WO2020053667A1 - Alternative oxidase activity stimulating chemical composition and a method for reducing hypoxia in plants - Google Patents
Alternative oxidase activity stimulating chemical composition and a method for reducing hypoxia in plants Download PDFInfo
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- WO2020053667A1 WO2020053667A1 PCT/IB2019/050380 IB2019050380W WO2020053667A1 WO 2020053667 A1 WO2020053667 A1 WO 2020053667A1 IB 2019050380 W IB2019050380 W IB 2019050380W WO 2020053667 A1 WO2020053667 A1 WO 2020053667A1
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/10—Fertilisers containing plant vitamins or hormones
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
- A01N33/02—Amines; Quaternary ammonium compounds
- A01N33/12—Quaternary ammonium compounds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
- A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
- C05C5/00—Fertilisers containing other nitrates
- C05C5/02—Fertilisers containing other nitrates containing sodium or potassium nitrate
Definitions
- the present invention relates generally to the field of plant physiology and biochemistry
- compositions, methods related to stimulation of Alternative Oxidase enzyme activity More particularly, it concerns compositions, methods related to stimulation of Alternative Oxidase enzyme activity
- the present invention extends to enhance survivability of and/or reduce damage to cells, tissues, organs, and organisms, particularly under stress induced hypoxic conditions.
- Plant tissue culture is the technique of maintaining and growing plant cells, tissues or organs especially on an artificial nutrient medium in suitable containers under controlled environmental conditions and is of great scientific, educational and commercial interest. The practice of growing plant in-vitro is well developed.
- Murashige T., Skoog F., Physiol. Plant. 15:437-497, 1962 (MS media) is widely used in plant tissue culture industry.
- MS media For normal growth and development of plants in in-vitro conditions requires various combinations of micronutrient, macronutrient, plant hormones (Phyto-hormones), vitamins and one or more simple sugars with additives like antioxidant and growth enhancers.
- Plant tissue culture technique generates abiotic stress in plant due to culturing conditions and frequent wounding to plant part during subculture. Plant propagated through plant tissue culture technique shows slow growth response, less response to fertilizer, low fruit quality and compromised plant immunity which results in great losses to farmer.
- Plants are obligate aerobes and therefore, oxygen deficiency (hypoxia) and especially its total absence (anoxia) cause dramatic ecological stress in plants. Meanwhile, plants often suffer from sudden molecular oxygen deprivation both under natural condition and as a result of human activity. Most often plants are subject to oxygen deprivation on hydromorphic and flooded soils for the oxygen’s poor solubility and low diffusion rate in the water.
- the first one is molecular adaptation which takes place at the absence or lack of oxygen through fundamental rearrangement of the entire cell metabolism.
- the second is adaptation of the plant as an entire body due to transportation of oxygen from aerial parts to parts localized in anoxic environment (roots), that is escape strategy to avoid anaerobiosis. It becomes more and more clear that cell energy metabolism is a key factor in both metabolic adaptation and plant damage under anaerobic stress.
- Vartapetian BB Dolgikh YI, Polyakova LI, Chichkova NV, Vartapetian AB. Biotechnological approaches to creation of hypoxia and anoxia tolerant plants. Acta Naturae. 2014;6(2):19-30. Acidification of the cytosol occurs and pyruvate, the product of glycolysis, is transformed to lactate and ethanol, which represent the main fermentation reactions in plants. 4
- plants being sessile are exposed to various environmental stressors, viz, drought, salinity, metal toxicity, low or high temperature, pathogen attack, nutrient deficiency, hypoxia etc; which aims to hamper their lifestyle and lifespan to a great extent.
- various environmental stressors viz, drought, salinity, metal toxicity, low or high temperature, pathogen attack, nutrient deficiency, hypoxia etc; which aims to hamper their lifestyle and lifespan to a great extent.
- it has developed certain inbuilt mechanism for perception of minute changes in the environment and responders which facilitates, either tolerance or avoidance responses to alleviate the stress.
- composition and method thereof needs to be developed in order to reduce hypoxia or increase tolerance to stress induced hypoxia in plants when cultured artificially.
- NADH Oxidase In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH Oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. 5
- Nitric oxide is a gaseous free radical which can have two opposite physiological roles in higher plants. While a high cellular production of NO can bring about extensive cellular damage, at low levels this compound is involved as a signal molecule in many important physiological processes. Under stress conditions, plants can undergo a de -regulated synthesis or overproduction of NO and NO derived products can have toxic physiological consequences.
- Nitric oxide (NO) a gaseous signaling molecule, plays important regulatory roles in higher plants, including seed dormancy and germination, root development and hypocotyl elongation, floral transition, senescence and cell death, phytohormone signaling, and responses to abiotic and biotic stresses.
- GSNO S-Nitrosoglutathione
- RNS reactive nitrogen species
- GSNO reductase GSNOR
- Mutations in the GSNOR gene cause the elevated GSNO level and consequently severe abnormalities under physiological and pathological conditions such as decreased chlorophyll content and altered photosynthetic properties. 6
- ROS reactive oxygen species
- Alternative oxidase is one of the terminal oxidases of the plant mitochondrial electron transport chain. AOX acts as a means to relax the highly coupled and tensed electron transport process in mitochondria thus providing and maintaining the much needed metabolic homeostasis by directly reducing oxygen to water. AOX provides a degree of metabolic homeostasis to carbon and energy metabolism. Beside their role in primary metabolism, plant mitochondria also act as signaling organelles which are able to influence processes such as nuclear gene expression. AOX activity can control the level of potential mitochondrial signaling molecules such as superoxide, nitric oxide and important redox couples .
- Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport and can prevent reactive oxygen species (ROS) and nitric oxide (NO) production under non-stressed, normoxic conditions.
- ROS reactive oxygen species
- NO nitric oxide
- Patent Publication No. US4846883A Succinic acid derivatives and plant growth regulating compositions containing them
- the prior art describes new mono- or di-(aryloxyethyl)-succinates and plant growth regulating compositions containing these compounds. It also relates to a process for the preparation of new mono- or di-(aryloxyethyl) succinates for regulation of plant growth.
- the invention applied for uses succinic acid and its derivatives along with other chemicals in a peculiar concentration so as to form a composition which is used to reduce stress induced hypoxia in plants and thus promote healthy plant growth.
- Patent Publication No. US5604177A Method for stimulating plant growth using GABA and succinic acid
- the prior art describes a process for increasing plant growth and productivity comprising treating the roots, stems and/or foliage of the plant with g-aminobutyric acid and succinic acid as a readily metabolized source of carbon.
- the present invention also provides for a process for increasing plant growth and productivity comprising treating the roots of the plant with g- aminobutyric acid and succinic acid, and a process for increasing the rate of root formation in a plant comprising treating the roots, stems and foliage of the plant with g-aminobutyric acid and succinic acid.
- the present invention provides composition for stimulation of Alternative Oxidase and adapting plant tissue, plant organ, plant cells to hypoxic condition in in-vitro plant tissue culture.
- the said chemical composition of present invention comprises Alternative Oxidase stimulator chemical agents and chemicals which are responsible for making plant hypoxia tolerance chemical such as succinic acid and its derivatives.
- Alternative Oxidase stimulator chemical agents and chemicals which are responsible for making plant hypoxia tolerance chemical such as succinic acid and its derivatives.
- the primary object of the present invention is to provide a chemical composition to reduce stress induced hypoxia in plants grown in in-vitro conditions;
- Further object of the present invention is to provide a chemical composition to induce activity of Alternative Oxidase in plants
- Further object of the present invention is to provide a chemical composition to generate pyruvate by way of stimulation of glycolysis in plants;
- Further object of the present invention is to provide chemical composition to induce hypoxia tolerance in plants cultivated in in-vitro conditions
- Another object of the present invention is to induce stress tolerance in plants without any gene manipulation
- object of the present invention is to induce biotic and abiotic stress tolerance in plants using a chemical composition along with growth hormones;
- Another object of the present invention is to reduce the level of reactive oxygen species (ROS) or to increase the level of NAD+, NADH+ or ATP in the cells of plants growing under adverse conditions;
- ROS reactive oxygen species
- Another object of the present invention is to overcome the problems or issues associated with cultivation of plants in in-vitro conditions as identified in the background above-mentioned;
- Further object of the present invention is to promote growth of healthy, disease free and stress tolerant plants.
- Embodiments of the present disclosure present technological improvements as solution to one or more of the above-mentioned technical problems recognized by the inventor in conventional practices.
- the present disclosure provides a chemical composition when used in effective concentration, yields plants which are tolerant to stress induced hypoxia.
- the disclosed chemical composition comprises - a mixture of Alternative Oxidase stimulator chemical agents; Potassium Nitrate; Sodium Silicate; choline chloride, glutamine acid, Glycine, L-Cysteine, Adenine Sulphate, D Ca pantothenate acid and Growth Hormones.
- alternative oxidase stimulating chemical are Succinic Acid, derivatives of Succinic Acid such as Monomethyl succinate, Di methyl Succinate, Monoethyl Succinate, Di-ethyl Succinate;
- Auxins and Cytokinins are to be used as growth hormones.
- the disclosed chemical composition may be added during any of the multiplication cycle of in-vitro plant cultivation;
- the disclosed chemical composition can be added at either shooting or rooting stage of in-vitro plant cultivation or plant tissue culture.
- the disclosed chemical composition induces hypoxia in plants and stimulates activity of Alternative Oxidase which effectively yields tolerance against hypoxia thus making the plants tolerant and healthier overcoming the adverse effect of conventional plant tissue culture methods.
- the present invention provides composition for stimulation of Alternative Oxidase and adapting plant tissue, plant organ, plant cells to hypoxic condition in in-vitro plant tissue culture.
- This composition achieves substantially normal growth and improved health of plants throughout plant tissue culture process and even in in-vivo conditions.
- This chemical composition is incorporated into the plant tissue culture medium in a concentration that is effective to stimulate alternative oxidase and making plant hypoxic tolerant.
- embodiments of the present invention are concerned with developing tolerance to stress induced hypoxia in plants, and specifically with stimulation of alternative oxidase by usage of a chemical composition comprising succinic acid, derivatives of succinic acid and growth hormones.
- the chemical agents useful in practicing the present invention preferably comprises a mixture of Alternative Oxidase stimulator chemical agents and chemical which is responsible for making plant hypoxia tolerance chemical such as Succinic acid and its derivatives such as but not limited to Monomethyl succinate, Di-methyl Succinate, Monoethyl Succinate, Di-ethyl Succinate or combination thereof choline chloride, glutamine acid, Glycine, L-Cysteine, Adenine Sulphate, D Ca pantothenate acid; high nitrate concentration in the form of potassium nitrate and sodium silicate as basic solution for PH adjustment in an effective concentration to plants in tissue culture along with growth hormones such as Auxin.
- Alternative Oxidase stimulator chemical agents and chemical which is responsible for making plant hypoxia tolerance chemical such as Succinic acid and its derivatives such as but not limited to Monomethyl succinate, Di-methyl Succinate, Monoethyl Succinate, Di-ethyl Succinate or combination thereof choline chloride, glutamine acid, Glycine
- Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes.
- Previous studies showed that the mitochondrial AOX is up-regulated in nitrate -grown cells in comparison with ammonium-grown cells. 9 This is why the present disclosure involves high concentration of nitrate in the disclosed composition.
- ROS have additional signalling roles in plant adaptation to the stress.
- the plant growth regulator Auxin may be the connecting link regulating the level of ROS and directing its role in oxidative damage or signalling in plants under stress. 10 This is the reason use of Auxin in the disclosed composition is essential.
- the relative concentration of the individual components comprising the chemical agents may be varied to produce a mixture that is optimally effective in practicing the method of the present invention for any particular plant tissue plant tissue culture medium.
- Preferred mixture of components in the chemical agents comprises such as succinic acid its derivatives like mono methyl succinate, dimethyl succinate, mono ethyl succinate, diethyl succinate in a concentration range of about lOOmg/l to 350mg/l, Choline chloride in a range about lmg/l to 15 mg/l, glutamine acid in a range about 20mg/l to 80mg/l, adenine sulphate in a range about 20mg/l to 80mg/l, glycine in a range about lmg/l to 2.5mg/l, D ca pantothenate in a range about 0.5 mg/l to lg/l, L - cysteine in a range about lmg/l to
- the components of the chemical agents are mixed to form a individual stock solution of chemical agents using any liquid in which the components will be dissolve but preferably in water and most preferably in distilled or de-ionized water (pH 6-7, water TDS 20-30 ppm).
- plant tissue culture or culturing plant tissue refers to any process carried out in vitro wherein plant organ plant tissue plant cells are propagated, differentiated, sub cultured or maintained on a culture medium defined or undefined which generally maintained in a sterile condition.
- plant tissue culture medium plant culture medium culture medium and medium refers to solid substrate or liquid solution in which a plant tissue organ cells maintained or grown propagated or differentiated one or more isolated plant cell plant cell aggregates may be maintained propagated or differentiated which is to be maintain sterile condition i.e substantially free of microbial contamination.
- plant tissue medium plant culture medium, culture medium, medium are further intended to refer to water containing an appropriate mixture of mineral salts the culture medium may further incorporate in appropriate concentrations including, for example.
- the components of the minerals salts mixtures may be selected and prepared in accordance with the requirement of the particular plant species being propagated the appropriate composition of minerals salts may either be empirically determined or selected from mineral salts compositions previously known in plant tissue culture art and prepared accordingly alternatively the mineral salts may be selected from any number of commercially available mixture (i.e HiMedia) in addition various micronutrients macronutrients and vitamins carbon source components known in art may be variously combined to produce a culture medium appropriate to plant species being propagated.
- HiMedia commercially available mixture
- various micronutrients macronutrients and vitamins carbon source components known in art may be variously combined to produce a culture medium appropriate to plant species being propagated.
- the optimal effective concentration of the chemical agents in the culture medium for use in the claimed methods may be determined empirically by growing said plant tissue plant organ plant cells in a plant tissue culture medium at a range of concentration at which of the chemical agents selecting one or more concentration at which induction of alternative oxidase and adaptation to hypoxic condition of plant tissue plant organ plant cells in vitro to such level that normal plant growth and development of plant at such an empirical determination may be carried out without undue experimentation using standard technique for germination or propagation of plant tissue plant organ plant cells in combination with routine screening technique known to be those skilled in the art.
- plant tissue culture media can be prepared according to any standard recipe for plant tissue culture media know in the art.
- chemical agents added in a range of concentrations for screening as well as without chemical agents (control) plant tissue may be planted in these media appropriately incubated and examined after a sufficient periods of time to determine growth of plant (response) in addition an evaluation of growth or development determined for example using any morphological anatomical or biochemical characteristic may be made by comparing plant tissue plant organ plant cells growth response on media comprising chemical agents to plant tissue plant organ plant cells to growth response on control media lacking chemical agents.
- a morphological analysis may comprise a comparison of the shape size or number of roots shoots leaves or reproductive organ or parts thereof an anatomical analysis may comprises size shape pattern or differentiation of cells the presence or absence of activity dividing meristems.
- Biochemical analysis may comprises analysis of proteins, DNA synthesis, chlorophyll content or presence or absence of any plant pigments one or more of these assays all of which are known to the skilled artisan can contribute to an empirical determination of the optimal concentration of the chemical agents in plant tissue medium to practice the method of the present invention on a particular plant species.
- the optimal concentration of chemical agents for practicing the method of the present invention may be added to a culture medium that is thereafter to be used in liquid or solidifying culture with gelling agent.
- the culture medium is then typically adjusted to an appropriate pH range of 5.7 - 5.9 using acidic solution as hydrochloric acid (HCL) basic solution or as Sodium silicate (NaoSio ⁇ ) selection of basic solution for PH adjustment will be depend on particular plant species like phytolith accumulating and not accumulating and then autoclaved at l2l°c for 20 min.
- acidic solution as hydrochloric acid (HCL) basic solution or as Sodium silicate (NaoSio ⁇ ) selection of basic solution for PH adjustment will be depend on particular plant species like phytolith accumulating and not accumulating and then autoclaved at l2l°c for 20 min.
- the present invention can be performed with known chemical combination in plant tissue culture or chemical composition made according to particular plant species requirement generally the present invention can be performed with (MS, WHITES, HELLERS, ER, B5, NITSCHS, NT )
- the chemical composition disclosed in the present invention has to be added to the plant tissue culture medium in order to induce alternative oxidase acitivity in plants.
- the example herewith describes preparation of 10 litre plant tissue culture medium using succinic acid as alternative oxidase inducer.
- the illustrated media preparation is for banana plant (MS media chemical compositions, G9 banana plant variety).
- Succinic acid 27g Succinic acid is dissolved in 1 litre water preferably distilled or de-ionized water;
- Sodium silicate as basic solution for pH adjustment 95g Sodium Silicate is dissolved in 1 litre distilled or de-ionized water;
- Adenine Sulphate 4 g Adenine sulphate is dissolved in 1 litre 0.5M HCL containing distilled or de-ionized water;
- Glutamine Acid 4 g Glutamine Acid is dissolved in 1 litre distilled or de-ionized water;
- Choline chloride lg Choline is dissolved in 500ml distilled or de-ionized water;
- Glycine 200mg glycine is dissolved in 500ml distilled or de-ionized water;
- L- Cysteine 200mg L - cysteine is dissolved in 500ml distilled or de-ionized water;
- Potassium nitrate (as high nitrate concentration): 250g Potassium nitrate is dissolved in 2 litre distilled or de-ionized water;
- D Ca-Pantothenate 50 mg D Ca - Pantothenate is dissolved in 500ml distilled or de-ionized water;
- Glutamine acid (lOOml), Choline (50ml) , Adenine sulphate (lOOml), Potassium nitrate (200ml), Glycine (50ml), L - Cysteine (50ml), D Ca pantothenate (50ml) is added after sodium silicate and Succinic acid in 8 litre distilled or deionized water.
- Agar Agar and l0.5g gelling agent After adjusting pH, 25g Agar Agar and l0.5g gelling agent is added. 10 litre media composition is well heated to dissolve Agar Agar and gelling agent. The heated media is poured in 300ml glass bottles or plastic container then caps tightened to it and then the media filled in containers is autoclaved at l2l°C for 20 min. Autoclaved media is cooled overnight and the same is used for subculture purpose.
- the only change in media preparation is that according to multiplication, growth hormones are added viz., - cytokinin 3mg /l and Auxins l.2mg/l.
- growth hormones are added viz., - cytokinin 3mg /l and Auxins l.2mg/l.
- Auxin is added to the media in the concentration 1.2 mg/l and for shooting the concentration of Auxins is maintained at 0.5mg/l.
- the effectiveness of the present disclosure was tested by propagating G-9 variety banana plants in combination of effective concentration of the disclosed composition with MS media. Thereafter, primary hardened banana plant leaf was tested for mitochondrial and cytosolic enzymes activity at BioEra Life Sciences. Pvt. Ltd. Pune (Reference No. BE/PNU/6254).
- the one healthy and disease free, banana plant sucker was selected for micro-propagation and sterilized according to best practiced procedure. It was initiated on MS media containing cytokinin (6-BAP) 1.5 mg/l and thereafter maintained or propagated for 4 cycles of multiplication on MS media containing cytokinin (6-BAP) 3mg/l, Auxin (IAA) 1.2 mg/l and Ascorbic Acid 5 mg/l as antioxidant.
- the plant culture thus obtained was divided in five groups. Lirst group was maintained or propagated on MS media supplemented with 3mg/l 6-BAP, l.2mg/l IAA, Ascorbic acid 5mg/l. Lor further 3 multiplications, the culture was transferred to shooting medium containing 0.5mg/l IAA and then to rooting medium containing lmg/l IB A and activated charcoal lg/l. pH was adjusted to 5.8. (Culture Group named as‘A’)
- Second plant culture group is maintained or propagated on media containing all the chemical composition disclosed in the present invention except succinic acid or its derivatives or combination of succinic acid derivatives, choline chloride lOmg/l, glutamine acid 40mg/l, adenine sulphate 40mg/l, glycine 2mg/l, L - Cysteine 2mg/l, D ca pantothenate 0.05mg/l, potassium nitrate 2500mg/l.
- cultures were transferred to shooting medium containing 0.5mg/l IAA and then to rooting medium containing lmg/l IBA and activated charcoal (lg/l ) and pH is adjusted to 5.8.
- Third plant culture group is maintained or propagated on media containing chemical agents composition disclosed in the present invention succinic acid 270mg/l, choline chloride lOmg/l , glutamine acid 40mg/l , adenine sulphate 40mg/l, glycine 2mg/l, L-cysteine 2mg/l, D Ca pantothenate 0.05mg/l and potassium nitrate 2500mg/l. After 3 multiplication cycles, the cultures were transferred to shooting medium containing 0.5mg/l IAA and then to rooting medium containing lmg/l IBA and activated charcoal (lg/l ) with pH adjusted to 5.8 with sodium silicate as basic solution. (Culture group named as‘C’).
- Fourth and fifth plant culture group were prepared like third culture group but with different concentrations of Succinic acid ranging from l50mg/l (Culture group named as‘D’), 450mg/l (Culture group named as ⁇ ’).
- the effectiveness of the present invention chemical composition of concentration was tested by observing mitochondrial enzymes activity (succinate dehydrogenase, fumarase, aconitase, citrate synthase, succinyl Co- A synthase) and Cytosolic enzymes activity (Nitrate reductase, ATP citrate lyase). Three plants out of hundred plants from each five groups were randomly selected for test.
- HEPES 25mM -(2-hydroxyethyl)-l -piperazine ethane sulfonic acid
- the Mitochondria pellet was resuspended in 10 ml of washing buffer, aliquots of 1.5 ml were layered over a 32 ml self-generating Percoll gradient containing 0.3 M sucrose, 10 mM KH 2 PO 4 , pH 7.2, 0.1% (w/v) BSA, 28% (v/v) Percoll and a linear gradient of 0-10% (w/v) PVP-25 (top to bottom) in a Beckman SW28 rotor (tube size 34 ml, swing out rotor) and centrifuged for 45 min at 40,000rpm. The mitochondria were localized in a yellow-white band near the bottom of the tube and were carefully removed.
- Protein concentration was determined by Lowry et al., using Bovine Serum Albumin (BSA) as standard.
- BSA Bovine Serum Albumin
- Citrate synthase activity was measured by following the reduction of APAD at 365 nm.
- Malate oxidation to Oxalo acetate (OAA) by commercial malate dehydrogenase was coupled to APAD reduction. This reaction only proceeds if OAA is being used by citrate synthase because the malate dehydrogenase reaction is strongly inhibited by OAA.
- the rate of APAD reduction is followed spectrophotometrically by measuring absorbance changes at 365 nm.
- the extinction coefficient of APADH at 365 nm (e365) is 9,100 M -1 cm -1 .
- the mitochondrial sample was initially incubated in reaction medium without Acetyl-CoA at 25 °C for 10-15 min. Assays is initiated by the addition of acetyl-CoA.
- the coupled reaction involves measuring the rate of NADP + - isocitrate dehydrogenase
- the rate of NADPH synthesis was measured by change of absorbance at 340 nm.
- the extinction coefficient of NADPH at 340 nm (e340) is 6,220 M 1 cm 1
- Pinal reaction concentrations 80 mM KH 2 PO 4 -NaOH (pH 7.8), 0.5 mM NADP + , 0.5 mM MnCF, 2 U/mL of NADP-isocitrate dehydrogenase, 0.05 % (v/v) Triton X-100, 8 mM aconitate.
- the reaction was initiated by adding the mitochondrial sample.
- the absorbance at 340 nm was measured over time.
- the reaction was initiated by adding mitochondrial sample and terminated by the addition of 50 mL 1 M HC1 and 50 mL 1 M NaOH.
- reaction mixture was heated at 95 °C for 20 min to destroy un-reacted 5',5'-diadenosin pentaphosphate and was then allowed to cool on ice.
- Succinate dehydrogenase (Complex II) is both an enzyme of the TCA cycle and a complex of the ETC complex. In this reaction, UQ is the receptor of electrons from succinate.
- PMS was used as an intermediate electron carrier and coupled with DCPIP as an electron acceptor. Succinate dehydrogenase activity was assayed spectrophotometrically by measuring the change of the absorbance of DCPIP at 600 nm. The extinction coefficient of DCPIP at 600 nm was 0.021 M -1 cm -1 .
- Assay was initiated by adding the mitochondrial sample.
- the absorbance at 600 nm was measured over time.
- the absorbance of fumarate produced in the reaction can be recorded at 240nm directly by spectrophotometry.
- the extinction coefficient of fumarate at 240 nm (e240) is 2,530 M 1 cm 1
- the reaction was initiated by adding the mitochondrial sample.
- the absorbance at 240nm was measured over time.
- ACL activity was measured in the cytosolic fractions using the malate-dehydrogenase-dependent coupled Spectrophotometric method.
- the assay mixture contained 50mM Tris/HCl, 0.2mM NADH, I Orn M MgCI 2 , 10mM KC1, 5mM dithiothreitol, 200m M CoA, 5mM ATP, and cytosolic extract.
- the assay was usually initiated by the addition of CoA. All assays were carried out at 30°C. Nitrate Reductase
- the enzyme activity in the cytosolic extracts was measured in lml assay mixture (pH 7.5) containing 50mM K-phosphate, 5mM KNO3, 0.2mM NADH, and enzyme extract.
- the reaction mixture was incubated at 25°C for 15 minutes and terminated by adding 0.1 ml alcohol dehydrogenase (140 units ml 1 for 100m M K-phosphate, pH 7.0) followed by O.lml of 1M acetaldehyde to oxidize excess NADH. After 5 minute nitrite produced was determined.
- Nitrite was determined by adding lml of 1% (W/V) sulphanilamide (in 1.5N HCL) followed by lml of 0.01% (W/V) N-(l-napthyl) ethylene diamine dihydrochlode to develop the red diazotized complex of N0 2 . After 15 minute, the absorbance was read at 540nm, in a spectrophotometer.
- the assay mix was centrifuged for 10 minutes at 5,000 rpm to clear the turbidity before taking absorbance.
- the growth characteristic was assessed by measuring the following parameters like plant height , leaf number, leaf area, length of bunch , number of hands and bunch weight at 10 th month of planting.
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AU2019337472A AU2019337472A1 (en) | 2018-09-11 | 2019-01-17 | Alternative oxidase activity stimulating chemical composition and a method for reducing hypoxia in plants |
PH12021550426A PH12021550426A1 (en) | 2018-09-11 | 2021-03-01 | Alternative oxidase activity stimulating chemical composition and a method for reducing hypoxia in plants |
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PCT/IB2019/050380 WO2020053667A1 (en) | 2018-09-11 | 2019-01-17 | Alternative oxidase activity stimulating chemical composition and a method for reducing hypoxia in plants |
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AU (1) | AU2019337472A1 (en) |
PH (1) | PH12021550426A1 (en) |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US4846883A (en) * | 1985-06-03 | 1989-07-11 | Reanal Finomvegyszergyar | Succinic acid derivatives and plant growth regulating compositions containing them |
WO1995022900A1 (en) * | 1994-02-23 | 1995-08-31 | Auxien Corporation | Method for simulating plant growth using gaba |
US6534446B1 (en) * | 1994-02-23 | 2003-03-18 | Emerald Bioagriculture Corporation | Method to mitigate plant stress |
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2019
- 2019-01-17 WO PCT/IB2019/050380 patent/WO2020053667A1/en active Application Filing
- 2019-01-17 AU AU2019337472A patent/AU2019337472A1/en not_active Abandoned
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2021
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4846883A (en) * | 1985-06-03 | 1989-07-11 | Reanal Finomvegyszergyar | Succinic acid derivatives and plant growth regulating compositions containing them |
WO1995022900A1 (en) * | 1994-02-23 | 1995-08-31 | Auxien Corporation | Method for simulating plant growth using gaba |
US6534446B1 (en) * | 1994-02-23 | 2003-03-18 | Emerald Bioagriculture Corporation | Method to mitigate plant stress |
Non-Patent Citations (2)
Title |
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MILLAR AH ET AL.: "Organic acid activation of the alternative oxidase of plant mitochondria", FEBS LETTERS, vol. 329, no. 3, 30 August 1993 (1993-08-30), pages 259 - 62, XP025602193, DOI: 10.1016/0014-5793(93)80233-K * |
WAGNER AM ET AL.: "Stimulation of the alternative pathway by succinate and malate", PLANT PHYSIOLOGY, vol. 108, no. 3, 1 July 1995 (1995-07-01), pages 1035 - 1042, XP055691113 * |
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AU2019337472A1 (en) | 2021-03-04 |
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