DE2129591A1 - Enzymes as plant growth regulators - Google Patents

Description

«REAL LAWYERS & JUNE! 971

DR. JUR. DIPL-CHEM. WALTER BEIL
ALFRED HOEPPENER
DR. JUR. DIPL-CHEM. HJ. WOLFF
DR. JUR. HANS CHR. AX

FRANKFURTAM MAJN-HOCHST ADELONSIRaSSE 5β ■ '

Our number 17 T48

The Procter & Gamble Company, Cincinnati, Ohio, USA

Enzymes as plant growth regulators

The invention relates to a method for regulating plant growth, in which oxidase or peroxidase enzymes, possibly in combination with their corresponding enzyme subarates, applied to plants.

It has long been known that ethylene is an important hormone which influences and regulates plant growth; it is also believed to be closely related to the ripening of fruits and vegetables and others relating to plants Appearances, e.g. the falling of fruits and leaves, the development of roots, the development of flowers, the rate of growth etc., is linked. These phenomena can be brought about with the help of externally applied means referred to in the present context as "plant growth regulation"; the funds used for this purpose will be accordingly Called "plant growth regulators". The impact

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of ethylene gas on growing plants and ripening fruits an already industrially carried out method for planting— Growth regulation.

It is believed that ethylene occurs naturally in plants by enzymatic action on certain compounds that are present in the plant tissues. So one has after Means sought by which the plant metabolism could be brought under control by taking that of the plant the amount of ethylene produced increased. It can be assumed that certain phosphonic acid derivatives, e.g. 2-chloroethylphosphonic " acid, ethylene release when brought into contact with the plant tissues, which then leads to a rapid and even Development of flowers as well as fruit ripeness leads to increased yields (see: South African patent application 681 036, deposited on February 16, 1968 by Amchem Products Inc.) The use of synthetic chemicals such as 2-Chloräthylphosphonsäure on crops to achieve certain the desired physiological effects are therefore known; however, it would be more desirable from an ecological standpoint if it would be possible to use naturally occurring, biologically active and easily biodegradable substances for this purpose.

It has already been pointed out that enzymes that naturally occur in plants at the biosynthetic Formation of ethylene through catalysis of the conversion of certain substrates, which are also present in the plants, involved could be. In the present context, “substrate” is understood to mean a chemical compound and its conversion to another compound is catalyzed by an enzyme. liebemann et al. have in Nature 204, pp. 343-34-5 (1964) given the assumption that the enzyme lipoxidase could be involved in the formation of ethylene in apples, namely via an oxidation of linolenic acid to an "active" form, which is then broken down into ethylene and other compounds.

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Mapson et al. give in Biochemical Journal 111 (1969), pp. 413-418, as an explanation for the natural formation of ethylene in cauliflower plants, the conversion of D-glucose, which has been activated by grlucose oxidase, with atmospheric oxygen to form HpOp. The HpO ₂ then reacts further with methional, the reaction being catalyzed by peroxidase; ethylene and other compounds are formed. However, the use of such enzymes on plants to achieve plant growth regulation or other purposes has not yet been attempted.

According to the invention, a method is therefore proposed which regulates plant growth with the aid of Enzymes allowed.

As has now been established, regulation is possible to achieve plant growth by applying a sufficient amount of certain enzymes to the plants, In particular, it has been shown that enzymes which react with the plant substrates in such a way that ethylene or an intermediate which ultimately leads to the formation of Ethylene appears as a plant growth regulator the plants can be applied. The inventive use of the enzymes includes the application of the same to seeds, Seedlings, roots, already severed fruits and the unearthly Parts of the growing plants. The application to seeds and roots can be carried out by using the same treating the enzyme before planting or by applying the enzymes to the soil either before or after planting.

The application of enzymes to plants according to the invention allows the growth of plants in very different ways regulate, taking factors such as type of plant, section of the plant's growth cycle at the time of application, Concentration of the applied enzyme, number of treatments with the enzyme, etc. play a role. Are the inventive

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if enzymes were applied to the plants in a manner known to the following remarkable and industrially valuable effects can be achieved: incentive and intensification of flowering, increased disease resistance, suppression of apical dominance, auxin activity, increased branching in certain plant species, increase in protein content in certain plants, leaf and fruit detachment effects, Reduction of storage in grain, beans and peas, interruption of rest in seeds, tubers and rhizomes, early Tires, increased yields, increased resistance to rust, reinforced Rooting etc. It was also possible to establish that the enzymes according to the invention, if they are applied to certain Plants. In certain growth stages, the plant growth prevent or reduce, so that the enzymes according to the invention as a means for suppressing an undesired Can use plant growth.

The enzymes used according to the invention are peroxidases and certain oxidases. A particularly useful one Oxidase is lipoxidase, which is believed to contain polyunsaturated fatty acids and their esters, which form the cis, cis-1,4-pentadiene system (e.g. linolenic acid, linoleic acid and arachidonic acid as well as their esters), in the corresponding Epk converts oxides or hydroperoxides, which in turn are then broken down into ethylene or other products. Another one, A particularly useful oxidase enzyme is glucose oxidase. ■ Other useful oxidases are listed in Table I, together with their respective substrates. The table also contains references to manuals on enzymes in which you can find out about the origin and manufacture of the enzymes. The note A acts it is "The Enzymes", first edition, edited by Sumner and Myrback, Academic Press N.Y. (1950); the Note B refers to The Enzymes, Second Edition, published by Bayer, Lardy and Myrback, Academic Press N.Y. (1963); Finally, note C relates to the work

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"iElie Enzyme Handbook" edited by Barman at Springer-Verlag NY (1969). These oxidase enzymes presumably act like glucose oxidase through the formation of H ₂ Op, which in turn forms the substrate for peroxidase in the synthesis of ethylene. The peroxidase can be that which is naturally present in the plant or one which has been applied to the plant according to the invention.

Table I.

Oxidases, which act as plant growth regularors are useful, as well as their corresponding substrates

enzyme

D-amino acid oxidase
L-amino acid oxidase
Monoamine oxidase
Diamine oxidase
Xanthine oxidase
Aldehyde oxidase
ß-galacto-oxidase
ßlyc ollat-Oxi das e
Acetate oxidase.

Substrate B. Vol. Ref. Reference. P.634-648 . 410-430 D-amino acid B. Vol. VII, P.611-634 110 L-amino acid B. Vol. VII, Pp. 337-349 111 Monoamines B. Vol. VIII, Pp. 313-335 Diamines B. Vol. VIII, Pp. 533-556 Xanthine A. Vol. VII, I, Part 1, 3. 355 aldehyde B. Vol. IV, p D-Galaotose. C. Vol. I, S. Glycollate 0. Vol. I, S. 1-lactate

Lipoxidase, the preferred oxidase enzyme according to the invention Can be used in a known manner from various plants, such as soybeans, green peas and wheat, or from Microorganisms are obtained. Works best for that Purposes of the invention Lipoxidase obtained from soybeans has been. Soybean lipoxidase is a well known, commercially available one and highly effective product, which in the invention Application brings about a wide variety of effects in relation to plant regulation. The lipoxidae according to the invention, e.g. soybean lipoxidase, can be used in both pure and can also be produced in impure form, the latter form being easier to obtain and easier to handle.

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Habitation is to be preferred. Pure crystalline lipoxidase can be made from Vegetables such as soybeans or green peas, pea seeds, wheat, green beans, green bean seeds and others in a known manner to be isolated. A suitable extraction method for soybean lipoxidase consists in the aqueous extraction of soybean meal at pH 4.5, which is followed by a precipitate Ammonium sulfate follows. Extraction methods that can be used are described in "The Enzymes", 1st edition, II, part 1, pp. 564-565.

Commercially available lipoxidase preparations usually contain lipoxidase in combination with inert vehicle substances such as carbohydrates, proteins that act as binders, inorganic salts such as calcium sulfate, trypsin inhibitors, proteases, etc. In such preparations, lipoxidase only makes up a smaller amount; in general, their proportion is from about 1 to about 50 percent by weight of the preparation. The remaining $ 50 to $ 99> consist of the listed vehicle substances. Commercially available preparations containing lipoxidase are preferred as the starting material for the lipoxidase according to the invention because they are more easily available than pure crystalline lipoxidase and because they have a satisfactory lipoxidase activity.

Lipoxidase of microbial origin suitable for the purposes of the invention includes both bacterial and fungal lipoxidase obtained from fermentation broth. Examples of such lipoxidases are those which are associated with Aspergillus soj-ae, Aspergillus flavus, Aspergillus glacus, Aspergillus niger, Aspergillus elegans, Rhizopus usamii, Rhizopus G. 34 Yamasake, Rhizopus dl 36 Yamazake, Rhizopus tritici, Penicillium rugillium 15 and cf. . Lukuba, Nippon Fayu Kayuku Kaishi 26, · 167, 1952). Lipoxidase obtained with Aspergillus parasiticus (ATOC 11906) and Aspergillus flavus (ATCC 1003) can also be used; these strains are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA.

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Another well-suited oxidase is glucose oxidase. This enzyme can be obtained in a known manner from plants such as cauliflower or obtained from microorganisms. A glucose oxidase which is also well suited for the purposes of the invention can be obtained with the help of Aspergillus niger. A.niger glucose oxidase is a well-known, commercially available and highly effective product when used according to the invention for regulating the Growth of various plants is used «, glucose oxidase can be obtained either in pure or in impure lorm The use of the latter 3-shape is preferred because it is easier to obtain and easier to handle is. Heine crystalline glucose oxidase can, for example, with the help of microorganisms such as Aspergillus niger, Penicillium Glaucum, Penicillium notatum and Penicillium amagasakiense i are isolated. A useful extraction method for glucose oxidase consists in the adsorption and elution of raw material on the resin Amberlite CG-50, to which the precipitation takes place Ammonium sulfate follows.

Corresponding extraction methods are described in the works "The Enzymes? ¹ second edition, Volume VII, pp 568-569. Commercially available glucose oxidase preparations normally contain glucose oxidase in combination with inert vehicle substances such as carbohydrates, proteins, inorganic salts, etc. The proportion of glucose oxidase makes in such preparations about 1 to about 50 wt .- # of · The remaining 50 to 99 % consist of the vehicle substances already listed above. The use of commercially available preparations containing glucose oxidase is preferred to the use of pure crystalline glucose oxidase, because the former are more readily available and yet have good glucose oxidase activity.

Peroxidases are also useful enzymes for the purposes of present invention. They are also commercially available preparations; these can be made from plants as well animal or microbiological materials are obtained.

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A description of the starting materials and the manufacturing methods the various types of peroxidase can be found in "The Enzymes", Second Edition, Volume VIII, pp. 227-274.

For practical use as plant growth regulators, the enzymes according to the invention are processed together with inert carrier materials to form appropriate preparations. In the present context, an "inert carrier material" is understood to mean in a solvent or a dry extender which itself has no effect on plant growth regulation, but which is suitable for mixing with pure enzymes or commercially available preparations containing enzymes. With the aid of such preparations, the enzymes The preparations can be in the form of solid substances, for example as dusts, granules or wettable powders, but also as liquids, for example solutions and aerosols. The solid preparations generally contain the active enzyme in amounts of around 0, 5 ppm to about 5 wt io \ the liquid preparations containing the active enzymes usually in amounts of from about 0.005 ppm to about 50,000 ppm.

Dusts can be produced by grinding and mixing the enzyme with a solid, inert carrier material such as talc, clay, silica, pyrophylite, etc. Granular preparations can be produced by impregnating the enzymes in and on granulated carrier materials such as attapulgite or vermiculite with a particle size between about 0.3 and 1.5 mm, generally in dispersion or solution in a suitable solvent (e.g. water). or applies. It is also possible to coat an inert carrier material with the compounds in the form of wettable powders. Suitable methods and aids for converting the enzymes into granular preparations are described, for example, in the USA Patent 4-τΗ> €? -. Since soybean lipoxidase is known to be found in soybean meal in an amount of $ 0.1 to $ 0.5, it is possible

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«. 9 -

129591

Unroasted soybean meal directly as a granular lipoxidase preparation to use. Wettable powders that can be dispersed in water or oil to "any desired enzyme concentration," can be obtained by incorporating wetting agents into concentrated dust preparations.

The carrier material preferably used for the enzymes is water. Some of the enzymes of the present invention are sufficient water soluble so that they can be used directly in this solvent. Often it is also possible to use this watery To disperse enzyme solutions under superatmospheric pressure to form aerosols. Usually, however, the enzyme is not in water sufficiently soluble and must be made soluble with the help of other materials. For example, the enzymes can be made soluble make by adding a water-soluble salt to the liquid preparation in an amount of about 0.5 to about 20 times, preferably about 0.1 to about 1 times the weight of the active enzyme. Salts suitable for this purpose are water-soluble Alkali and alkaline earth metal salts of inorganic acids such as hydrochloric acid, nitric acid and sulfuric acid. Preferably suitable for the purposes of the invention are sodium chloride and / or Potassium chloride; a liquid enzyme preparation which is suitable for direct application to plants should preferably be about Contain 0.005 to about 50,000 ppm enzyme and about 0.0005 to about 5,000 ppm sodium chloride and / or potassium chloride in water. To simplify the shipping and handling of liquid preparations, they are initially placed in an approx. 100 to 2,000 times the concentration - based on the concentration for later use - and dilutes the preparation to the Desired concentration of 0.005 to 50,000 ppm active enzyme just before use.

To improve the spread of the liquid preparations on the The plant surface and to increase the penetration of the enzyme into the plant tissue can be added to the liquid preparations also add surfactants. Suitable surface

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active agents are, for example, those from the anionic, cationic, non-ionic, ampholytic or zwitterionic Type.

Examples of suitable anionic surfactants are the sodium salts of fatty alcohol sulfates having 8 to 18 carbon atoms in the chain as well as sodium salts of alkylbenzenesulfonates with 9 to 15 carbon atoms in the alkyl chain. Examples of suitable nonionic surfactants are polyethylene oxide condensates of alkylphenols in which the alkyl chain contains about 6 to 12 carbon atoms and which condensed amount of ethylene oxide per mole of alkylphenol is about 5 to 25 moles, as well as polyethylene oxide condensates of sorbitan monolaurate, in which about 10 to 40 moles of ethylene oxide per mole of sorbitan monolaurate are condensed. Examples of suitable cationic surfactants are quaternary dimethyldialkylammonium salts, in which the alkyl chain contains about 8 to 18 carbon atoms and the salt-forming anion a halogen is. Examples of useful ampholytic surfactants Agents are derivatives of aliphatic secondary or tertiary amines in which one of the aliphatic substituents is about Contains 8 to 18 carbon atoms and another one an anionic water-solubilizing group, e.g. a sulfate or sulfonate represents. Specific examples of ampholytic surfactants are sodium jj-dodecylaminopropionate and sodium 3-dodecylaminopropanesulfonate. Examples of suitable zwitterionic surfactants are derivatives of aliphatic ones quaternary. Ammonium compounds, in which one of the aliphatic Substituents about 8 to 18 carbon atoms and one another contains an anionic water-solubilizing group. Specific examples of zwitterionic surfactants Remedies are 3- (H, N-Dimethyl-H-hexadecylammonio) -propane-1— sulfonate and 3- (N, N-dimethyl-N-hexadecylammonio) -2-hydroxypropane-1-sulfonate. Other suitable surface-active agents are in the work "Detergents and Emulsifiers - 1969 Annual" by John W. McCutcheon Inc. When used in the

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liquid enzyme preparations according to the invention directly on
When applied to plants, the surfactant should be in amounts of from about 0.001 to about 0.5 °, preferably-about
0.0t to about 0.1 wt .- ^ of the preparation may be present. Amounts above about 0.5 wt .- ^ disrupt the enzyme activity.

The enzymes described here should be like this with all
Plant growth regulators are commonly used to be applied to the plants concerned in certain optimal amounts (eg weight per unit of cultivated soil) and in certain stages of the plant's growth cycle if a certain growth-regulating effect is achieved in this
target. The practically unlimited number of plant species and the very different effects that can be seen in the plants make it impossible, if you also add the different climatic conditions that may be encountered in individual cases, to provide exact information about the im
To make the quantities to be used on a case-by-case basis. These amounts can, however, be determined very easily by the person skilled in the art with the aid of customary methods. You can get clues as to the quantities to be used by considering the use of other ethylene
forming plant growth regulators considered. In this
In connection with this, reference is again made to the above-mentioned South African patent application by Amchem Products, Inc. and to the "ETHREL" product catalog from the same company. Quite generally it can be said that the amount to be applied - when applied widely to standing vegetation or to the ground - is based on the weight of the active enzyme, normally about 0.23 to 11.3 kg, preferably about 0.23
to about 6.8 kg per 4000 m (1 acre). In many cases, repeated application is beneficial. Becomes a liquid
Solution or dispersion for watering trees or soaking
When used from seeds or tubers, the concentration of active enzyme in the liquid should usually be between about
0.005 and about 50,000 parts per million.

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In many cases there can be an improved effect regarding to achieve plant growth regulation when working with the enzyme also applies the corresponding enzyme substrate to the plant at the same time. "Together" means herein Relation that the enzyme and its corresponding substrate are applied to the plant within about 3 hours. Optionally, the enzyme and the substrate can also be contained in the same preparation, so that they can act simultaneously on the Plant can be applied. The amount of substrate should be about 0.01 to 10 times, preferably about 0.1 to about 5 times the weight of the enzyme used. It is assumed that increasing the amount of substrate for the enzyme beyond the amount naturally present in the plant is, causes a further increase in the formation of ethylene and thus an increase in the regulation of plant growth.

The following examples serve to further illustrate the invention. In these examples the enzymes consisted of commercially available preparations containing vehicle substances. The specified However, enzyme concentrations refer to the amount by weight of the active enzyme.

example 1

* 15 to 20 seeds of red radish (Early Scarlet Qlobe) 'were planted in 4 plastic pots with a diameter of 8 inches and a depth of 12.7 cm, which was a mixture of 1/3 soil, 1/3 Peat dust and 1/3 Pearlitef which were then placed in a greenhouse. Nine days after planting, the plants that had emerged were thinned or warped to 10 per pot, the spatial distance between the plants in the pots being kept approximately the same. The plants were fertilized with a 7: 40: 6 N: P ₂ 0n: K ₂ 0 fertilizer when planting and were then watered daily during the entire course of the experiment with enough water that the soil was moistened. The plants in the 4 pots were 3, 10, 14, 21 and 24 days after the

"contained

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pull treats. Treatment B was repeated "for 2 pots, whereas treatments A and C were carried out on one pot each. The treatments were as follows:

A. Treatment with water containing 58 ppm NaGl (control) ,

B. Treatment with water containing 58 ppm NaCl + 500 ppm Contained lipoxidase *,

C. Treatment with water containing 58 ppm NaCl + 500 ppm lipoxidase * + 280 ppm linoleic acid.

* Soybean lipoxidase produced by PL Biochemioals, Milwaukee, Wisconsin, USA.

The treatment was carried out so that each pot with plants was sprayed with a 100 ecm portion of the test solution until it ran down the leaves; the remaining amount of the portion was added to the soil. The radishes were harvested 7 days after the last application of the test solution. The plants were washed, the tips were cut off, and the radishes and foliage from each pot were weighed together, whereupon the average fresh weight per plant was calculated. The radishes and foliage were then 16 hours long held in an oven at 105 C and then reweighed to determine dry weight. The results (in grams) are included in Table II along with the calculated percentage increases with treatment with the solutions to be tested compared to the control experiment.

Table II

Fri'sch weight dry weight

Average / plant average / plant

(g) (g)

Treatment root foliage root foliage

A (control) 2.75 6.06 0.155 0.380

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-H-

Fresh Weight Dry Weight Avg.sciin.itt / Plant Avg / Plant U) UI

Treatment root foliage root foliage

* B (enzyme) 5.0 ($ 82); $ 6.99 ($ 15) .0.260 ($ 68) .460

G (Enzyme + $ 5.24 ($ 90) $ 6.73 ($ 11) $ 0.280 ($ 80) $ 0.440 ($ 16) Substrate)

^ combined results from multiple treatments

The results clearly explain the regulating plant growth Effect of lipoxidase as well as the somewhat greater effect of using lipoxidase together with its linoleic acid substrate is achieved. It should be noted that both the enzyme and the enzyme + substrate are primarily the Increased plant growth in the valuable root part of the radish plant and not in the course of it. The dry weight results also prove that the treatments actually increased the weight of the tissue and not just an increase in water intake.

Virtually the same results are obtained when the enzyme in treatment B is one of the following oxidases: D-amino acid oxidase, L-amino acid oxidase, monoamine oxidase, Diamine oxidase, xanthine oxidase, aldehyde oxidase, ß-galaetosidase, Glycollate Oxidase or Lactate Oxidase. Corresponding results can also be achieved if, in treatment C, one of the The following enzyme / substrate combinations can be used instead of lipoxidase / malic acid: glufcose oxidase / D-glucose, D-amino acid oxidase / D-alanine, L-amino acid oxidase / L-alanine, monoamine oxidase / propylamine, Diamine oxidase / 1,5-diaminopropane, xanthine oxidase / xanthine, Aldehyde oxidase / benzaldehyde, ß-galactosidase / D-galactose, glycolate oxidase / ethyl glycollate, lactate oxidase / Ethyl lactate.

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Example 2

2-5 to 30 seeds of white radishes (White Icicle variety) were planted in 32 clay pots with a diameter of 20.3 om. 10 days after planting, the emerged plants were moved to 10 per pot, the spatial distances between the plants in the pots being kept approximately the same; the pots were then divided into 2 groups of 16 pots each. The plants were fertilized with 20; 20: 2 (VNrP ₂ O ₅ : K ₃ O fertilizer (2.2 g / pot) on the 24th and 31st day after planting and then watered daily with enough water that the Control and experimental treatments as described below were applied to the two groups of plants on days 10, 17, 21 and 28 after planting, respectively.

Control: 58 ppm NaCl in water Experiment: 58 ppm NaCl + 500 ppm lipoxidase * in water

♦ Soybean lipoxidase, obtained from! Company Pl Bi ο chemicals ..

The treatments were carried out by spraying each pot with a 100 cc portion of a solution until it ran down the leaves and the remainder of the portion was placed on the floor. The radishes were harvested 4 days after the last treatment. The plants were washed, the tops were cut off, and the radishes and foliage from the plants were weighed together so that the fresh weight per plant could be calculated. The radishes and the leaves were kept for 24 hours at 105 ⁰ C in an oven, whereupon then the dry weight was gbestimmt. The results (in grams) are summarized in Table III, together with the calculated percentage weight increase that resulted from the test treatments compared to the control treatment.

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Table ΙΣΙ

Fresh weight dry weight

Average / plant Average / plant

() eat}

Treatment of root foliage root foliage

Control 24.2 57.2 1.7 3.7 Enzyme 31.0 (28 ^) 63.0 (11/0 $ 18.0) 4.1 (1150

The results clearly explain the regulating plant growth Effect of lipoxidase on white radishes. Here, too, the enzyme mainly caused an increase in the growth of the valuable Root part of the plant, stronger than in the foliage. The results of the dry weight measurements further show that it is actually an increase in the weight of the tissue and not just an increase in water absorption.

A preferred stimulation of root growth as opposed to stimulation of foliage growth as in the present one Attempt could be achieved is particularly important in the case of radishes, because here the root is valuable and usable It is part of the plant, but the stimulation of root growth is also quite general in other plants favorable 'and worth striving for, because this improves the state of health the plant is improved; the plants can survive dry periods more easily because they have more soil moisture is accessible.

Example 3

Equal amounts of "Merion Bluegrass" (sedge) were added to the Surfaces of fertilized soil in two pots, labeled A and B were marked, sown; each pot was 30.5 cm in diameter and 30.5 cm in depth. The pots were watered at intervals so that the soil in a weakly moist

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Condition was maintained. After the grass started in "both pots" had to grow, the following treatments were carried out at intervals of 7 days, each with 100 com solution per application:

A. 58 ppm NaOL in water

B. 58 ppm NaCl + 500 ppm lipoxidase in water.

Six weeks after the first application of the solutions, the pots were broken off from the earth and the root structure in the Earth was examined. It was found that the grass in the pot that had been treated with lipoxidase was a great deal had developed stronger root structure than the grass grown in the pot which was the control treatment only had received.

Example 4

Eight tomato plant seedlings of the Hutgers variety of approximately the same size were individually planted in a greenhouse in pots with a diameter of 25.4 cm. All plants were initially fertilized with 7:40: 6 NtPoOCtKpO fertilizer and then three more times in the course of the experiment with 5: 10: 5 N: P ₂ O ^ K ₂ O fertilizer. The temperature was kept between 12.8 and 18.3 ^° C .; all plants were watered daily with a lot of water so that the soil was moistened. The potted plants were divided into 4 groups of 2 each and treated as follows on the day after planting and then at 7-day intervals over the following 98 days:

Control 58 ppm NaOL in water

T-1 58 ppm NaQl + 50 ppm lipoxidase * in water

T-2 58 ppm NaCl + 500 ppm lipoxidase * in water

T-3 58 ppm NaCl + 2000 ppm lipoxidase * in water

■ ♦ Soybean lipoxidase, obtained from PL Biochemie as _a

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The solutions were each in amounts of 100 ecm on the Plants sprayed until they were thoroughly wetted; the rest of the 100 cc was poured onto the floor. Green fruits First, 21 days after the start of the experiment, the plants that had received lipoxidase treatment showed “ripe tomatoes” could of the plants treated with lipoxidase for the first time on the 82nd day of the experiment and only about 2 weeks later from the control plants are harvested. The trial was started on day 137 (i.e. 55 days after the first ripe tomatoes were harvested had been ended. The results of the experiment are shown in Table IV.

treatment

Table IV

Days after cumulative of the first number of ripe harvest tomatoes

Cumulative Total Cumulative Weight Average Weight

control 30th 8th 564 grams 71 Gram Il Il T ₇ I. 30th 12th 1693 ^a 141 Il Il T-2 30th 15th 1609 ■ 107 Il T-3 30th 14th 1032 " 74 Il control 37 17th 1059 " * 62 Il T _- 1 37 14th 1921 " 137 Il T-2 37 21 1881 " 90 It T-3 37 20th 1219 " 61 It control 55 37 1556 " 42 It T-1 55 23 2172 ⁿ 94 T-2 55 34 2461 » 73 T-3 55 44 1821 ■ 41

The above values clearly show an enlarged Total yield at each concentration of lipoxidase treatment on fruits, the optimum seems to be around 50 ppm ... The Mpoxidase treatment also accelerated the onset the maturity.

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Example 5

Ripe green avocado fruits were purchased from a local trader concerned and stored at 16 ° C until use. Some of the fruits were immersed in a 500 ppm aqueous solution for one hour Lipoxidase (soybean lipoxidase from PL Biochemicals), which had been solubilized with 58 ppm NaCl, immersed. Me fruits were then placed in a closed desiccator, which was equipped with separating lids. Corresponding fruit samples that had not been treated with lipoxidase solution, were simultaneously placed in desiccators for control. From the test desiccator and from the control desiccators gas samples were taken periodically and checked for their ethylene content checked with the help of gas chromatography »The values of the α-fchylene formation determined by these measurements after Hour and after 18 hours in the desiccator are given in Table V, in microliters of ethylene per kilogram of fruit per hour.

Table V

Ethylene evolution from avocados

1 h 18 h

Untreated fruit

Repetition 1 0 / Ul C ₃ H ₄ AgA 0.068 / Ul C ₃ H ₄ 0 2 AgA repetition / ul C ₃ H ₄ AgA 0,091 / ul O ₂ H ₄ AeA treated fruit lipoxidase

Repeat 1 0.25 / _{µL C 3} H ₄ AgA 0.29 / µL C ₃ H ₄ AgA Repeat 2 0.24 / µL C ₃ H ₄ AgA 0.31 / µL C ₃ H ₄ AgA

The values clearly show the increased formation of ethylene that occurs in the fruits that have already been separated from the plant, if these are treated with lipoxidase. The fourfold increase in ethylene formation in those treated with lipoxidase Fruits after 18 hours was a distinct one

109 & 52/1861

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Table VI

Red radishes

Fresh weight clrfc! Dry weight

Average / Plant Average / Plant

Treatment root foliage root foliage

Water, control 2.82 3.01 0.18 0.25

Glucose Oxidase 4.25 ($ 50) 2.83 (-6 $) 0.25 ($ 39) 0.23 (-

The values clearly show that glucose oxidase regulates growth Has an effect; they show in particular that glucose oxidase is mainly the growth of the root part of the Plant favors, and more so than that of the foliage. The dry weight results further demonstrate that the Glucose oxidase treatment actually increases the size of the Weight of the root tissue was reached, and not just an increase in water absorption.

The previous examples provide proof of the ability of the Enzymes, positive effects on plant growth regulation, i.e. greater fruiting, increased root growth and early ripeness. The following examples in contrast, are intended to show that the enzymes, especially when used in certain concentrations, also can have negative effects on plant growth regulation in certain plant species; as a result, can the invention also serve to reduce or suppress the growth of certain plants. What amounts to achieve a positive or negative influence on growth regulation are necessary in individual cases, cannot be said without preliminary experiments; however, these preliminary tests can be carried out easily Carry out in a known manner.

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Example 7

Heirs (Pisum sativum from Alaska) were 3.5 before planting Sterilized in a 0.1% HgC ^ solution for hours. In 12 Τοη pots With a diameter of 15 cm, 4 peas were sown per pot. All plants were daily during the experiment watered by soaking the soil. As soon as all the plants showed the first flowers, they were divided into two groups A and B divided, which were treated once a week as follows:

A. 58 ppm FaCl in water

B. 58 ppm NaCl + 500 ppm lipoxidase * in water

* Soybean lipoxidase, obtained from PL Biochemie as.

Each pot was treated with a 100 ccm portion of the corresponding Solution sprayed until the plants were well moistened; the rest of the 100 cc was poured onto the floor. peas pods containing were harvested on the 36th, 43rd and 49th days after the start of treatment. The total weight of the pods harvested of the plants that had received the control treatment was 179.9 gj compared with the weight for those with Lipoxidase treated plants at 117.9 g. With the LipoxitlaBe treatment of the pea plants, a 35% decrease in yield could be achieved in the concentrations tested.

Example 8

20 to 25 red radish seeds (Early Scarlet Globe) were sown each in 4 clay pots with a diameter of 20 cm. 10 days after sowing, the plants were pulled to 10 per pot. All plants were fertilized during planting with 7:40 : 6-Ή: Έ ₂ 0 ^: K ₂ O and then watered daily during the entire course of the experiment with enough water that the soil was moistened. Starting on day 10 after planting, the plants became

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once a week in the, following 4 weeks as prime follows brehandelte "2 ESpf e were com with It) O _r foesprffirfc an aqueous solution containing 80 ppm lieEcet-feioh-Peroxtaase (iianm Research li ^ orairoisfcBaas. ,, ,, Orangeburg Hew Yorifc, . IFSA), contained. Until other tireinern i ait · 100 ecm of water "oesiKPatiti, 4 days ^; Bach ά & τ

became the Pflansea; geBrntei * The EÜLaiiaea were Wash the tops and the radishes and dae Iiatih from each pot were weighed together "on what one that ! • Average weight per plant removed .. The; Hadiesoiieii and dös Leaves were then "placed in an oven at 105 ° C for 24 hours," whereupon one then, the: Epockengewichti "bestiiBiEite; *, the results, in grams ,, are put together in iaBelXe ¥ 11 ^ namely together with the weight percentage differences, that is, see Feei of the treatment according to the invention in the Terglseich sur Köntroll- "treatment result.

Hot »R&D slide viewing

Treatment of the root EauSb ¥ ur «el:

Control, 2.82 % Q1 \ 0 _t m 0.25 t

Peroxidase 1

The results show that the Berosälaß ^ enzyme in the lower We looked for centrifugal factors, eüiec negative effects on the growth of the roots and the Itauües · from rotesi Eaöiesehesti hat «.

Bedapieä. 9

itt 4 clay pots. «· with; einiBif BrnriohmiesÄer: from ZQ am mxF && BL jjät 20 25 weifi & RettiohsaBiien: (iKhitfeiee Ecdiai: ©) «iögef« ätv «10 Tj & g»: after; desr

The plants were sown to 10 per pot. All plants were fertilized with 7:40: 6-ItPpOc: KpO during planting and then irrigated daily during the entire course of the experiment with enough water that the soil was well moistened. Starting on the 10th day after planting, all plants were treated once a week for the following 4 weeks as follows: 2 pots were sprayed with 100 ecm of an "aqueous solution" containing 93 ppm of glucose oxidase (as described in Example 6), The other two pots were sprayed with 100 cc of water, the plants were harvested 4 days after the last application, the plants were washed, the tops were cut off and the radishes and foliage from each pot were weighed together and the fresh weight per The radishes and foliage were then placed in an oven at 105 ° C. for 24 hours, after which the dry weight was determined The results, in grams, are summarized in Table VIII, and again together with the weight percentage differences ₉ that arise in result in treatment according to the invention compared to the control treatment.

Table VIII

White radishes

Basic weight dry weight

Average / Plant Average / Plant (*) '(* )

Treatment root foliage root foliage

Water, control 4.13 5.51 0.23 0.43

Glucose Oxidase 2.90 (-30 #) 5.83 ($ 6) 0.1S (-22 $) O ₉ 45 (5 $)

The above values show that glucose oxidase is present in the tested Concentrations has a negative effect on the growth of the roots of white radishes.

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Example 10

Pumpkin (Acorn squash) was planted outdoors in parallel rows *. About 4 weeks after planting (length of the tendrils about 0.6 m), a number of individual pumpkin plants with a Sprayed solution containing 7.4 g per liter of lipoxidase (equivalent to about 500 ppm active lipoxidase); the aqueous treatment solution Also contained $ 0.1 "Tween 20" as an emulsifier. The plants were sprayed until the solution ran down, i. E. with about 10 to 20 ml per plant. The control plants were not treated. When harvesting, the pumpkins were made with the same Number of treatments and the control plants removed and weighed. The control plants provided 16.2 kg of ripe pumpkin, the plants treated with lipoxidase, on the other hand, 21.1 kg of ripe pumpkin. These results clearly show that by the lipoxidase an increase in the amount of pumpkin harvested could be achieved.

Pumpkin seeds (Jack-o'Lantern) were planted on 5 hills, namely 4 plants per hill; after the tendrils were about 0.6 m long, they were sprayed with lipoxidase (7.4 g per liter; 500 ppm active lipoxidase; 0.1 ^ S "Tween 20" in water), namely in the manner already described. 5 mounds with control plants, each of which also carried 4 plants per mound, were filled with water treated. After this one-time treatment, the pumpkins were allowed to ripen; the control plants yielded at harvest 654 kg of ripe pumpkin, whereas the lipoxidase-treated plants yielded 1035 kg of ripe pumpkin.

The procedure described above was applied to soybeans, sugar beets, sugar cane, pines, pineapples and chrysanthemums repeated. In the case of soybeans, the overall yield increased as a result of the lipoxidase treatment. With sugar beets and sugar cane, ' who had been treated with lipoxidase, an increased sugar content could be determined. With the pines could as a result of the lipoxidase treatment an increase in callulose

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Education to be noted. In the case of pineapple, lipoxidase solution was used poured into the petals, which led to an increase in flowers. In the case of chrysanthemums, the application of Increased branching "observed in lipoxidase solutions.

Example 11

Carrot seeds were sown in a row in a field. After the seeds have been in the ground for about 5 weeks (plant size a bit 7.6 TdIs 12.7 cm) the plants were treated with a mixture of lipoxidase in water "sprayed (7.4 g lipoxidase per liter; approx. 500 ppm active lipoxidase; 0.1 wt .- ^ "Tween 20" as an emulsifier). The treatment was carried out on a 12 m long row of 5 week old carrot plants as follows: the first 3 m the row were sprayed with lipoxidase solution, the next 3 m remained untreated; another 3 m were sprayed with lipoxidase and the last 3 m of the row were not treated. When harvested, the untreated carrots gave an average weight of 757 g per 0.3 m of row. The lipoxidase treated carrots gave 826 g / 0.3 m row. These results show that lipoxidase in the concentrations used already resulted in a considerable increase in the amount of fruit in carrots causes a single treatment of the plants.

Cucumbers were planted in a greenhouse and allowed to ripen to the "3 full leaf stage". At this point in time 6 plants were sprayed with water, 6 plants with a 0.1 percent by weight solution of "Tween 20" and 6 plants with an aqueous solution containing 1 g per liter of glucose oxidase (approx. 95 ppm of active glucose oxidase) and 0.1 fo "Tween 20". The flowers formed on the plants within a period of 12 weeks were counted and the number of female flowers was determined. (The female flowers are those which produce cucumbers.) In the water control treatment, 24.2% of the flowers were female; in the "Tween 20" control treatment, 31% of the flowers were female; in glucose oxidase Beh _a MINATION

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48 ^C J> of the flowers were female. These data clearly show that the number of female flowers on the cucumber plants could be increased significantly by the glucose oxidase treatment in the specified concentrations.

Example 12

Apple trees (Red Delicious) were arbitrarily made from two rows

—5 growing trees selected and treated with aqueous 1 × 10 molar lipoxidase solutions which also contained 0.5 $ "Tween 20". Another group of apple trees was treated with a mixture consisting of 1 × 10 molar glucose oxidase (approx. 95 ppm active enzyme) and 0.5 ° "Tween 20", control groups of apple trees were given 0.5% "Tween 20" in Yfasser or not dealt with at all. The treatment was carried out in such a way that the test solutions and the control solutions were applied once 13 days before harvest until they were completely wetted, ie until the leaves had run off. Doctrines were used at harvest to measure the force required to separate the fruit from the tree. On average, the "Tween 20" treated apples and the untreated apples required about 1.4 kg of force to remove an apple from the tree. The fruits treated with lipoxidase and glucose oxidase solutions required less force to remove from the tree than the control fruits. Glucose oxidase and lipoxidase in the applied concentrations reduce the force required to detach them from the trees in growing apples.

The procedure described above was carried out with solutions containing lipoxidase and glucose oxidase in appropriate concentrations contained, repeatedly, on citrus trees, i.e. Orange trees, lemon trees, lemon trees, mandarin trees and grapefruit trees; in either case, the fruit was easier to separate from the tree. Corresponding results, i.e. a Easier separation of the fruits could also be achieved with stone fruits if they were treated with aqueous lipoxidase or glucose

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Oxidase solutions have been treated; Corresponding tests were carried out on cherries, peaches, apricots as well as on grapes and olives. Corresponding effects with regard to the detachment of the fruit could also be achieved in pome trees and bushes. For example, in the case of pear trees, plum trees and pomegranate trees, when treated with aqueous solutions with both lipid oxidase and glucose oxidase (50,000 ppm in each case in aqueous solution), a considerable reduction in strength can be achieved about 2 weeks before harvest is necessary to separate the corresponding fruits from the branches.

The type of application described above both by lipoxidase as well as glucose oxidase on nut trees such as walnut trees, pecan trees and macadamias about 14 days before the Harvesting also resulted in a significant reduction in the forces necessary to remove the ripe fruit from the branches to replace.

The application of aqueous solutions of lipoxidase or glucose oxidase on grain crops such as wheat, rye, and rice Oats in a V / achs turns stage of the plant, in which they are about 20 cm high, (about 2 weeks after the tips are over appearing on the ground) leads to a significant increase in the amount of grain that can be harvested.

Example 13

4 treated rubber trees and 4 untreated control trees of the species Hevea Brasiliensis were used for the experiments. A 5 cm area under the tenon was scratched free; at this point the test chemicals were applied. The enzyme was dissolved in 40% glycerin, namely at a concentration of 2.5 $ active enzyme. The test solutions were deleted at the place mentioned. After the applied solution dried, palm oil was put over the spot painted, the trees were tapped and the latex was made of

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collected from each tree according to a predetermined scheme. The rubber was separated from the latex and dried. The following results have been achieved, which increase production, based on the dry rubber analyzes.

treatment average
lich yield
(G)
per tree / tap
Job % Increase Time after
use
(Weeks) Glucose oxidase 153.1 88 2 control 81.3 Lipoxidase 185.7 128 1 control 81.6 Ascorbic acid
Oxidase 176.7 77 2 control 40.8 Peroxidase 138.4 70 2 control 81.3

The vVerts clearly show that an increase in the Rubber flow occurs when the trees are exposed to lipoxidase resp. Glucose oxidase enzymes are treated according to the invention,

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Claims (8)

Claims 1. A method for plant growth regulation, characterized in that that the plants with an enzyme from the group of peroxidase, lipoxidase, glucose oxidase, monoamine oxidase, Diamine oxidase, xanthine oxidase, aldehyde oxidase, ß-Gslactosidase, glycollate oxidase and lactate oxidase. 2. The method according to claim 1, characterized in that the enzyme is lipoxidase. 3. The method according to claim 1, characterized in that the Enzyme is glucose oxidase. 4. The method according to claim 1, characterized in that the enzyme is peroxidase. 5. A method for regulating plant growth, characterized in that that the plants with one of the enzymes according to claim 1 together with the enzyme corresponding to the selected Substrate which is used in about 0.01 to about 10 times the amount by weight of the enzyme. 6. The method according to claim 5, characterized in that the ) enzyme and the substrate are applied simultaneously. 7. The method according to claim 6, characterized in that the enzyme is lipoxidase and that the substrate consists of linoleic acid, Linolenic acid or arachidonic acid. 8. The method according to claim 7, characterized in that the enzyme is lipoxidase and that the substrate is made of linoleic acid which is used in about 0.1 to 5 times the amount of the weight of enzyme. For The Procter & Gamble Company, _v Cincinnati, Ohio, USA 109852/1861 Lawyer