become sufficiently damp, we carry out the harvest using a towed or self-propelled harvester.

The tubers are stored at a temperature compatible with the technology.

The tubers, which were heated to 14-16 ° C before planting in the cold store, are planted in properly prepared soil.

The planting date should be scheduled for the first decade of July. After preparation for the installation of the vases, the isolation passages from the spring planting areas are moved to the summer planting areas. The work is done during the period between the planting and the treatment, as in the case of spring planting, as in the lilies. Care, irrigation, virological monitoring, selection and harvesting of the herd should be carried out in the same way as spring planting.

Propagation of seed potatoes produced in the spring and summer cycles will be repeated in the spring next year, and a difference in the type of irrigation can be suggested.

Another difference is in the virology control system. In both cycles, standard selection and. of the produced marrow guinea pigs according to the standard.

An advantage of the present invention is to obtain varieties of seedless, virus-free seedlings which are suitable for climatic conditions and which yield the best yield under ndotl conditions.

Claims (2)

I. A process for the large-scale production of seed potatoes of virusszegériy, which is known to be placed in a culture vessel, e.g. vial, containing meristemat broth in a manner known per se, sterilized in the medium at about 120 ° C. ) culture pots, e.g. vials are placed in a periodically darkened room at about 20-25 'C, 1500-2000 lux illuminated, periodically darkened, until the meristem has developed 4-6 leaf shoots, then the meristem-derived plants are preferably cut into petri dishes containing nodules and containing plant hormones. 6-8 leaf shoots are grown on propagation medium in culture room, preferably in 16/8 hour propagation / darkening periods, characterized in that the shoots from said 6-8 leaflets are sliced to form additional nodal segment isolates, and the process is repeated until sufficient plants were reached, and the plants formed in the in vitro phase were shoots and roots in insulated microclimate for a period of 14-21 days in an intermediate phase, followed by 8-10 l. - in omblevcles state, in soil prepared under an isolator tent, - which is preferably enriched with fertilizers supplemented with micro and macroelements - - we transfer plants from the swabs to the soil of the isolator trough, tubers obtained from in vitro propagated plants are optionally further propagated, preferably in two cycles, in additional isolator housings or under similar conditions in lanes which exclude the presence of viral vectors under virological control of the herd. harvested and stored until storage. -619 288 2. Λ: ·. The β-Zorint method according to claim 1, characterized in that, for the cultivation of the meristem, virus-free or virus-poor tubers of the species are selected by bioassay and serological assay, cultivated at room temperature without illumination, and cultured for about 3 hours. ————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————