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    • 专利摘要:
    Method for the regeneration of damaged plant tissues. The present invention provides a method for regenerating damaged plant tissues. Said method is based on the use of cellulose, preferably bacterial cellulose (cb), which is applied directly on the damaged area allowing its rapid healing. The present invention thus demonstrates the regenerative potential of cellulose, preferably bacterial cellulose, on wounds in plants. In a preferred embodiment of the method of the invention, cellulose is used in combination with silver nanoparticles to, in addition to regenerating the tissue, prevent and/or treat bacterial infections therein. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2645757A1
    申请号:ES201630592
    申请日:2016-05-06
    公开日:2017-12-07
    发明作者:Anna Laromaine Sagué；Nuria SÁNCHEZ COLL
    申请人:Centro De Investigacion En Agrigenomica；Centro De Investig En Agrigenomica；Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    5 The present invention falls within the field of agriculture and botany,specifically within the methods and materials useful for the regeneration ofplant wounds
    STATE OF THE TECHNIQUE
    Wounds in plants are frequently caused by cuts during the process of harvesting their fruits and, in general, during the activity of the agricultural industry. Although such wounds can also be caused by various other causes such as, for example, natural aggressions, infections by phytopathogenic organisms or human manipulation. Wounds in plants represent a dangerous route of entry for pathogenic organisms, thus facilitating the development of serious infections in plant tissues that can be extended to the entire plant,
    which can ultimately lead to serious losses in agricultural crops. The healing of wounds in plant tissues is therefore a challenge of great relevance for the agricultural and grafting industry.
    In this sense, various approaches have been developed to solve this problem. One of them is that described in ES2163977A1, where a waterproofing-sealant composition of tree cuts is disclosed, which has as its main products an ethene-vinyl copolymer emulsion, isolating barrier products with respect to ozone, fatty acid esters and glycerin; a protective colloid based on a cellulose hydroxyethyl ether colloid and a quartz-based cap-pore. On the other hand, JPH1033069A describes a patch for treating plant wounds, which is obtained by mixing chitin powder with a vulcanized natural rubber, in addition to components such as an antibacterial agent, an antifungal agent, a plasticizer and a natural material such as powder. of cellulose as filler material. EP0290155A2 refers to an absorbent material in sheet form for application in wounds of plants comprising several layers of different materials. Finally, CN102334514A refers to an agent to cure
    35 wounds on trees that, among other components, contain hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl cellulose.
    Cellulose constitutes an almost inexhaustible biopolymer, being the most abundant natural polysaccharide in the biosphere. Cellulose, with a complex hierarchical structure, and more recently nanocellulose or cellulose nanocrystals, are actively being investigated in the design of new (bio) nanocomposites.
    Although cellulose is predominantly obtained from plants, it can also be synthesized by bacteria, algae and fungi. In particular, bacterial cellulose (eS) produced by microbes has the same molecular formula as plant cellulose but has the particularity of being a pure biopolymer that has a higher degree
    10 polymerization and better crystallinity. The CS also has high porosity, is transparent in the UV-NIR and has a high water retention capacity. In addition, a unique feature of CS is the possibility of modifying its micro (nano) structure and its shape during biosynthesis.
    15 CS has been observed to have important effects on wound regeneration in humans, especially in burn recovery (Czaja W., et al., 2006, Biomaterials, 27: 145; Rajwade JM., Al., 2015 , Apptied Microbiology and Biotechnotogy. 99: 2491).
    20 Therefore, new treatments are required for the regeneration of wounds in plant tissues that allow correct tissue healing and, preferably, also help prevent and / or treat infections caused by pathogenic organisms that may be favored by the existence of tissue structure damage.
    25 DESCRIPTION OF THE INVENTION
    The present invention provides a method for effectively regenerating damaged plant tissues. Said method is based on the use of cellulose, preferably
    30 bacterial cellulose (CS), which is applied directly to the damaged area allowing rapid healing or healing. The present invention thus demonstrates the regenerative potential of cellulose, preferably bacterial cellulose, on plant wounds. The method described in the present invention provides a chemical composition material to the structure of the plant.
    This method has the advantage that it allows a correct and complete healing of the damaged area in a short period of time since, as the examples described below demonstrate, the creation of new tissue in the damaged areas can be observed after approximately, although not limited, 48h.
    In addition, said method is applicable in the regeneration of wounds caused by any cause, from cuts to damage caused by abrasion, in any type of fabric, although preferably in non-lignified tissues, and in any type of plant.
    On the other hand, the cellulose used in the method of the invention can be used in combination with one or more antibacterial, antifungal and / or antiviral components, such as silver nanoparticles, thus further allowing the prevention and / or treatment of bacterial, fungal infections. or viral over the area
    15 damaged from the plant.
    Thus, in a first aspect, the present invention relates to a method for
    healing, healing or regeneration of wounds in plants, for regeneration
    plant tissue or to regenerate damaged plant tissues, from now on
    20 "method of the invention ~, comprising:
    to. contacting, preferably directly, the damaged plant tissue with cellulose, preferably with a cellulose sheet, and
    b. allow regeneration of damaged plant tissue.
    Preferably, in step (a) of the method of the invention, the cellulose is applied directly to the damaged plant tissue, ex vivo or in vivo, so that said cellulose completely or partially covers, more preferably totally, the affected area.
    30 "Cellulose ~ is the main component of plant cell walls. From a biochemical point of view, cellulose (C6H, 00 5) n with a minimum value of n = 200, is a natural polymer, consisting of a long chain of polysaccharide carbohydrates The structure of cellulose is formed by the union of B-glucose molecules through B-1,4-glucosidic bonds, which makes it insoluble in water.
    35 has a linear or fibrous structure, in which multiple hydrogen bonds are established between the hydroxyl groups of different juxtaposed glucose chains,
    making them very resistant and insoluble to water. In this way, compact fibers that constitute the cell wall of plant cells originate, thus giving them the necessary stiffness. Cellulose can be extracted or obtained from plant sources, particularly from woody plants, such as, for example, but not limited to, 5 trees, such as pine or eucalyptus, or other plants, such as grasses, bamboos, starch, wheat, corn, barley, sorghum, sugarcane bagasse, cottons, linens, hemp or others. Cellulose can also be obtained from other non-plant sources, such as bacteria, algae or fungi, or by in vitro enzymatic synthesis or in vitro chemical synthesis from benzylated glucose derivatives. Examples of 10 bacteria from which bacterial cellulose can be obtained are, but not limited to, bacteria of the genera Dictyostelium, Acetobacter, Alcali-genes, Pseudomonas, Aerobacter, Achromobacter, Azotobacter, Salmonella, Sarcina, Agrobacterium, Rhizobium or Gluconacetobacter. An example of fungi from which fungal cellulose can be obtained is, but not limited to, Trichoderma
    15 reesei. Examples of algae from which cellulose can be obtained are, although not limited to, Cladophora, Rhodophyta, Pyrrophyta, Chrysophycease, Xanthophyceae, Phaeophyta or Chlorophyta.
    Cellulose as defined in the present invention can be, although without
    20 limit ourselves, lignocellulose, vegetable cellulose, cellulose of bacterial origin, cellulose of fungal origin, cellulose fibers, nanocomposite cellulose or cellulose crystals, preferably nanocellulose, microcrystalline cellulose or cellulose nanocrystals.
    In a preferred embodiment of the method of the invention, cellulose is cellulose.
    25 plant or bacterial. Bacterial cellulose, produced by microorganisms, has the same molecular formula as plant cellulose, so both can be applied interchangeably in the method described in the present invention by presenting similar physicochemical properties. The examples shown below demonstrate the usefulness of both types of cellulose, plant and bacterial, in healing
    30 wounds in plant tissues. However, bacterial cellulose has the advantages that it has a higher degree of polymerization than plant cellulose and a better crystallinity and purity. In addition, it has high porosity and high water retention capacity. Therefore, this type of cellulose is preferably used in the regeneration method of damaged plant tissue described herein.
    Invention. Thus, in a more preferred embodiment, the cellulose is bacterial cellulose.
    "Bacterial cellulose" or "cs" is that cellulose formed and secreted by bacteria, preferably in culture. The CS, for its purity and highly crystalline structure, stands out as an alternative source to that of plant origin. The production of CS in the Monera kingdom is diversified, the synthesis is observed in species within the 5 genera, but without limiting ourselves, Achromobacter, Agrobacterium, Rhizobium, Sarcina, Zoogloea and Gluconacetobacter, to the latter belongs G. xylinus, the species with Greater production capacity. This bacterium allows the biogenesis of CS from a wide variety of substrates, yielding a product of high purity (free of lignin and hemicellulose) and of similar structure to that of plant origin. This microorganism 10 also has the advantage of easy handling. Therefore, in an even more preferred embodiment, bacterial cellulose is produced by bacteria of the Gluconacetobacter genus. In a particular embodiment, the bacterium of the genus Gluconacetobacter is selected from among Gluconacetobacter xylinus or Gluconacetobacter europaeus. Among these two bacteria, G. xylinus is the most
    15 preferred in the present invention, since it has a high cellulose production rate.
    Gluconacetobacter xylinus (formerly Acetobacter xylinum), is a Gram negative bacterium belonging to the Acetobactereaceae family; strict aerobic that performs the incomplete oxidation of various sugars and alcohols (process known as oxidative fermentation). Its natural habitat is fruits and vegetables in the process of decomposition; It is capable of producing CS on liquid and solid media by forming a "film" or "cream" on the surface. The CB film functions as a "flotation" mechanism, allowing G. xylinus to be at the air / liquid interface 25 to more easily obtain the O2 necessary for its growth. The film is a physical barrier that protects the bacteria from UV radiation, increases the ability to colonize substrates and its highly hygroscopic nature allows it to retain moisture preventing the drying of the substrate. Two characteristics are particular to CB microfibrils: their polarity is unidirectional and they are variable in thickness. The crystallization mechanism of microfibrils in G. xylinus can give rise to two cellulose aloforms: if the microfibrils are oriented in parallel, cellulose 1 is synthesized, while if the arrangement of the microfibrils is antiparallel, cellulose is obtained 11. Both Cellulose I as cellulose II are within the scope of the present invention. The microstructure of the CB is made up of microfibrils 35 with a diameter of 4 to 7 nm and a polymerization degree of 2,000 to 14,000 glucose molecules. The microfibrils in turn crystallize in packages and tapes, which reach a thickness of 1 to 9 IJm and form an extensive cross-linked structure stabilized by hydrogen bonds. The condensation of the tapes gives rise to the three-dimensional structure or macrostructure of the CS. The macrostructure of the eB is totally dependent on growing conditions; Thus, under static conditions 5 a "film" or "cream" is generated at the air-liquid interface of the culture medium. The microfibrils, which are continuously released by the bacteria, crystallize into tapes, which overlap forming parallel planes. In agitated culture, a lower degree of clustering is achieved, the amount of parallel planes is smaller and consequently irregular granules, fibrous chains or
    10 branched CB. In the context of the present invention, CS is preferably produced under static culture conditions, thus generating a "film" at the air / substrate interface of the culture medium.
    As already mentioned, the CS presents a purity superior to that present in the
    15 cellulose from any plant source, which gives it a series of advantageous characteristics, such as, high degree of crystallization, high resistance to pressure, elasticity and durability. Cellulose has a high capacity to absorb water and due to a smaller diameter of microfibrils, the CS has a greater surface area than that present in wood pulp. In addition to these properties
    20 physicochemicals of industrial importance, CS is metabolically inert, non-toxic, nor causes allergic reaction to contact, properties of particular importance for the purpose described in the present invention.
    CB can be produced by chemical synthesis in vitro or by, for example
    25 but not limited to, the culture of the producing bacterium in the presence of a culture medium and culture conditions (pH, TO, oxygenation, light / dark, CO2, etc.) suitable for the production of cellulose. Those skilled in the art will recognize the bacterial culture media and the culture conditions that can be used for this purpose.
    The "culture medium" is a suitable nutritive medium, that is, it comprises the nutrients necessary for the maintenance and in vitro growth of the producing bacterium, for the development of its metabolic activity and, therefore, for the production of CS. Said culture medium may be liquid, solid or semi-solid, although preferably it is liquid. For the bacteria to grow properly in the culture medium, it must meet a series of conditions such as temperature,
    adequate degree of humidity, light / dark and oxygen pressure, as well as a correct degree of acidity or alkalinity (pH). Likewise, the culture medium must be free of all contaminating microorganisms.
    5 Therefore, the producing bacterium can be grown in a solid, semi-solid or liquid culture medium, preferably liquid, in the presence of suitable salts and nutrients to favor the growth and proliferation of bacterial cells. The suitable nutrient medium comprises, for example, but not limited to, agar or gelatin or albumin, carbon sources (for example, glucose, sucrose or mannitol),
    10 sources of nitrogen (for example, peptones), sulfur, phosphorus, sources of vitamins, amino acids and hormones and / or growth factors (for example, meat extract or yeast extract), inorganic salts (for example, sodium or potassium ), hydrogen ions, citric acid, etc.
    15 The bacteria can be grown, for example, but not limited to, by flask or Erlenmeyer culture and small or large scale growth carried out in a laboratory or industrial bioreactor, in a suitable medium and under conditions that allow the production of CS. Preferably, the bacteria is initially grown in solid medium, for example, but not limited to agar, and subsequently
    20 transfers the culture to a liquid medium to be expanded.
    The culture of the bacteria, preferably of G. xylinus, for the production of cellulose is more preferably practiced under static conditions. The optimum temperature range for crops is 20 to 350 C, the temperature at which production takes place. The pH of the culture medium may vary from preferably 4.0 to 6.0, the optimum pH being dependent on the producing strain. The composition of the culture medium is variable according to the strain used, carbohydrates are the appropriate carbon source for the synthesis of CS, so the production of CS can occur in media containing, for example, but not limited to, sucrose, glucose, fructose, lactose or mannitol. The bacteria can synthesize de novo glucose from lactic acid
    or succinic acid. Yeast extract is the most commonly used nitrogen source for the growth of G. xylinus, and therefore preferred in the present invention; Peptone, polypeptone, tryptone, corn liquor and ammonium sulfate can also be used in the production of CS. It is also common to add sodium phosphate or
    35 potassium to buffer pH changes, and the addition of sulfate or magnesium chloride. The incubation periods are dependent on the culture system, in static culture long periods of culture are preferred, preferably ranging from
    one to two weeks, although periods of 5 or more days are also suitable.
    Cellulose is thus formed in the nutrient medium, preferably in the form of a film, and
    5 can be recovered directly from the medium, from the air / liquid interface in the case ofthat the crop develops in liquid medium. Thus, in another preferred embodiment of themethod of the invention, cellulose is in the form of a film or sheet.
    Preferably, the film of is formed is collected and subjected to a treatment
    10 comprising the washing steps and optionally drying. More preferably, the washing comprises immersion of the film of is in alcohol, preferably ethanol, its transfer to a deionized aqueous medium and a step in which the es is boiled and subsequently neutralized. The boiling step takes place for a time of preferably between 30 and 50 min, more preferably 40 min. This step can
    Repeated several times, preferably four more times, in the presence of NaOH at a temperature between 80 and 100oe, preferably gOoe, for a period of
    between 10 and 30 min, preferably 20 min.
    The es can then undergo an optional drying step, in which
    The cellulose is cut to the desired shape and size and dried at room temperature for preferably between 3 and 5 days, more preferably 4 days.
    The method of the invention also allows regenerating damaged plant tissues as a result of an infection by some pathogen. This effect will be seen.
    25 particularly enhanced if the cellulose used is combined with one or more antimicrobial, antifungal and / or antiviral agents, since this approach will allow not only the regeneration of the tissue affected by the infection but also the disappearance or reduction of the pathogenic load present in the affected area. Therefore, in another preferred embodiment, the cellulose further comprises an agent.
    30 antibacterial, antifungal and / or antiviral. Said agent may be found, but not limited to, coating the cellulose piece or it may be embedded in it.
    In a more preferred embodiment, said agent is an antibacterial agent, such as, but not limited to, antibiotics, nanoparticles, microparticles, 35 milparticles, beads or spheres, etc., loaded with at least one antibacterial agent, natural products, such as lactic acid. , citric acid, acetic acid, and its salts, oils
    essentials, etc. In an even more preferred embodiment, the antibacterial agent are nanoparticles, even more preferably silver and / or copper nanoparticles.
    The nanoparticles, such as silver and / or copper nanoparticles, are
    5 may produce procedures well known to those skilled in the art.matter. For example, although not limited, Ag nanoparticles can bemanufacture by laser ablation or thermal decomposition of AgN03 in situ undermicrowave irradiation, as described in the examples below.
    On the other hand, cellulose films, preferably of CB, as described in the present invention, adhere better to the damaged plant surface if they are moistened. Therefore, in another preferred embodiment of the method of the invention, the cellulose is wet or wet.
    When the cellulose used in the method of the invention is vegetable cellulose, it must have a high relative humidity, "high relative humidity" being a high water content, preferably a relative humidity greater than 60%, more preferably a higher relative humidity at 80% or 90%, even more preferably a relative humidity around 99% or 99%. Conversely,
    When the cellulose used in the method of the invention is bacterial cellulose, it can have a high or low relative humidity, "low relative humidity" being a low water content, preferably a relative humidity of less than 60% or 60%, more preferably a relative humidity of 60%.
    In another preferred embodiment, the plant tissue referred to in the method of the invention is non-lignified plant tissue.
    "Non-lignified plant tissue" or "non-woody tissue" means plant tissue that has not undergone lignification, or deposit of lignin or oxygenated derivatives of the
    30 cellulose and xylanases, in the cell wall. That is, that plant tissue that does not include lignin. In a more preferred embodiment, the plant tissue is selected from the list consisting of: leaf, fruit, stem, branch, flower or root. In an even more preferred embodiment, the plant tissue is leaves.
    The method of the invention is useful for the healing or regeneration of wounds or damaged plant tissue in any plant, however, in another preferred embodiment the plant tissue belongs to a plant of the Solanaceae family. More preferably, the plant tissue belongs to Nicotiana benlhamiana or So / anum Iycopersicum.
    The method of the invention is useful for the healing or regeneration of wounds or of
    5 plant tissue damaged by any cause, however, in another preferred embodiment the tissue damage is caused by cutting, abrasion, puncture, pressure, breakage or infection, more preferably by cutting or infection.
    As previously mentioned, when cellulose is associated with
    10 an antibacterial, antifungal and / or antiviral agent, this is useful not only in the regeneration of tissue damage caused as a result of a bacterial, fungal and / or viral infection, but also in the treatment and / or prevention of infection in herself. Particularly, when the cellulose is bound to silver and / or copper nanoparticles as described above, it is capable of
    15 regenerate the damage produced in the tissue as a result of a bacterial infection and in addition to preventing and / or treating said infection in the sense of causing the disappearance or reduction of the bacterial load in the tissue. Therefore, in a more preferred embodiment, tissue damage is caused by an infection, more preferably by a bacterial infection, even more preferably by a
    20 bacterial infection caused by one or more Gram-negative bacteria. In an even more preferred embodiment, the bacterial infection is caused by E. coli or Pseudomonas syringae. As the examples described below show, cellulose, preferably CB, associated with Ag nanoparticles in contact with plant tissue in the presence of a P. syringae infection prevents and treats said
    25 infection, detecting low bacterial load, tissue regeneration and little necrotized tissue.
    The "infections" referred to in the present invention are those caused by any phytopathogenic organism.
    In step (b) of the method of the invention, the damaged plant tissue is allowed to heal in the presence (in contact, preferably direct) of the cellulose for the necessary time. This step, therefore, will take place over a period of time such that it allows healing, preferably total, of the damaged area to occur. Thus, the person skilled in the art will know how to recognize the necessary time during which the damaged plant tissue and cellulose must keep in contact. The degree of
    Tissue regeneration may be determined, for example, but not limited to, by visualizing the amount of new tissue created in the affected area. In the examples shown below, tissue regeneration is demonstrated after, but not limited to, 48h. Therefore, in another preferred embodiment of the method of the invention, the
    5 step (b) takes place over a period of at least 24 hours. In amost preferred embodiment, step (b) takes place over a period of time of atminus 48h.
    Another aspect of the invention relates to a composition comprising cellulose,
    10 preferably CB, more preferably in the form of one or more sheet / s or film / s, and nanoparticles, preferably silver and / or copper nanoparticles. In a preferred embodiment, the cellulose is coated with said nanoparticles, although they could also be in any other arrangement, such as embedded in said cellulose. In another preferred embodiment, the eB is obtained from
    15 bacteria of the genus Gluconacetobacter. In a particular embodiment, the bacterium of the genus Gluconacetobacter is selected from among Gluconacetobacter xylinus or Gluconacetobacter europaeus, more preferably G. xylinus.
    Another aspect of the invention relates to the use of the composition as described in
    20 the preceding paragraph for the regeneration of damaged plant tissues, preferably of non-lignified tissues, more preferably of tissues selected from the list consisting of: leaf, fruit, stem, branch, flower or root, even more preferably of leaves. Preferably tissue damage is caused by an infection, more preferably bacterial, even more preferably by E. coli or Pseudomonas.
    25 syringae.
    Another aspect of the invention relates to the use of the composition as described above for the treatment and / or prevention of infections, preferably bacterial, more preferably by E. coli or Pseudomonas syringae, in plants or
    30 plant tissues, preferably not lignified, more preferably in leaf, fruit, stem, branch, flower or root, even more preferably in leaves.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the
    invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    DESCRIPTION OF THE FIGURES
    FIG. 1. TEM images of bacterial cellulose (CB) films coated with Ag nanoparticles (Ag-CB) (top and bottom-left). Bottom-right, size distribution of Ag nanoparticles (AgNPs).
    FIG. 2. Toxicity test using the biomarker Eseherichia eoli. The degree of toxicity of the different substances! Materials are determined based on their ability to eliminate the bacteria (aura).
    FIG. 3. Test of regeneration of cuts in Nicotiana benthamiana leaves, at 48h post-treatment with bacterial cellulose (CB) films.
    FIG. 4. Regeneration test for damage caused by puncture on Nicotiana benthamiana leaf in the presence and absence of bacterial cellulose (eB).
    FIG. 5. Scanning electron microscopy (SEM) showing regeneration of damage caused by cutting on sheets of Nicotiana benthamiana in the presence and absence of bacterial cellulose (CB).
    FIG. 6. Effects of bacterial cellulose (CB) or bacterial cellulose with silver nanoparticles (Ag-CB) on cuts in benthamian Nieotiana leaves that also include infection by Pseudomonas syringae. Micropore tape (3M Deutschland GmbH, Neuss, Germany) (Tape), Ag-CB, CS or nothing was added over the cuts.
    FIG. 7. Effects of bacterial cellulose (CB) and plant cellulose on leaf sections of Nicotiana benthamiana. Micropore tape (3M Deutschland GmbH, Neuss, Germany) (Tape), filter paper (vegetable cellulose), is added or not added to the cuts.

    EXAMPLES
    Next, the invention will be illustrated by tests carried out by theinventors who demonstrate the effectiveness of cellulose films,5 preferably of bacterial cellulose, in the regeneration of plant tissuesdamaged as a result of various attacks of different nature.
    EXAMPLE 1. Production of bacterial cellulose films (eB).
    10 The bacterial strains used were: Gluconacetobacter xylinum (GX) (ATCC 11142) (Yamada Y, Hoshino K, Ishikawa T (1997) Biosci, Biotechnol, Biochem 61 (8): 1244-1251) and Gluconacetobacter europaeus (GE) (MF03 ) (Yamada Y, Hoshino K, Ishikawa T (1997) Biosci, Biotechnol, Biochem 61 (8): 1244-1251) And were acquired from the Spanish Type Culture Collection (Spain).
    15 Glucose, peptone, yeast extract and agar were purchased from Canda Lab, NaOH, Na2HP04 • 12H20 and citric acid monohydrate were purchased from Sigma Aldrich and used as received.
    20 GX is the bacteria most widely used to produce cellulose, due to its high production speed. GX was cultured in solid agar and an isolated colony was expanded in liquid medium for 3 days. Then, 8 ml of GX solution was transferred to an Erlenmeyer with 200 ml of liquid medium. A thin layer of bacterial cellulose grew on top of the liquid media of more than 5
    25 days for GX. The culture media for GX consisted of 20 gIl glucose, 5 gIl peptone, 5 gIl yeast extract, 1.15 gIl citric acid monohydrate and 6.8 gIL Na2HP04 • 12H20. The culture conditions were 25 ° C and pH 7 initially.
    30 The CB films harvested from the liquid air I interface were immersed in ethanol. Subsequently, they were transferred to deionized water (DI) and boiled for 40 min, four times more with 0.1 M NaOH at 90 e e for 20 min and, finally, neutralized with DI water for 24 h.
    35 Films of bacterial cellulose not previously dried, were cut in the form of rectangular pieces of 1 x 2 cm.
    Drying at room temperature (ROl
    The CB films were kept at room temperature for 4 days until they dried completely. When drying at room temperature (RD), the pressures
    5 water meniscus capillaries exert a compressive force on the pores of the films that can induce the modification of the structure, density and porosity of the eB films.
    Synthesis of silver nanoparticles
    The silver nanoparticles were manufactured by thermal decomposition of AgNOJ in situ under microwave irradiation as follows: AgNOJ and polyvinyl pyrrolidone (PVP) were mixed in the 1: 10 ratio in 4.5 ml of DI water.
    15 Microwave (MW) experiments were carried out using a CEM Discover reactor (12-Hybrid Explorer) operating at a frequency of 2.45 GHz and with a maximum power of 300 W. Conventionally, the power was adjusted automatically to heat the sample to the reaction parameters (temperature and time). The temperature and pressure were controlled by using a sensor
    20 volume-independent infrared.
    The solutions containing AgN03 and PVP were heated at 90 ° C for 2 minutes in the microwave. Then, the solution was automatically cooled to 50 ° C by using compressed nitrogen for approximately 3 min. The 25 cellulose silver (Ag-CB) films were harvested from the solution, cleaned in 10 ml of water and sonicated for 3 min. CB film changed from transparent color - white to brown, indicating the incorporation of AgNP (Ag nanoparticle) into the structure. The suspended nanoparticles were separated by adding 40 ml of acetone with 20 III of PVP (used as electrostatic surfactant) and centrifugation at
    30 6000 rpm for 20 min
    Characterization techniques
    Thermogravimetric analysis fTGA): The TGA of the Ag-cellulose films was performed with a TGA-DSC I DTA (NETZSCH STA 449 F1 Jupiter, ICMAB) analyzer with a heating rate of 10 oC I min from room temperature to 800
    .oc in air. A weight reduction of 96.79% was obtained, which indicated a weight of Ag
    NPs of approx. 0.047mg Since a cellulose band of 1cm2 was used for this analysis, with a weighted weight of 1 mg, it can be estimated that the concentration of AgNPs per unit area of 0.0321mg / cm2 cellulose.
    Scanning electron microscopy (SEM): Samples placed on an SEM aluminum substrate on a carbon tape adhesive allowed imaging with a scanning electron microscope (OUANTA FEI 200 FEG-ESEM) under high vacuum conditions, a Acceleration voltage of 10 to 30 kV, a point of the 10 electron beam of 3.0, a pressure of 2-9 x 10-4 Pa, and a distance of 4-4.5 mm. The homogeneous distribution of AgNPs along the surface of
    the CB.
    Transmission electron microscopy (TEM): TEM images were obtained with
    15 an electronic microscope JEOL JEM-1210, which operates at 120 kV. The average size of the NPs was calculated by adjusting a histogram of the sizes (of at least 200 nanoparticles) to a Gaussian function. The standard deviation (o) is defined as the root of the mean of the squares of the differences of all the observations of the average size, and the polydispersity of the distribution (P) is defined as the percentage
    20 of the standard deviation (o) related to the average size (P = o / (mean · value)) (100). A CB size distribution was obtained: 17.3 ± 8.9 nm (Fig. 1).
    UV-Vis Spectroscopy: Ag-CB films and Ag solutions were analyzed in a range of 300 to 800 nm and a peak was observed around 407-415 nm, indicative of the
    25 presence of nanoparticles. Based on the literature, this peak indicates the presence of 15 nm Ag-NPs.
    EXAMPLE 2. Toxicity test of bacterial cellulose films.
    30 Escherichia coN was grown in LB-agar medium overnight at 37 oC. A colony was collected and incubated in 4 ml of liquid LB-agar medium overnight at 37 oC. 2 ml of the liquid culture were seeded with a spreader in LB-agar medium and incubated overnight at 37 ° C. The following materials / solutions were added:
    35 -Antibiotic: 5 1-11 of a 10 mg I mi solution of gentamicin.
    - Water: 5 fd.
    -Ag-CB: a piece of Ag-CB.-CB: a piece of CB.-Ag nano: 5¡LI of a solution 62.7 mg / ml Ag nanoparticles (15 nm ofdiameter).-Ag nano 1:10: 5) .11 of a 1: 10 dilution of the previous nanoparticle solution ofAg.-AgN03: 5 ~ d of a solution of 635 mg I ml of AgN03.-AgN03 1:10: 5 .LI of a 1: 10 dilution of the AgN03 solution of 635 mg I mi.-AgN03 1: 100: 5 ~ d of a 1: 100 dilution of the AgN03 solution of 635 mg I ml.
    Digital photographs were taken after 24 hours. The results are shown inFig. 2, where it is clearly observed that CS alone is not toxic to cellslive The toxicity experiment consisted of applying on a plate withconfluent growth of Escherichia coli, biomarker bacteria, CB, nanoparticlesof silver (Ag nano), Ag-CB or the compound AgN03. As a positive toxicity controlThe antibiotic gentamicin was used and as a negative water control. Meanwhile heantibiotic, Ag nano or AgN03 are toxic to bacteria - a halo is observedaround the place of application - neither the water nor the CS are, not altering theBacterial growth after application.
    EXAMPLE 3. Plant tissue regeneration assay with cellulose filmsbacterial
    Cuts
    Nicotiana benthamiana plants were planted in alveoli tray duringone week. The seedlings were transferred to individual pots (5 ti) filled withSphagnum peat substrate (Gramoflor) for 3 weeks under conditionsconstants: 23-26 ° C day and 21-22 ° C in darkness, 50-60% humidity and a regimeof darkness-photoperiod of 16 h of light I 8 h of darkness. Two leaves per plantThey were used for the trial.
    The cuts were made with sterile surgical blades number 10 (AlbionSurgicals Limited, Sheffield, England). Particularly a 1 cm cut was made.
    Each cut was immediately covered with a 1 cm long band of the material to be tested:
    -Bacterial cellulose (CB).5-Micropore tape (3M Deutschland GmbH, Neuss, Germany).-No material.
    The obselVation was done daily and photographs were taken 48 hours after making the cuts, with a digital camera or with an LCD camera connected to an Olympus
    10 DP71 binocular (Zeiss, Oberkochen, Germany). The results are shown in Figure 3. The cuts covered with CB show a complete tissue regeneration, sealing the wound. On the other hand, those cuts in which the CB was not applied remained open and unregenerated.
    15 Scanning electron microscopy (SEM): After the curing process, the samples were cut and placed in an SEM aluminum substrate on a carbon tape adhesive. Then, they were metallized using (Au I Pd, 25 mA, 4 '). Subsequently, images were taken with a scanning electron microscope (FEI Quanta 200 FEG-ESEM) under high vacuum conditions or low vacuum conditions. Be
    20 performed an elementary analysis using the SEM microscope in the selected areas. The results are shown in Fig. 5, where it is possible to observe the appearance of new tissue in the area adjacent to the cut in the sample treated with CB.
    Puncture
    25 Nicotiana benthamiana plants were planted in alveoli tray for one week. The seedlings were transferred to individual pots (5 ti) filled with Sphagnum peat substrate (Gramoflor) for 3 weeks under constant conditions: 23-26 ° C day and 21-22 ° C in darkness, 50-60% humidity and A regime
    30 of darkness-photoperiod of 16 h of light I 8 h of darkness. Two leaves per plant were used for the test.
    The punctures were made with 21G x 1112 "needles (Terumo, Shibuya, Japan). Each puncture was immediately covered with a 1 cm long band of the material to be tested:
    -Bacterial cellulose (CS).-Micropore tape (3M Deutschland GmbH, Neuss, Germany).-No material.Scanning electron microscopy (SEM): After the healing process, thesamples were cut and placed in an SEM aluminum substrate on acarbon tape adhesive. Then, they were metallized using (Au! Pd, 25 mA, 4 ').Subsequently, images were taken with a scanning electron microscope (FEIQuanta 200 FEG-ESEM) under high vacuum conditions or low vacuum conditions. Beperformed an elementary analysis using the SEM microscope in the areasselected. The results are shown in Fig. 4, where you can observe theappearance of new tissue in the area adjacent to the puncture in the sample treated withCB.
    Cut and infection
    Nicotiana benthamiana plants were planted in alveoli tray duringone week. The seedlings were transferred to individual pots (5 ") filled withSphagnum peat substrate (Gramoflor) for 3 weeks under conditionsconstants: 23-26 ° C day and 21-22 ° C in darkness, 50-60% humidity and a regimeof darkness-photoperiod of 16 h of light! 8 h of darkness. Two leaves per plantThey were used for the trial.
    The cuts were made with sterile surgical blades number 10 (AlbionSurgicals Limited, Sheffield, England). Particularly a 1 cm cut was made.
    The virulent bacteria Pseudomonas syringae DC3000 was inoculated through the cutusing a syringe with plunger. The contour of the inoculated area was marked with awaterproof marker. Each inoculated cut was immediately covered with a1 cm length band of the material to be tested:
    -Bacterial cellulose (eS).-Bacterial cellulose with Ag nanoparticles (Ag-CB)-Micropore tape (3M Deutschland GmbH, Neuss, Germany) (Tape).-No material.
    The observation was made daily and photographs were taken 48 hours after making the cuts, with a digital camera or with an LCD camera connected to a binocular Olympus DP71 (Zeiss, Oberkochen, Germany). The results are shown in Figure 6. Infected sections covered with Ag-CS show levels of
    5 lower infection, as can be seen by the lower chlorosis in the areainoculated The CS without silver or the micropore tape (Tape) showed no effectAbout the infection
    EXAMPLE 4. Plant tissue regeneration test with vegetable cellulose films.
    Nicotiana benthamiana plants were planted in alveoli tray during
    one week. The seedlings were transferred to individual pots (5 ") filled with
    Sphagnum peat substrate (Gramoflor) for 3 weeks under conditions
    15 constants: 23-26 ° C day and 21-22 ° C in darkness, 50-60% humidity and a dark-photoperiod regime of 16 hours of light I 8 hours of darkness. Two leaves per plant were used for the test.
    The cuts were made with sterile surgical blades number 10 (Albion
    20 Surgicals Limited, Sheffield, England). Particularly a 1 cm cut was made. Each cut was immediately covered with a 1 cm long band of the material to be tested:
    - Vegetable pulp: filter paper (PrimeSource, Morgan Scott Group Inc., USA).
    25-Micropore tape: tape (3M Deutschland GmbH, Neuss, Germany). -CB. -No material
    After 5 days, the micropore tape showed no wound recovery and the filter paper showed wound recovery under a controlled relative humidity of 99% (as shown in Fig. 7).
    However, under low relative humidity (chamber growth, 60%) only the CS remained attached to the leaf and was able to regenerate the wound. The filter paper quickly detached from the wound and regeneration could not occur.
    Therefore, these studies indicate that the percentage of moisture has a great impact on water within the structure and on tissue regeneration. Vegetable cellulose (filter paper) when used under conditions of high relative humidity (high water content) was also able to regenerate wounds in plants (Fig. 7).
    权利要求:
    Claims (16)
    [1]
    1. A method to regenerate damaged plant tissues comprising:5 a. contact the damaged plant tissue with cellulose, and
    b. allow regeneration of damaged plant tissue.
    [2]
    2. The method according to claim 1, wherein the cellulose is plant or bacterial cellulose.
    [3]
    3. The method according to claim 2, wherein the cellulose is bacterial cellulose.
    [4]
    Four. The method according to claim 3, wherein the bacterial cellulose is produced by bacteria of the genus Gluconacetobacter.
    [5]
    5. The method according to claim 4, wherein the bacterium of the genus Gluconacetobacter is selected from among Gluconacetobacter xylinus or Gluconacetobacter europaeus.
    The method according to any one of claims 1 to 5, wherein the cellulose is in the form of a film.
    [7]
    7. The method according to any of claims 1 to 6, wherein the cellulose further comprises an antibacterial, antifungal and / or antiviral agent.
    [8]
    8. The method according to claim 7, wherein the agent is silver and / or copper nanoparticles.
    [9]
    9. The method according to any one of claims 1 to 8, wherein the cellulose is wet.
    [10]
    10. The method according to any of claims 1 to 9, wherein the plant tissue is non-lignified plant tissue.
    The method according to any of claims 1 to 10, wherein the plant tissue is selected from the list consisting of: leaf, fruit, stem, branch, flower or root.
    [12]
    12. The method according to claim 11, wherein the plant tissue is leaves.
    [13]
    13. The method according to any of claims 1 to 12, wherein the plant tissue belongs to a plant of the Solanaceae family.
    [14]
    14. The method according to any of claims 1 to 13, wherein the plant tissue belongs to Nicotiana benthamiana or Solanum Iycopersicum.
    [15]
    fifteen. The method according to any of claims 1 to 14, wherein the damage to the tissue is caused by cutting, abrasion, puncture, pressure, breakage or infection.
    [16]
    16. The method according to claim 15, wherein the infection is bacterial.
    [17]
    17. The method according to claim 16, wherein the bacterial infection is caused by E. coli or Pseudomonas syringae.
    [18]
    18. The method according to any of claims 1 to 17, wherein step (b) takes place over a period of at least 24 hours.
    The method according to claim 18, wherein step (b) takes place over a period of at least 48 hours.
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    同族专利:
    公开号 | 公开日
    WO2017191350A1|2017-11-09|
    ES2645757B1|2018-09-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB8709802D0|1987-04-24|1987-05-28|Haircloth Weaving & Finishing|Sheet material|
    JPH1033069A|1996-07-22|1998-02-10|Nishikawa Rubber Co Ltd|Patch for wound of plant|
    ES2163977B1|1999-06-10|2003-05-01|Garcia Jose An Fernandez-Pello|WATERPROOF-SEALING COMPOUND OF CUTS IN TREES.|
    CN102334514B|2011-07-19|2014-05-07|陕西喜嘉旺生物科技有限责任公司|Tree wound healing protective agent and preparation method thereof|
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