WO2019055022A1 - Systèmes et méthodes d'administration d'acides nucléiques à une plante - Google Patents

Systèmes et méthodes d'administration d'acides nucléiques à une plante Download PDF

Info

Publication number
WO2019055022A1
WO2019055022A1 PCT/US2017/051628 US2017051628W WO2019055022A1 WO 2019055022 A1 WO2019055022 A1 WO 2019055022A1 US 2017051628 W US2017051628 W US 2017051628W WO 2019055022 A1 WO2019055022 A1 WO 2019055022A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
plant
rna
pore
applying
Prior art date
Application number
PCT/US2017/051628
Other languages
English (en)
Inventor
Gregory P. DROUILLARD
Original Assignee
Premier Citrus Apz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Premier Citrus Apz filed Critical Premier Citrus Apz
Priority to PCT/US2017/051628 priority Critical patent/WO2019055022A1/fr
Publication of WO2019055022A1 publication Critical patent/WO2019055022A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • This invention is directed to devices and methods for delivering nucleic acids into a plant using a laser.
  • HLB Huanglongbing
  • citrus greening disease has led to a loss of many citrus trees. Consequently, HLB may be very damaging to the crop yield in large scale operations.
  • RNA interference (hereinafter referred to as "RNAi") to improve the characteristics of a plant.
  • RNAi RNA interference
  • Younis et al. contemplates RNAi as a tool to engineer pathogen resistant plants, insect/pest resistant plants, and improved quality plants.
  • Adnan Younis et al. RNA Interference (RNAi) Induced Gene Silencing: A Promising
  • RNAi for insect-proof plants, Nature Biotechnology 2007, Vol.
  • Nucleic acids may be used to enhance a plant as briefly described above. Some have contemplated delivering RNA into a plant to control pathogen and/or pest control damage.
  • Tang contemplates spraying or brushing the surface of a plant with a double- stranded RNA (hereinafter referred to as "dsRNA") to promote penetration of dsRNA into plant cells.
  • dsRNA double- stranded RNA
  • spraying or brushing is less invasive than other delivery methods, it appears that the uptake of RNA by spraying or brushing may be compromised due to the relatively low permeability of the outer layer of some plants.
  • nucleic acids e.g. , RNAi
  • the present disclosure is directed to an apparatus, systems, and methods in which light energy may be used to enhance uptake of nucleic acids in plants.
  • plant means any type of plant life, including a tree, vines, forage, perennial crops, row crops, bush crops, an ornamental plant, annual plants, and grasses.
  • a light energy may be applied to a surface of a plant to create a pore, and the nucleic acid is applied to the pore. It should be appreciated that a pore may be a rupture, an ablation, a disruption or a minor incision. Thus, genetic activity within the plant may be efficiently and effectively modified.
  • a plant having a disease may be treated.
  • light energy may be applied to a treatment area to create a pore
  • nucleic acid may be applied to the pore in an amount effective to treat a disease in an infected site.
  • Contemplated diseases are typically systemic in nature, which means that a pathogen occurs within plant tissue.
  • surface diseases e.g. , citrus canker
  • the dosage amount of the nucleic acid may be greatly reduced by feeding the nucleic acid to a pore created by the light energy. It should be appreciated that treating a plant does not require a complete cure of the plant from the disease, but may also include a reduction in the disease state or a reduction in a symptom of the disease.
  • the step of applying a light energy includes creating a pore pattern.
  • the pore pattern may be a single pore or a plurality of pores, wherein the plurality of pores may be arranged in a single line, a plurality of lines, a continuously bending line (e.g., swirls, random curves, etc.), a plurality of continuous bending lines, and combinations thereof. It is contemplated that the pore pattern may influence the amount of the first dosage of the nucleic acid that may be required to be effective to induce a therapeutic response. Furthermore, the first pore may have a diameter of approximately 100 to 300 ⁇ .
  • pore depths, widths, lengths, surface areas, and patterns may be used to promote a therapeutic response in a minimally invasive manner. It should be understood that the terms “pore,” “first pore,” “second pore”, or the like may refer to single pore or a plurality or grouping of pores.
  • the step of applying a light energy may be before the step of applying the first dosage.
  • the reverse order may also be performed to treat plant.
  • a predetermined time may also be used to regulate the performance of such steps to ensure effective uptake of the nucleic acid into the plant via the first indentation.
  • the step of applying a light energy and the step of applying a first dosage may be performed in less than an hour, less than 30 minutes, or even less than 1 minute from one another.
  • the infected site may be in a phloem.
  • suitable treatment areas include a surface of at least one of a leaf, a stem, and a bark of the plant.
  • a plant can be a citrus tree having HLB .
  • the citrus tree having HLB has an infected site of the phloem.
  • light energy can be applied to create a first pore or pores on a treatment area, which can include at least one of a leaf, a steam and a bark.
  • a first dosage of a nucleic acid may be applied to the treatment area in an amount effective to induce a therapeutic response (e.g., reduce a symptom of HLB).
  • a therapeutic response e.g., reduce a symptom of HLB.
  • the first pore may comprise a rupture of a cuticle on the leaf to allow introduction of the nucleic acid.
  • the step of applying the light energy to the treatment area may include creating a second pore.
  • the first dosage of the nucleic acid may be beneficially received by the first and second pores.
  • additional pores may be contemplated so long as the amount of pores created is minimally invasive to prevent or reduce damage to the plant.
  • wax may be applied to the treatment area in an amount effective to seal the first pore after the steps of applying the light energy to the treatment area and applying the first dosage of a nucleic acid to the treatment area are complete.
  • an antifungal spray may be applied to the treatment area.
  • the treatment area and the infected site may be in distinct plant systems within the plant.
  • the treatment area may be in the dermal system of the plant (e.g., cuticle, cork) while the infected site may be in the vascular system of the plant (e.g. , phloem).
  • the treatment area and infected site may be distal.
  • distal means situated away from. In some instances, distal may refer to a physical distance between the treatment area and the infected area, for example, a distance of between 0.01 cm to 100 cm, more preferably, between 0.05 cm to 50 cm, and most preferably, between 0.1 cm to 25 cm.
  • distal may refer to a distance between treatment area and infected site in terms of a systemic pathway.
  • the treatment area and the infected site are distal by one to two layers of a plant epidermis or one to two layers of bark tissues.
  • distal could refer to a systemic distance that crosses multiple systems (e.g., dermal system, vascular system, ground system).
  • distal could refer to locations on different branches and/or leaves of a plant.
  • a first nucleic acid may be delivered to a first site in a plant.
  • a light energy may be applied to a second site located on a plant surface.
  • the first site e.g., phloem, xylem
  • the second site e.g., cuticle, cork
  • the first site and the second site may be different plant systems (e.g., dermal system, vascular system) or different plant tissues.
  • a first dosage of the first nucleic acid may be applied to the second site in an amount effective to promote uptake of the first nucleic acid and delivery to the first site via the second pore.
  • the plant may include a tree, row crop, a bush crop, and an ornamental plant. Furthermore, as a precautionary measure, a wax may be applied to the second site in an amount effective to seal the second pore after the steps of applying the light energy to the second site and applying the first dosage of the nucleic acid to the second site.
  • FIG. 1 is a flow diagram of a method of treating a plant having a disease in an infected site of the plant.
  • FIGS. 2A-2F are perspective views of an embodiment showing deliver of the first nucleic acid from a treatment area to an infected site.
  • FIGS. 3A-3F are perspective views of an embodiment showing deliver of a first nucleic acid from a treatment area to an infected site separated by a systemic pathway.
  • FIGS. 4A-4D illustrates various embodiments of pore patterns.
  • FIGS. 5A-5B is a top and perspective view of embodiments for delivering a nucleic acid to a plant.
  • the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
  • the use of “substantially” is also equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.
  • the terms “approximately” and “about” generally refer to a deviance of within 5% of the indicated number or range of numbers. In one embodiment, the term “approximately” and “about”, may refer to a deviance of between 0.01-10% from the indicated number or range of numbers.
  • FIG. 1 shows a schematic of a method 100 for treating a plant having an infected site.
  • the method 100 may comprises a step 102 of applying a light energy to a treatment area on the plant to create a first pore.
  • the treatment area may be distal from the infected site.
  • a first dosage of a nucleic acid may be applied to the treatment area in an amount effective to induce a therapeutic response in the infected site.
  • Suitable therapeutic responses may include, but are not limited to, a complete cure of a disease causing the infected area, a reduction in the infected area, a reduction in a symptom of the disease causing the infected area, a health stimulant, and combinations thereof.
  • step 102 of applying the light energy to the treatment area may occur prior to step 104 of applying the first dosage of the nucleic acid.
  • the reverse order may be performed to treat a plant whereby step 104 occurs prior to step 102.
  • a predetermined time may be used to determine when each step may be completed with respect to one another. For example, step 102 and step 104 may be completed within less than 1 hour, more preferably less than 30 minutes, and most preferably within 1 minute of one another. Therefore, uptake of the nucleic acid via the first indentation may be enhanced by performing steps 102 and 104 within the predetermined time.
  • An additional step 108 of applying a light energy may include creating a pore pattern. Suitable pore patterns may be as simple as one pore or a line of pores. Pore patterns may be more complex and may include a plurality of pores arranged in lines, continuously curved lines, or a combination thereof. It should be appreciated that pore patterns may influence the uptake of the nucleic into the plant by providing different dimensional shapes and areas for absorption.
  • the method 100 may include an additional step 110 of applying the light energy to create a second pore, or any number of additional pores.
  • the method 100 may also include an additional step 106 of applying a wax to the treatment area in an amount effective to seal the first pore and any additional pores.
  • the step of applying the wax may be performed after the steps of applying the light energy to the treatment area and applying the first dosage of a nucleic acid to the treatment area.
  • the first pore may be sealed from the outside environment to prevent harmful contaminants from accessing the plant through the first pore.
  • the wax may reduce the amount of perspiration of the substance to help its absorption into the plant.
  • FIGS. 2A-2F shows a cross section of a plant 200, which has an infected site 206.
  • plant 200 may comprise any type of plant life, including a tree, vines, forage, perennial crops, row crops, bush crops, an ornamental plant, annual plants, and grasses.
  • plant 200 is a citrus tree.
  • Infected site 206 may have an area affected by various known diseases/pathogens. However, it is contemplated that a plant may have a plurality of infected sites. Contemplated diseases are typically systemic in nature, which means that the pathogen occurs within plant tissue. Consequently, the treatment of such diseases is difficult to control using conventional surface treatments.
  • contemplated diseases include HLB, Citrus Tristeza Virus (CTV), Citrus Variegated Chlorosis (CVC), Laurel wilt disease, Fusarium wilt, Phytoplasmas, Zebra chip disease, bacterial kiwifruit vine disease, Chestnut blight, Oak wilt, Fusarium wilt and Pierce's disease.
  • Infected site 206 may be an area in a plant affected by a disease, such as HLB, Citrus Tristeza Virus (CTV), Citrus Variegated Chlorosis (CVC) Laurel wilt disease, Fusarium wilt, Phytoplasmas, Zebra chip disease, bacterial kiwifruit vine disease, Chestnut blight, Oak wilt, Fusarium wilt, and Pierce's disease. While FIG. 2A shows infected site 206 within plant 200, it is contemplated that infected site 206 may reside on the outside surface of plant 200, such as in the case of citrus canker. It should also be noted that while the methods disclosed thus far relate to treating diseased plants, the methods herein may also be applied to healthy plants for preventive care or to promote overall health (e.g., fertilizer application).
  • a disease such as HLB, Citrus Tristeza Virus (CTV), Citrus Variegated Chlorosis (CVC) Laure
  • FIG. 2A shows a light energy 202 may be applied to a treatment area 204 of plant 200.
  • Treatment area 204 is typically on a surface of plant 200. However, it is contemplated that treatment area 204 may also be within plant 200 (i.e. , beyond the outer epidermis).
  • Treatment area 204 may be a region of a plant or a specific organ of a plant (e.g. , leaf, bark, stem, etc.).
  • treatment area 204 may comprise a targeted coverage area of a plant.
  • treatment area 204 may comprise less than 25% of the surface area of the plant (e.g., leaf, stem, trunk, etc.), and more preferably less than 20% of the surface area of a plant.
  • Treatment area 204 is typically distal from infected site 206.
  • treatment area 204 may be a surface of at least one of a leaf, a stem, and a bark, while the infected site is in a phloem of plant 200.
  • treatment area 204 may be in the dermal system of plant 200 while the infected site 206 may be in the vascular system of plant 200. Therefore, it is contemplated that the treatment area and the infected site are in distinct plant systems (e.g. , dermis, vascular, ground).
  • Light energy 202 may be used to create a first pore 208 as shown in Fig. 2B.
  • Co- invented U.S. Pat. Nos. 5,660,747 and 5,897,797, and U.S. Pat. Pub. 2005/0226975 describe various techniques for etching the skin of produce using energy from a C0 2 laser. It is contemplated that the present inventive subject manner may employ some of the techniques in these references, or modifications thereof, in combination with suitable operational parameters, to accomplish the objectives of the present inventive subject matter.
  • light energy 202 may comprise a CO2 laser that may be configured with suitable parameters (e.g.
  • suitable parameters for light energy 202 may include a wavelength having about 10 micrometers (e.g. , 10.6 micrometers) and a power output range between 20-90% at 30-2000 microseconds. It is contemplated that the power output of light energy 202 may range from 10-100 watts. It should be appreciated that the suitable parameters may be adjusted in real-time to accommodate various environmental factors that could affect light energy 202.
  • treatment area 204 may comprise different parts of plant 200 (e.g. , leaf and bark), it is contemplated that different parameters of light energy 202 may be used for different parts of plant 200.
  • light energy 202 applied to a bark to create a pore typically requires more power and dwell than light energy 202 applied to a leaf to create a pore.
  • Suitable power settings for applying light energy 202 to a bark may be in the range of 80-90% power at a dwell of 1200-2000 microseconds depending on the age of plant 200.
  • grasses would typically require less power in the range of 20-50% power at a dwell of 30- 120 microseconds.
  • Jump speeds may range from 1 to 3000mm/s, and more typically between 1000-2000 mm/s.
  • Marking speeds may range from 100 to 3000 mm/s, and more typically from 1000-2000 mm/s.
  • Marking intensity may range from 20- 100%, and more typically between 30-70%.
  • Pulse Frequency may range from 15 to 20000 Hz, and more typically between 1000-20000 Hz. It should be appreciated that the jump speeds, marking speeds, marking intensities, and pulse frequencies may be applied to all areas of plant 200, including the leaves, branches, stems, bark.
  • light energy 202 may be generated using a 400mm output lens, which provides a scan window of approximately 14 inches by 14 inches without changing the beam characteristics and energy.
  • the scan window is the area that may be lased by light energy 202.
  • output lens provides an optimum focal distance of 15 inches and an effective depth of focus of 9 inches.
  • the focal point and depth of focus may be changed based on the size of the output lens used.
  • a 200mm lens provides a focal point of 7 inches and a depth of focus of 4 inches
  • a 800 mm lens provides a focal point of 23 inches and a depth of focus of 15 inches.
  • a "scribing" laser or continuous wave laser is contemplated, which may be air cooled and designed for the outdoor environment.
  • a galvanometer may be used to control a scribing path of the light energy 102.
  • the galvanometer is placed behind the laser output lens, such that light energy 102 travels to the galvanometer before the laser output lens.
  • more than one galvanometer may be used to control the direction of light energy 102 in two directions (e.g. , x and y directions).
  • suitable parameters for light energy 202 may vary depending on environmental factors, among other things.
  • a control system e.g. , a feedback loop
  • the sensors may be used to detect various parameters affecting the application of light energy 202, such as the different parts of plant 200 (e.g., bark vs. leaf), weather, age of plant 200, depth and size of first pore 208, and the distance from the light energy source and treatment area 204.
  • the control system may adjust light energy 202 to create a pore.
  • a sensor may detect that treatment area 204 is part of the bark of plant 200 and adjust the power settings of light energy 202 to create a pore, and at a subsequent point in time the sensor may detect that treatment area 204 is on the surface of grass and adjust the power settings by reducing power of light energy 202 to create a pore.
  • light energy 202 may have a default setting (e.g., power setting to create a pore on a leaf of plant 200) and, upon sensing another part (e.g. , stem, bark) of plant 100, control system may adjust light energy 202 to create a pore before returning to the default setting.
  • a sensor may be integrated within light energy 202 to determine whether first pore 208 has reached a pre-determined tissue layer.
  • light energy 202 may be applied having the light energy source in contact with treatment area 204.
  • light energy 202 may be applied having the light energy source at a distance from treatment area 204.
  • the light energy source may be between 4 to 14 inches (e.g. , 7 inches) from treatment area 204.
  • light energy 202 may comprise a C0 2 laser and treatment area 204 may be on a surface of a leaf whereby the distance between the CO2 laser and the leaf is between 4 to 14 inches, and more preferably, 5 to 8 inches.
  • First pore 208 may be removed or disrupted portion of treatment area 204.
  • first pore 208 may comprise an opening through the treatment area 104.
  • first pore 208 may extend through treatment area 204 to expose a surface underneath treatment area 204.
  • first pore 208 may be a rupture, ablation, or disruption of a cuticle on the leaf.
  • typical diameters for first pore 208 may be approximately between 100 and 300 ⁇ , and typically have a depth of approximately 50 ⁇ . It should be noted that first pore 208 may have a greater or lesser depth, but the depth should generally not exceed 75 ⁇ as it may destroy too many cells.
  • first pore 208 may extend through treatment area 204, it is also contemplated that first pore 208 may extend partially through treatment area 204. In other words, first pore 208 need not be a complete breach through treatment area 204 (e.g., the depth of first pore 208 does not extend into the entire thickness of the cuticle). On the other hand, it is also contemplated that first pore 208 may extend through various layers past treatment area 204. Preferably, first pore 208 may be configured to achieve maximum uptake of the nucleic acid while minimizing harm to the plant.
  • a first dosage of a nucleic acid 310 may be applied to treatment area 204 as shown in FIG. 2C.
  • Nucleic acid 210 typically promotes the overall health or treatment of plant 200. It is contemplated that nucleic acid 210 may comprise RNA. Suitable RNA for nucleic acid 210 may include at least one of a RNA interference (RNAi), an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a double- stranded RNA (dsRNA), a non-coding RNA (ncRNA), and mitochondrial RNA.
  • RNAi RNA interference
  • asRNA antisense RNA
  • miRNA micro RNA
  • siRNA small interfering RNA
  • dsRNA double- stranded RNA
  • ncRNA non-coding RNA
  • nucleic acid 210 may be RNAi that at least one of (i) modifies a gene expression to increase resistance to a pathogen in the plant (e.g., interfere with the metabolism or development process, affect the development of an insect (e.g., citrus psyllid) that feeds on the plant (wings, gut, offspring, etc.)), (ii) suppresses a virus-based vector in the plant, and (iii) modifies a gene expression to obtain a favorable trait in the plant (e.g. , color, flavor, reduced degradation, reduced abiotic stress, etc.).
  • RNAi may be applied via foliar application with enhanced uptake due to the pores created by the light energy.
  • nucleic acid 210 may comprises at least one of DNA and a nucleic acid analogue. Nucleic acid 210 may also comprise a tag for detection to determine whether there was uptake of nucleic acid 210 into plant 200. Moreover, nucleic acid 210 may also comprise a pharmaceutical tag to enhance or complement the action of the nucleic acid 210. Nucleic acid 210 may have any number of base or base pairs. For example, nucleic acid 110 may have 1-10, 8-20, 12-30, 25-50, 40-100, 100- 1,000, 1,000- 10,000, and 10,000-1,000,000 base pairs.
  • Nucleic acid 210 may have various suitable structures and characteristics.
  • nucleic acid 210 may be single or double stranded and circular or linear.
  • Nucleic acid 210 may be coding or noncoding (e.g., noncoding DNA, noncoding RNA). Where protein is encoded by nucleic acid 210, it is contemplated that such proteins are under the control of the regulatory mechanisms specific to plant 210 or a disease or pathogen or to neither.
  • nucleic acid 210 may comprise at least one of yeast artificial chromosomes (YACs) and a phagemid plasmid.
  • Nucleic acid 210 may be with or without sequences that support replication. It is also contemplated that the sequences may be from non-plants (e.g. , animal, bacteria, yeast, fungi, etc.). The sequences may also be chimeric (e.g., part animal and part plant, part plant and part bacteria, etc.).
  • First dosage of nucleic acid 210 will depend on the type of nucleic acid 210 being applied.
  • nucleic acid 210 may have a concentration of 0.1- 1 pmol, 1- 10 pmol, 10 pmol - 1 nmol, 1- 10 nmol, 10 nmol - 1 ⁇ , 1- 10 ⁇ , 10 ⁇ - 1 mmol, and even higher.
  • first dosage comprises 0.1-1 pmol/ml, 1- 10 pmol/ml, 10 pmol/ml - 1 nmol/ml, 1- 10 nmol/ml, 10 nmol/ml - 1 ⁇ /ml, 1-10 ⁇ mol/ml, 10 ⁇ /ml - 1 mmol/ml, and even higher.
  • First dosage of nucleic acid 210 may be buffered or non-buffered solution.
  • First dosage of nucleic acid 210 may comprise additional components. It is contemplated that first dosage of nucleic acid 210 may comprise at least one of stabilizers, surfactants, and a nucleic acid degradation inhibitor (e.g., DNAse inhibitor, RNAse inhibitor). Thus, first dosage of nucleic acid 210 may be co-administered with other compounds. It is contemplated that first dosage of nucleic acid 210 may comprise counter ions if, for example, at neutral pH.
  • a nucleic acid degradation inhibitor e.g., DNAse inhibitor, RNAse inhibitor
  • Nucleic acid 210 may be applied to first pore 208 using various methods, including spraying, dusting, sprinkling, brushing, smearing and drenching. Application of the first dosage may also be accomplished by introducing nucleic acid 210 into an irrigation system (e.g. , sprinkler system) that delivers water to plant 200. It should be appreciated that first dosage of nucleic acid 210 may comprise a surfactant to increase absorption of nucleic acid 210 in plant 200. Using these application techniques, at least some of nucleic acid 210 enters first pore 208. It is also contemplated that a more targeted application technique may be used to focus the application of nucleic acid 210 onto the area of first pore 208.
  • an irrigation system e.g. , sprinkler system
  • nucleic acid 210 may produce a harmful response to plant 200 to thereby kill and eliminate plant 200.
  • the methods and devices disclosed herein may be used to selectively remove certain plants from a crop, such as to remove weeds, infected plants, older plants, etc.
  • first pore 208 may be configured to optimize uptake without concern of being minimally invasive.
  • nucleic acid 210 is packaged into a variety of lipid soluble nano-particles. These nano-particles should provide for higher retention rates and for improved cuticle penetration. When combined with the use of light energy to create a pore for nucleic acid 210, penetration of nano-particles (or nucleic acids in solution) may be exceptionally enhanced.
  • nucleic acid 210 is better absorbed through first pore 208 as shown in FIG. 2D.
  • nucleic acid 210 within first pore 208 has a more direct path into plant 200 as opposed to nucleic acid 210 that is on the outer layer (i.e., outer surface of treatment area 204) of plant 200.
  • the first dosage of nucleic acid 210 applied to treatment area 204 is in an amount effective to induce a response (e.g., therapeutic response, biological response) in infected site 206.
  • Plant 200 may respond in various manners.
  • plant 200 may have a therapeutic response or biological that comprises a reduction in the infected site 206 as shown in FIG. 2E.
  • the therapeutic response may comprise at least one of reducing a symptom of a pathogen, reducing a cause of the disease, and completely curing a disease.
  • nucleic acid 210 may be RNAi and the therapeutic response comprises at least one of (i) modifying a gene expression to increase resistance to a pathogen in plant 200 (e.g.
  • nucleic acid 210 may affect (i) one or more components of plant 200, (ii) one or more components of a symbiotic organism, and (iii) one or more components of a pathogen.
  • a wax 212 may be applied to treatment area 204 as shown in FIG. 2F. It should be appreciated that wax 212 has several benefits, including preventing pathogen or other harmful contaminants from entering first pore 208, reducing water loss, and reducing perspiration of nucleic acid 210 from first pore 208. Thus, water loss will be mainly localized and should not affect the overall tree water relations.
  • FIGS. 3A-3F shows a cross section of a plant 300, which has an infected site 306 that is distally located from a treatment area 304 across a systemic pathway 305.
  • treatment area 304 could be located on a first leaf and infected site 306 could be located on a second leaf, wherein the first leaf and separate leaf are located on different stems and/or branches.
  • Light energy 302 may be applied to treatment area 304 to create first pore 308 as shown in FIGS. 3A-3B.
  • a first dosage of nucleic acid 310 may be applied to treatment area 304 as shown in FIG. 3C.
  • nucleic acid 310 may be applied using various application methods and may further comprise a surfactant to increase absorption.
  • Nucleic acid 310 may absorbed by plant 300 through first pore 308 as shown in FIG. 3D. While nucleic acid 310 has been introduced within plant 300, it should be noted that in some circumstances the infected site 306 is distal from treatment area 304, such that nucleic acid 310 is not immediately in contact with infected site 306 and is separated by a systemic pathway. Thus, first dosage of nucleic acid 310 and first pore 308 may be configured (e.g.
  • the quantity of first dosage, number of applications of dosage, size and depth of first pore 308, percentage of coverage of first pore 308, etc. to allow for sufficient uptake and delivery of the nucleic acid 310 to the infected site 306 to achieve a therapeutic response at the infected site 306.
  • nucleic acid 310 may travel through a systemic pathway 305 to arrive at infected site 306 as shown in FIG. 3E. While nucleic acid 310 is shown to be present in the area immediately adjacent to first pore 308, it is contemplated that nucleic acid 310 does not have an effect (e.g., therapeutic or biological response) until it travels through systemic pathway 305. FIG. 3F shows first pore 308 and nucleic acid 310 may be sufficient to induce a therapeutic response, such as reducing the area of infected site 306. However, it is also contemplated that nucleic acid 310 remains localized in the area adjacent to treatment area 304 (e.g. , within the same leaf, or within the leaves on a single branch, etc.).
  • nucleic acid 310 may be absorbed by at least one of the phloem and the xylem in plant 300.
  • nucleic acid 310 may be localized in the phloem adjacent to treatment area 304 and not travel to the roots.
  • nucleic acid 310 may be absorbed by the phloem and/or xylem to thereby travel through the vascular system of plant 300.
  • nucleic acid 310 may generate an effect throughout 1-5%, 5- 15%, 15-35%, 35-60%, 60-90%, and 90-100% of the plant within 1 hour, 2 hours, 5 hours, 10 hours, 18 hours, 1 day, or 2 days.
  • a treatment area may have more than one pore and may comprise a pore pattern.
  • FIGS. 4A-4D show a treatment area 404 that may have various pore patterns 403a- d.
  • pattern 403a is a single solid line
  • pattern 403b comprises multiple solid lines
  • pattern 404c comprises a plurality of circular dots equally distanced from one another
  • pattern 404d comprises a plurality of randomly spaced dots.
  • pattern 403a is a single solid line
  • pattern 403b comprises multiple solid lines
  • pattern 404c comprises a plurality of circular dots equally distanced from one another
  • pattern 404d comprises a plurality of randomly spaced dots.
  • more than 25% of the surface area of the plant may comprise pores, but it is generally recommended that an additional step of waxing is applied on the pores to protect the plant from harmful substances.
  • a treatment area may span to various parts of a plant.
  • treatment area 404 may encompass the leaves, stems and the trunk of a plant.
  • treatment area 404 may be limited to just one region or part of the plant (e.g. , leaves, stems, bark, roots, etc.).
  • light energy may be applied to a first site and a second site on a plant that are different in kind (e.g. , different tissues, cells, organs, systems of plant, distal).
  • the pores may vary in size and shape to account for differences in the absorption rate between various parts of the treatment area (e.g., the bark and the cuticle). For example, if it is determined that nucleic acids on a pore on the bark are absorbed slower than nucleic acids on a pore on the cuticle, then the pore on the bark may be larger to compensate for the slower absorption rate. Moreover, pores may be created to promote capillary action of the nucleic acids.
  • light energy may be used to create a first pore pattern.
  • a first dosage of nucleic acid may be applied to the first pore pattern and a second dosage of nucleic acid may be applied to the first pore pattern after the first dosage is applied.
  • a first dosage of the first nucleic acid may be applied, then after a pre-determined time period, a second dosage of the first nucleic acid may be applied.
  • a first dosage of the first nucleic acid may be applied, and then a first dosage of a second nucleic acid may be applied after a predetermined time period.
  • the second nucleic acid may be any of the various nucleic acids described above. Additionally, the second substance may further include a booster or a catalyst for the first nucleic acid to further activate the first nucleic acid. It is contemplated that the second nucleic acid may produce a second biological response, wherein the second biological response may be connected to the first biological response or distinct from the first biological response. It is contemplated that the first and second dosage amounts are equal. In other embodiments, the first and second dosage amounts vary depending on the effective dosage amount required to produce a biological response.
  • nucleic acids may be effectively delivered to a plant using light energy.
  • the nucleic acids may be effective to interact with pathogens in the plant.
  • effective delivery of nucleic acids may also interact with plant metabolism. Therefore, the delivery methods described herein may also be used to regulate genes within the plant. Due to the ability to modify genetic activity of the plant, nucleic acids applied to the plant may interact with nucleic acids that are normally found in the plant or with nucleic acids that are not normally found in a plant (e.g., transgenes). It is contemplated that nucleic acids may be delivered to a plant to activate or deactivate transgenes, which may include nucleic acids previously introduced into the plant.
  • FIGS. 5A-5B show an embodiment of an apparatus 500 configured for delivering at least one nucleic acid to a plant 502.
  • the apparatus may be mounted on a movable platform 504 for ease of use.
  • the basic unit may comprise an electronically controlled robotic arm 506 containing: (i) a laser light emitter 508 to disperse a light to a treatment area or second site on a plant 502; (ii) a nucleic acid nozzle applicator 510; and (iii) a wax applicator 512 to seal the treated area as shown in FIG. 5A.
  • the laser light emitter 508, nucleic acid nozzle applicator 510, and wax applicator 512 may be nozzles.
  • All these nozzles may be mounted at the end of the arm on a movable bracket.
  • the laser light emitter 508 may emit light energy to create a momentary breach in the treatment area or second site on the plant 502
  • the nucleic acid nozzle applicator 510 may deliver the nucleic acid over the treatment area or second site on the plant 502
  • the wax applicator 512 may reseal the treatment area or second site on the plant as the system moves forward.
  • the system design may reduce the amount of substance needed by applying it only over the treatment area or second site on the plant, and not over the entire canopy/area and also ensures that the pores may be sealed to protect against water loss and other pathogens.
  • the apparatus 500 may further comprise five individual arms 520, 522, 524, 526, 528 as shown in FIG. 5B, each independently controlled and electronically actuated. These arms may be stacked vertically, with each laser having a scan window of approximately 14"xl4". This will give a maximum laser treated height of approximately 6 ft.
  • the design of the system may be modular so that more laser arms may be added vertically to accommodate larger trees if needed.
  • the entire apparatus 500 may be mounted on the movable platform 504 which will be pulled through the grove by a tractor 540 at 2.3-2.7 mph. As the apparatus moves down the grove, optics will guide each arm to determined positions on the canopy, avoiding entanglement with branches of uneven length.
  • Each independently controlled arm 520, 522, 524, 526, 528, with the laser system and attached applicator nozzle assembly may be designed to typically move in the horizontal direction. However, each laser system and assembly arm may move independently from the other arms. This independent scheme may allow each laser system to follow the contour of the tree canopy in its scan path. Multiple sensors on the articulating arm and laser system may ensure that it traces the tree canopy. The entire laser system may also be raised or lowered vertically depending on tree height. In addition, because of tree canopy irregularities and differing tree height, sensors on the laser may turn it "off and on" based on the presence of canopy to be treated within its scan path.
  • the laser system and applicator nozzles may be attached on the arm using a rotating mount (not shown). This rotating mount may allow the lasers to be pointed at an angle to the tree canopy to better laser the adaxial (top) of the leaves.
  • the platform 504, on which the entire laser system is mounted on may have an isolation design so that it will eliminate (as much as possible) the transfer of motion from the platform 504 to the apparatus 500 as it moves through the grove.
  • the horizontal articulating robotic arms may also have isolation mounts 530 that are designed to minimize any further vibrations from affecting the operation of the laser system in the field.
  • a generator 532 and tanks with pumps 534 may be mounted on the platform 504, as shown in FIG. 5A.
  • the generator 532 provides sufficient power for the laser systems, pumps and associated electronic equipment.
  • the tanks provide the storage for at least one of the substance and wax.
  • the two pumps deliver the substance and wax to the applicator nozzles.
  • the overall system design preferably takes into account environmental working conditions, heat, humidity, rain and varying grove conditions.
  • RNAi may be introduced into a plant by administering RNAi directly to the phloem through pores created by a laser.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne une méthode d'administration d'au moins un acide nucléique à une plante, comprenant : l'application d'une énergie lumineuse à une surface de la plante pour créer un pore; et l'application d'au moins un acide nucléique au pore, ledit ou lesdits acides nucléiques comprenant de l'ARN, ledit ou lesdits acides nucléiques comprenant au moins une interférence ARN (ARNi), un ARN antisens (ARNas), un micro-ARN (miARN), un petit ARN interférent (ARNsi), un ARN double brin (ARNdb), un ARN non codant (ARNnc), un ARN mitochondrial (ADNmt), et des combinaisons de ceux-ci.
PCT/US2017/051628 2017-09-14 2017-09-14 Systèmes et méthodes d'administration d'acides nucléiques à une plante WO2019055022A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2017/051628 WO2019055022A1 (fr) 2017-09-14 2017-09-14 Systèmes et méthodes d'administration d'acides nucléiques à une plante

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2017/051628 WO2019055022A1 (fr) 2017-09-14 2017-09-14 Systèmes et méthodes d'administration d'acides nucléiques à une plante

Publications (1)

Publication Number Publication Date
WO2019055022A1 true WO2019055022A1 (fr) 2019-03-21

Family

ID=65723814

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/051628 WO2019055022A1 (fr) 2017-09-14 2017-09-14 Systèmes et méthodes d'administration d'acides nucléiques à une plante

Country Status (1)

Country Link
WO (1) WO2019055022A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802164A (zh) * 2023-12-29 2024-04-02 华中农业大学 基于植物内原核生物基因沉默系统的黄龙病防治方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7758561B2 (en) * 1997-12-30 2010-07-20 Altea Therapeutics Corporation Microporation of tissue for delivery of bioactive agents
US20130052738A1 (en) * 2011-08-30 2013-02-28 General Electric Company Optical based delivery of exogenous molecules to cells
US20140303546A1 (en) * 2011-06-24 2014-10-09 University Of Miami Laser assisted delivery of functional cells, peptides and nucleotides
US9265260B1 (en) * 2014-04-07 2016-02-23 Gpd Technologies Llc Systems and methods for using light energy to facilitate penetration of substances in plants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7758561B2 (en) * 1997-12-30 2010-07-20 Altea Therapeutics Corporation Microporation of tissue for delivery of bioactive agents
US20140303546A1 (en) * 2011-06-24 2014-10-09 University Of Miami Laser assisted delivery of functional cells, peptides and nucleotides
US20130052738A1 (en) * 2011-08-30 2013-02-28 General Electric Company Optical based delivery of exogenous molecules to cells
US9265260B1 (en) * 2014-04-07 2016-02-23 Gpd Technologies Llc Systems and methods for using light energy to facilitate penetration of substances in plants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BADR, Y. A. ET AL.: "Production of fertile transgenic wheat plants by laser micropuncture", PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, vol. 4, 2005, pages 803 - 807, XP055584946 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802164A (zh) * 2023-12-29 2024-04-02 华中农业大学 基于植物内原核生物基因沉默系统的黄龙病防治方法

Similar Documents

Publication Publication Date Title
US10219443B2 (en) Systems and methods for using light energy to facilitate penetration of substances in plants
US11191278B2 (en) Systems and methods for delivering nucleic acids to a plant
US11178823B2 (en) Systems and methods for using light energy to facilitate penetration of substances in plants
Andreasen et al. Laser weeding with small autonomous vehicles: Friends or foes?
US20230048111A1 (en) Apparatus for Applying Contact Resistance-Reducing Media and Applying Current to Plants
US20160174475A1 (en) Indiscriminately On-the-GO Tree Heat Treatment Apparatus and its Effective Methods
US10278334B2 (en) Systems and methods for delivering nucleic acids to a plant
AU2014290850A1 (en) Improvements in, or relating to, plant maintenance
Meadows NEWS OVERVIEW: Researchers develop alternatives to methyl bromide fumigation
Li et al. Precision trunk injection technology for treatment of huanglongbing (HLB)-affected citrus trees—A review
WO2019055022A1 (fr) Systèmes et méthodes d'administration d'acides nucléiques à une plante
BR102020024021A2 (pt) Dispositivo para combater organismos indesejáveis
EP3386289B1 (fr) Procédé pour utiliser l'énergie lumineuse pour faciliter la pénétration de substances dans des plantes
US11116144B2 (en) Plant treatment system and method
US8365464B1 (en) Plant eradication using unnatural mechanical and thermal trauma
CN103109712A (zh) 一种林间减少松褐天牛密度和增加花绒寄甲密度的方法
US20200037607A1 (en) Materials and Methods for Delivering a Substance to a Plant
CN107006270A (zh) 一种金叶榆嫁接及培育方法
Egorov et al. Control of Sosnovsky’s hogweed (Heracleum sosnowskyi Manden.) in forests using herbicides
WO2024040293A1 (fr) Dispositif et procédé de lutte contre les mauvaises herbes
Sunitha Studies on management of grape stem borer Celosterna scabrator Fab.(Cerambycidae: Coleoptera)
TH1801003382A (th) ระบบและวิธีการสำหรับการใช้พลังงานแสงเพื่อทำให้การซึมผ่านของสารในพืชง่ายขึ้น
RU2567367C2 (ru) Способ уничтожения пней от деревьев на приусадебном участке
Gilrein Late Summer Pests at the Entomology Diagnostic Lab
Wilson Technical aspects of weed management in Australian rangelands.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17925093

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17925093

Country of ref document: EP

Kind code of ref document: A1