HK1023033A - Biological material embedded in hydrogels, a process for its embedding, as well as its use as artificial seed material - Google Patents

Description

Hydrogel coated biological material, method for coating said material and use thereof as artificial seed

The invention relates to fully biodegradable hydrogels containing polyester polyurethane polyureas and polysaccharides and/or derivatives thereof, and plant materials that can be isolated.

The invention also relates to a method for coating the biological material and generating and forming the hydrogel from the aqueous solution, and an artificial seed made of the biological material coated by the hydrogel.

Plants are propagated sexually by seeds and asexually by vegetative propagation of plant meristems. Both propagation modes are economically important. Although a large number of natural seeds are sown mechanically, vegetative propagation requires a large amount of manual labor, and thus takes a lot of time and labor intensity compared to seed propagation.

Plant varieties, lines, cultivars, and cell lines (e.g., clonal propagation of elite plants) are clonally propagated, and a particular genome is important for them. Vegetative propagation can also be used for plants which have been able to form seeds over a long period of time, form only a small number of seeds, or whose seed germination capacity has been impaired.

In order to simplify the vegetative propagation of plants, in addition to the development of automated methods for large-scale cultivation, there is a need for suitable substances and methods for coating friable material, which can be further processed into seed boxes.

In large-scale cultivation methods, it is now possible to produce, for certain types of plants, miniaturized and isolatable regenerable plants (tissues) (e.g. WO95/19102, US5294549, US 5334530). Without mechanical protection and/or protection against desiccation, these plant parts can only be transported and stored to a limited extent. For this purpose, it is necessary to coat or cover the plant parts as separate units so that they can be stored and/or transported, in sufficient dosage and as such be used as natural plant seeds.

DE2103873, EP141374, EP107141, US4562663, WO8502972, US4779376, WO9207457 describe hydrogel-coated plant materials which are produced with ionic crosslinkable polysaccharides such as alginates, gelatin, carrageenan or locust bean gum.

The aforementioned materials, in combination with the materials and methods described in the prior art, are not yet fully satisfactory, since in some cases they neither impart sufficient mechanical stability to the coated structure nor protect the plant tissue against too rapid and excessive water loss under the conditions of use. This is particularly true of the polysaccharide derivatives mentioned above. At the same time, a significant shrinkage of the material during drying is also observed, which seriously affects the protective function of the seed box. A further problem consists in particular in that the coating hitherto applied on the basis of polysaccharides, such as alginic acid or a salt thereof, or further ionic polysaccharide derivatives, is not rehydrated to a sufficient extent after the drying stage. These materials can only be stored at a suitable air humidity.

Subsequent application of a fat, oil, wax or water-insoluble polymer coating to retard dehydration and to maintain mechanical stability is also undesirable, as disclosed for example in US4562663, WO9217422, US 5190797. They also are not suitable if they have to be processed at non-physiological elevated temperatures, requiring the use of organic solvents, or if they adversely affect the oxygen supply of the enclosed biological material.

Besides polysaccharide-based hydrogels, polyurethanane hydrogels (PU) are also described. DE3312578 and DE4217891 describe the use of polyureauranes for segregating immobile cells. In this application, the PU hydrogels act as cell support materials and biocatalysts in aqueous suspensions, but the PU hydrogels described for this purpose are not biodegradable.

It is an object of the present invention to provide a detachable coated/packaged form of biological material to protect said material from substantial delays in dehydration during storage, transport and handling, to remain dimensionally stable, to re-swell to a sufficient extent after partial dehydration, to be biodegradable and to be easily manufactured.

Additives such as nutrients or active and protective substances may also be added.

The materials required must operate under sterile conditions and avoid the use of toxic solvents or physiologically unacceptable conditions.

The foregoing objects are surprisingly achieved by the use of fully biodegradable polyester polyurethaneureas (polyester polyurethane polyhardnstoff) and polysaccharides or polysaccharide derivatives, which can be used as aqueous dispersions or as aqueous solutions.

In accordance with the foregoing objects of the present invention, it has surprisingly been found that polyester polyurethaneureas are suitable for coating biomaterials and can be used in coatings with biodegradable polysaccharides or their derivatives.

The invention relates to biodegradable hydrogels containing at least A) a polyester-polyurea, and B) a polysaccharide and/or polysaccharide derivative, and C) a biological material, preferably a plant material that can be isolated, in particular a plant cell, a callus, a protoplast, a plant tissue or a plant organ, such as an adventitious branch, a micro-nodule, an axillary bud, a terminal bud, a scion, and a zygote or a somatic embryo or a proembryo analogue.

The plant material may be obtained from the following plants: plants which provide nutrients and raw materials, such as cereals (rice, maize, wheat, barley, rye, millet), potatoes, legumes (e.g., alfalfa and soybean), rapeseed, sunflower, oil palm, sugarcane, sugar beet, sisal, cotton, miscanthus and tobacco; vegetables and root crops (e.g., tomatoes, various cabbage, lettuce, carrot, eggplant, melon, cucumber, asparagus, onion, parsley, ginger); medicinal plants such as Ginseng radix, belladonna, and Digitalis purpurea; fruit trees (e.g., apples, pears, cherries, grapes, strawberries, citrus fruit, mangoes, papayas, bananas, nuts); tea trees, cocoa trees, coffee shrubs; forest trees, such as conifers, spruces, pines, larch, foliage trees, such as populus plants, beech, oak plants; ornamental plants, such as roses, chrysanthemum, lilies, flowers of all flowers, orchids, plants of the genus geranium, begonia, dianthus, and plants of the genus candelilla.

Furthermore, it is preferred to use those separable biological materials, particularly preferably transgenic plants, which no longer make it possible to propagate by means of seeds or vegetative organs or only bring about difficulties in propagation of seeds or vegetative organs due to the nature of the genetic improvement, for example the specific expression of seeds or nodules of the product.

Hereinafter the term "coating" denotes all possible methods of encapsulating, covering, coating, packaging biological material etc. according to the invention.

The biodegradability of the material meets the requirements under standard conditions (see example 6).

The polyester polyurea polyureas according to the invention can be used in one step or in several steps in admixture with ionic or neutral biodegradable polysaccharides and their derivatives to form shaped bodies such as spheres, fibers, sheets, films and the like.

According to the invention, the aqueous matrix (hydrogel) is formed by a polysaccharide, the mechanical properties of the hydrogel are surprisingly improved by the polyester polyurethanane polyureas in order to produce simple shaped bodies, such as spheres, and the water loss of the hydrogel and the biomaterial can be controlled.

The polyester polyurethaneureas used according to the invention are known and are disclosed in DE 19517185.

The aforementioned polyureas are prepared by the reaction while maintaining the equivalent ratio of isocyanate species to isocyanate reactive ranges from 1: 1 to 2: 1.

a) The diisocyanate component comprises

a1)1, 6-hexamethylene diisocyanate or

a2)1, 6-hexamethylene diisocyanate and up to 60% by weight, based on the total mixture, of 1-isocyanato-3, 3, 5-trimethyl-5-isocyanatomethyl-cyclohexane and/or 4, 4' -diisocyanatodicyclohexylmethane and/or 1-methyl-2, 4(6) -diisocyanatocyclohexane

b) The diol component comprises

b1) Is at least one polyester diol having a molecular weight of 500 to 10000, calculated on the hydroxyl group content, from (i) adipic acid and/or succinic acid and (ii) at least one alkanediol having 2 to 6 carbon atoms, or

b2) A mixture of the above polyester diols and, based on the total weight of component b), 32 equivalent% of alkanediols optionally having ether groups and having from 2 to 6 carbon atoms.

c) Comprising a hydrazine component in an amount of from 2 to 50 equivalent%, based on the total amount of isocyanate-reactive groups present in components (b) and (c).

c1) Diaminosulphonate of the general formula

H₂N-(-CH₂-)_n-NH-(-CH₂-)_m-SO₃Me

c2) Mixtures of salts of diaminosulphonic acid c1) and 70% by weight, based on the total weight of component c), of 1, 2-ethylenediamine

d) 10% by weight, based on the total weight of b), c) and d), of an optional hydrophilic component polyetherol of the formula

H-X-O-R

And

e) optionally water, not included in the calculation of the isocyanate groups and the equivalent weight of groups reactive with isocyanate groups in equal proportions,

wherein in the aforementioned formula

m and n each independently represent an integer of 2 to 6,

me represents potassium or sodium, and Me represents potassium or sodium,

r represents a monovalent hydrocarbon group having 1 to 12 carbon atoms, and

x represents a polyalkylene oxide chain having a molecular weight of 88 to 4000, the alkylene oxide units of which comprise at least 40% ethylene oxide units and the balance propylene oxide units.

An aqueous dispersion of a polyester polyurethaneurea is thus obtained.

The term "aqueous dispersion" is intended to include aqueous solutions if the concentration of hydrophilic centers in the urea group-containing polyurethane is sufficiently high to ensure stability in water. Often these dispersions are aqueous solutions containing polyurethanes with urea groups that are dispersible and soluble.

To prepare this aqueous dispersion, the aforementioned starting materials a), b), c) and optionally d) and/or optionally e) are mixed in the quantitative ratios mentioned above.

The diisocyanate component a) preferably consists of 1, 6-hexamethylene diisocyanate or a mixture of 1, 6-hexamethylene diisocyanates with up to 60% by weight of 1-isocyanato-3, 3, 5-trimethyl-5-isocyanatomethyl-cyclohexane and/or 4, 4' -diisocyanatodicyclohexylmethane and/or 1-methyl-2, 4(6) -diisocyanatocyclohexane.

The diol component b) comprises either b1) of at least one polyester diol or a b2) mixture of at least one b1) and up to 32% by weight, preferably up to 10% by weight, of an alkyl diol optionally containing ether groups and 2 to 6 carbon atoms.

Suitable polyester diols b1) are those based on (i) adipic acid and/or succinic acid and (ii) optionally alkanediols having 2 to 6 carbon atoms and containing ether groups, such as 1, 2-ethanediol, diethylene glycol, 1, 4-butanediol, neopentyl glycol and/or 1, 6-hexanediol, having a molecular weight of 500 to 10000, preferably 1000 to 2500, calculated as hydroxyl group content. Particular preference is given to using only 1, 2-ethylene glycol and/or 1, 4-butanediol as diol.

These optional ether-containing alkane diols having 2 to 6 carbon atoms which can optionally be used as the hydroxyl-containing chain extenders are those of the type exemplified above.

The hydrazine component c) comprises or is c1) a diaminosulphonate of the formula mentioned above or c2) a mixture of these diaminosulphonates and 1, 2-ethylenediamine, where the amount of 1, 2-ethylenediamine, if used, is up to 90%, preferably 70%, equivalents based on the number of amino groups which have been reacted in the component c) to neutralize the cyanic acid groups. Particularly preferred salts of diaminosulphonic acid are the potassium or sodium salts of N- (2-aminoethyl) -2-aminoethylsulphonic acid.

The diamine component c) is generally used in an amount of from 1 to 10% by weight, preferably from 2 to 5% by weight, based on the weight of component b).

The optionally used structural component d) is a hydrophilic, monohydric polyether alcohol of the formula

H-X-O-R

Wherein

R and X are the same as described above.

Preference is given to polyether alcohols of the type in which

R represents an aliphatic hydrocarbon group having 1 to 4 carbon atoms, and

x represents polyalkylene oxide chains having a molecular weight of 500 to 4000, of which at least 40%, in particular at least 70% and particularly preferably 100%, of the alkylene oxide units are ethylene oxide units and the remainder alkylene oxide units are propylene oxide units.

The forming and simultaneous coating of the biomaterial is effected by ion-induced coagulation of the polyester polyurethanane polyurea wherein the polysaccharide component is blocked. The coating process may be carried out in one step or in multiple steps. In a one-step process the biomaterial and the polyester polyurea are mixed together and added to a salt solution to coagulate. This coating method is essentially determined by the viscosity of the polyester polyurethane polyurea/polysaccharide mixture used in the solution used.

In a two-step process in which the hydrogel spheres comprise a polysaccharide, a suitable polysaccharide production, for example alginate, may first be selected. These hydrogel particles can provide a mechanically stable coating by immersion in an aqueous solution of a polyester polyurethane polyurea.

All biodegradable polysaccharides or their derivatives can be used according to the invention alone or as the polysaccharide component of the hydrogel of the mixture. Suitable polysaccharides are, for example, native and soluble starch obtained from any suitable material, amylose, amylopectin, alginic acid, hydrochloric acid, alginates, carrageenans, chitin, chitosan, dextran, glycogen, guar gum, carob seed flour, laevan, pectin, pullulan, tamarind powder, synthetic biopolymers and hylan, as well as cellulose obtained from any suitable material. Suitable cellulose derivatives are, for example, cellulose ethers, cellulose esters and cellulose carbamates.

Particularly suitable are, for example, cellulose ethers such as methylcellulose, ethylcellulose or benzylcellulose having an average degree of substitution of 2.5 or less, hydroxyethylcellulose, hydroxypropylcellulose, dihydroxypropylcellulose, hydroxybutylcellulose, methylhydroxyethylcellulose, methylhydroxypropylcellulose, methylhydroxybutylcellulose, ethylhydroxypropylcellulose, ethylhydroxyethylcellulose, carboxyalkyl cellulose, sulfoalkyl cellulose, cyanoethylcellulose and mixed ethers thereof. Particularly preferred is methylcellulose, hydroxyethylcellulose or hydroxypropylcellulose. Also suitable are polysaccharide derivatives, in particular cellulose derivatives and any mixtures of ether, ester and carbamate substituted celluloses thereof.

The term "mixing" in the following may be sterilization by autoclaving and complete biodegradation, according to the invention of the polyurethanes in combination with the polysaccharides.

These mixtures can also be examined and adjusted for further properties such as moisture content and balance, size stability, permeability to oxygen and nutrients, regulation of physiological conditions, such as mechanical breakdown of the germinating plants, and binding and permeability of nutrients, protectants and active ingredients.

Surprisingly, such a mixture has a combined property which is beneficial for the purpose of the invention, i.e. coating the biological material capable of being split. These combined properties include:

the mixing can be carried out in aqueous solution.

The mixing can be carried out at physiological temperatures (18-30 ℃).

The mixing can be sterilized by autoclaving without affecting their properties.

The mixture is completely biodegradable and compostable.

The mixed coating method is simple and economical to use.

The mixture is non-toxic to plants.

The exchange of moisture and gas is ensured after the mixing process.

Satisfactory germination is obtained after mixing.

The invention also provides an aqueous coated biomaterial composition comprising a fully biodegradable polyester polyurethaneurea and at least one fully biodegradable ionic or neutral polysaccharide or polysaccharide derivative.

The coating composition preferably comprises at least 20% by weight of the aforementioned polyester polyurea and at least 0.1% by weight of a polysaccharide component, such as starch, starch derivatives, cellulose ethers, or any mixtures thereof.

Preference is given to water-soluble or at least readily swellable polysaccharide derivatives, such as starch, starch ethers or cellulose ethers, and also aqueous dispersions of 5 to 50% by weight of polyester polyurethane polyureas. Particularly preferred are soluble starch, alginates, methylcellulose, hydroxyethylcellulose, methylhydroxypropylcellulose, methylhydroxyethylcellulose and/or hydroxypropylcellulose.

The invention also provides a process for coating biological material, wherein the biological material is mixed with a polysaccharide and/or polysaccharide derivative in the presence of an aqueous dispersion of a polyester polyurethanane and the mixture is coagulated by contact with an aqueous salt solution. The polysaccharide component and the added biomaterial are coated by means of ion-induced coacervation of the polyester polyurethanepolyurea, this coating step also being substantially determined by the viscosity of the applied polyester polyurethanepolyurea/polysaccharide mixture in the solution used.

The dynamic viscosity of the ionomer solution is preferably greater than 1.1X 10⁶m²/sec。

The coating process may be carried out in one or more steps. In the case of a one-step coating of the biomaterial, the polyester polyurethane polyurea and polysaccharide components are mixed together and the mixture is added to an aqueous solution of salt to coagulate. The hydrogel particles form a spherical shape, a barrel shape, etc. according to this method. The hydrogel coating the biomaterial comprises a mixture of a polysaccharide and a polyester polyurea.

In the two-stage process, hydrogel spheres consisting of polysaccharides can be produced by first selecting suitable polysaccharides, for example alginates. These hydrogel spheres are obtained by dropwise addition of a salt solution to a mixture of polysaccharide and biomaterial. The hydrogel particles in the coacervation of the polyester polyurethanepolyurea also contain a sufficient amount of ions. These hydrogel particles are then impregnated in an aqueous solution of a polyurethanane polyurea to obtain a mechanically stable coating.

Thus there are at least 2 possible ways to coat biomaterials with polysaccharide/polyester polyurethanane polyurea hydrogels in general.

In general this method will vary with the mix of biomaterial, polysaccharide, polyester polyurethanepolyurea and ions, although any mixture of A and B may be used, the interaction between the polyester polyurethanepolyurea and ions will always lead to agglomeration and coating and shaping, as a result of which this step has to be carried out last, where mixture A may comprise polysaccharide, polyester polyurethanepolyurea and/or biomaterial, and mixture B may comprise ion, biomaterial and polysaccharide.

In a particularly preferred one-step embodiment the polysaccharide component is swollen or dissolved in an aqueous polyester polyurethanane polyurea dispersion, the biological material is added, and the resulting mixture is coagulated by adding ions, preferably polyvalent ions, in particular Ca²⁺,Mg²⁺Or Al³⁺The chloride form, in the concentration range of 10-1000mM, will affect shaping into spheres, fibers, flakes or other shaped bodies in this step. Finally, a hydrogel comprising a mixture of polysaccharide and polyester polyurethanepolyurea is produced.

In another preferred two-step embodiment the biomaterial is mixed in aqueous solution with ions and polysaccharides and coated with a polysaccharide hydrogel, which is sequentially coated with the polyurethanepolyurea by adding the mixture to the polyurethanepolyurea dispersion.

In the coating process, nutrients, protective substances and active agents which promote the growth or metabolism of the biological material, which protect the biological material against harmful influences, may also be added to the coating composition.

In a preferred embodiment the coating composition may be prepared as a semi-concentrated nutritional liquid as described by Murashige and Skoog (published in Physiol.plant.15,473,1962) to which 5-20g/l, preferably 10g/l, of sucrose is added.

Other nutrient salt mixtures, as commercially applied, and sugars may also be used depending on the coated plant material. To affect development, the nutrients may include plant hormones known to those skilled in the art. Depending on the plant material, nutrients include nutrient salt mixtures and vitamin mixtures which are convenient and commercially available, also optionally commercially available natural or synthetic phytohormones, such as those derived from auxins, cytokinins, gibberellins, abscisic acid, and ethylene formers. In addition, compounds having vitamin-like or plant hormone-like effects such as chlormequat chloride, lipo-oligosaccharides, salicylic acid derivatives may also be used.

In a particular embodiment, bactericides, fungicides, insecticides, acaricides, nematicides and suitable natural tolerance or tolerance obtained by genetic technology, and also herbicidal active substances can also be added to the coating material to protect the isolated plant material. The protective substances include, for example, insecticides, such as those selected from the group consisting of phosphates, carbamates, in particular imidacloprid, or fungicides such as those selected from the group consisting of pyrroles, in particular triadimenol and tebuconazole.

The following are examples that may be used as fungicides:

2-aminobutane; 2-anilino-4-methyl-6-cyclopropyl-pyrimidine; 2,6 ' -dibromo-2-methyl-4 ' -fluoromethoxy-4 ' -trifluoromethyl-1, 3-thiazole-5-carboxanilide; 2, 6-dichloro-N- (4-trifluoromethylbenzyl) -benzamide; (E) -2-methoxyimino-N-methyl-2- (2-phenoxyphenyl) -acetamide; 8-hydroxyquinoline sulfate; methyl- (E) -2- {2- [6- (2-cyanophenoxy) -pyrimidin-4-yloxy ] -phenyl } -3-methoxyacrylate; methyl- (E) methoxyimino [ α - (o-tolyloxy) -o-tolyl ] acetate; 2-biphenol (OPP), Aldimorph, Ampropylfos, benomyl, penconazole, benalaxyl, mefenoxam, benomyl, Binapracyl, biphenyl, bitertanol, blasticidin, bromuconazole, sulfopyrimethanil, flutriafol, lime-thion, captafol, captan, carbendazim, carboxin, benzoquinone, chloroneb, chlorothalonil, chlozolinate, thiabendazole, Cymomanil, cyproconazole, Cyprofuranm, dichlorobenzene, chlorotriazole, dichlorotrimazole, dichlofluanid, pyridaben, 2, 6-dichloro-4-nitroaniline, diethofencarb, difenoconazole, dimethomorph, diniconazole, dinocap, diphenylamine, pyridaben, dithianon, dodine, fenaminostrobilurin, fenzophos, etoxazone, ethionzazole, ethion, fenarimol, Fenbuconazole, fenflurazole, ferbamate, azoxanizone, fluazinam, Fludioxonil, flufenadine, Fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, Fosetyl-aluminum, tetrafluorophthalide, furylbenzimidazole, furamectin, furmecylox, iminoctam acetate, hexachlorobenzene, hexaconazole, hymexazol, imazalil, iminoctal, iminoctam acetate, iprobenfos (ibp), imazalil, isoprothiolane, kasugamycin, copper preparations such as: copper hydroxide, copper naphthenate, copper oxychlorolide, copper sulfate, copper oxide, copper 8-hydroxyquinoline and bordeaux mixture, mancopper mixture, mancozeb, maneb, mepanipyrim, mefenoxamine, Metconazol, sulbencarb, furam, metiram, tiadinil, myclobutanil, nickel dimethyldithiocarbamate, iprodione, ciclesonide, methylfuroamide, oxadixyl, Oxamocarb, carboxin, pefurazoate, penconazole, pencycuron, Phosdiphen, polymalexin, piplaline, polyoxin, probenazole, prochloraz, folpet, pavil, propiconazole, propineb, fosinophos, pyriproxyfen, Pyrimethanil, pyroquilon, quintozene, penclonitrobenzene, sulfur and sulfur preparations, tebuconazole, phthalein, tetrachlorfenpyrazote, fluconazole, thiabendazole, triadimefon, trimethoprim, pyrazofos, triazophos, pyraclostrobin, trifloxystrobin, triazophone, pyraclostrobin, trifloxystrobin, triazophone, triazophos, trichlamid, tricyclazole, tridemorph, tefurazone, azinam, Triticonazol, validamycin A, vinclozolin, zineb, ziram, 8-tert-butyl-2- (N-ethyl-N-N-propyl-amino) -methyl-1, 4-dioxo-spiro [4,5] decane, N- (R) - (1- (4-chlorophenyl) -ethyl) -2, 2-dichloro-1-ethyl-3 t-methyl-1R-cyclopropanecarboxylic acid amide (mixture of diastereomers or each individual isomer), [ 2-methyl-1- [ [ [1- (4-methylphenyl) -ethyl ] -amino ] -carbonyl ] -propyl ] -carbamic acid-1-methylethyl ester and 1-methyl- Cyclohexyl-1-carboxylic acid- (2, 3-dichloro-4-hydroxy) -anilide.

The following are examples of bactericides that can be used:

bronopol, dichlorobenzene, chlordine, nickel dimethyldithiocarbamate, kasugamycin, octyl isothiazolone, furancarboxylic acid, oxytetracycline, probenazole, streptomycin, phyllo-cumylphthalein, copper sulfate and other copper preparations.

The following are examples that may be used as insecticides, acaricides and nematicides:

oleamycin, acephate, fluthrin, gossypocarb, aldicarb, alphamethrin, amitraz, avermectins, AZ60541, Azadirachtin, oryzophos a, oryzophos M, azocyclotin, bacillus thuringiensis, 4-bromo-2- (4-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) -1H-pyrrole-3-carbonitrile, bendiocarb, amgac, Bensultap, cyfluthrin, bifenthrin, fenobuconazole, bromophos a, carboxim, buprofezin, methylthiobutanone methylaminocarbonyl oxime, butypyridaben, cadusafos, carbaryl, carbofuran, carbostyryl, chlorcarb, trithion, carbosulfan, chlorfenthion, chlorhexyd, chlorhexydos, chlorfenvinphos, N- [ (6-chloro-3-cyano) -N' -methyl-amide-ethane, chlorpyrifos, Chlorpyrifos M, cis-pyrethrum, Clocythrin, clocytazine, cyanophos, cycloprothrin, cyfluthrin, cyhalothrin, fenhexatin, cypermethrin, cyromazine, deltamethrin, systemic phosphorus-M, systemic phosphorus-S, methyl systemic phosphorus thionate isomers, diafenthiuron, diazinon, dichlofenthion, dichlorvos, dichliphos, chlormephos, ethion, diflubenzuron, dimethoate, dioxathion, ethoprophos, dichlorphos, Emamectin, cis-fenvalerate. Bendiocarb, ethiofen, toleron, ethoprophos, oxypyrimidine, fenamiphos, fenazaquin, fenbutazone, Fenobucarb, fenoxycarb, fenpropathrin, fenpyrd, fenpyroximate, fenthion, esfenvalerate, Fipronil, fluazinam, Fluazuron, flucycloxuron, flucythrinate, flufenoxuron, Flufenprox, flufenthion, difenofos, fosthiazate, furathiocarb, HCH, heptenophos, hexaflumuron, nilosone, imidacloprid, Iprobenfos, isoxathion, isofenpropaphos, isofenthion, abamectin, lambda-cyhalothrin, Lufenuron, malathion, imazapyr, methidathion, Mesulfenphos, mesulofos, tetramethon, methidathion, metocloprid, phenthoate, phorate, phosmet, phosphamidon, phoxim, pirimicarb, pirimiphos-methyl, pirimiphos-a, profenofos, mecarb, bendiocarb, propoxur, prothioate, Pymetrozin, pyrazothion, pyridaphenthion, pyrethrum, pyridaben, Pyrimidifen, pyriproxyfen, quinalphos, pinoxaden, clotrimaran, Silafluofen, sulfotepa, ethoprophos, Tebufenozide, Tebufenpyrad, Tebupirimiphos, tebuthioron, tefluthrin, temephos, terbufos, thiofenphos, thiofenox, thiodicarbar, tetramethoprophos, thiametphos, thiamethoxam, thionin, tetrabromthrin, triathlaspin, triazophos, triasulfuron, thifenuron, dimeuron, dimeglumauron, thiuron, monocarb, xylarb, monocarb, and xylarb.

Chemical or biological agents which induce resistance and protect plants against phytopathogenic microorganisms, such as fungi, bacteria, viruses or viroids, may also be used as protective substances. Many compounds with resistance-inducing effects provide protection against insects or nematodes. Examples of such substances having an inducing resistance effect include benzodithiazole and derivatives thereof, mono-and dichloroisonicotinic acid and derivatives thereof, dichloroisothiazole and derivatives thereof, dibromothiophenecarboxylic acid and derivatives thereof, salicylic acid and derivatives thereof, and allylisothiazole. Organisms which induce resistance include microorganisms, such as fungi, bacteria or viruses which are useful for plants, and provide protection for plants against pathogens, such as harmful fungi, bacteria, viruses or nematodes.

Furthermore, these microorganisms can also be used according to the invention in artificial seeds, as symbiont organisms such as mycorrhizal fungi, or to promote plant growth such as rhizobia in connection with nitrogen fixation. Also, the use of specific metabolites formed by microorganisms in combination with plant materials can promote germination and growth of plants and protect plants against pathogen and pest attacks.

The hydrogel coating composition according to the present invention can be used as a means for storing or transporting biological materials.

The invention also provides the use of the product-coated biological material as an artificial seed.

The biodegradability of the polyester polyurea and of the mixture containing polysaccharide derivatives according to the invention is demonstrated in the description below. The biodegradability of the coating compositions formed from the material according to the invention is also demonstrated in compost and in soil. The control test with material completely degraded after up to 4 weeks without bioactive matrix did not show any decomposition and correspondingly excluded the hydrolysis or mechanical influence of the coating composition. According to the invention, degradation also occurs when specific additives, such as active ingredients, nutrients, etc., are present.

The compounds were tested, buried in a fully rotted compost mixture of a height of 2cm in a suitable box, selected from composting units of degree of rotting iv. The filled boxes were incubated in a constant temperature incubator at 60 ℃,50 ℃ and 37 ℃ for 4 weeks in succession. The water loss is determined by weight loss and is replenished. The pH of the compost was measured periodically during the incubation. If the measured value deviates from pH7 by more than 1 unit, the pH was adjusted to 7.0 by adding 100mM potassium phosphate. One week apart, the incubation batch was discontinued, the material removed, washed, dried to constant weight at 80 deg.CAnd recording. The weight loss of the dried material was determined immediately as the make-up weight. In the poison control, the incubated batch was completely dried at 105 ℃ and then evaporated water was added with 0.1% HgCl₂And (4) replacing the solution. The control sample was placed in HgCl before being added to the compost mixture₂The solution is then dried. The control group was incubated in exactly the same manner as the test group. If classified as a biodegradable substance, the sample material was no longer detected in the non-toxic group and the sample remained unchanged in the toxic group after 4 weeks.

The invention will now be explained in more detail with the following examples, without being limited thereto.

Examples

In these examples a polyester polyurethane polyurea according to DE1951185 is used as polyester polyurethane polyurea. Hydroxyalkyl cellulose ethers having an average molecular weight (number average) of about 10000 to 200000 g/molecule and a degree of substitution of water-soluble, biodegradable ether groups of about 0.5 to 1.5 are used as hydroxyethyl cellulose or hydroxypropyl cellulose in these examples.

Example 1

Potato plants propagated in glass test tubes. For this purpose, the stem with 2-6 lobules was cut and placed in a BM liquid medium containing 20g/l and every 12 hours day/night rhythm, and incubated at 22 ℃ day and 19 ℃ night in a plant incubator. BM medium was composed of salts and vitamins of gammorg medium B5 (gammorhage o.l., Miller r.a., Ojima k., exp.cell.res.50,151,1968, gammorrago.l., muhip t.a., visipe t.a., Vasil i.k., Vitro 12473,1976) according to Murashige/Skoog (see Murashige t.a., Skoog, physiol.plant.15,473-479,1962). After 3-4 weeks, stem sections were taken from these plants for coating trials.

The stem segments were suspended in 3% hydroxypropyl cellulose dispersion (HPC; 0.2MCaCl was added according to the semi-concentrated nutrient solution) under sterile conditions₂) And added dropwise while stirring to a 1% alginate (Alginat) solution. The spheres are re-cleaned, and0.2MCaCl₂the solution was stirred. The spheres were then added to a 5% aqueous dispersion of a polyester polyurethaneurea and gently stirred, and a thin, elastic film of the polyester polyurethaneurea was formed on the surface of the spheres. After 5 minutes the spheres had a diameter of about 5mm and were removed from the solution with 0.2M CaCl₂And (4) cleaning with the solution. The spheroids were then germinated on agar plates containing Murashige-Skoog semi-concentrated nutrient medium. The cells were incubated in a plant incubator at 20 ℃ under light for 12 hours a day.

Plantlets grew on polymer spheres after about 2-3 weeks. The germination rate was 66%.

Example 2

The coated biological material, taken from potato (cultivated according to example 1) plants, was suspended aseptically in a 3% sodium alginate solution. The suspension was added dropwise to 0.2M CaCl₂Solution to form alginate spheres. After 30 minutes the spheres were aspirated into a gently stirred 5% aqueous polyester-polyurethane-polyurea dispersion. Thin, elastic films of polyester polyurethaneureas are formed on the surface of alginate hydrogels. After 5 minutes the spheres were removed from solution and reused with 0.1M CaCl if necessary₂The solution is washed once. Seeds with a diameter of about 5mm for germination were placed on a Murashige-Skoog semi-concentrated nutrient agar plate. The incubation was carried out in a plant incubator as described in example 1.

Example 3

75ml of a 40% polyester polyurethane polyurea dispersion and 75ml of a 2% hydroxyethyl cellulose dispersion were each autoclaved at 121 ℃ for 20 minutes and then mixed 1: 1 under aseptic conditions.

The potato stem pieces were aseptically placed on the surface of the hydroxyethylcellulose and polyester polyurethanane polyurea mixture and separately aspirated by pipette.

Then 0.2M CaCl was added dropwise₂The solution is added into the mixture of the stem segment and the hydroxyethyl cellulose and polyester polyurea alkane polyurea coating the stem segment. After 10 minutes of residence, spheres of about 5mm diameter were removed and placed on the agar plate of MS semi-concentrated medium. In a plant incubator for 12 hours a dayIncubation at 20 ℃ with light. The germination rate was 90% in 2-3 weeks.

Example 4

75ml of a 40% polyester polyurethane polyurea dispersion and 75ml of a 2% hydroxypropyl cellulose dispersion were each autoclaved at 121 ℃ for 20 minutes and then mixed 1: 1 under aseptic conditions.

Potatoes were propagated in glass tubes (see example 1). Stem segments were taken from these plants after 3-4 weeks for coating trials. The stem segments were aseptically placed on the surface of the polyester polyurethane polyurea and hydroxypropyl cellulose mixture and separately aspirated by pipette.

Then 0.2M CaCl was added dropwise₂The solution is added to the mixture of the stem segments and the hydroxypropyl cellulose and polyester polyurethanane polyurea coating the stem segments. After 10 minutes of residence, spheres of about 5mm diameter were removed and placed on the agar plate of MS semi-concentrated medium.

The cells were incubated in a plant incubator at 20 ℃ under light for 12 hours a day. The germination rate is 90-100% in 2-3 weeks. Example 5

Carrot cell suspensions were incubated in 50ml of Murashige-Skoog medium (MS medium, see Murashige T.A., Skoog, F.Physiol.plant.15,473-479,1962) containing the hormone at 25 ℃ in the absence of light at 100 rpm on a mechanical shaker.

After 8 days 150ml of cell suspension was sieved through a series of 500 μm,75 μm and 30 μm wide sieves. The 30 μm-75 μm cell fraction was washed with hormone-free medium, centrifuged at 100g and pelleted, washed twice with hormone-free MS medium, and centrifuged again to take 20ml of hormone-free MS medium. Cell count was 0.5X 10⁴-10⁵Cell/ml is standard.

These cells are used to induce embryogenesis. Further incubating said selected washed cells on a mechanical shaker; after 2-5 days the medium changed and the cells were centrifuged off and resuspended in hormone-free medium. The cells were then incubated for an additional 9 days. The suspension contained 10-100 embryoid bodies/ml after a total of 14 days.

Carrot somatic embryos are placed on the surface of a mixture of hydroxypropyl cellulose and polyester polyurethanane polyurea in the "torpedo stage" and the "cotelydonary stage". These embryos are separately pipetted and added dropwise to 0.2M CaCl together with their surrounding polymer mixture₂And (3) solution. After 10 minutes of residence, spheres of about 5mm diameter were removed and placed on the agar plate of MS semi-concentrated medium. The cells were incubated in a plant incubator at 20 ℃ under light for 12 hours a day. After 2 weeks 20% of the spheres germinated.

Example 6

The biodegradability of the coating was determined.

The coatings obtained in examples 1 to 5 described above were tested for their full biodegradability for composting tests. The degradation was checked at intervals of several days. The toxic compost control experiment showed that microbial degradation occurred.

Examples	7 days	20 days	32 days	42 days	Control
						1	Is not changed	Color change	Start decomposition	Decomposition of	Is not changed
2	Is not changed	Color change	Start decomposition	Decomposition of	Is not changed
						3	Is not changed	Color change	Start decomposition	Decomposition of	Is not changed
4	Unchanged and changed color	Color change	Start decomposition	Decomposition of	Is not changed
						5	Unchanged and changed color	Color change	Start decomposition	Decomposition of	Is not changed

Example 7

75ml 40% polyester Polyurethane polyurea DispersionAnd 75ml of 2% hydroxypropyl cellulose dispersion containing 2% imidacloprid were autoclaved at 121 ℃ for 20 minutes and then mixed under sterile conditions 1: 1. This mixture was added dropwise to 0.2M CaCl₂And (3) solution.

Approximately 5mm large spheres containing approximately 30mg/g active ingredient were obtained.

Example 8

Drying/rehydration.

The spheres prepared in examples 1-5 were dried under normal air conditions 7 and weighed after drying. When the cells were stored in water for 24 hours, the weight was increased by about 45%, and the weight did not increase even when the storage time in water was prolonged.

Example 9

In combination with active ingredients

75ml of a 40% polyester polyurethane polyurea dispersion and 75ml of a 2% hydroxypropyl cellulose dispersion were each autoclaved at 121 ℃ for 20 minutes and then mixed 1: 1 under aseptic conditions.

The herbicide imidacloprid solution (1 mol/l in DMF) was filter sterilized (pore size 0.2 μm) with a membrane filter and then diluted 1: 10 with sterile water. The resulting imidacloprid stock suspension was added to a mixture of polyester polyurethaneurea and hydroxypropyl cellulose to a final concentration of 0.1 mM/l. Sterilized 0.2MCaCl containing imidacloprid₂The final concentration of the solution was the same.

The potato stem sections (see example 1) were aseptically placed on the surface of a mixture of polyester polyurethane polyurea and hydroxypropyl cellulose and separately pipetted off.

The stem segments were added dropwise to 0.2MCaCl together with the surrounding mixture of polyester polyurethaneurea and hydroxypropyl cellulose₂And (3) solution. Potato stalks coated without imidacloprid were used as a control experiment. After 10 minutes of residence, the spheres were removed and placed on a MS semi-concentrated medium agar plate. Incubate at 20 ℃ in a plant incubator with 12 hours of light and 70% air temperature per day.

The germination rate was 64% in 4 weeks; the control germination rate without imidacloprid was 57%.

Claims (13)

1. A hydrogel comprising at least one polyester polyurethaneurea, a polysaccharide and/or polysaccharide derivative, and a biomaterial.

2. Hydrogel according to claim 1, characterized in that it comprises as biological material an isolatable plant material, in particular a plant material selected from the group comprising plant cells, callus tissue, protoplasts, plant tissue, plant organs, zygotic embryos, somatic embryos, proembryogenic body analogues.

3. The hydrogel of claim 2, which comprises adventitious branches, micro nodules, axillary buds, terminal buds or scions as plant organs.

4. The hydrogel according to claim 1, characterized in that it comprises as biological material an isolatable material selected from the group consisting of transgenic plants.

5. The hydrogels according to any of the preceding claims, characterized in that they contain polyester polyurethane polyureas formed by the reaction of a diisocyanate component a) and a diol component b), a diamine component c), optionally a hydrophilic polyether alcohol d), optionally water e), which is not included in the calculation of the equivalent ratio of isocyanate groups and groups reactive with isocyanate groups.

6. The hydrogels according to claim 5, characterized in that they comprise polyester-polyurea polyureas formed by a reaction in which 1, 6-hexamethylene diisocyanate or a mixture of 1, 6-hexamethylene diisocyanates up to 60% by weight, based on the total amount, of 1-isocyanato-3, 3, 5-trimethyl-5-isocyanatomethyl-cyclohexane and/or 4, 4' -diisocyanatodicyclohexylmethane and/or 1-methyl-2, 4(6) -diisocyanatocyclohexane is used as diisocyanate component a).

7. Hydrogels according to any of the preceding claims, characterised in that they contain as polysaccharide and/or polysaccharide derivative water-soluble starch, alginate, methyl cellulose, hydroxyethyl cellulose, methylhydroxypropyl cellulose, methylhydroxyethyl cellulose and/or hydroxypropyl cellulose.

8. Hydrogels according to any of the preceding claims, characterised in that they contain nutrient salt mixtures suitable for plant development, bactericidal, fungicidal, insecticidal, acaricidal, nematicidal and resistance-inducing and/or herbicidal active components.

9. Coating composition suitable for biological materials comprising a polyester polyurethaneurea and a polysaccharide and/or polysaccharide derivative.

10. Coating composition according to claim 9, characterized in that the coating composition comprises an aqueous dispersion of the polyester polyurethanane polyurea in an amount of 5 to 50% by weight and the polysaccharide and/or polysaccharide derivative in an amount of at least 0.1% by weight.

11. A process for the production of a biomaterial coated with a hydrogel, characterized in that the biomaterial is mixed in the presence of an aqueous dispersion of a polyester-polyurethane polyurea and a polysaccharide and/or polysaccharide derivative and the mixture is coagulated by contact with a salt solution.

12. A method according to claim 10, characterized in that a salt solution of a multivalent ion is used.

13. Use of a hydrogel-containing biomaterial according to any one of claims 1 to 8 as an artificial seed.