EP0726702A1 - Soilless propagation method and device - Google Patents

Soilless propagation method and device

Info

Publication number
EP0726702A1
EP0726702A1 EP94928735A EP94928735A EP0726702A1 EP 0726702 A1 EP0726702 A1 EP 0726702A1 EP 94928735 A EP94928735 A EP 94928735A EP 94928735 A EP94928735 A EP 94928735A EP 0726702 A1 EP0726702 A1 EP 0726702A1
Authority
EP
European Patent Office
Prior art keywords
tuberization
cuttings
carried out
vitro
seedlings
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94928735A
Other languages
German (de)
French (fr)
Inventor
Alain Billet
Jacques Viseur
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0726702A1 publication Critical patent/EP0726702A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A01G31/02—Special apparatus therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures

Definitions

  • the present invention relates to a process for obtaining plant reproductive organs combining in vitro culture and above-ground multiplication of seedlings, bulbs, bulbils, rhizomes and tubers (minitubercules). This process finds an application in obtaining seed or pre-base plants of irreproachable phytosanitary quality, in particular in the context of the cultivation of potatoes.
  • the invention also relates to a device for implementing the method.
  • Processes of multiplication and tuberization are known by in vitro techniques, the only techniques which can currently guarantee the production of seedlings, tubers or other organs of vegetative reproduction free of pathogenic agents such as viruses, viroids, bacteria, fungi or nematodes by example.
  • microplantules obtained in vitro are either acclimatized in a greenhouse, or maintained in in vitro culture for the production of reproductive organs (bulbs or microtubercules).
  • microtubers In the particular case of the potato, for example, the methods of in vitro production of microtubers have been described frequently, which consist in stimulating tuberization after several weeks on the stems of microplantules by transferring the microplantules into a nutritive medium, generally used in liquid form, containing a higher concentration of sucrose than in multiplication media and / or containing a complement of biologically active substances such as hormones (cytokinins), growth retardants (CCC), absorbers ethylene or by applying carbon dioxide C0 2 (patent EP 476,141), the seedlings then being placed under a diffuse light or at
  • micro-tubers These processes are characterized by a low yield of micro-tubers, by small microtubers (2 to 7 mm in diameter), by a high dormancy of 4 to G months and by a limited reserve of organic and mineral substances in them . They are often cultivated in a greenhouse before sowing in the field in order to obtain mini-tubers.
  • Patent EP-A-0 476 141 Process for producing tuber (1991).
  • mini-tubers obtained after a subculture in a greenhouse or under shelter of the seedlings or microtubers from Vin vitro.
  • techniques have been proposed for increasing the rate of production of minitubercules in soil (patent WO 88/04137) by transplanting propagated plants to the greenhouse and having undergone an induction treatment in vitro. The plants are treated with a culture medium containing hormonal additives, in order to promote the development of mini-tubers, which can be harvested G at 8 weeks after transplantation.
  • plants are never immune to contamination, since the culture is done either in soil or in growing medium in the greenhouse.
  • the method according to the invention makes it possible to increase the crop produced by seedling, to reduce the duration of the process of obtaining this crop, to arrive precisely at a seed with the desired diameter, to ensure the aptitude for seeding in the open ground and obtaining crops all year round by radically modifying the propagation techniques and the known conditions of implementation.
  • the present invention consists in multiplying and inducing above-ground tuberization by an aero-hydroponic technique, to replace the techniques of in vitro culture in the multiplication and tuberization phases.
  • This new aero-hydroponic technique for producing seedlings, bulbs, bulbils, rhizomes and tubers, guarantees quality health of equipment at costs significantly lower than those of products derived from in vitro techniques.
  • the method according to the invention is characterized in that it comprises a first step of in vitro propagation of cuttings from the merisrasi, a second step of multiplication and elongation of said cuttings in order to obtain seedlings then a third step of tuberization of said seedlings, the second and third stages being carried out in aero-hydroponics with a nutritive solution substantially identical to the solutions suitable for the conditions of culture in vitro, with the proviso that it is substantially free of an assimilable carbon source.
  • meristem is generally meant the plant tissue formed of undifferentiated cells, seat of rapid and numerous divisions, located in the growth regions of the plant.
  • assistant carbon source in particular all the sugars, in particular sucrose, glucose.
  • the amount possibly present is very much lower than that normally used in solutions for in vitro cultures. At least 10 times less. Preferably at least, 50 times less.
  • the solution may not contain said assimilable carbon source at all. In other words, plants are such that they work in autotrophic conditions.
  • the nutrient solution is also remarkable for the fact that it contains an effective amount of vitamin, in particular thiamine HC1, pyridoxine, glycine, casein hydrorysat.
  • this composition will include the anions and cations commonly used, in particular the ingredients of the formula of Muraschige and Skoog (1962) added with a mixture of amino acids obtained by acid hydrolysis of casein, for example the cations, potassium , calcium, magnesium, sodium, iron, copper, zinc, cobalt, nitrate, sulfate anions etc.
  • this composition will comprise in part by weight: Ammonium nitrate 1650 mg
  • Nicotinic acid 0.5 mg
  • hydro-hydroponic denotes a method of hydroponic culture in which the water loaded with different additives which allow the growth of plants is conveyed in nebulized form.
  • the cuttings have a portion of stem and carry at least one node, preferably two nodes, that is to say approximately 10 to 20 weeks after the differentiation of the meristems, these are subjected to the step of multiplication and elongation.
  • a first multiplication can be carried out in vitro using techniques identical to those described in the above-mentioned prior art.
  • the cuttings obtained at the end of the first phase are placed on a support, in particular in synthetic foam, preferably sheets of polyphenol foam, under suitable lighting in particular from 60 to 150 ⁇ E / m 2 / s at 16 hours of light, a knot that can be inserted into the openings for this purpose in the support.
  • the cuttings are cultivated on the support until at least one axillary stem has been formed, the apical portion of which is transplanted in order to obtain a complete seedling comprising at least five nodes.
  • the intermediate portions of the axillary rod comprising at least one node are transplanted to form new axillary rods.
  • the apical and intermediate portions of the new stems are treated as above: this operation is repeated the appropriate number of times.
  • the apical buds are removed approximately two to three weeks after transplanting.
  • the nutritive solution, during the second stage, is that used for the propagation of the cuttings, possibly modified by additives to favor the elongation of the cuttings and the appearance of the axillary stems, in particular benzyladenine (B, A) and 2 -isopentenyladenine (2-iP).
  • B, A benzyladenine
  • 2-iP 2 -isopentenyladenine
  • the tuberization stage can be carried out according to two methods. According to a first method, the tuberization stage is carried out with a nutritive solution of reduced content of nitrogenous compound and under conditions of short days or long days, depending on the physiology of the variety used.
  • the leaves of the pre-induced plants are removed with a portion of the stem and transplanted onto the support.
  • the tuberization is carried out with a nutritive solution comprising the elements described previously with the exception of ammonium nitrate and disodium EDTA.
  • the method according to the invention is particularly suitable for obtaining mini-tubers of potatoes.
  • a variant of this process relating to the cultivation of potato cuttings is described below.
  • Cuttings of seed potatoes from meristems in vitro are placed on polyphenol foam plates in an enclosure fed by spraying with a culture medium containing the components of the culture medium used for the initial propagation of the seedlings, optionally supplemented by additives promoting growth and transfer into non-sterile conditions of explants.
  • Each cutting comprises a portion of stem carrying two nodes, one of them being pressed into the polyphenol foam support.
  • the cuttings are placed under a lighting of 60 to 150 ⁇ E / mVs at 16 hours of light.
  • the seedlings After three weeks, the seedlings have formed one or two axillary stems with at least five nodes.
  • the axillary stems obtained are divided into three: two portions comprising two nodes and a portion comprising a node and the apex.
  • the portions comprising two nodes are transplanted as above and form the cuttings of type A.
  • the apical portion, also transplanted in the support in polyphenol, constitutes a cuttings of type D.
  • the type A cuttings can be successively multiplied while the type D cuttings, form after 15 days a complete seedling, of a size greater than 15 cm, comprising more than five nodes.
  • the elongated plants of a size greater than 15 cm are transplanted into a new support under conditions of short days at 8 hours of light under a lighting of 60-150 ⁇ E / n.2 / s for 10 to 21 days.
  • various inducers of tuberization can be used (jasmonic acid, salicilic acid and derivatives, coumarin, ancymidol as well as growth retardants (CCC, Alar, TIBA) or ethylene absorbing agents such as permanganate.
  • Alar succinic acid 2-2 dimethyl hydrazine
  • CCC chlorocholine hydrochloride
  • carbon dioxide (CO 2 ) at a concentration of 1 to 40% can also be applied in the culture chambers, in particular at the level of the roots after the induction phase of the tuberization, the plants can be maintained in short days on a medium of the same composition, except for a reduced content of nitrogen compound.
  • the minitubercules therefore form, after six to nine weeks, on the runners which develop in the lower part of the aero-hydroponic device.
  • the leaves of the pre-induced plants are removed and cut with a portion of stem, to be transplanted, vertically on the polyphenol substrate.
  • the plants are placed in continuous light or in long day, under aero-hydroponic conditions, according to the procedure described by EWING E.E. on leaves of plants in culture in vitro (Potato physiology, 1985).
  • the induced tuberization on the seedlings or leaves is carried out in an air-conditioned room by modifying the photoperiod and the temperature.
  • the tubers are harvested as soon as they have reached the desired size.
  • the harvest continues for 10 to 12 weeks, depending on the species or variety. Continuous removal of part of the tubers promotes tuberization.
  • the photoperiod for the tuberization of the seed potatoes is then 12 to 20 hours for a temperature of 15 to 25 ° C, depending on the variety used.
  • 1 or 2 mini-tubers are collected at the base of each leaf after 15 to 18 days.
  • the multiplication and tuberization cycles for the seed potatoes are carried out in air-conditioned rooms which eliminate any risk of contamination by aphids.
  • the nutritive solution recycled and subjected to UV radiation as well as the absence of soil or potting soil guarantees the sanitary property of the products.
  • the apparatuses used for the culture above-ground can be of different types.
  • a device particularly suitable for implementing the method according to the invention comprises a container on which a culture support is placed, said container containing inside a nebulizer, connected to a means for supplying a nutritive solution and the container being connected to a means for discharging the nutrient solution, said supply and discharge means being connected to a nutrient solution tank and to one or more control means.
  • FIG. 1 represents a vertical section of a production channel and the figure 2 shows schematically a device for implementing the method according to the invention.
  • Figure 1 shows a cross section of a channel 2 for producing seedlings 1 and tubers or bulbils 18.
  • the nutrient solution is distributed by means of line 17 and is nebulized at the root part of the seedlings.
  • the seedlings 1 are arranged above a channel 2, on and through a support 16, their lower parts being in the channel, and are fed 23 periodically with a solution mist.
  • nutrient pressurized by a pump 4 and distributed by a nebulizer 17.
  • the excess solution is recycled 24 and sterilized by a UV device before returning to the tank 5 containing the nutrient solution 3.
  • the tubers or bulbils produced will appear in channel 2 and are fed in the same way as the seedlings 1. Compensation for losses of reservoir 5, in water and nutrients is done automatically, by level sensors 6 and / or concentration 7 and 8 and regulating organs 9 and 10.
  • a group of metering pumps 14 pours into the reservoir 5 of nutritive solution 3 the quantities of acid and nutrients 11 and 12 calculated by the regulators 9 and 10.
  • a sensor 21 making it possible to measure the concentration of C0 2 acts on a regulating valve 20 which injects, from a compressed CO 2 reservoir 22, the required quantities of CO 2 into the channel 2 for producing seedlings.
  • the control unit 15 manages the correction of the pH, the conductivity of the CO 2 concentration and other level. It can also manage the entire circuit in all phases of the process.
  • the multiplication is carried out in an air-conditioned room at a temperature of 20-24 ° C, the cuttings receive an assimilation illumination of approximately 60-100 ⁇ E / mVs, thanks to fluorescent tubes, with a photoperiod of 16 hours.
  • Each transplanting of autotrophic cuttings is carried out at regular intervals.
  • the culture medium is renewed every three weeks.
  • the aim of the test was to compare the quality of the potato seedlings and the yield of the micropropagation carried out according to the process of the invention on supports of polyphenol foam and in autotrophic condition, compared with the conventional technique in vitro.
  • the yields as a function of the type of cuttings cultivated were compared: a) 1 node provided with an axillary bud inserted into the culture substrate, b) 1 node provided with an axillary bud placed outside the substrate of culture, c) 1 upper node provided with a terminal bud.
  • This test is carried out with a view to highlighting the advantage of developing an automatic method of cutting or chopping seedlings to transplant under industrial production conditions.
  • the culture support is brought to saturation, then the spraying of the culture medium is stopped.
  • the capillary retention of the substrate used makes it possible to space two saturation cycles over a period of 8 hours.
  • Lengthening, vigor, leaf development and growth speed are favored in non-sterile conditions.
  • the conditions of culture were the same (temperature of 24 ° C and light of 60 ⁇ E / mVs for a photoperiod of 16 hours.
  • Plants obtained under non-sterile conditions had, on average, a height of 10.9 cm of had 9.5 leaves while those cultivated under sterile conditions had a height of 9.4 cm and had 6.3 leaves.
  • transplanting process and the type of explant determines the quality of the plants obtained at the end of the culture.
  • Type of cuttings put total length of the Average number of P u rce n of Coefficient of in crop planting (cm) leaves per seedling developed seedlings multiplication (*) after transplanting
  • the multiplication coefficient is defined as the number of axillary buds developed per cutting, after transplanting.
  • the apical segments also allow a multiplication greater than or equal to 4 in two weeks of culture.
  • the supports were placed at a temperature of 23 "C under the condition of short days (8 hours of light). Under these conditions, after three weeks of incubation, the formation of minitubercules is observed on the stems of the plants located in the part bottom of the device, kept in the dark
  • the quantity of minitubers produced was between 0.5 to 3 tubers per plant for a size varying from 0.7 to 1.5 cm.
  • Tests for the production of minitubers were carried out by comparing techniques 1 and 2 described above. Plants from type A and type D cuttings were placed in induction conditions in short days (8 hours of light) for 3 weeks on the tuberization medium, then placed under 20 hours of light, at a temperature of
  • Method 1 Type A cuttings 59.6 225 plants whole plants

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Hydroponics (AREA)

Abstract

A method for obtaining plant reproductive organs, comprising a first step wherein cuttings are propagated in vitro from meristems, a second step wherein said cuttings are propagated and lengthened to give seedlings, and a third step wherein said seedlings are grown into tubers. The second and third steps are carried out under aero-hydroponic conditions with a nutrient solution that is substantially identical to solutions suitable for use under in vitro culture conditions. Said method is particularly suitable for preparing potato minitubers. A device for carrying out the method is also disclosed.

Description

PROCEDE ET DISPOSITIF DE MULTIPLICATION HORS-SOL METHOD AND DEVICE FOR ABOVE GROUND MULTIPLICATION
La présente invention concerne un procédé d'obtention d'organes de reproduction de végétaux associant la culture in vitro et la multiplication hors sol de plantules, de bulbes, de bulbilles, de rhizomes et de tubercules (minitubercules). Ce procédé trouve une application dans l'obtention de semailles ou de plants de pré-bases de qualité phytosanitaire irréprochable, notamment dans le cadre de la culture des pommes de terre. L'invention concerne également un dispositif pour mettre en oeuvre le procédé.The present invention relates to a process for obtaining plant reproductive organs combining in vitro culture and above-ground multiplication of seedlings, bulbs, bulbils, rhizomes and tubers (minitubercules). This process finds an application in obtaining seed or pre-base plants of irreproachable phytosanitary quality, in particular in the context of the cultivation of potatoes. The invention also relates to a device for implementing the method.
On connaît les procédés de multiplication et de tubérisation par les technique in vitro, seules techniques pouvant actuellement garantir la production de plantules, de tubercules ou autres organes de reproduction végétative exempts d'agents pathogènes tels que virus, viroïdes, bactéries, champignons ou nématodes par exemple.Processes of multiplication and tuberization are known by in vitro techniques, the only techniques which can currently guarantee the production of seedlings, tubers or other organs of vegetative reproduction free of pathogenic agents such as viruses, viroids, bacteria, fungi or nematodes by example.
Actuellement, le point de départ de toutes les filières de production de semailles, de boutures saines ou plants de pré-base est le processus qui consiste à utiliser des plantes microbouturées in vitro de diverses espèces assainies contre l'infection virale par exemple par culture de méristèmes, ensuite de les développer et de les multiplier par clonage jusqu'au nombre désiré de microplantules. Toutes ces étapes se font sur un milieu enrichi d'une source de carbone (glucose, saccharose) et en condition d'hétérotrophie et de stricte stérilité : désinfection des plantes, autoclavage des milieux nutritifs et de la vaisselle de culture, travail de repiquage sous hotte à flux laminaire, air stérile, instruments désinfectés, bocaux de culture fermés.Currently, the starting point for all production chains for sowing, healthy cuttings or pre-basic plants is the process which consists in using microbouturé plants in vitro of various sanitized species against viral infection for example by cultivation of meristems, then to develop and multiply them by cloning to the desired number of microplantules. All these steps are carried out on a medium enriched with a carbon source (glucose, sucrose) and under conditions of heterotrophy and strict sterility: disinfection of plants, autoclaving of nutrient media and culture dishes, transplanting work under laminar flow hood, sterile air, disinfected instruments, closed culture jars.
Les microplantules obtenues in vitro sont soit acclimatées en serre, soit maintenues en culture in vitro pour la production d'organes de reproduction (bulbilles ou microtubercules).The microplantules obtained in vitro are either acclimatized in a greenhouse, or maintained in in vitro culture for the production of reproductive organs (bulbs or microtubercules).
Dans le cas particulier de la pomme de terre, par exemple, on a décrit fréquemment les procédés de production in vitro de microtubercules qui consistent à stimuler la tubérisation au bout de plusieurs semaines sur les tiges de microplantules en transférant les microplantules dans un milieu nutritif, utilisé en général sous forme liquide, contenant une concentration plus élevée de saccharose que dans les milieux de multiplication et/ou contenant un complément de substances biologiquement actives telles que des hormones (cytokinines), des retardateurs de croissance (CCC), des absorbeurs d'éthylène ou par l'application de gaz carbonique C02 (brevet EP 476 141), les plantules étant ensuite placées sous une lumière diffuse ou àIn the particular case of the potato, for example, the methods of in vitro production of microtubers have been described frequently, which consist in stimulating tuberization after several weeks on the stems of microplantules by transferring the microplantules into a nutritive medium, generally used in liquid form, containing a higher concentration of sucrose than in multiplication media and / or containing a complement of biologically active substances such as hormones (cytokinins), growth retardants (CCC), absorbers ethylene or by applying carbon dioxide C0 2 (patent EP 476,141), the seedlings then being placed under a diffuse light or at
ENT l'obscurité.ENT darkness.
Ces procédés sont caractérisés par un faible rendement en micro tubercules, par de microtubercules de petite taille (2 à 7 mm de diamètre), par une dormance élevée de 4 à G mois et par une réserve limitée de substances organiques et minérales dans ceux-ci. On pratique souvent leur culture en serre avant l'ensemencement au champ en vue d'obtenir des minitubercules.These processes are characterized by a low yield of micro-tubers, by small microtubers (2 to 7 mm in diameter), by a high dormancy of 4 to G months and by a limited reserve of organic and mineral substances in them . They are often cultivated in a greenhouse before sowing in the field in order to obtain mini-tubers.
De nombreuses publications décrivent ces techniques habituellement utilisées de micropropagation et de microtubérisaiion in notamment :Many publications describe these commonly used techniques of micropropagation and microtubérisaiion in particular:
- Abott A.J. and Belcher R. Potato tuber formation in vitro. In: In vitro Plant tissue culture and its Agricultural Applications (pp. 113-122), Butteπvarih, London, 1986,- Abott A.J. and Belcher R. Potato tuber formation in vitro. In: In vitro Plant tissue culture and its Agricultural Applications (pp. 113-122), Butteπvarih, London, 1986,
- Debergh P.C. and Zimmerman R.H. Micropropagation, Technology and Application, Klu er Académie Publishers, 1991,- Debergh P.C. and Zimmerman R.H. Micropropagation, Technology and Application, Klu er Académie Publishers, 1991,
- Wang Po-Jen and HU Ching-yeh. Potato Tissue Culture and its applications in Agriculture, Potato Physiology, 1985,- Wang Po-Jen and HU Ching-yeh. Potato Tissue Culture and its applications in Agriculture, Potato Physiology, 1985,
- Hussey G and Stacey NJ. Factors affecting the formation in vitro tubers of potato (Solanum Tuberosum L) Annals of Botany 53, 565-578, 1984. - Brevet WO 89/10399 : Microtuber propagation of potatoes ( 1989)- Hussey G and Stacey NJ. Factors affecting the formation in vitro tubers of potato (Solanum Tuberosum L) Annals of Botany 53, 565-578, 1984. - Patent WO 89/10399: Microtuber propagation of potatoes (1989)
- Brevet EP-A-0 292 488 : Method of multiplying tubers ( 1987)- Patent EP-A-0 292 488: Method of multiplying tubers (1987)
- Brevet EP-A-0 476 141 : Process for producing tuber ( 1991).- Patent EP-A-0 476 141: Process for producing tuber (1991).
Ces techniques nécessitent des équipements de culture sophistiqués et comportent certaines limitations dues notamment aux contraintes suivantes :These techniques require sophisticated cultivation equipment and include certain limitations due in particular to the following constraints:
- Les conditions de stérilité nécessitent la répartition et le repiquage fréquent des explants végétaux dans des éprouvettes ou des petites récipients stérilisés de façon individuelle,- The sterility conditions require the frequent distribution and transplanting of plant explants in test tubes or small containers sterilized individually,
- La maîtrise des problèmes physiologiques liés au confinement des plantules sur milieux artificiels (approvisionnement en CO2, petit calibre et dormance des microtubercules, malformations, problèmes de croissance...) s'est avérée difficile,- Controlling physiological problems linked to the confinement of seedlings on artificial media (CO 2 supply, small size and dormancy of microtubers, malformations, growth problems ...) has proven difficult,
- Les pertes subies lors de l'acclimatation des plantules aux conditions de pleine terre sont importantes, - Enfin, la mécanisation de la culture in vitro ainsi que le transfert des microplantules au champ est difficile à mettre en oeuvre.- The losses suffered during the acclimatization of the seedlings to open ground conditions are significant, - Finally, the mechanization of the in vitro culture as well as the transfer of microplantules to the field is difficult to implement.
Ces contraintes ont pour conséquence d'imposer un prix de revient élevé aux produits issus de Vin vitro (microplantules, microtubercules ou minitubercules).These constraints result in imposing a price of is a high cost of products produced from Vin vitro (microplantules, microtubercules or minitubercules).
Etant donné les limites de conservation de ces plantules issues de Vin vitro et leur difficulté d'acclimatation directe en plein champ, aussi bien que la fragilité des microtubercules produits in vitro, des méthodes intermédiaires ont été proposées afin d'obtenir du matériel de propagation de la pomme de terre sous forme de "minitubercules", obtenus après une subculture en serre ou sous abri des plantules ou des microtubercules issues de Vin vitro. Par exemple, des techniques ont été proposées pour accroître le taux de production de minitubercules en terre (brevet WO 88/04137) en transplantant en serre des plantes multipliées et ayant subi un traitement d'induction in vitro. Les plantes sont traitées avec un milieu de culture contenant des additifs hormonaux, afin de favoriser le développement des minitubercules, qui peuvent être récoltés G à 8 semaines après la transplantation.Given the limits of conservation of these seedlings from Vin vitro and their difficulty of direct acclimatization in the open field, as well as the fragility of the microtubercules produced in vitro, intermediate methods have been proposed in order to obtain propagation material for the potato in the form of "mini-tubers", obtained after a subculture in a greenhouse or under shelter of the seedlings or microtubers from Vin vitro. For example, techniques have been proposed for increasing the rate of production of minitubercules in soil (patent WO 88/04137) by transplanting propagated plants to the greenhouse and having undergone an induction treatment in vitro. The plants are treated with a culture medium containing hormonal additives, in order to promote the development of mini-tubers, which can be harvested G at 8 weeks after transplantation.
Cette technique nécessite une subculture des plants de pommes de terre dans le sol à des densités de plantations élevées, dans des conditions contrôlées et à l'abri des contaminations par les agents pathogènes et les insectes. Toutefois, ces cultures intermédiaires dépendent des cycles saisonniers, n'autorisent qu'une récolte de tubercules limitée en quantité, sur l'espace d'une seule génération et ne permettent pas d'obtenir des tubercules de taille et de qualité uniformes.This technique requires a subculture of seed potatoes in the soil at high planting densities, under controlled conditions and protected from contamination by pathogens and insects. However, these intermediate crops depend on seasonal cycles, allow only a limited quantity of tubers over a single generation and do not allow tubers of uniform size and quality to be obtained.
Par ailleurs, les plantes ne sont jamais à l'abri des contaminations, étant donné que la culture se fait soit en terre ou dans du substrat de culture en serre.In addition, plants are never immune to contamination, since the culture is done either in soil or in growing medium in the greenhouse.
Le procédé selon l'invention permet d'accroître la récolte produite par plantule, de réduire la durée du processus d'obtention de cette récolte, d'arriver avec précision à une semence au diamètre souhaité, d'assurer l'aptitude à l'ensemencement en pleine terre et d'obtenir des récoltes toute l'année en modifiant radicalement les techniques de multiplication et les conditions de mise en oeuvre connues.The method according to the invention makes it possible to increase the crop produced by seedling, to reduce the duration of the process of obtaining this crop, to arrive precisely at a seed with the desired diameter, to ensure the aptitude for seeding in the open ground and obtaining crops all year round by radically modifying the propagation techniques and the known conditions of implementation.
La présente invention consiste à multiplier et à induire la tubérisation hors-sol par une technique aéro-hydroponique, pour remplacer les techniques de la culture in vitro dans les phases de multiplication et de tubérisation. Cette nouvelle technique aéro-hydroponique de production de plantules, de bulbes, bulbilles, rhizomes et tubercules, garantit la qualité sanitaire du matériel à des coûts nettement inférieurs à ceux des produits issus des techniques in vitro.The present invention consists in multiplying and inducing above-ground tuberization by an aero-hydroponic technique, to replace the techniques of in vitro culture in the multiplication and tuberization phases. This new aero-hydroponic technique for producing seedlings, bulbs, bulbils, rhizomes and tubers, guarantees quality health of equipment at costs significantly lower than those of products derived from in vitro techniques.
Le procédé selon l'invention est caractérisé en ce qu'il comprend une première étape de propagation in vitro de bouture à partir de mérisième, une seconde étape de multiplication et d'allongement desdites boutures afin d'obtenir des plantules puis une troisième étape de tubérisation desdites plantules, les deuxième et troisième étapes étant effectuées en aéro- hydroponie avec une solution nutritive substantiellement identique aux solutions appropriées aux conditions de culture in vitro, avec la reserve qu'elle est sensiblement exempte d'une source de carbone assimilable.The method according to the invention is characterized in that it comprises a first step of in vitro propagation of cuttings from the merisième, a second step of multiplication and elongation of said cuttings in order to obtain seedlings then a third step of tuberization of said seedlings, the second and third stages being carried out in aero-hydroponics with a nutritive solution substantially identical to the solutions suitable for the conditions of culture in vitro, with the proviso that it is substantially free of an assimilable carbon source.
Par méristème, on désigne de façon générale le tissu végétal formé de cellules indifférenciées, siège de divisions rapides et nombreuses, situé dans les régions de croissance de la plante.By meristem, is generally meant the plant tissue formed of undifferentiated cells, seat of rapid and numerous divisions, located in the growth regions of the plant.
Par l'expression "substantiellement identique", on entend que cette composition est substantiellement identique à celles décrites dans l'art antérieur sus-mentionné.By the expression "substantially identical" is meant that this composition is substantially identical to those described in the above-mentioned prior art.
Par l'expression "source de carbone assimilable", on entend en particulier tous les sucres notamment le saccharose, le glucose.By the expression "assimilable carbon source" is meant in particular all the sugars, in particular sucrose, glucose.
Par l'expression "sensiblement exempte" on entend que la quantité éventuellement présente est très nettement inférieure à celle normalement utilisée dans les solutions pour les cultures in vitro. Au moins 10 fois moins. De préférence au moins, 50 fois moins. La solution peut ne pas contenir du tout de ladite source de carbone assimilable. En d'autres termes, les plantes sont telles qu'elles fonctionnent en conditions autotrophes. La solution nutritive est remarquable également par le fait qu'elle contient une quantité efficace de vitamine, notamment la thiamine HC1, la pyridoxine, la glycine, l'hydrorysat de caséine.By the expression "substantially free" is meant that the amount possibly present is very much lower than that normally used in solutions for in vitro cultures. At least 10 times less. Preferably at least, 50 times less. The solution may not contain said assimilable carbon source at all. In other words, plants are such that they work in autotrophic conditions. The nutrient solution is also remarkable for the fact that it contains an effective amount of vitamin, in particular thiamine HC1, pyridoxine, glycine, casein hydrorysat.
Par quantité efficace on entend la quantité qui permet la mise en oeuvre convenable des deuxième et troisième étapes. De façon générale, cette composition comprendra les anions et cations communément utilisés, notamment les ingrédients de la formule de Muraschige et Skoog ( 1962) additionnés d'un mélange d'acides aminés obtenus par hydrolyse acide de la caséine, par exemple les cations, potassium, calcium, magnésium, sodium, fer, cuivre, zinc, cobalt, les anions nitrate, sulfate etc.By effective amount means the amount which allows the proper implementation of the second and third steps. In general, this composition will include the anions and cations commonly used, in particular the ingredients of the formula of Muraschige and Skoog (1962) added with a mixture of amino acids obtained by acid hydrolysis of casein, for example the cations, potassium , calcium, magnesium, sodium, iron, copper, zinc, cobalt, nitrate, sulfate anions etc.
Avantageusement, cette composition comprendra en partie en poids : Nitrate d'ammonium 1650 mgAdvantageously, this composition will comprise in part by weight: Ammonium nitrate 1650 mg
Nitrate de potassium 2000 mgPotassium nitrate 2000 mg
Chlorure de calcium, 2H2O 440 mgCalcium chloride, 2H 2 O 440 mg
Sulfate de magnésium 370 mgMagnesium sulfate 370 mg
Monophosphate de potassium 170 mgPotassium monophosphate 170 mg
Disodium EDTA 37,250 mgDisodium EDTA 37.250 mg
Sulfate ferreux, 7H20 27,850 mgFerrous sulfate, 7H 2 0 27.850 mg
Sulfate de zinc, 7H20 8,600 mgZinc sulfate, 7H 2 0 8.600 mg
Acide borique 6,200 mgBoric acid 6.200 mg
Sulfate de manganèse, 7I-bO 22,300 mgManganese sulfate, 7I-bO 22.300 mg
Sulfate de cuivre, SH O 0,025 mgCopper sulfate, SH O 0.025 mg
Iodure de potassium 0,830 mgPotassium iodide 0.830 mg
Molybdate de sodium, 2H2O 0,250 mgSodium molybdate, 2H 2 O 0.250 mg
Chlorure de cobalt, 6 H2O 0,025 mgCobalt chloride, 6 H 2 O 0.025 mg
Myo-inositol 100 mgMyo-inositol 100 mg
Thiamine HC1 0,1 mgThiamine HC1 0.1 mg
Pyridoxine 0,5 mgPyridoxine 0.5 mg
Acide nicotinique 0,5 mgNicotinic acid 0.5 mg
Glycine 2,0 mgGlycine 2.0 mg
Hydrolysat de caséine 500 mgCasein hydrolyzate 500 mg
eau 1 Q.S.D. 1000 ml avec une fourchette variant pour chaque ingrédient de 50 %.water 1 Q.S.D. 1000 ml with a range varying for each ingredient by 50%.
Par l'expression aéro-hydroponique, on désigne une méthode de culture hydroponique dans laquelle l'eau chargée en différents additifs qui permettent la croissance des plantes est véhiculée sous forme nébulisée.The expression “aero-hydroponic” denotes a method of hydroponic culture in which the water loaded with different additives which allow the growth of plants is conveyed in nebulized form.
Une fois que les boutures comportent une portion de tige et portent au moins un noeud, de préférence deux noeuds, c'est-à-dire 10 à 20 semaines environ après la différenciation des méristèmes, celles-ci sont soumises à l'étape de multiplication et d'allongement.Once the cuttings have a portion of stem and carry at least one node, preferably two nodes, that is to say approximately 10 to 20 weeks after the differentiation of the meristems, these are subjected to the step of multiplication and elongation.
Une première multiplication peut être réalisée in vitro selon des techniques identiques à celles décrites dans l'art antérieur sus-mentionné.A first multiplication can be carried out in vitro using techniques identical to those described in the above-mentioned prior art.
Les boutures obtenues à l'issue de la première phase sont placées sur un support, notamment en mousse synthétique, de préférence des plaques de mousse de polyphenol, sous un éclairage approprié notamment de 60 à 150 μE/m2/s à 16 heures de lumière, un noeud pouvant être enfoncé dans les ouvertures ménagées à cet effet dans le support.The cuttings obtained at the end of the first phase are placed on a support, in particular in synthetic foam, preferably sheets of polyphenol foam, under suitable lighting in particular from 60 to 150 μE / m 2 / s at 16 hours of light, a knot that can be inserted into the openings for this purpose in the support.
Les boutures sont cultivées sur le support jusqu'à ce que se soit formée au moins une tige axillaire dont la portion apicale est repiquée afin d'obtenir une plantule complète comportant au moins cinq noeuds. Les portions intermédiaires de la tige axillaire comportant au moins un noeud sont repiquées pour former de nouvelles tiges axillaires. Les portions apicales et intermédiaires des nouvelles tiges sont traitées comme précédemment : cette opération est répétée le nombre de fois approprié. De préférence, le prélèvement des bourgeons apicaux est effectué deux à trois semaines environ après le repiquage.The cuttings are cultivated on the support until at least one axillary stem has been formed, the apical portion of which is transplanted in order to obtain a complete seedling comprising at least five nodes. The intermediate portions of the axillary rod comprising at least one node are transplanted to form new axillary rods. The apical and intermediate portions of the new stems are treated as above: this operation is repeated the appropriate number of times. Preferably, the apical buds are removed approximately two to three weeks after transplanting.
La solution nutritive, lors de la seconde étape, est celle utilisée pour la propagation des boutures, modifiée éventuellement par des additifs pour favoriser l'allongement des boutures et l'apparition des tiges axillaires, notamment la benzyladenine (B,A) et la 2-isopentenyladenine (2-iP). La troisième étape de tubérisation correspond à la formation des minitubercules.The nutritive solution, during the second stage, is that used for the propagation of the cuttings, possibly modified by additives to favor the elongation of the cuttings and the appearance of the axillary stems, in particular benzyladenine (B, A) and 2 -isopentenyladenine (2-iP). The third stage of tuberization corresponds to the formation of mini-tubers.
L'étape de tubérisation peut être effectuée selon deux procédés. Selon un premier procédé, l'étape de tubérisation est effectuée avec une solution nutritive de teneur réduite en composé azoté et dans des conditions de jours courts ou de jours longs, en fonction de la physiologie de la variété utilisée.The tuberization stage can be carried out according to two methods. According to a first method, the tuberization stage is carried out with a nutritive solution of reduced content of nitrogenous compound and under conditions of short days or long days, depending on the physiology of the variety used.
Selon un second procédé, les feuilles des plantes pré-induites sont prélevées avec une portion de tige et repiquées sur le support. De préférence, la tubérisation est effectuée avec une solution nutritive comprenant les éléments décrits antérieurement à l'exception du nitrate d'ammonium et le disodium EDTA.According to a second method, the leaves of the pre-induced plants are removed with a portion of the stem and transplanted onto the support. Preferably, the tuberization is carried out with a nutritive solution comprising the elements described previously with the exception of ammonium nitrate and disodium EDTA.
La troisième étape de tubérisation peut de préférence être précédée d'une phase d'induction de la tubérisation notamment dans des conditions de jour court (8 heures de lumière) et éventuellement au moyen d'inducteurs de tubérisation tels que la coumarine, l'ancymidol, l'acide salycilique et des régulateurs de croissance (chlorocholine hydrochloride = C.C.C., Alar, TIBA, etc..) présents dans la solution nutritive ou appliqués en pulvérisation sur le feuillage.The third stage of tuberization can preferably be preceded by a phase of induction of tuberization in particular in short daylight conditions (8 hours of light) and optionally by means of tuberization inducers such as coumarin, ancymidol , salycilic acid and growth regulators (chlorocholine hydrochloride = CCC, Alar, TIBA, etc.) present in the nutrient solution or applied as a spray on the foliage.
Le procédé selon l'invention est particulièrement approprie à l'obtention de minitubercules de pommes de terre. On décrit ci-après une variante de ce procédé relative à la culture de boutures de pommes de terre.The method according to the invention is particularly suitable for obtaining mini-tubers of potatoes. A variant of this process relating to the cultivation of potato cuttings is described below.
Des boutures de plantules de pommes de terre issues de méristèmes in vitro sont placées sur des plaques de mousse de polyphenol dans une enceinte alimentée par pulvérisation d'un milieu de culture contenant les composants du milieu de culture utilisé pour la propagation initiale des plantules, éventuellement complété par des additifs favorisant la croissance et le transfert en conditions non stériles des explants.Cuttings of seed potatoes from meristems in vitro are placed on polyphenol foam plates in an enclosure fed by spraying with a culture medium containing the components of the culture medium used for the initial propagation of the seedlings, optionally supplemented by additives promoting growth and transfer into non-sterile conditions of explants.
Chaque bouture comprend une portion de tige portant deux noeuds, l'un d'entre eux étant enfoncé dans le support en mousse de polyphenol.Each cutting comprises a portion of stem carrying two nodes, one of them being pressed into the polyphenol foam support.
Les boutures sont placées sous un éclairage de 60 à 150 μE/mVs à 16 heures de lumière.The cuttings are placed under a lighting of 60 to 150 μE / mVs at 16 hours of light.
Après trois semaines, les plantules ont formé une ou deux tiges axillaires comportant au minimum cinq noeuds.After three weeks, the seedlings have formed one or two axillary stems with at least five nodes.
Les tiges axillaires obtenues sont divisées en trois : deux portions comportant deux noeuds et une portion comportant un noeuf et l'apex. Les portions comportant deux noeuds sont repiquées comme précédemment et forment les boutures de type A. La portion apicale, également repiquée dans le support en polyphenol, constitue une bouture de type D.The axillary stems obtained are divided into three: two portions comprising two nodes and a portion comprising a node and the apex. The portions comprising two nodes are transplanted as above and form the cuttings of type A. The apical portion, also transplanted in the support in polyphenol, constitutes a cuttings of type D.
Les boutures de type A peuvent être remultipliées successivement tandis que les boutures de type D, forment après 15 jours une plantule complète, d'une taille supérieure à 15 cm, comportant plus de cinq noeuds.The type A cuttings can be successively multiplied while the type D cuttings, form after 15 days a complete seedling, of a size greater than 15 cm, comprising more than five nodes.
Les plantes allongées d'une taille supérieure à 15 cm (issues de boutures D) sont repiquées dans un nouveau support dans des conditions de- jours court à 8 heures de lumière sous un éclairage de 60- 150 μE/n.2/s pendant 10 à 21 jours. A ce stade, différents inducteurs de la tubérisation peuvent être utilisés (acide jasmonique, acide salicilique et dérivés, coumarine, ancymidol ainsi que des retardateurs de la croissance (C.C.C., Alar, TIBA) ou des agents absorbants l'éthylène tel que le permanganate de potassium (TIBA = acide triiodobenzoïque, Alar = acide succinique 2-2 diméthyl hydrazine, CCC = chlorocholine hydrochloride)). Selon une première méthode, du gaz carbonique (CO2) à la concentration de 1 à 40 % peut être également appliqué dans les enceintes de culture, notamment au niveau des racines après la phase d'induction de la tubérisation, les plantes peuvent être maintenues en jours courts sur un milieu de même composition, excepté une teneur réduite en composé azoté. Les minitubercules se forment dès lors, après six à neuf semaines, sûr des stolons qui se développent dans la partie inférieure du dispositif aéro- hydroponique. Selon une seconde méthode, les feuilles des plantes pré-induites sont prélevées et découpées avec une portion de tige, pour être repiquées, verticalement sur le substrat en polyphenol.The elongated plants of a size greater than 15 cm (from cuttings D) are transplanted into a new support under conditions of short days at 8 hours of light under a lighting of 60-150 μE / n.2 / s for 10 to 21 days. At this stage, various inducers of tuberization can be used (jasmonic acid, salicilic acid and derivatives, coumarin, ancymidol as well as growth retardants (CCC, Alar, TIBA) or ethylene absorbing agents such as permanganate. potassium (TIBA = triiodobenzoic acid, Alar = succinic acid 2-2 dimethyl hydrazine, CCC = chlorocholine hydrochloride)). According to a first method, carbon dioxide (CO 2 ) at a concentration of 1 to 40% can also be applied in the culture chambers, in particular at the level of the roots after the induction phase of the tuberization, the plants can be maintained in short days on a medium of the same composition, except for a reduced content of nitrogen compound. The minitubercules therefore form, after six to nine weeks, on the runners which develop in the lower part of the aero-hydroponic device. According to a second method, the leaves of the pre-induced plants are removed and cut with a portion of stem, to be transplanted, vertically on the polyphenol substrate.
Les plantes sont placées en lumière continue ou en jour long, en conditions aéro-hydroponiques, selon la procédure décrite par EWING E.E. sur des feuilles de plantes en culture in vitro (Potato physiology, 1985).The plants are placed in continuous light or in long day, under aero-hydroponic conditions, according to the procedure described by EWING E.E. on leaves of plants in culture in vitro (Potato physiology, 1985).
La tubérisation induite sur les plantules ou les feuilles est effectuée dans une chambre climatisée en modifiant la photopériode et la température. Les tubercules sont récoltés dès qu'ils ont atteint la taille souhaitée.The induced tuberization on the seedlings or leaves is carried out in an air-conditioned room by modifying the photoperiod and the temperature. The tubers are harvested as soon as they have reached the desired size.
Selon la première méthode, la récolte se poursuit pendant 10 à 12 semaines, selon l'espèce ou la variété. Le prélèvement continu d'une partie des tubercules favorise la tubérisation. La photopériode pour la tubérisation des plantules de pommes de terre est alors de 12 à 20 heures pour une température de 15 à 25°C, en fonction de la variété utilisée.According to the first method, the harvest continues for 10 to 12 weeks, depending on the species or variety. Continuous removal of part of the tubers promotes tuberization. The photoperiod for the tuberization of the seed potatoes is then 12 to 20 hours for a temperature of 15 to 25 ° C, depending on the variety used.
Selon la deuxième méthode, 1 ou 2 minitubercules sont recueillis à la base de chaque feuille au bout de 15 à 18 jours.According to the second method, 1 or 2 mini-tubers are collected at the base of each leaf after 15 to 18 days.
On obtient ainsi une production de tubercules de taille uniforme répartie sur toute l'année, dont la dormance est rapidement levée, pouvant être semés au champ ou stockés pendant plusieurs mois.We thus obtain a production of tubers of uniform size distributed throughout the year, the dormancy of which is quickly broken, which can be sown in the field or stored for several months.
Les cycles de multiplication et de tubérisation pour les plantules de pommes de terre sont réalisés dans des chambres climatisées qui éliminent tous risques de contamination par des pucerons. La solution nutritive recyclée et soumise à un rayonnement U.V.c ainsi que l'absence de terre ou terreau garantit la propriété sanitaire des produits.The multiplication and tuberization cycles for the seed potatoes are carried out in air-conditioned rooms which eliminate any risk of contamination by aphids. The nutritive solution recycled and subjected to UV radiation as well as the absence of soil or potting soil guarantees the sanitary property of the products.
Les appareillages utilisés pour la culture hors-sol peuvent être de différents types. On peut utilement se référer à la description d'appareillages antérieurement décrits tels que celui figurant au brevet US A 5 136 804 (système pour la germination, la propagation et la croissance des plantes en conditions de brouillard formé par ultrason) ou le brevet US 4 332 105 (document qui revendique l'utilisation d'un dispositif pour la culture et le développement de plantes faisant appel au spray du milieu de culture sur les racines aériennes des plantes et associant un ensemble de bacs de culture et un circuit de récupération et de filtration du milieu de culture, ainsi que les instruments de contrôle et de réglage).The apparatuses used for the culture above-ground can be of different types. One can usefully refer to the description of previously described apparatuses such as that appearing in US patent A 5,136,804 (system for the germination, propagation and growth of plants under conditions of mist formed by ultrasound) or US patent 4 332 105 (document which claims the use of a device for the cultivation and development of plants using a spray of the culture medium on the aerial roots of the plants and associating a set of culture tanks and a circuit for recovery and filtration of the culture medium, as well as control and adjustment instruments).
Un dispositif particulièrement approprié à la mise en oeuvre du procédé selon l'invention comporte un récipient sur lequel est posé un support de culture, ledit récipient contenant à l'intérieur un nébulisateur, relié à un moyen d'alimentation en solution nutritive et le récipient étant relié à un moyen d'évacuation de la solution nutritive, lesdits moyens d'alimentation et d'évacuation étant reliés à un réservoir de solution nutritive et à un ou plusieurs moyens de contrôle.A device particularly suitable for implementing the method according to the invention comprises a container on which a culture support is placed, said container containing inside a nebulizer, connected to a means for supplying a nutritive solution and the container being connected to a means for discharging the nutrient solution, said supply and discharge means being connected to a nutrient solution tank and to one or more control means.
L'invention est décrite ci-après à l'aide d'un exemple relatif à la multiplication de la pomme de terre et en référence aux figures jointes, dans lesquelles la figure 1 représente une coupe verticale d'un canal de production et la figure 2 schématise un dispositif permettant de mettre en oeuvre le procédé selon l'invention.The invention is described below using an example relating to the multiplication of the potato and with reference to the attached figures, in which FIG. 1 represents a vertical section of a production channel and the figure 2 shows schematically a device for implementing the method according to the invention.
La figure 1 représente une coupe transversale d'un canal 2 de production de plantules 1 et de tubercules ou bulbilles 18. La solution nutritive est distribuée au moyen de la conduite 17 et est nébulisée au niveau de la partie racinaire des plantules.Figure 1 shows a cross section of a channel 2 for producing seedlings 1 and tubers or bulbils 18. The nutrient solution is distributed by means of line 17 and is nebulized at the root part of the seedlings.
Selon les figures 1 et 2, les plantules 1 sont disposées au-dessus d'un canal 2, sur et au travers d'un support 16, leurs parties inférieures se trouvant dans le canal, et sont alimentées 23 périodiquement par un brouillard de solution nutritive mis sous pression par une pompe 4 et distribué par un nébulisateur 17. L'excédent de solution est recyclé 24 et stérilisé par un dispositif à rayonnement UV avant son retour au réservoir 5 contenant la solution nutritive 3.According to FIGS. 1 and 2, the seedlings 1 are arranged above a channel 2, on and through a support 16, their lower parts being in the channel, and are fed 23 periodically with a solution mist. nutrient pressurized by a pump 4 and distributed by a nebulizer 17. The excess solution is recycled 24 and sterilized by a UV device before returning to the tank 5 containing the nutrient solution 3.
Les tubercules ou bulbilles produits apparaîtront dans le canal 2 et sont alimentés de la même façon que les plantules 1. La compensation des pertes du réservoir 5, en eau et en nutriments se fait de façon automatisée, par capteurs de niveau 6 et/ou de concentration 7 et 8 et des organes de régulation 9 et 10. Un groupe de pompes doseuses 14 déverse dans le réservoir 5 de solution nutritive 3 les quantités d'acide et d'éléments nutritifs 11 et 12 calculées par les régulateurs 9 et 10. Un capteur 21 permettant de mesurer la concentration en C02 agit sur une vanne de régulation 20 qui injecte, à partir d'un réservoir de CO2 comprimé 22, les quantités requises de CO2 dans le canal 2 de production de plantules. L'unité de contrôle 15 gère la correction du pH, de la conductivité de la concentration en CO2 et autre niveau. Elle peut également gérer l'ensemble du circuit dans toutes les phases du processus.The tubers or bulbils produced will appear in channel 2 and are fed in the same way as the seedlings 1. Compensation for losses of reservoir 5, in water and nutrients is done automatically, by level sensors 6 and / or concentration 7 and 8 and regulating organs 9 and 10. A group of metering pumps 14 pours into the reservoir 5 of nutritive solution 3 the quantities of acid and nutrients 11 and 12 calculated by the regulators 9 and 10. A sensor 21 making it possible to measure the concentration of C0 2 acts on a regulating valve 20 which injects, from a compressed CO 2 reservoir 22, the required quantities of CO 2 into the channel 2 for producing seedlings. The control unit 15 manages the correction of the pH, the conductivity of the CO 2 concentration and other level. It can also manage the entire circuit in all phases of the process.
La multiplication est réalisée dans une chambre climatisée à une température de 20-24°C, les boutures reçoivent un éclairement d'assimilation d'environ 60-100 μE/mVs, grâce à des tubes fluorescents, avec une photopériode de 16 heures.The multiplication is carried out in an air-conditioned room at a temperature of 20-24 ° C, the cuttings receive an assimilation illumination of approximately 60-100 μE / mVs, thanks to fluorescent tubes, with a photoperiod of 16 hours.
Chaque repiquage des boutures autotrophes est réalisé à intervalle régulier.Each transplanting of autotrophic cuttings is carried out at regular intervals.
Le milieu de culture est renouvelé toutes les trois semaines.The culture medium is renewed every three weeks.
Essais de multiplication et de tubérisation des plantes de pommes de terre en filière in vitro/hors-sol. I - Multiplication des bouturesMultiplication and tuberization tests of potato plants in in vitro / above-ground supply chain. I - Multiplication of cuttings
Comparaison de la croissance et du taux de multiplication de la pomme de terre en culture classique in vitro et en culture hors-sol en conditions non stériles.Comparison of the growth and multiplication rate of the potato in conventional in vitro culture and in soilless culture in non-sterile conditions.
L'essai visait à comparer la qualité des plantules de pommes de terre et le rendement de la micropropagation réalisée selon le procédé de l'invention sur des supports en mousse de polyphenol et en condition autotrophe, par rapport à la technique classique in vitro.The aim of the test was to compare the quality of the potato seedlings and the yield of the micropropagation carried out according to the process of the invention on supports of polyphenol foam and in autotrophic condition, compared with the conventional technique in vitro.
Par ailleurs, les rendements en fonction du type de bouture mis en culture ont été comparés : a) 1 noeud muni d'un bourgeon axillaire enfoncé dans le substrat de culture, b) 1 noeud muni d'un bourgeon axillaire placé en dehors du substrat de culture, c) 1 noeud supérieur muni d'un bourgeon terminal. Ce test est réalisé en vue de mettre en évidence l'intérêt de développer une méthode de découpe automatique ou de hachage des plantules pour réaliser le repiquage en conditions de production industrielle.Furthermore, the yields as a function of the type of cuttings cultivated were compared: a) 1 node provided with an axillary bud inserted into the culture substrate, b) 1 node provided with an axillary bud placed outside the substrate of culture, c) 1 upper node provided with a terminal bud. This test is carried out with a view to highlighting the advantage of developing an automatic method of cutting or chopping seedlings to transplant under industrial production conditions.
Afin de maintenir un équilibre hydrique optimal, le support de culture est amené à saturation, puis la pulvérisation du milieu de culture est arrêtée. La rétention capillaire du substrat utilisé permet d'espacer deux cycles de saturation par une période de 8 heures.In order to maintain an optimal water balance, the culture support is brought to saturation, then the spraying of the culture medium is stopped. The capillary retention of the substrate used makes it possible to space two saturation cycles over a period of 8 hours.
L'allongement, la vigueur, le développement foliaire et la vitesse de croissance sont favorisés en conditions non stériles.Lengthening, vigor, leaf development and growth speed are favored in non-sterile conditions.
Un essai a été réalisé visant à comparer l'allongement, le nombre de noeuds produits et le coefficient de multiplication de boutures de pomme de terre (variété "Désirée") mise en culture en conditions non stériles par rapport aux boutures du même type multipliées in vitro. Les conditions de culture étaient les mêmes (température de 24°C et lumière de 60 μE/mVs pour une photopériode de 16 heures.A test was carried out to compare the elongation, the number of knots produced and the multiplication coefficient of potato cuttings ("Désirée" variety) cultivated in non-sterile conditions compared to cuttings of the same type multiplied in vitro. The conditions of culture were the same (temperature of 24 ° C and light of 60 μE / mVs for a photoperiod of 16 hours.
Sur un total de 600 plantes, nous avons observé dans les mêmes conditions expérimentales de l'essai un coefficient de multiplication d'une valeur de 5,7 en 28 jours en conditions non stériles pour un coefficient de 4,9 en 28 jours en conditions stériles.On a total of 600 plants, we observed under the same experimental conditions of the test a multiplication coefficient of a value of 5.7 in 28 days in non-sterile conditions for a coefficient of 4.9 in 28 days in conditions sterile.
Les plantes obtenues en conditions non stériles avaient, en moyenne, une hauteur de 10,9 cm de possédaient 9,5 feuilles tandis que celles cultivées en conditions stériles avaient une hauteur de 9,4 cm et possédaient 6,3 feuilles.Plants obtained under non-sterile conditions had, on average, a height of 10.9 cm of had 9.5 leaves while those cultivated under sterile conditions had a height of 9.4 cm and had 6.3 leaves.
Par ailleurs, le procédé de repiquage et le type d'expiant détermine la qualité des plantes obtenues à l'issue de la culture.Furthermore, the transplanting process and the type of explant determines the quality of the plants obtained at the end of the culture.
Au cours d'un autre essai, la différence de rendement a été comparée entre des boutures comprenant un seul bourgeon axillaire placé en dehors du milieu de culture, des boutures munies d'un bourgeon apical et celles qui comprennent un noeud enfoncé dans le support en mousse de polyphenol. Les résultats figurent au tableau 1. In another test, the difference in yield was compared between cuttings comprising a single axillary bud placed outside the culture medium, cuttings provided with an apical bud and those comprising a knot pressed into the support in polyphenol foam. The results are shown in Table 1.
TΛBLEΛU 1TΛBLEΛU 1
Qualité des plantules de pomme de terre (variété "Désirée") en fonction du mode de repiquage des boutures en culture aéro- hvdroponique.Quality of the seed potatoes ("Desiree" variety) according to the mode of transplanting the cuttings in aeroproponic culture.
Type de boutures mises longueur totale de la Nombre moyen de Po u rce n tag e de Coefficient de en culture planture (cm) feuilles par plantule plantules développées multiplication (*) après repiquageType of cuttings put total length of the Average number of P u rce n of Coefficient of in crop planting (cm) leaves per seedling developed seedlings multiplication (*) after transplanting
1 bouture comprenant 1 seul bourgeon axillaire 5,6 6,7 72,00 % 2,7 placé en dehors du substrat1 cutting comprising 1 single axillary bud 5.6 6.7 72.00% 2.7 placed outside the substrate
1 bouture comprenant 1 bourg eon axill ai re 9,2 9 94,00 % 3,3 enfoncé dans le substrat et 1 bourgeon aérien1 cutting comprising 1 boron eon axill ai re 9.2 9 94.00% 3.3 pressed into the substrate and 1 aerial bud
1 bouture supérieure munie du bourgeon 10,5 8,9 98,00 % 4 terminal1 upper cutting with bud 10.5 8.9 98.00% 4 terminal
Légende : (*) Le coefïîcient de multiplication est défini comme le nombre de bourgeons axillaires développés par bouture, après repiquage. Caption: (*) The multiplication coefficient is defined as the number of axillary buds developed per cutting, after transplanting.
Les segments apicaux permettent par ailleurs d'obtenir en deux semaines de culture une multiplication supérieure ou égale à 4.The apical segments also allow a multiplication greater than or equal to 4 in two weeks of culture.
Cette observation permet de recommander un premier prélèvement des bourgeons apicaux deux semaines après le repiquage, ce qui conduit à améliorer les taux de multiplication globaux de 30 %.This observation makes it possible to recommend a first removal of the apical buds two weeks after transplanting, which leads to improving the overall multiplication rates by 30%.
1 1 - Production de minitubercules sur des plantes issues de culture in vitro, en croissance en condition hors-sol1 1 - Production of mini-tubers on plants grown in vitro, growing in above-ground conditions
Les plantes obtenues après trois semaines de croissance dans les conditions décrites précédemment, ont été placées en présence d'un milieu de tubérisation. Les supports ont été placés à une température de 23 "C sous condition de jours courts (8 heures de lumière). Dans ces conditions, après trois semaines d'incubation, on observe la formation de minitubercules sur les tiges des plantes situées dans la partie inférieure du dispositif, maintenue à l'obscurité. La quantité de minitubercules produits se situait entre 0,5 à 3 tubercules par plante pour une taille variant de 0,7 à 1,5 cm.The plants obtained after three weeks of growth under the conditions described above, were placed in the presence of a tuberization medium. The supports were placed at a temperature of 23 "C under the condition of short days (8 hours of light). Under these conditions, after three weeks of incubation, the formation of minitubercules is observed on the stems of the plants located in the part bottom of the device, kept in the dark The quantity of minitubers produced was between 0.5 to 3 tubers per plant for a size varying from 0.7 to 1.5 cm.
Des essais de production de minitubercules ont été réalisés en comparant les techniques 1 et 2 décrites ci-dessus. Des plantes provenant de boutures de type A et de type D ont été mises en conditions d'induction en jours courts (8 heures de lumière) pendant 3 semaines sur le milieu de tubérisation, puis placées sous 20 heures de lumière, à une température deTests for the production of minitubers were carried out by comparing techniques 1 and 2 described above. Plants from type A and type D cuttings were placed in induction conditions in short days (8 hours of light) for 3 weeks on the tuberization medium, then placed under 20 hours of light, at a temperature of
23°C, pendant 7 semaines.23 ° C, for 7 weeks.
Une autre partie des plantes ont été découpées et les segments de tiges comportant une feuille ont été découpés dans les mêmes conditions (220 segments ont été obtenus à partir de 750 plantes). Les résultats de la tubérisation sont détaillés au tableau 2. Another part of the plants were cut and the stem segments comprising a leaf were cut under the same conditions (220 segments were obtained from 750 plants). The results of the tuberization are detailed in Table 2.
TABLEAU 2TABLE 2
Nombre de minitubercules produits en aéro-hvdroponie (variété "Désirée") en fonction du tvpe de boutures du tvpe de boutures repiquées et du mode de productionNumber of mini-tubers produced in aero-hydroponics ("Desired" variety) according to the type of cuttings, the type of transplanted cuttings and the mode of production
Mode de production Type de bouture N o m b r e d e Nombre de plantes initiale mi ni t uberc u l es testées pour 100 bouturesMethod of production Type of cuttings N o m b r e of Number of initial half-grown plants tested for 100 cuttings
Méthode 1 : Bouture de type A 59,6 225 plantes plantes entièresMethod 1: Type A cuttings 59.6 225 plants whole plants
Bouture de type D 99,6 250 plantesType D cuttings 99.6 250 plants
Méthode 2 : par segment découpé 45,4 750 plantesMethod 2: per segment cut 45.4 750 plants
Portions de tigesRod portions
+ 1 feuille par plante entière 133,3 750 plantes+ 1 leaf per whole plant 133.3 750 plants
Légende : Minitubercules récoltés 7 semaines après la mise en tubérisationCaption: Mini-tubers harvested 7 weeks after setting in tuber
La qualité et le rendement élevé de plantules et de minitubercules obtenus au cours de ces essais démontrent que le procédé selon l'invention permet de réaliser l'ensemble des étapes de production de semences de pommes de terre en conditions non stériles tout en garantissant une qualité de produits comparables à celles obtenues par les techniques de Vin vitro. Bien entendu, l'invention n'est pas limitée au mode de réalisation décrit ci-dessus qui constitue qu'un exemple représentatif du procédé mais s'étend au contraire aux autres plantes susceptibles d'être obtenues par cette méthode. The quality and high yield of seedlings and mini-tubers obtained during these tests demonstrate that the method according to the invention makes it possible to carry out all of the steps for producing potato seeds under non-sterile conditions while guaranteeing quality of products comparable to those obtained by the techniques of Vin vitro. Of course, the invention is not limited to the embodiment described above, which constitutes only a representative example of the process, but on the contrary extends to the other plants capable of being obtained by this method.

Claims

REVENDICATIONS
1. Procédé d'obtention d'organes de reproduction de végétaux caractérisé en ce qu'il comprend une première étape de propagation in vitro de bouture à partir de méristème, une seconde étape de multiplication et d'allongement desdites boutures afin d'obtenir des plantules puis une troisième étape de tubérisation desdites plantules et en ce que les deuxième et troisième étapes sont effectuées en aéro-hydroponie avec une solution nutritive substantiellement identique aux solutions appropriées aux conditions de culture in vitro, à la condition qu'elle ne contienne sensiblement pas de source de carbone assimilable.1. Method for obtaining plant reproductive organs, characterized in that it comprises a first step of in vitro propagation of cuttings from meristem, a second step of multiplication and elongation of said cuttings in order to obtain seedlings then a third stage of tuberization of said seedlings and in that the second and third stages are carried out in aero-hydroponics with a nutritive solution substantially identical to the solutions suitable for the conditions of culture in vitro, on the condition that it does not contain substantially from assimilable carbon source.
2. Procédé selon la revendication 1, caractérisé en ce que lors de la seconde étape, les boutures sont cultivées sur un support jusqu'à ce que se soient formées au moins une tige axillaire dont la portion apicale est repiquée afin d'obtenir une plantule complète comportant notamment au moins cinq noeuds.2. Method according to claim 1, characterized in that during the second step, the cuttings are cultivated on a support until at least one axillary stem has formed, the apical portion of which is transplanted in order to obtain a seedling complete including at least five knots.
3. Procédé selon la revendication 2, caractérisé en ce que les portions intermédiaires de la tige comportant au moins un noeud sont repiquées pour former de nouvelles tiges axillaires et en ce que les portions apicales formées sont traitées selon la revendication 2 alors que les portions intermédiaires sont repiquées, cette opération étant répétée le nombre de fois approprié.3. Method according to claim 2, characterized in that the intermediate portions of the rod comprising at least one knot are transplanted to form new axillary rods and in that the apical portions formed are treated according to claim 2 while the intermediate portions are transplanted, this operation being repeated the appropriate number of times.
4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce que le support est une mousse synthétique.4. Method according to one of claims 1 to 3, characterized in that the support is a synthetic foam.
5. Procédé selon la revendication 4, caractérisé en ce que la mousse synthétique est choisie parmi la mousse de polyphenol, de polyuréthane.5. Method according to claim 4, characterized in that the synthetic foam is chosen from polyphenol foam, polyurethane.
6. Procédé selon l'une des revendications 1 à 5, caractérisé en ce que la solution nutritive lors de la seconde étape est celle utilisée pour la propagation des boutures complétée éventuellement par des additifs favorisant la croissance.6. Method according to one of claims 1 to 5, characterized in that the nutritive solution during the second step is that used for the propagation of the cuttings possibly supplemented by additives promoting growth.
7. Procédé selon l'une des revendications précédentes, caractérisé en ce que l'étape de tubérisation est précédée d'une phase d'induction de la tubérisation, notamment dans des conditions de jour court.7. Method according to one of the preceding claims, characterized in that the tuberization step is preceded by a phase of induction of tuberization, in particular under short day conditions.
8. Procédé selon la revendication 7, caractérisé en ce que la phase d'induction est effectuée en présence d'inducteurs de tubérisation présents dans la solution nutritive. 8. Method according to claim 7, characterized in that the induction phase is carried out in the presence of inducers of tuberization present in the nutrient solution.
9. Procédé selon l'une des revendications précédentes, caractérisé en ce que l'étape de tubérisation est effectuée avec une solution nutritive de teneur réduite en composé azoté.9. Method according to one of the preceding claims, characterized in that the tuberization step is carried out with a nutritive solution of reduced content of nitrogenous compound.
10. Procédé selon l'une des revendications 1 à 8, caractérisé en ce que l'étape de tubérisation est effectuée par prélèvement des feuilles de plantes pré-induites avec une portion de tige puis repiquage.10. Method according to one of claims 1 to 8, characterized in that the tuberization step is carried out by removing the leaves of pre-induced plants with a portion of stem then transplanting.
11. Procédé selon la revendication 10, caractérisé en ce que la tubérisation est effectuée avec une solution nutritive ne comprenant sensiblement pas du nitrate d'ammonium et le disodium EDTA. 11. Method according to claim 10, characterized in that the tuberization is carried out with a nutritive solution not comprising substantially ammonium nitrate and disodium EDTA.
12. Procédé d'obtention de minitubercules de pommes de terre, caractérisé en ce qu'il consiste à mettre en oeuvre le procédé de l'une des revendications 1 à 11 à partir de méristèmes de pommes de terre.12. A method for obtaining potato minitubers, characterized in that it consists in implementing the method of one of claims 1 to 11 from potato meristems.
13. Dispositif convenant pour la mise en oeuvre du procédé selon l'une des revendications 1 à 12, caractérisé en ce qu'il comporte un récipient (2) sur lesquel est posé un support de culture (16), ledit récipient contenant à l'intérieur un nébulisateur ( 17) relié à un moyen d'alimentation (23 ) en solution nutritive et le récipient étant relié à un moyen d'évacuation (24) de la solution nutritive, lesdits moyens d'alimentation et d'évacuation étant reliés à un réservoir de solution nutritive et à un ou plusieurs moyens de contrôle. 13. Device suitable for implementing the method according to one of claims 1 to 12, characterized in that it comprises a container (2) on which is placed a culture support (16), said container containing the inside a nebulizer (17) connected to a means of feeding (23) in nutritive solution and the container being connected to a means of evacuation (24) of the nutritive solution, said means of supply and evacuation being connected to a reservoir of nutrient solution and to one or more means of control.
EP94928735A 1993-10-27 1994-10-21 Soilless propagation method and device Withdrawn EP0726702A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
BE9301152 1993-10-27
BE9301152A BE1007666A3 (en) 1993-10-27 1993-10-27 MULTIPLICATION PROCESS ABOVE GROUND OF SEEDLINGS, BULBS, bulbils, RHIZOMES AND TUBERS.
PCT/BE1994/000071 WO1995011587A1 (en) 1993-10-27 1994-10-21 Soilless propagation method and device

Publications (1)

Publication Number Publication Date
EP0726702A1 true EP0726702A1 (en) 1996-08-21

Family

ID=3887480

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94928735A Withdrawn EP0726702A1 (en) 1993-10-27 1994-10-21 Soilless propagation method and device

Country Status (4)

Country Link
EP (1) EP0726702A1 (en)
AU (1) AU7805694A (en)
BE (1) BE1007666A3 (en)
WO (1) WO1995011587A1 (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10113085A (en) * 1996-10-14 1998-05-06 Japan Tobacco Inc Production of potato tuber
WO2000005942A1 (en) * 1998-07-28 2000-02-10 Institute Of Genetics, Chinese Academy Of Sciences Culture container and process for producing potato microtubers by using the same
NL2004976C2 (en) * 2010-06-28 2011-12-29 Living Foods B V SYSTEM, CASH AND METHOD FOR GROWING AND / OR INCREASING PRODUCTS.
DE202010012739U1 (en) 2010-09-17 2011-12-19 Kamal Daas Device for growing one or more plants
DE102010050367A1 (en) 2010-11-03 2012-05-03 Kamal Daas Method for growing one or more plants
DE202011002791U1 (en) * 2011-02-16 2012-06-12 Kamal Daas Device for growing one or more plants
DE202011004908U1 (en) * 2011-04-06 2012-07-09 Kamal Daas Device for growing one or more plants
JPWO2016147577A1 (en) * 2015-03-19 2017-11-24 パナソニックＩｐマネジメント株式会社 Hydroponics equipment

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4332105A (en) * 1976-07-26 1982-06-01 Adi-Aeroponics Growth Ltd. Apparatus and method for plant growth in aeroponic conditions
HU206012B (en) * 1986-12-01 1992-08-28 Novotrade R T In vitro - in vivo method of high activity for producing potato small sized tubers
WO1988004136A1 (en) * 1986-12-02 1988-06-16 Kyowa Hakko Kogyo Co., Ltd. Method of multiplying tubers
US5047343A (en) * 1988-04-29 1991-09-10 Wisconsin Alumni Research Foundation Microtuber propagation of potatoes
IL88105A0 (en) * 1988-10-20 1989-06-30 Shira Aeroponics 1984 Ltd System for germination,propagation and growing plants in ultrasonic-fog conditions
KR920001196B1 (en) * 1989-03-11 1992-02-06 한국과학기술원 Propagation of potato by microtuber using petridish
ES2190444T3 (en) * 1990-03-23 2003-08-01 Kirin Brewery PROCESS TO PRODUCE TUBERCULOS.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9511587A1 *

Also Published As

Publication number Publication date
WO1995011587A1 (en) 1995-05-04
BE1007666A3 (en) 1995-09-12
AU7805694A (en) 1995-05-22

Similar Documents

Publication Publication Date Title
Aighewi et al. Improved propagation methods to raise the productivity of yam (Dioscorea rotundata Poir.)
Khan et al. Direct shoot regeneration system for date palm (Phoenix dactylifera L.) cv. Dhakki as a means of micropropagation
Dhawan et al. In vitro vegetative propagation of Leucaena leucocephala (Lam.) de Wit
SU1590029A3 (en) Method of producing plant material for plant reproduction
CN108770695B (en) Method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya
JPH03195427A (en) Rapid mass production method for artificial seed potato by tissue culture technique, and use thereof
EP0726702A1 (en) Soilless propagation method and device
EP0266287B1 (en) Method of sunflower regeneration by embryogenesis
Albers et al. Micropropagation of paeonia
Yemataw et al. Traditional enset [Ensete ventricosum (Welw.) Cheesman] sucker propagation methods and opportunities for crop improvement
EP0691073B1 (en) Process for producing tuber
CN112042477A (en) Jujun grass seedling cultivation method
KR101740571B1 (en) Circulation type deep flow technique apparatus for producing seed potatoes and cultivation method using the same
CN107873518B (en) A kind of tissue culture method of Fourstamen Stephania Root seedling
EP0223624B1 (en) Process for cultivating water hyacinths, plants and their uses
Bustami et al. Somatic Embryogenesis in elite indonesian cacao (Theobroma cacao L.)
JP4278183B2 (en) Sugarcane production
CN109792925A (en) West safflower bulbil breeds bulb method
CN100362096C (en) Method for obtaining amphihaploid plant of pumpkin ovule and its special culture medium
WO2019048382A1 (en) Method for production of sugarcane seedlings
EP0615683B1 (en) Method for producing plants with the flotation system, particularly for transplanting for example plants of the genus Nicotiana
FR2510354A1 (en) PROCESS FOR THE PREPARATION OF THE PROPAGATION MATERIAL OF DIGITAL (DIGITALIS LANATA EHRH.) IN A CULTURE OF TISSUE
Batukaev et al. In vitro microclonal propagation of strawberries and ex vitro adaptation
HU206012B (en) In vitro - in vivo method of high activity for producing potato small sized tubers
RU2824883C1 (en) Method of growing cloudberries (rubus chamaemorus linnaeus)

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19960524

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE FR NL

17Q First examination report despatched

Effective date: 19961122

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19990109