DE102013101452A1 - Method for in-vitro production of VA mycorrhizae in e.g. bio agriculture, involves mechanically reducing spores containing nutrient medium, and harvesting roots based on incubation of roots multiple weeks in darkness and room temperature - Google Patents

Abstract

The method involves performing inoculation of roots (D), performing proliferation of roots generated by natural transformation of dicotyledonous plants in root lines, and selecting a set of roots of highest growth performance. The selected root lines are pre-cultivated in successive propagation steps. A set of root pieces is removed from the recent proliferation of the root line. A set of spores containing solid nutrient medium is mechanically reduced, and a set of mycorrhizae roots is harvested based on incubation of the roots multiple weeks in darkness and room temperature. An independent claim is also included for a plant for in-vitro production of VA mycorrhizae.

Description

The invention relates to a process for the in-vitro production of VA mycorrhizal spores in mass production and to a plant suitable for carrying out the process.

The use of the symbiosis between mycorrhizal fungi and higher plants has long been known and is becoming increasingly important, especially in the areas of reforestation, recultivation, organic farming, horticulture and horticulture.

Conventional VA mycorrhizal production is based on in vivo methods on plants by fixing the VA mycorrhizal spores on support materials. The inocula thus produced contain up to 200 infection units / ml vehicle, of which about one third are spores or spore groups. Expanded clay, vermiculite or other porous granular materials may be considered as carriers. In this methodology, whole plants, e.g. Corn, dressed in beds. The plants root through the expanded clay or similar. and the inoculated VA mycorrhizal fungi multiply in the root-expanded clay mixture. The expanded clay is harvested, dried, optionally ground and then used as an inoculum.

The disadvantage of this in vivo procedure is the required amounts of plant and carrier materials. In addition, there is a high risk of contamination of the acquired mycorrhiza spores.

Methods have already been proposed for in vitro production of VA mycorrhizal spores, but these are not suitable for mass production.

A method is known for the monoxenic in vitro cultivation of VA mycorrhizal fungi ( WO2005 / 000008 A2 ) with the process of growing the roots and the inoculum, inoculating and growing the spore root culture, wherein the cultivation phase of the inoculum in the presence of a rhizogenic callus takes place under sterile conditions. Inoculum, nutrient medium and rhizogenic callus are prepared under sterile culture conditions. The nutrient medium contains macroelements, microelements, vitamins, hormones and at least one sugar. Starting from 10 Petri dishes with a diameter of 10 cm as a culture vessel for inoculation, several thousand fungal spores can be produced in three months.

In the WO2007 / 014974 A1 For example, for in vitro production of a VA mycorrhizaloculum, a semi-solid culture medium with a very specific viscosity is used. It can be recovered 2000 to 5000 infection units / ml. Infection units are spores, mycorrhizal roots and extra-radicular mycelium.

From the EP 1 231 854 B1 a process is known for the production of at least one premy-cured whole plant with the following process steps:
Providing petri dishes having two compartments with a bottom and a lid, wherein a root compartment (RC) is physically separated from a hyphal compartment (HC); Inserting a hole in the bottom and in the lid of the Petri dishes on the RC side; Filling RC and HC with a suitable medium; Transferring at least one sterilized plant to the RC department, with the roots on the surface of the medium and the shoot outside the petri dish; Inoculating the spores of an arbuscular mycorrhizal (AM) fungus near the plant roots and sealing the shells; Darkening of the petri dishes and their conversion into a growth chamber; after colonization, additional holes are placed in the bottom and top of the HC and used in these sterilized plants to grow additional plants in the mycelium mesh, as premycorrhizal plants.

This procedure is based on the use of whole plants, wherein the roots are kept under aseptic conditions, and is very expensive and expensive for large-scale application.

The general disadvantage of the known methods for in vitro production of VA mycorrhizal spores is that the harvested spores have no assured symbiotic properties.

As a result, when using these spores, z. B. in agriculture, the growth of the treated plants not or only slightly improved.

The invention has for its object to develop a method for the in-vitro production of VA mycorrhizaspores, which is suitable for mass production and with the durable spore product with consistently good symbiosis properties can be produced.

Furthermore, a plant for carrying out the method is to be created, which allows an economic mass production.

According to the invention the object is achieved by the features specified in claim 1. Advantageous developments of the procedure are the subject matter of claims 2 to 10. A suitable for carrying out the process plant is specified in claim 11. Advantageous embodiments are subject matter of claims 12 to 14.

• a) Vermehrung von durch natürliche Transformation erzeugten Wurzeln zweikeimblättriger Pflanzen in Wurzellinien und deren Selektion nach der höchsten Wachstumsleistung (Hochleistungswurzellinie);
• b) Vorkultivierung der selektierten Wurzellinien in aufeinanderfolgenden Vermehrungsschritten in Petrischalen, Entnahme von Wurzelstücken aus der letzten Vermehrung der Wurzellinie zur Massenanzucht in ersten Kulturgefäßen in einem flüssigen, sterilen Nährmedium im Ruhezustand, wobei die einzelnen Kulturgefäße mit Wurzeln unterschiedlicher Wurzellinien beschickt werden;
• c) Überführung von in-vivo-VA-Mykorrhizasporen in die in-vitro-Kultur in Petrischalen an transformierten Wurzeln zweikeimblättriger Pflanzen auf festem Nährmedium (Sporen-Wurzel-Medium), wobei die Vorkultivierung in mehreren Passagen durchgeführt wird und in jeder Passage ein Wirtswechsel erfolgt;
• d) Produktion des Inokulums mykorrhizierte Wurzeln aus nach Verfahrensschritt b) erhaltenen Wurzeln und nach Verfahrensschritt c) erhaltenen VA-Mykorrhizasporen in zweiten Kulturgefäßen in einem festen sterilen Nährmedium, wobei jedes Kulturgefäß einem Inokulationsrahmen mit Wurzelfixierungsgewebe zur Aufnahme der Wurzeln und VA-Mykorrhizasporen besitzt und nach mehrwöchiger Inkubation bei Raumtemperatur (26 °C) und Dunkelheit mykorrhizierte Wurzeln geerntet und auf ca. 0,5 bis 2 cm lange Stücke zerkleinert werden;
• e) Inokulation der nach Verfahrensschritt d) erhaltenen mykorrhizierten Wurzelstücke in dritten Kulturgefäßen mit festem Nährmedium und mit einer auf diesem aufliegenden siebartigen Unterlage (Inokulationsrahmen) zur Aufnahme der Wurzelstücke und nachfolgender Inkubation während einer Zeitdauer von mindestens 90 Tagen, bei Dunkelheit und Raumtemperatur, und abschließender Trennung der Wurzeln vom Nährmedium durch Abheben des Inokulationsrahmens;
• f) mechanische Zerkleinerung des festen Nährmediums zur schonenden Teilung der Hyphen, Auflösung des Restnährmediums einschließlich Gelbestandteilen, Waschen der VA-Mykorrhizasporen und Hyphen und Resuspendieren der Sporen.

For the purposes of the present invention, mass production means a contamination-free annual production of at least 1 billion in vitro VA mycorrhizal spores. The method according to the invention comprises the following method steps:

• a) Propagation of naturally-transformed roots of dicotyledonous plants in root lines and their selection for the highest growth performance (high-performance root-line);
• b) precultivation of the selected root lines in successive propagation steps in Petri dishes, removal of root pieces from the last propagation of the root line for mass cultivation in first culture vessels in a liquid, sterile nutrient medium at rest, wherein the individual culture vessels are fed with roots of different root lines;
• c) transfer of in vivo VA mycorrhizaspores in the in vitro culture in petri dishes on transformed roots of dicotyledonous plants on solid nutrient medium (spore root medium), wherein the pre-cultivation is carried out in several passages and a host change in each passage he follows;
• d) production of the inoculum mycorrhiz roots from roots obtained in step b) and VA mycorrhizas pores obtained in step c) in second culture vessels in a solid sterile nutrient medium, each culture vessel having an inoculation frame with root fixation tissue for uptake of the roots and VA mycorrhizal spores several weeks of incubation at room temperature (26 ° C) and dark mycorrhizal roots are harvested and minced to about 0.5 to 2 cm long pieces;
• e) inoculation of the mycorrhizal root pieces obtained according to method step d) in third culture vessels with solid nutrient medium and with a sieve-like support (inoculation frame) for receiving the root pieces and subsequent incubation for a period of at least 90 days, in the dark and room temperature, and finally Separation of the roots from the nutrient medium by lifting the inoculation frame;
• f) mechanical comminution of the solid nutrient medium for gentle division of the hyphae, dissolution of the residual nutrient medium including gel constituents, washing of the VA mycorrhizaspores and hyphae and resuspension of the spores.

With this procedure, a mass production of in vitro VA mycorrhizal spores is possible and thus a large-scale use of mycorrhizaspores for main crops, such as corn, wheat, soybean or rice, at relatively low cost.

According to the proposed procedure, spores are obtained which have consistently good symbiosis properties. The entire process is free of genetic engineering and the resulting waste products can be used for further use. All required roots for the preculture were transferred to the in vitro culture by known methods. For root crops carrots (Daucus carota), tobacco (Nicotiana tabacum), tomato (Lycopersicon lycopersicon), lotus corniculatum and strawberries (Fragaria ssp.) Can be used. All roots can be transformed using the bacterial strain Agrobacterium rhizogenes A4 (origin INRA Versailles Cedex, France). From Daucus carota various accessions can be used, such as DAU 268 origin Germany, DAU 160 origin Netherlands, DAU 169 origin Hungary, DAU 9842 origin Uzbekistan.

The following types of tomatoes are particularly useful: "Tamina" (LYC 2411) "Lukullus" (LYC 127), "Bonner's Best". Roots of tobacco originate from the wild type Nicotiana tabaccum Samsun NN. Lotus corniculatum is available from seeds of the strain collections of various universities.

Throughout the study, it was found that all roots are free from genetic modification.

The spores of the VA mycorrhizal fungi are especially fungi of the order Glomerales and the genera Glomus and Gigaspora in question, especially fungi of the species Glomus intraradices, Glomus Irregulare, Glomus proliferum, Glomus etunicatum, Glomus clarum, Glomus manihotis, Glomus aggregatum, Glomus caledonium, Glomus claroideum, Glomus fasciculatum and Glomus hoi.

To secure the symbiosis ability of in vitro VA mycorrhizaspores across many propagation passages, it has been found in the course of many years of research and extensive research that a host-switching system and an in vivo / in vitro change in the cultivation of VA mycorrhizal spores to very good results leads. Already with two passages sufficient results can be achieved to ensure the symbiosis of in vitro VA mycorrhizal spores.

The term passage is an amplification step from an origin culture. This term is used for both root cultures and spore root cultures.

• Passage 1: in die in-vitro-Kultur überführte VA-Mykorrhizasporen aus Substratisolaten mykorrhizierter Pflanzen mit der höchsten Biomasse aus Pflanzentests auf transformierte Wurzeln von Daucus carota;
• Passage 2: in-vitro-VA-Mykorrhizasporen aus Passage 1 auf transformierte Wurzeln von Lotus corniculatus oder Nicotiana tabacum;
• Passage 3: in-vitro-VA-Mykorrhizasporen aus Passage 2 auf transformierte Wurzeln von Daucus carota;
• Passage 4: in-vitro-VA-Mykorrhizasporen aus Passage 2 auf transformierte Wurzeln anderer zweikeimblättriger Pflanzen wie Nicotiana tabacum oder Lotus corniculatus;
• Passage 5: in-vitro-VA-Mykorrhizasporen aus Passage 4 auf transformierte Wurzeln von Daucus carota;

Host change is understood to mean a change of plant species in host roots for the spore root culture, ie the plant root is the host for the fungus and the host change is realized by the alternation of roots of different plant species. Very good results with regard to the symbiosis properties of the spores were achieved, for example, with five passages for the cultivation of the spore inoculum:

• Passage 1: in vitro culture transferred VA mycorrhizal spores from substrate solates mycorrhizated plants with the highest biomass from plant tests on transformed roots of Daucus carota;
• Passage 2: in vitro VA mycorrhizal spas of passage 1 on transformed roots of Lotus corniculatus or Nicotiana tabacum;
• Passage 3: in vitro VA mycorrhizal spas from passage 2 on transformed roots of Daucus carota;
• Passage 4: in vitro VA mycorrhizal spas from passage 2 to transformed roots of other dicotyledonous plants such as Nicotiana tabacum or Lotus corniculatus;
• Passage 5: in vitro VA mycorrhizal spas from passage 4 on transformed roots of Daucus carota;

With Passage 5, the pre-cultivation is completed and the harvested spores serve as inoculum for the cultivation of mycorrhizierter roots in the second culture vessel. There was a change of hosts in each passage.

Throughout the trial, it was found that all spores are free of genetic modification.

The nutrient or culture media used are gelatinous or aqueous solutions based on sucrose, which contain macroelements, microelements and vitamins.

To grow the transformed roots in the first culture vessel, a liquid nutrient medium root medium (R medium) is used. The specific composition of this medium, as indicated in the example below, makes it possible to obtain a residue-free, further-processable root network. In addition, the root harvest is facilitated and the growth of the roots in the next process step, in the second culture vessel (INO-BOX), favors.

For the production of the inoculum mycorrhizal roots in the second culture vessel and for inoculation with mycorrhizal roots to produce the VA mycorrhizal spores in the third culture vessel, gelatinous nutrient media with phytohormones as functionalizing additives, e.g. Indole butyric acid (IBA) and / or indole acetic acid (IAA) abscisic acid (ABA), naphthylacetic acid (NAA) used.

The respective nutrient or culture media differ slightly in their compositions.

For the gelatinous culture media, the inoculum (I) and spore production (SP) medium, the use of phytohormones (IBA, ABA, IAA and NAA) is crucial, because through them the factors spore germination, root length growth, root branching, hyphal growth, number of Secondary spores (spore yield) and time optimization of the overall process are positively influenced. For mass production, the use of these phytohormones in spore root culture and their beneficial effect on the growth of VA mycorrhizal fungi is important.

Another advantage of the composition of the gelatinous culture media is their simple and inexpensive conversion to a liquid state. This is achieved by comminution and Citratzusatz, with the optimal ratio of medium to be dissolved and citrate buffer is 1: 1.5.

The spores present in the liquid mixture of nutrient medium and citrate buffer (pH 6) in suspended form can be inexpensively filtered under vacuum and stored in alginate or Tylose.

A plant for carrying out the process according to the invention includes at least one feed unit with the following assemblies:
A circulating conveyor belt for holding culture vessels.

A culture vessel consists of a transparent, lightweight, biocompatible, sterilizable plastic container, preferably made of polycarbonate, with a rectangular or square cross-sectional area, a height of 50 to 150 mm and a capacity of at least 1.5 l culture medium with a filling height in centimeters of 10% 20% of the amount of culture medium. Further, the culture vessel include an insertable into the container frame with mesh and a easily aufsetz- and removable lid with a circumferential overhanging edge.

The frame consists of two identical frame members between which the mesh is clamped. The two frame components are held together by means of clip-on brackets. The frame components are made of stainless steel and the polyester mesh with a mesh size of 0.5 to 2 mm.

When using approx. 25,000 MYKO-BOXES for an annual production, these are subject to special process-technical requirements. The essential factor is the inoculation of the MYKO-BOXES by means of a feeding unit. The feed unit includes a movable in the vertical direction pressure roller which presses in the operating state on the mesh of the incoming culture vessel resting root pieces. Above the circulating conveyor belt, above the culture vessel located thereon, a circulating root conveyor belt is arranged in a kind of multi-level construction, which serves to feed the culture vessels with root pieces.

For this purpose, a Wurzelaufgabe- and metering device is arranged above the root conveyor belt. In front of this, a sterilization unit for sterilizing the assembly line is installed. At the front end of the root conveyor belt is a Wurzelverteilvorrichtung.

The entire system includes other aggregates, such as for the media preparation, sterilization, spore harvest, work-up of the media residues, storage shelves for incubation and transport and conveying means, which, however, in the context of the invention submitted are of minor importance.

The invention will be explained in more detail using an exemplary embodiment.

In the accompanying drawing show:

1 the individual process steps for the mass production of VA mycorrhizaspores as a flow diagram,

2 a loading unit as a simplified schematic diagram as a side view,

3 the system according to 2 as a top view,

4 an execution of the culture vessel (INO-BOX or MYKO-BOX) as a side view

5 the usable frame as a side view and

6 the culture vessel according to 4 as a top view.

In the 1 the flow of production from the preparation of the media to the shipment of the VA mycorrhizal spores is shown as a flow chart.

The main process steps (A to G) for the mass production of in vitro VA mycorrhizal spores of Glomus intraradices on roots of Daucus carota are explained below.

• A) Anzucht von Wurzeln (Vorkultivierung) in einem ersten Kulturgefäß, einer sogenannten ROOT-BOX;
• B) Anzucht der Sporen (Vorkultivierung);
• C) Produktion des Inokulums (mykorrhizierte Wurzeln) in einem zweiten Kulturgefäß, einer sogenannten INO-BOX;
• D) Inokulation in einem dritten Kulturgefäß, einer sogenannten MYKO-BOX, mit mykorrhizierten Wurzeln;
• E) Sporenernte;
• F) Endproduktprüfung und
• G) Konfektionierung und Produktlagerung.

The in vitro VA mycorrhizal production method according to the invention comprises the following stages or process sections:

• A) growing of roots (precultivation) in a first culture vessel, a so-called ROOT-BOX;
• B) cultivation of the spores (precultivation);
• C) production of the inoculum (mycorrhizal roots) in a second culture vessel, a so-called INO-BOX;
• D) inoculation in a third culture vessel, a so-called MYKO-BOX, with mycorrhizal roots;
• E) spore harvest;
• F) final product testing and
• G) packaging and product storage.

The overall process includes further steps or sections, such as preparation of the culture or nutrient media, sterilization of the equipment and materials and process control, which are not discussed in detail below.

A) Cultivation of transformed roots

As a preculture roots of carrots (Daucus carota) are used. These were naturally transformed for the subsequent culture in the first culture vessel, a so-called ROOT-BOX, in a known manner according to Becard and Fortin with the aid of the bacterial strain Agrobacterium rhizogenes A4 (origin INRA Versailles Cedex, France).

From the transformed root pieces obtained, 20 petri dishes each containing one root piece on gel-containing MSR medium ("modified Strullu-Romand medium; WO2005 / 000008 A2 ) And selected for the highest growth performance, ie the largest biomass formation.

A root line is a root culture that has emerged from a single root piece in the transformation process. The root lines differ in their properties, even if they are from the same source, e.g. a carrot slice, come from. Both symbiosis and growth potential are different. The best growing lines with good symbiosis properties are referred to as high performance root lines.

A pool of high performance root lines is formed and propagated, from which the roots for mass production are taken.

A passage refers to a growth phase in a culture vessel. After completion of a passage, a selection of the root components and their implementation is carried out in another culture vessel.

All obtained high-performance root lines are propagated in five Petri dishes each over a period of 3 weeks and form a pool of root cultures. This serves as a source for a change of the root lines. The change takes place in the subsequent precultivation of the roots from cultivation to cultivation in the first culture vessel (ROOT-BOX) in order to ensure the symbiosis ability of the in vitro spores of Glomus intraradices to be produced.

15 to 20 root pieces, each 3 cm in length, are removed from the high-performance root line selected for the process and transferred to the first culture vessel (ROOT-BOX). The ROOT-BOX is a glass container with a capacity of 1 liter of the liquid nutrient medium (R-medium). The composition of the nutrient medium is given in Table 1 below.

For the production of one liter of R medium, the ingredients are mixed according to the amounts given in this table. For simpler dosing, 4 stock solutions of constituent groups are needed: macroelements (# 1-4), microelements (# 5-10), vitamins (# 11-16), and EDTA (# 17). These components and sucrose are dissolved in demineralized / deionized water, adjusted to pH 4.5 with KOH, sterilized at 121 ° C. for 20 minutes in the heat exchanger and filled into the sterile first culture vessel (ROOT-BOX). Table 1 Composition of the R medium

The propagation of the root pieces takes place in the dark at room temperature in complete silence. After 5 weeks of incubation, the roots are harvestable.

The roots propagated in the liquid nutrient medium represent an intact network of white roots which, unlike the MSR medium, are not uncontrollably damaged during harvesting and are shredded specifically for the further cultivation of mycorrhizated roots. The obtained roots were subjected to an official test confirming that they are free of genetic modification.

B) Precultivation of the spores

For the precultivation of the spores, the spores of Glomus intraradices are isolated from substrate solates of the mycorrhizal plants with the highest biomass from the spore testing plant test. These spores are cultured as single spore lines on leek (Allium porrum) in vivo. The spores obtained serve as a spore pool for pre-cultivation.

From this in vivo spore pool of Glomus intraradices spores are removed, disinfected and precultivated in the Petri dish on transformed roots of Daucus carota in vitro on solid nutrient medium (spore root medium).

For each preculture of in vitro mass production of Glomus intraradices, an in vivo / in vitro transition of spore delivery occurs. This preserves the symbiosis of the in vitro Glomus intraradices spores across many propagating passages. To further safeguard this symbiosis ability In addition, a host change system from passage to passage in the context of pre-cultivation is carried out as follows.

In the first passage, spores of Glomus intraradices on Daucus carota are pre-cultured as hosts. In the second passage, a host change takes place on Nicotiana tabacum. In the third passage again Daucus carota is used as a host. The landlord changes from passage to passage.

The resulting spores were subjected to an official test and confirmed to be free of genetic modification.

C) Production of the inoculum

The production of the inoculum (mycorrhizal roots) takes place in a second culture vessel, a so-called INO-BOX. For the inoculum production in the INO-BOX a solid nutrient medium (I-medium) with the composition given in Table 2 is used.

For the production of one liter of I-medium, the ingredients are mixed according to the amounts given in Table 3.2. For simpler dosing, 6 stock solutions of constituent groups are necessary: macroelements (# 1-4), microelements (# 5-10), vitamins (# 11-16), EDTA (# 17), IBA (# 19) and IAA (# 20) as functionalizing additives that provide for increased spore formation and increased root elongation. These components and sucrose are dissolved in demineralized / deionized water, adjusted to pH 5.6 with KOH, heated to 70 ° C, phytagel added and dissolved. This solution is sterilized at 121 ° C for 20 min in the heat exchanger and filled into the sterile INO BOX's. After cooling, a gel of 1 liter volume is formed. Table 2 Composition of the I-medium

The inoculation takes place in eight INO-BOX's, wherein on the inoculation frame of each box about 30 root pieces (length 3 cm) obtained after method step A) and 300 in vitro spores of Glomus intraradices are given. After 3 weeks incubation at room temperature (26 ° C) and darkness, the roots are mycorrhizal and harvestable. After comminution, they are used for inoculation.

D) Spore production

The in vitro spore production of Glomus intraradices takes place in a third culture vessel, a so-called MYKO-BOX. The structure of the MYKO-BOX is analogous to that of the INO-BOX. The differences exist in the useful volume and in the composition of the nutrient or culture medium used.

The MYKO-BOX is a culture vessel with the standardized dimensions height: 130 mm, width: 530 mm, depth: 330 mm. The MYKO-BOX consists of a transparent, lightweight, biocompatible, sterilizable plastic as an outer vessel and an insertable frame with mesh (inoculation frame) for fixing mycorrhizal roots in the vessel. The transparency of the material allows a visual control of the growth process. The inert surface properties of the material ensure biocompatible production without adversely affecting the organisms. The low weight and high stability of the MYKO-BOX facilitate the handling, storage and transport of the vessels in the production process. The requirements for the MYKO-BOX and the INO-BOX are best met by the material Polycarbonate caliber. The MYKO-BOX is a cuboid culture vessel with a fitting lid. MYKO-BOX and lid are made of the same material. The lid is 2 cm overhanging on the sides to eliminate the risk of contamination and to prevent the culture from drying out. If required, peripheral connections can be retrofitted with additional notches in the cover. The inside of the MYKO-BOX is finished without corners. The transitions of the side walls into each other and the transition of the side walls to the bottom of the vessel are fluid.

The MYKO-BOX is fed with a nutrient medium (SP medium) at a layer height of approx. 5 cm, which changes to the solid state after filling.

The chemical composition of the SP medium is given in Table 3 below.

*FZ: Funktionalisierender Zusatz zur Erhöhung der Sporenausbeute For the production of 5 liters of SP medium, the ingredients are mixed according to the amounts indicated in the table. For simpler dosing, 5 stock solutions of constituent groups are necessary: macroelements (# 1-4), microelements (# 5-10), vitamins (# 11-16), EDTA (# 17), and IBA (# 19) , These components and sucrose are dissolved in demineralised / deionized water, adjusted to pH 5.6 with KOH, heated to 70 ° C, phytagel added and dissolved. This solution is sterilized at 121 ° C for 20 min in the heat exchanger and filled into the sterile MYKO-BOX. Table 3 Composition of the SP medium

* FZ: Functionalizing additive to increase the spore yield

The inoculation of a MYKO-BOX is carried out with about 30 root pieces a 3 cm, which were obtained after process step C). According to this example, eight MYKO BOX's are used.

E) spore harvest

• • Trennen der Wurzeln vom Nährmedium durch Abheben des Inokulationsrahmens;
• • Zerkleinerung des festen Nährmediums und Vereinzeln der Sporen;
• • Auflösen der Gelbestandteile des Restnährmediums;
• • Waschprozess der Sporen und Hyphen mittels Filtration und
• • Resuspendieren der Sporen in Suspensionen zur Weiterverarbeitung.

After completion of the incubation period in the MYKO BOXs, the spore harvest comprises the following steps:

• • separating the roots from the nutrient medium by lifting the inoculation frame;
• • comminution of the solid nutrient medium and separation of the spores;
• Dissolving the gel components of the residual nutrient medium;
• • Washing process of spores and hyphae by means of filtration and
• • Resuspending the spores in suspensions for further processing.

During the slow removal of the inoculation frame from the MYKO-BOX, the root braid is lifted off the media surface. To separate the spores and hyphae, the gel-like residual nutrient medium is filled into a container and comminuted by means of rotating knives, the hyphae being cut gently. Through this procedure, the vitality of the spores is retained to 99%. The mechanical comminution breaks up the gel structure of the culture medium and achieves partial liquefaction. The addition of 7.5 l of citrate buffer with a pH of 6.0 per 5000 cm ³ amount of gel results in a complete dissolution of the gel. The resulting liquid mixture of residual medium and citrate buffer, in which the spores produced are suspended and optimally isolated, is filtered through a biocompatible polyester mesh (mesh size 20 μm) with vacuum to separate the spores and hyphae and washed several times with demineralized / deionized water. The filter cake of spores and hyphae is to keep sufficiently moist, in order not to expose the spores to any drought stress. The spore cake is carefully removed from the mesh with a suitable tool. The filter cake obtained is resuspended for the subsequent steps in a suitable liquid and stored. The storage of the in vitro spores of Glomus intraradices in 1% alginate or 0.5% Tyloselösung has proven to be particularly favorable. These spore suspensions are homogeneous and prevent the spores from settling over an extended period of time.

F) End product test

The determination of the number of spores in the resuspended harvest solution is carried out by microscopic counting of a subset (aliquots) of 10 × 10μl and serves to determine the overall yield.

The determination of the vitality of the harvested in-vitro Glomus intraradices spores in a known manner due to the enzymatic reduction of colorless iodo-nitrotetrazoliumchlorid (INT) to red formazan. The spores are incubated in a 0.5 mg / ml INT solution for 72 h at 26 ° C. Thereafter, vital Glomus intraradices spores appear red and damaged remain unstained. To determine the vitality rate, the percentage of red-colored spores is determined microscopically. The average vitality rate of the total technology described is 99%.

The symbiosis of the in vitro Glomus intraradices spores is determined in the in vivo plant test. The biomass increase is the decisive criterion here. In the application example maize, a biomass increase in the greenhouse> 20% and under field conditions> 10% can be detected.

G) packaging and product storage

The filling of the in vitro spore suspensions of Glomus intraradices into the volumes of the commercial forms is realized by means of pump systems with compressed air portioning. The single dosage depends on the end customer requirement and field of application.

The overall method described allows the widest possible use of all resulting end products. The spore harvest roots contain infectious components (propagules) of Glomus intraradices and are processed by suitable methods. Likewise, the media residues from the steps of precultivation of roots and in vitro spores of Glomus intraradices, inoculum production and spore production are worked up.

Under the conditions mentioned above, 30 million in vitro spores of Glomus intraradices, approx. 200 g of mycorrhized roots (wet mass) with> 4000 spores / g, 12.5 liters of liquid media residues with usable metabolites (glomalin, root exudates) per MYKO-BOX and fertilizer components.

For a mass production, 100 MYKO BOXES (one lot) can be processed daily in a hall on a production line with the indicated process steps. Each batch goes through three production cycles per year. Thus, 9 billion in vitro spores of Glomus intraradices can be produced per lot and year. With 250 batches per year, an annual production of 2.25 trillion spores is possible.

For the major agricultural crops corn, wheat, soy, rice and others, the application rates / ha and forms of application of in vitro spores are different. For corn seed killing, 70,000 grains / ha require 700,000 in vitro VA mycorrhizal spores / ha. With an annual production of 2.25 trillion spores, corn seed can be inoculated for approximately 3 million hectares of cultivated land.

The resulting culture media can be used after processing and transfer into the liquid phase as a valuable fertilizer.

An essential part of a plant for the mass production of VA mycorrhizal spores is in the 2 and 3 shown feeding unit, which can be used for both the feeding of the second culture vessels (INO-BOXES) and the third culture vessels (MYKO-BOXEN) with roots.

The number of feed units depends on the size of the entire system, ie the number and size of the required culture vessels for the production volume of VA mycorrhizaspores to be achieved. The size of the culture vessels depends on the one hand on their ability to manipulate in the overall process and on the other hand on the growth conditions. Culture vessels for uptake of 5 l of culture medium have proven to be the most effective for mass production, since in these the hyphae could form to the bottom and the secondary spores were evenly distributed throughout the medium. In addition, after completion of the incubation, the largest number of VA mycorrhizal spores per liter of medium were determined.

Depending on the size of the culture vessels is also the lower conveyor belt 1 designed and dimensioned. The conveyor belt 1 at defined intervals successively supplied culture vessels 2 , without lid, are already with the respective culture medium 3 filled, which turns into a solid consistency after filling. After that, the frames become 4 with mesh 5 used such that the screen mesh 5 on the solid culture medium 3 rests.

Above the on the assembly line 1 located culture vessels 2 is a shorter in its length root conveyor belt 6 arranged in a kind of floor construction. At the beginning of the root conveyor belt 6 is a sterilization unit 7 intended for sterilization of the conveyor belt. Depending on the width of the band, one or more UV sterilization lamps are arranged. After the sterilization unit 7 is a rooting and dosing device 8th Installed. These become already shredded root pieces 9 fed via a metering lock on the root conveyor belt 6 fall and from this into the underlying culture vessel 2 , By means of a at the end of the root conveyor belt 6 arranged distribution device 10 become the falling root pieces 9 evenly on the mesh located in the culture vessel 5 distributed. The transport direction of the conveyor belt 1 is marked by arrows. During the filling process for the next culture vessel 2 Be the ones on the mesh 5 of the previous culture vessel resting root pieces 9 by means of a lowerable pressure roller 11 firmly attached to the mesh 5 pressed so that they come into contact with the culture medium. The lowering and lifting mechanism for the pressure roller is equipped with a sensor, so that shortly before reaching the rear wall of the culture vessel 2 the pressure roller 11 automatically moved up again. The directions of movement are indicated by an arrow. If the feeding device is intended for feeding the third culture vessels (MYKO-BOXES), these are after the completion of the feed by means of the associated lid 12 closed and subsequently by a gripper from the assembly line 1 removed and inserted into a shelf unit not shown in detail for subsequent incubation.

After an incubation period of at least 90 days at a temperature of 26 ° C and darkness, a count of the spores in the aliquot, after the result of the harvesting process is initiated.

During the incubation process, the culture vessels (MYKO-BOXES) are visually inspected for contamination after 14 and 28 days. After 65 days, a first assessment of root growth and spore formation takes place.

By using frames with mesh during cultivation in MYKO-BOXEN, it is possible to largely separate the roots from the medium without affecting the growth of any of the biological components. This is a decisive advantage in the later processing of the final product.

For successful mass production of VA mycorrhizal spores, the development of appropriate culture vessels (see 4 to 6 ) proved to be essential. In the developed culture vessels 2 for the cultivation of inoculation material (INO-BOX) or for the formation of VA mycorrhiza spores (MYKO-BOX) are rectangular plastic containers 12 ( 4 ) with usable frame 4 with mesh 5 ( 5 ) and a loosely attachable and removable cover 13 ,

The frame 4 ( 5 ), which on the solid culture medium 3 rests and receives the inoculum (mycorrhizal roots) consists of two congruent, for example 15 mm wide, frame parts 4a and 4b made of stainless steel. Between these is the tissue 5 clamped to fix the root culture. The attachment of the tissue 5 between the frame components 4a and 4b takes place by means of symmetrically arranged brackets 15 made of stainless steel. This frame construction ensures by the weight of the steel an intensive surface contact of the mycorrhizal roots with the surface of the solid culture medium 3 ,

The tissue 5 consists of a biocompatible, sterilizable polyester that is reusable several times. For optimum fixation, the mesh size should not exceed 2 mm. The fabric serves as an anchoring and growth aid. Root growth occurs along the tissue on the media surface of the culture. The root growth into the depth of the nutrient medium is thereby significantly reduced, but not hindered hyphal growth. The spores are mainly formed in the medium below the frame.

That in the 4 and 6 shown culture vessel 2 is a cuboid container, for. B. in the dimensions 130 × 530 × 330 mm (height × width × depth), with a tight fit lid 13 , The container is designed to hold 5 liters of culture medium with a height of 5 cm. Container and lid are made of the same material, preferably polycarbonate caliber. It has been found that this material has the required requirements for compatibility in spore root culture (no adverse effects on spore germination, root and hyphae growth, formation of secondary spores).

The lid 13 has on the sides of about 2 cm overhanging edge 14 to eliminate the risk of contamination and prevent the culture from drying out. If required, peripheral connections can be retrofitted with additional notches in the cover. The transitions from the bottom to the side walls and corner areas in the interior of the container are designed to be round in order to facilitate the removal of the gel for spore harvest. The culture vessel and lid consist of a transparent, lightweight, biocompatible, sterilisable plastic that can be steam-sterilized at a temperature of 121 ° C. The transparency of the material allows a visual control of the growth process. The inert surface properties of the material ensure biocompatible production without adversely affecting the organisms. The low weight and high stability facilitate the handling, storage and transport of the vessels in the production process.

Depending on the size of culture vessels for receiving 1.5 l and 5 l culture medium are determined, at a media layer thickness of 1.5 cm and 5 cm.

The culture vessels described above are suitable for the cultivation of inoculation material in the so-called second culture vessel (INO-BOX) as well as for the process for the formation of VA mycorrhizaspores in the so-called third culture vessel (MYKO-BOX).

The technical difference is that the associated frame component 4b for the second culture vessel (INO-BOX) feet 4c are arranged, whose height is matched to the height of the culture medium.

With the new culture vessel for 5 liter culture medium, a production of 3,000 to 6,000 spores / ml medium could be statistically reliably achieved in experiments.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2005/000008 A2 [0006, 0052]
• WO 2007/014974 A1 [0007]
• EP 1231854 B1 [0008]

Claims (14)

 Method for the in-vitro production of VA mycorrhizal spores with the following method steps: a) propagation of roots of dicotyledonous plants in root lines produced by natural transformation and selection of the roots for the highest growth performance, b) precultivation of the selected root lines in successive propagation steps in Petri dishes, removal of root pieces from the last propagation of the root line for mass cultivation in first culture vessels in a liquid, sterile nutrient medium at rest, wherein the individual culture vessels are fed with roots of different root lines; c) transfer of in vivo VA mycorrhizaspores in the in vitro culture in petri dishes on transformed roots of dicotyledonous plants on solid nutrient medium (spore root medium), wherein the precultivation in several passages carried out, wherein in each passage a host change he follows; d) production of the inoculum of mycorrhizal roots from roots obtained according to method step b) and VA obtained according to method step c) in second culture vessels in a solid sterile nutrient medium, each culture vessel having an inoculation frame with root fixation tissue for uptake of the roots and VA mycorrhizal spores several weeks of incubation at room temperature and darkness, mycorrhizal roots are harvested and minced to about 0.5 to 2 cm long pieces; e) inoculation of the mycorrhizal root pieces obtained according to method step d) in third culture vessels with solid nutrient medium and with a sieve-like support for root pieces and subsequent incubation for a period of at least 90 days, in darkness and room temperature, and finally separating the roots from the nutrient medium by lifting the inoculation frame, f) mechanical comminution of the solid nutrient medium containing the produced spores and transfer into the liquid phase, gentle division of the hyphae, dissolution of the residual nutrient medium including gel components, washing the VA mycorrhizaspores and hyphae and resuspending the spores. A method according to claim 1, characterized in that the spores of the VA mycorrhizal fungi are selected from fungi of the order Glomerales and the genera Glomus and Gigaspora. A method according to claim 2, characterized in that the fungi are species Glomus intraradices, Glomus irregular, Glomus proliferum, Glomus etunicatum, Glomus clarum, Glomus manihotis, Glomus aggregatum, Glomus caledonium, Glomus claroideum, Glomus fasciculatum and Glomus hoi. Method according to one of claims 1 to 3, characterized in that the root lines come from different accessions of Daucus carota. Method according to one of claims 1 to 4, characterized in that the root lines of various varieties of Nicotiana tabacum, Lotus corniculatus, Lycopersicon lycopersicon or Fragaria ananassa originate. Method according to one of claims 1 to 5, characterized in that for preculturing according to method step c) three, four, five, six or seven passages are performed. Method according to one of claims 1 to 6, characterized in that are used as nutrient media gel-like and / or aqueous solutions based on sucrose containing macroelements, microelements and vitamins and optionally other functionalizing additives. Method according to one of claims 1 to 7, characterized in that the gelatinous nutrient media contain as functionalizing additives at least one or more phytohormones in the ppm range in order to improve root growth (IAA), spore germination and spore formation. Method according to one of claims 1 to 8, characterized in that a complete dissolution of the gelatinous nutrient medium is achieved by comminution and Citratzusatz, wherein the ratio of medium to be dissolved and citrate buffer is 1: 1.5. Method according to one of claims 1 to 9, characterized in that in the liquid mixture of nutrient medium and citrate buffer in a suspended form before lying spores are filtered under vacuum and stored in alginate or Tylose. Plant for carrying out the method according to one of claims 1 to 10, comprising at least one charging unit with a) a circulating conveyor belt ( 1 ) for receiving culture vessels ( 2 ), wherein a culture vessel made of a transparent, lightweight, biocompatible, sterilizable plastic container ( 12 ) having a rectangular or square cross-sectional area, a height of 50 to 150 mm and a capacity of at least 1.5 l of culture medium with a filling level in centimeters of 10% to 20% of the amount of culture medium, one in the container ( 12 ) usable framework ( 4 ) with mesh ( 5 ) and a easily attachable and removable cover ( 13 ) with a circumferential overhanging edge ( 14 ), b) a vertically movable pressure roller ( 11 ), which in the operating state on the screen mesh ( 5 ) of the incoming culture vessel ( 2 ) resting root pieces ( 9 c) one above the culture vessels ( 2 ), circulating root conveyor belt ( 6 ) for feeding the culture vessels ( 2 ) with root pieces ( 9 ), d) one above the root conveyor belt ( 6 ) arranged rooting and dosing device ( 8th ), e) one before the rooting and dosing device ( 8th ) arranged sterilization unit ( 7 ) for sterilizing the assembly line ( 1 ) and f) one at the front end of the root conveyor belt ( 6 ) arranged root distribution device ( 10 ). Plant according to claim 11, characterized in that the frame 4 from two identical frame components ( 4a . 4b ), between which the mesh ( 5 ) and the two frame components ( 4a . 4b ) are held together by means of clip-on brackets. Installation according to one of claims 11 or 12, characterized in that the frame components ( 4a . 4b ) made of stainless steel. Installation according to one of claims 11 to 13, characterized in that the screen fabric ( 5 ) consists of polyester with a mesh size of 0.5 to 2 mm.

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