Method for Improving Shelf Life by Regulating Expression of Polyphenol Oxidase
The present invention provides novel compositions for use to enhance crop performance. Specifically, the present invention provides for improved shelf life and reduced postharvest losses enhancement in various crops. The present invention also provides for combinations of compositions and methods that provide for improved shelf life and reduced postharvest losses.
This application claims the benefit of U.S. Provisional Patent Application No. 61/704,602, filed on Sep. 24, 2012, which is incorporated herein by reference in its entirety.
INCORPORATION OF SEQUENCE LISTINGSA sequence listing containing the file named “40—77—58676_PCT.txt”, which is 204,990 bytes (measured in MS-Windows®), contains 348 sequences, and was created on Sep. 24, 2013, is provided herewith via the USPTO's EFS system, and is incorporated herein by reference in its entirety. The sequence listing contained in the file “40—75 (58676)B.txt” (file size of 172,076 bytes (measured in MS-Windows®)) comprising SEQ ID NO:1-303 and filed with U.S. Provisional Patent Application No. 61/704,602 on Sep. 24, 2012 is also incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTIONPolyphenol oxidases (PPOs) are a group of copper-binding proteins, widely distributed phylogenetically from bacteria to mammals, that catalyze the oxidation of phenolics to quinones which produce brown pigments in wounded tissues. PPO has been implicated in the formation of pigments, oxygen scavanging and defense mechanism against plant pathogens and herbivorous insects. The oxidation of phenolic substrates by PPO is thought to be the major cause of browning coloration of many fruits and vegetables during ripening, handling, storage and processing. This problem is of considerable importance to the food industry as it affects the nutritional quality and appearance, reduces the consumer's acceptability and can therefore also cause significant economic impact, both to the food producers and to the food processing industry.
SUMMARY OF THE INVENTIONProvided herein are compositions and methods that provide for improved shelf life and reduced postharvest losses that results from suppression of Polyphenol oxidase (PPO) expression. In certain embodiments, improved fruit and/or vegetable shelf life and reduced postharvest losses that results from suppression of Polyphenol oxidase (PPO) expression is obtained by topical applications of compositions comprising polynucleotides and transfer agents to plants. For the purpose of improved fruit and/or vegetable shelf life and reduced postharvest losses, the ability to control the timing of application so that Polyphenol oxidase (PPO) genes are suppressed only during desired times or conditions makes the compositions and methods provided herein particularly useful. Many crops will derive benefit from this method, including but not limited to, corn, wheat, rice, soybean, cotton, Canola, tomato, alfalfa, melon, lettuce, cucumber and broccoli.
Polynucleotides that can be used to suppress Polyphenol oxidase (PPO) include, but are not limited to, any of: i) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of the gene of SEQ ID NO:1-23; ii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of 24-295; or, iii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of SEQ ID NO:296-303. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a transcript of the Polyphenol oxidase (PPO) gene of SEQ ID NOs: 324-334. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 304-307. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 318-323. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 310-317. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 335-348. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 304-307. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 310-317. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 318-323. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 335-348. In some embodiments, the polynucleotides used to suppress PPO are antisense single stranded DNA (ssDNA). In some embodiments, the polynucleotides used to suppress PPO are single-stranded RNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded RNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded DNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded DNA/RNA hybrids.
In an aspect of the invention, the polynucleotide molecules are provided in compositions that can permeate or be absorbed into living plant tissue to initiate systemic gene inhibition or regulation. In certain embodiments of the invention, the polynucleotide molecules ultimately provide to a plant, or allow the in planta production of, RNA that is capable of hybridizing under physiological conditions in a plant cell to RNA transcribed from a target endogenous PPO gene or target PPO transgene in the plant cell, thereby effecting regulation of the target gene. In some embodiments, the target endogenous PPO gene is PPO 9, 10, or 11. In other embodiments of the invention, the polynucleotide molecules disclosed herein are useful for ultimately providing to a plant, or allowing the in planta production of, RNA that is capable of hybridizing under physiological conditions to RNA transcribed from a PPO target gene of the plant, thereby effecting regulation of the target gene. In certain embodiments, regulation of the target genes, such as by silencing or suppression of the target gene, leads to the upregulation of another gene that is itself affected or regulated by the target gene's expression.
In certain aspects or embodiments of the invention, the topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant or plant part according to the methods described herein does not necessarily result in nor require the exogenous polynucleotide's integration into a chromosome of the plant. In certain aspects or embodiments of the invention, the topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant or plant part according to the methods described herein does not necessarily result in nor require transcription of the exogenous polynucleotide from DNA integrated into a chromosome of the plant. In certain embodiments, topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant according to the methods described herein also does not require that the exogenous polynucleotide be physically bound to a particle, such as in biolistic mediated introduction of polynucleotides associated with gold or tungsten particles into internal portions of a plant, plant part, or plant cell. An exogenous polynucleotide used in certain methods and compositions provided herein can optionally be associated with an operably linked promoter sequence in certain embodiments of the methods provided herein. However, in other embodiments, an exogenous polynucleotide used in certain methods and compositions provided herein is not associated with an operably linked promoter sequence. Also, in certain embodiments, an exogenous polynucleotide used in certain methods and compositions provided herein is not operably linked to a viral vector.
In certain embodiments, methods for improved shelf life and reduced postharvest losses in a plant comprising topically applying compositions comprising a polynucleotide and a transfer agent that suppress the target PPO gene are provided. In some embodiments, methods for reducing or inhibiting discoloration of a plant or plant part following harvest comprising topically applying compositions comprising a polynucleotide and a transfer agent that suppress the target PPO gene are provided. In certain embodiments, methods for selectively suppressing the target PPO gene by topically applying the polynucleotide composition to a plant surface at one or more selected seed, vegetative, or reproductive stage(s) of plant growth are provided. In some embodiments, the target PPO gene is PPO 9, 10 or 11. Such methods can provide for PPO gene suppression in a plant or plant part on an as needed or as desired basis. In certain embodiments, methods for selectively suppressing the target PPO gene by topically applying the polynucleotide composition to a plant surface at one or more pre-determined seed, vegetative, or reproductive stage(s) of plant growth are also provided. Such methods can provide for PPO gene suppression in a plant or plant part that obviates any undesired or unnecessary effects of suppressing the PPO genes expression at certain seed, vegetative, or reproductive stage(s) of plant development. In some embodiments, the polynucleotide composition is applied to a plant surface prior to harvest. In some embodiments, the polynucleotide composition is applied to a plant surface after harvest.
In certain embodiments, methods for selectively improving shelf life and reducing postharvest losses in a plant by topically applying the polynucleotide composition to the plant surface at one or more selected seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for improved shelf life and reduced postharvest losses in a plant or plant part on an as needed or as desired basis. In certain embodiments, methods for selectively improved shelf life and reduced postharvest losses in a plant by topically applying the polynucleotide composition to the plant surface at one or more predetermined seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for the improved shelf life and reduced postharvest losses in a plant or plant part that obviates any undesired or unnecessary effects of providing the improved shelf life and reduced postharvest losses at certain seed, vegetative, or reproductive stage(s) of plant development.
In certain embodiments, methods for reducing or delaying discoloration of a plant or plant part following harvest by topically applying the polynucleotide composition to a plant surface at one or more selected seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for reducing or delaying discoloration in a plant or plant part on an as needed or as desired basis. In certain embodiments, methods for reducing or delaying discoloration of a plant or plant part by topically applying the polynucleotide composition to the plant surface at one or more predetermined seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for the reduced or delayed discoloration in a plant or plant part that obviates any undesired or unnecessary effects of providing for the reduced or delayed discoloration at certain seed, vegetative, or reproductive stage(s) of plant development. In some embodiments, the polynucleotide composition is applied before harvest to reduce or delay discoloration. In some embodiments, the polynucleotide composition is applied after harvest to reduce or delay discoloration.
Polynucleotides that can be used to suppress a Polyphenol oxidase (PPO) gene include, but are not limited to, any of: i) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to Polyphenol oxidase (PPO) gene or to a transcript of the gene of SEQ ID NO:1-23; ii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of SEQ ID NO:24-295; or, iii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of SEQ ID NO:296-303. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene transcript of SEQ ID NOs: 324-334. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 304-307. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 318-323. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 310-317. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 335-348. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 304-307. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 310-317. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 318-323. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 335-348. In some embodiments, the polynucleotides used to suppress PPO are antisense single stranded DNA (ssDNA). In some embodiments, the polynucleotides used to suppress PPO are single-stranded RNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded RNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded DNA. In some embodiments, the polynucleotides used to suppress PPO are double-stranded DNA/RNA hybrids.
Certain embodiments of the invention are directed to methods for producing a plant exhibiting improved shelf life and reduced postharvest losses comprising topically applying to a plant surface a composition that comprises:
a. at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of said gene; and
b. a transfer agent, wherein said plant exhibits an improvement in improved shelf life and reduced postharvest losses that results from suppression of said Polyphenol oxidase (PPO) gene.
Certain embodiments are directed to methods for producing a plant exhibiting reduced or delayed discoloration following harvest comprising topically applying to a plant surface a composition that comprises:
a. at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of said gene; and
b. a transfer agent, wherein said plant exhibits reduced or delayed discoloration that results from suppression of said Polyphenol oxidase (PPO) gene.
In certain embodiments of the methods, the polynucleotide molecule comprises sense ssDNA, sense ssRNA, dsRNA, dsDNA, a double stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23. In certain embodiments of the methods, the plant is a potato, apple, spinach, or lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO:24-302, and 303, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene transcript of SEQ ID NOs: 324-334. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 304-307. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 318-323. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 310-317. In some embodiments, polynucleotides that can be used to suppress PPO include polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NOs: 335-348. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 304-307. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 310-317. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 318-323. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO: 335-348. In certain embodiments of the methods, the composition comprises any combination of two or more polynucleotide molecules. In certain embodiments of the methods, the polynucleotide is at least 18 to about 24, about 25 to about 50, about 51 to about 100, about 101 to about 300, about 301 to about 500, or at least about 500 or more residues in length. In certain embodiments of the methods, the composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof. In certain embodiments of the methods, the composition further comprises a non-polynucleotide herbicidal molecule and said plant is resistant to said herbicidal molecule. In certain embodiments of the methods, the transfer agent comprises an organosilicone preparation. In certain embodiments of the methods, the polynucleotide is not operably linked to a viral vector. In certain embodiments of the methods, the polynucleotide is not integrated into the plant chromosome. Further embodiments of the invention are directed to: a plant made according to the above-described method that exhibits improved shelf life and reduced postharvest losses; progeny of said plant, wherein the progeny plant exhibits improved shelf life and reduced postharvest losses; seed of said plant, wherein seed from said plant exhibits improved shelf life and reduced postharvest losses; and a processed product of said plant, said progeny plant, or said seed, wherein said processed product exhibits improved shelf life and reduced postharvest losses.
An additional embodiment of the invention is directed to a composition comprising a polynucleotide molecule that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene, wherein said polynucleotide is not operably linked to a promoter; and, a transfer agent. In some embodiments, the PPO gene is PPO 9, 10 or 11. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 304-307. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 310-317. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 318-323. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 335-348. In certain embodiments, the plant is a potato, apple, spinach, or lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO:24-302, and 303, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23. In certain embodiments, the plant is a potato, apple, spinach, or lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO: 312-317, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO: 304-307 or 319-334. In certain embodiments, the polynucleotide is at least 18 to about 24, about 25 to about 50, about 51 to about 100, about 101 to about 300, about 301 to about 500, or at least about 500 or more residues in length. In certain embodiments, the composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof. In certain embodiments, the transfer agent is an organosilicone preparation. In certain embodiments, the polynucleotide is not physically bound to a biolistic particle.
Another embodiment of the invention is directed to a method of making a composition comprising the step of combining at least:
a) a polynucleotide molecule comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or a transcript of said gene, wherein said polynucleotide is not operably linked to a promoter or a viral vector; and,
b) a transfer agent. In some embodiments, the PPO gene is selected from the group consisting of PPO 9, 10 and 11.
In certain embodiments of the methods, the polynucleotide is obtained by in vivo biosynthesis, in vitro enzymatic synthesis, or chemical synthesis. In certain embodiments of the methods, the method further comprises combining with said polynucleotide and said transfer agent at least one of a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, an insecticide, a fungicide, and/or a nematocide. In certain embodiments of the methods, the transfer agent is an organosilicone preparation.
Yet another embodiment of the invention is directed to a method of identifying a polynucleotide for improved shelf life and reduced postharvest losses in a plant comprising:
a) selecting a population of polynucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene;
b) topically applying to a surface of at least one of said plants a composition comprising at least one polynucleotide from said population and a transfer agent to obtain a treated plant; and,
c) identifying a treated plant that exhibits suppression of the Polyphenol oxidase (PPO) gene or exhibits improved shelf life and reduced postharvest losses, thereby identifying a polynucleotide that improves shelf life and reduces postharvest losses in a plant. In certain embodiments of the methods, the polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23. In certain embodiments of the methods, the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO: 304-307 or 319-334. In certain embodiments of the methods, the plant is a potato, apple, spinach, or lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO:24-303 or 312-317, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23, 304-307 or 319-334. In certain embodiments, the plant further comprises an organosilicone compound or a component thereof. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO:24-303, and 312-317, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23, 304-307 or 319-334. In certain embodiments, the plant is a potato, apple, spinach, or lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO: 24-303, and 312-317, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23, 304-307 or 319-334.
A further embodiment of the invention is directed to a plant comprising an exogenous polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene, wherein said exogenous polynucleotide is not operably linked to a promoter or to a viral vector, is not integrated into the chromosomal DNA of the plant, and is not found in a non-transgenic plant; and, wherein said plant exhibits improved shelf life and reduced postharvest losses that results from suppression of the Polyphenol oxidase (PPO) gene. A further embodiment is directed to a plant comprising an exogenous polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene, wherein said exogenous polynucleotide is not operably linked to a promoter or to a viral vector, is not integrated into the chromosomal DNA of the plant, and is not found in a non-transgenic plant; and, wherein said plant exhibits reduced or delayed discoloration following harvest.
An additional embodiment of the invention is directed to a plant part comprising an exogenous polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene, wherein said exogenous polynucleotide is not operably linked to a promoter or to a viral vector and is not found in a non-transgenic plant; and, wherein said plant part exhibits improved shelf life and reduced postharvest losses that results from suppression of the Polyphenol oxidase (PPO) gene. In certain embodiments, the plant part further comprises an organosilicone compound or a metabolite thereof. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 24-303, and 312-317, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO: 1-23, 304-307 or 319-334. In certain embodiments, the plant part is a potato, apple, spinach, and lettuce plant part, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO: 24-303, and 312-317, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO: 1-23, 304-307 or 319-334. In certain embodiments, the plant part is a flower, stem, meristem, ovule, tuber, fruit, anther, pollen, leaf, root, or seed. In certain embodiments, the plant part is a seed. Also provided is a processed plant product obtained from any of the aforementioned plant parts. In certain embodiments, the product is a meal, a pulp, a feed, or a food product.
Another embodiment of the invention is directed to a plant that exhibits improved shelf life and reduced postharvest losses, wherein said plant was topically treated with a composition that comprises:
(a) at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of said gene; and,
(b) a transfer agent; and,
wherein said plant exhibits improved shelf life and reduced postharvest losses that results from suppression of said Polyphenol oxidase (PPO) gene. In some embodiments, the PPO gene is PPO 9, 10 or 11.
Another embodiment of the invention is directed to a plant that exhibits reduced or delayed discoloration following harvest, wherein said plant was topically treated with a composition that comprises:
(a) at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of said gene; and,
(b) a transfer agent; and,
wherein said plant exhibits reduced or delayed discoloration that results from suppression of said Polyphenol oxidase (PPO) gene. In some embodiments, the PPO gene is PPO 9, 10 or 11.
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.
As used herein, the terms “DNA,” “DNA molecule,” and “DNA polynucleotide molecule” refer to a single-stranded DNA or double-stranded DNA molecule of genomic or synthetic origin, such as, a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule.
As used herein, the terms “DNA sequence,” “DNA nucleotide sequence,” and “DNA polynucleotide sequence” refer to the nucleotide sequence of a DNA molecule.
As used herein, the term “gene” refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript. A “gene” thus includes, but is not limited to, a promoter region, 5′ untranslated regions, transcript encoding regions that can include intronic regions, and 3′ untranslated regions.
As used herein, the terms “RNA,” “RNA molecule,” and “RNA polynucleotide molecule” refer to a single-stranded RNA or double-stranded RNA molecule of genomic or synthetic origin, such as, a polymer of ribonucleotide bases that comprise single or double stranded regions.
Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations §1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, a “plant surface” refers to any exterior portion of a plant. Plant surfaces thus include, but are not limited to, the surfaces of flowers, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. A plant surface can be on a portion of a plant that is attached to other portions of a plant or on a portion of a plant that is detached from the plant.
As used herein, the phrase “polynucleotide is not operably linked to a promoter” refers to a polynucleotide that is not covalently linked to a polynucleotide promoter sequence that is specifically recognized by either a DNA dependent RNA polymerase II protein or by a viral RNA dependent RNA polymerase in such a manner that the polynucleotide will be transcribed by the DNA dependent RNA polymerase II protein or viral RNA dependent RNA polymerase. A polynucleotide that is not operably linked to a promoter can be transcribed by a plant RNA dependent RNA polymerase.
As used herein, any polynucleotides displayed in SEQ ID NO:24-295 or 3 in the form of ssDNA, encompass dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNA complements, ssDNA as shown, and ssDNA complements.
As used herein, a first nucleic-acid sequence is “operably” connected or “linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to an RNA and/or protein-coding sequence if the promoter provides for transcription or expression of the RNA or coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.
As used herein, the phrase “organosilicone preparation” refers to a liquid comprising one or more organosilicone compounds, wherein the liquid or components contained therein, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enable the polynucleotide to enter a plant cell. Exemplary organosilicone preparations include, but are not limited to, preparations marketed under the trade names “Silwet®” or “BREAK-THRU®” and preparations provided in the following Table. In certain embodiments, an organosilicone preparation can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of target gene expression in the plant cell.
As used herein, the phrases “improved shelf life and reduced postharvest losses” or “improving shelf life and reducing postharvest losses” refer to any measurable increase in shelf life or reduction in postharvest loss. In certain embodiments, an increase in shelf life or reduction in postharvest loss in a plant or plant part can be determined in a comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant is a plant that has not undergone treatment with polynucleotide and a transfer agent. Such control plants would include, but are not limited to, untreated plants or mock treated plants.
As used herein, the phrase “provides for a reduction”, when used in the context of a transcript or a protein in a plant or plant part, refers to any measurable decrease in the level of transcript or protein in a plant or plant part. In certain embodiments, a reduction of the level of a transcript or protein in a plant or plant part can be determined in a comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant or plant part is a plant or plant part that has not undergone treatment with polynucleotide and a transfer agent. Such control plants or plant parts would include, but are not limited to, untreated or mock treated plants and plant parts.
As used herein, the phrase “wherein said plant does not comprise a transgene” refers to a plant that lacks either a DNA molecule comprising a promoter that is operably linked to a polynucleotide or a recombinant viral vector.
As used herein, the phrase “suppressing expression” or “suppression”, when used in the context of a gene, refers to any measurable decrease in the amount and/or activity of a product encoded by the gene. Thus, expression of a gene can be suppressed when there is a reduction in levels of a transcript from the gene, a reduction in levels of a protein encoded by the gene, a reduction in the activity of the transcript from the gene, a reduction in the activity of a protein encoded by the gene, any one of the preceding conditions, or any combination of the preceding conditions. In this context, the activity of a transcript includes, but is not limited to, its ability to be translated into a protein and/or to exert any RNA-mediated biologic or biochemical effect. In this context, the activity of a protein includes, but is not limited to, its ability to exert any protein-mediated biologic or biochemical effect. In certain embodiments, a suppression of gene expression in a plant or plant part can be determined in a comparison of gene product levels or activities in a treated plant to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant or plant part is a plant or plant part that has not undergone treatment with polynucleotide and a transfer agent. Such control plants or plant parts would include, but are not limited to, untreated or mock treated plants and plant parts.
As used herein, the term “transcript” corresponds to any RNA that is produced from a gene by the process of transcription. A transcript of a gene can thus comprise a primary transcription product which can contain introns or can comprise a mature RNA that lacks introns.
As used herein, the term “liquid” refers to both homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions.
II. OverviewProvided herein are certain methods and polynucleotide compositions that can be applied to living plant cells/tissues to suppress expression of target Polyphenol oxidase (PPO) genes and that provide improved shelf life and reduced postharvest losses to a crop plant in need of the benefit. Also provided herein are plants and plant parts exhibiting improved shelf life and reduced postharvest losses as well as processed products of such plants or plant parts. The compositions may be topically applied to the surface of a plant, such as to the surface of a leaf, and include a transfer agent. Aspects of the method can be applied to various crops, for example, including but not limited to: i) row crop plants including, but not limited to, corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, potato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; or, vi) an ornamental plant (e.g., an ornamental flowering plant or shrub or turf grass). The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i.e., a plant not grown from a seed) that include fruit trees and plants. Fruit trees produced by such processes include, but are not limited to, citrus and apple trees. Plants produced by such processes include, but are not limited to, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants.
Without being bound by theory, the compositions and methods of the present invention are believed to operate through one or more of the several natural cellular pathways involved in RNA-mediated gene suppression as generally described in Brodersen and Voinnet (2006), Trends Genetics, 22:268-280; Tomari and Zamore (2005) Genes & Dev., 19:517-529; Vaucheret (2006) Genes Dev., 20:759-771; Meins et al. (2005) Annu. Rev. Cell Dev. Biol., 21:297-318; and Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53. RNA-mediated gene suppression generally involves a double-stranded RNA (dsRNA) intermediate that is formed intra-molecularly within a single RNA molecule or inter-molecularly between two RNA molecules. This longer dsRNA intermediate is processed by a ribonuclease of the RNAase III family (Dicer or Dicer-like ribonuclease) to one or more shorter double-stranded RNAs, one strand of which is incorporated into the RNA-induced silencing complex (“RISC”). For example, the siRNA pathway involves the cleavage of a longer double-stranded RNA intermediate to small interfering RNAs (“siRNAs”). The size of siRNAs is believed to range from about 19 to about 25 base pairs, but the most common classes of siRNAs in plants include those containing 21 to 24 base pairs (See, Hamilton et al. (2002) EMBO J., 21:4671-4679).
PolynucleotidesAs used herein, “polynucleotide” refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Embodiments of this invention include compositions including oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e.g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target Polyphenol oxidase (PPO) gene including coding or non-coding or both coding and non-coding portions of the target Polyphenol oxidase (PPO) gene). Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.
Polynucleotide compositions used in the various embodiments of this invention include compositions including oligonucleotides, polynucleotides, or a mixture of both, including: RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof. In certain embodiments, the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e.g., fluorescein or rhodamine) or other label (e.g., biotin).
Polynucleotides can be single- or double-stranded RNA, single- or double-stranded DNA, double-stranded DNA/RNA hybrids, and modified analogues thereof. In certain embodiments of the invention, the polynucleotides that provide single-stranded RNA in the plant cell may be: (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, and (i) a double-stranded, hybridized RNA/DNA molecule, or combinations thereof. In certain embodiments, these polynucleotides can comprise both ribonucleic acid residues and deoxyribonucleic acid residues. In certain embodiments, these polynucleotides include chemically modified nucleotides or non-canonical nucleotides. In certain embodiments of the methods, the polynucleotides include double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In certain embodiments where the polynucleotide is a dsRNA, the anti-sense strand will comprise at least 18 nucleotides that are essentially complementary to the target Polyphenol oxidase (PPO) gene. In certain embodiments the polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having an at least partially double-stranded structure including at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. Not intending to be bound by any mechanism, it is believed that such polynucleotides are or will produce single-stranded RNA with at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. In certain embodiments, the polynucleotides can be operably linked to a promoter—generally a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
The polynucleotide molecules of the present invention are designed to modulate expression by inducing regulation or suppression of an endogenous Polyphenol oxidase (PPO) gene in a plant and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of an endogenous Polyphenol oxidase (PPO) gene of a plant or to the sequence of RNA transcribed from an endogenous Polyphenol oxidase (PPO) gene of a plant, which can be coding sequence or non-coding sequence. These effective polynucleotide molecules that modulate expression are referred to herein as “a trigger, or triggers”. By “essentially identical” or “essentially complementary” it is meant that the trigger polynucleotides (or at least one strand of a double-stranded polynucleotide) have sufficient identity or complementarity to the endogenous gene or to the RNA transcribed from the endogenous Polyphenol oxidase (PPO) gene (e.g., the transcript) to suppress expression of the endogenous Polyphenol oxidase (PPO) gene (e.g., to effect a reduction in levels or activity of the gene transcript and/or encoded protein). In certain embodiments, the trigger polynucleotides provided herein can be directed to a Polyphenol oxidase (PPO) transgene present in the plant. Polynucleotides of the methods and compositions provided herein need not have 100 percent identity to a complementarity to the endogenous Polyphenol oxidase (PPO) gene or to the RNA transcribed from the endogenous Polyphenol oxidase (PPO) gene (i.e., the transcript) to suppress expression of the endogenous Polyphenol oxidase (PPO) gene (i.e., to effect a reduction in levels or activity of the gene transcript or encoded protein). Thus, in certain embodiments, the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or messenger RNA transcribed from the target gene (e.g., the transcript). In certain embodiments, an “essentially identical” polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides in either the endogenous target gene or to an RNA transcribed from the target gene (e.g., the transcript). In certain embodiments, an “essentially complementary” polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.
In certain embodiments, polynucleotides used in the methods and compositions provided herein can be essentially identical or essentially complementary to any of: i) conserved regions of Polyphenol oxidase (PPO) genes of both monocot and dicot plants; ii) conserved regions of Polyphenol oxidase (PPO) genes of monocot plants; or iii) conserved regions of Polyphenol oxidase (PPO) genes of dicot plants. Such polynucleotides that are essentially identical or essentially complementary to such conserved regions can be used to improve shelf life and reduce postharvest losses by suppressing expression of Polyphenol oxidase (PPO) genes in various dicot and/or monocot plants.
Polynucleotides containing mismatches to the target gene or transcript can thus be used in certain embodiments of the compositions and methods provided herein. In certain embodiments, a polynucleotide can comprise at least 19 contiguous nucleotides that are essentially identical or essentially complementary to said gene or said transcript or comprises at least 19 contiguous nucleotides that are essentially identical or essentially complementary to the target gene or target gene transcript. In certain embodiments, a polynucleotide of 19 continuous nucleotides that is essentially identical or essentially complementary to the endogenous target gene or to a RNA transcribed from the target gene (e.g., the transcript) can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 20 or more nucleotides that contains a contiguous 19 nucleotide span of identity or complementarity to the endogenous target gene or to an RNA transcribed from the target gene can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to the endogenous target gene or to an RNA transcribed from the target gene (e.g., the transcript) can have 1, 2, or 3 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to the endogenous target gene or to an RNA transcribed from the target gene can have 1, 2, or 3 mismatches to the target gene or transcript. In designing polynucleotides with mismatches to an endogenous target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain exemplary embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al. Nucleic Acids Research, 2005, Vol. 33, No. 5 1671-1677. In certain exemplary embodiments, mismatches in 19 base pair overlap regions can be at the low tolerance positions 5, 7, 8 or 11 (from the 5′ end of a 19 nucleotide target) with well tolerated nucleotide mismatch residues, at medium tolerance positions 3, 4, and 12-17, and/or at the high tolerance nucleotide positions at either end of the region of complementarity (i.e., positions 1, 2, 18, and 19) as described by Du et al. Nucleic Acids Research, 2005, Vol. 33, No. 5 1671-1677. It is further anticipated that tolerated mismatches can be empirically determined in assays where the polynucleotide is applied to the plants via the methods provided herein and the treated plants assayed for suppression of Polyphenol oxidase (PPO) gene expression or appearance of improved shelf life and reduced postharvest losses.
In certain embodiments, polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to one allele or one family member of a given target Polyphenol oxidase (PPO) gene coding or non-coding sequence. Target Polyphenol oxidase (PPO) genes include both the Polyphenol oxidase (PPO) genes of SEQ ID NO:1-23 as well as orthologous Polyphenol oxidase (PPO) genes obtainable from other crops. In other embodiments, the polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
In certain embodiments, polynucleotide compositions and methods provided herein typically effect regulation or modulation (e. g., suppression) of gene expression during a period during the life of the treated plant of at least 1 week or longer and typically in systemic fashion. For instance, within days of treating a plant leaf with a polynucleotide composition of this invention, primary and transitive siRNAs can be detected in other leaves lateral to and above the treated leaf and in apical tissue. In certain embodiments, methods of systemically suppressing expression of a gene in a plant, the methods comprising treating said plant with a composition comprising at least one polynucleotide and a transfer agent, wherein said polynucleotide comprises at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a gene or a transcript encoding a Polyphenol oxidase (PPO) gene of the plant are provided, whereby expression of the gene in said plant or progeny thereof is systemically suppressed in comparison to a control plant that has not been treated with the composition.
Compositions used to suppress a target gene can comprise one or more polynucleotides that are essentially identical or essentially complementary to multiple genes, or to multiple segments of one or more genes. In certain embodiments, compositions used to suppress a target gene can comprise one or more polynucleotides that are essentially identical or essentially complementary to multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.
In certain embodiments, the polynucleotide includes two or more copies of a nucleotide sequence (of 18 or more nucleotides) where the copies are arranged in tandem fashion. In another embodiment, the polynucleotide includes two or more copies of a nucleotide sequence (of 18 or more nucleotides) where the copies are arranged in inverted repeat fashion (forming an at least partially self-complementary strand). The polynucleotide can include both tandem and inverted-repeat copies. Whether arranged in tandem or inverted repeat fashion, each copy can be directly contiguous to the next, or pairs of copies can be separated by an optional spacer of one or more nucleotides. The optional spacer can be unrelated sequence (i.e., not essentially identical to or essentially complementary to the copies, nor essentially identical to, or essentially complementary to, a sequence of 18 or more contiguous nucleotides of the endogenous target gene or RNA transcribed from the endogenous target gene). Alternatively the optional spacer can include sequence that is complementary to a segment of the endogenous target gene adjacent to the segment that is targeted by the copies. In certain embodiments, the polynucleotide includes two copies of a nucleotide sequence of between about 20 to about 30 nucleotides, where the two copies are separated by a spacer no longer than the length of the nucleotide sequence.
Tiling
Polynucleotide trigger molecules can be identified by “tiling” gene targets in random length fragments, e.g., 200-300 polynucleotides in length, with partially overlapping regions, e.g., 25 or so nucleotide overlapping regions along the length of the target gene. Multiple gene target sequences can be aligned and polynucleotide sequence regions with homology in common are identified as potential trigger molecules for multiple targets. Multiple target sequences can be aligned and sequence regions with poor homology are identified as potential trigger molecules for selectively distinguishing targets. To selectively suppress a single gene, trigger sequences may be chosen from regions that are unique to the target gene either from the transcribed region or the non-coding regions, e.g., promoter regions, 3′ untranslated regions, introns and the like.
Polynucleotide fragments are designed along the length of the full length coding and untranslated regions of a Polyphenol oxidase (PPO) gene or family member as contiguous overlapping fragments of 200-300 polynucleotides in length or fragment lengths representing a percentage of the target Polyphenol oxidase (PPO) gene. These fragments are applied topically (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine the relative effectiveness in providing the improved shelf life and reduced postharvest losses phenotype. Fragments providing the desired activity may be further subdivided into 50-60 polynucleotide fragments which are evaluated for providing the improved shelf life and reduced postharvest losses phenotype. The 50-60 base fragments with the desired activity may then be further subdivided into 19-30 base fragments which are evaluated for providing the improved shelf life and reduced postharvest losses phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or in combination in one or more pools to determine effective trigger composition or mixture of trigger polynucleotides for providing the improved shelf life and reduced postharvest losses phenotype.
Coding and/or non-coding sequences of Polyphenol oxidase (PPO) gene families in the crop of interest are aligned and 200-300 polynucleotide fragments from the least homologous regions amongst the aligned sequences are evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in providing the improved shelf life and reduced postharvest losses phenotype. The effective segments are further subdivided into 50-60 polynucleotide fragments, prioritized by least homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by least homology, and again evaluated for induction of the improved shelf life and reduced postharvest losses phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or again evaluated in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
Coding and/or non-coding sequences of Polyphenol oxidase (PPO) gene families in the crop of interest are aligned and 200-300 polynucleotide fragments from the most homologous regions amongst the aligned sequences are evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the improved shelf life and reduced postharvest losses phenotype. The effective segments are subdivided into 50-60 polynucleotide fragments, prioritized by most homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by most homology, and again evaluated for induction of the improved shelf life and reduced postharvest losses phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the improved shelf life and reduced postharvest losses phenotype.
Also, provided herein are methods for identifying a preferred polynucleotide for providing improved shelf life and reduced postharvest losses in a plant. Populations of candidate polynucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of the Polyphenol oxidase (PPO) gene can be generated by a variety of approaches, including but not limited to, any of the tiling, least homology, or most homology approaches provided herein. Such populations of polynucleotides can also be generated or obtained from any of the polynucleotides or genes provided herewith in SEQ ID NO:1-303. Such populations of polynucleotides can also be generated or obtained from any genes that are orthologous to the genes provided herewith in SEQ ID NO:1-23. Such polynucleotides can be topically applied to a surface of plants in a composition comprising at least one polynucleotide from said population and a transfer agent to obtain treated plants. Treated plants that exhibit suppression of the Polyphenol oxidase (PPO) gene and/or exhibit an improvement in improved shelf life and reduced postharvest losses are identified, thus identifying a preferred polynucleotide that improves improved shelf life and reduced postharvest losses in a plant. Suppression of the Polyphenol oxidase (PPO) gene can be determined by any assay for the levels and/or activity of a Polyphenol oxidase (PPO) gene product (i.e., transcript or protein). Suitable assays for transcripts include, but are not limited to, semi-quantitative or quantitative reverse transcriptase PCR® (qRT-PCR) assays. Suitable assays for proteins include, but are not limited to, semi-quantitative or quantitative immunoassays, biochemical activity assays, or biological activity assays. In certain embodiments, the polynucleotides can be applied alone. In other embodiments, the polynucleotides can be applied in pools of multiple polynucleotides. When a pool of polynucleotides provides for suppression of the Polyphenol oxidase (PPO) gene and/or an improvement in improved shelf life and reduced postharvest losses are identified, the pool can be de-replicated and retested as necessary or desired to identify one or more preferred polynucleotide(s) that improve improved shelf life and reduced postharvest losses in a plant.
Methods of making polynucleotides are well known in the art. Such methods of making polynucleotides can include in vivo biosynthesis, in vitro enzymatic synthesis, or chemical synthesis. In certain embodiments, RNA molecules can be made by either in vivo or in vitro synthesis from DNA templates where a suitable promoter is operably linked to the polynucleotide and a suitable DNA-dependent RNA polymerase is provided. DNA-dependent RNA polymerases include, but are not limited to, E. coli or other bacterial RNA polymerases as well as the bacteriophage RNA polymerases such as the T7, T3, and SP6 RNA polymerases. Commercial preparation of oligonucleotides often provides two deoxyribonucleotides on the 3′ end of the sense strand. Long polynucleotide molecules can be synthesized from commercially available kits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.) have DNA ligated on the 5′ end that encodes a bacteriophage T7 polymerase promoter that makes RNA strands that can be assembled into a dsRNA. Alternatively, dsRNA molecules can be produced from expression cassettes in bacterial cells that have regulated or deficient RNase III enzyme activity. Long polynucleotide molecules can also be assembled from multiple RNA or DNA fragments. In some embodiments design parameters such as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326-330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676, 2006) are known in the art and are used in selecting polynucleotide sequences effective in gene silencing. In some embodiments random design or empirical selection of polynucleotide sequences is used in selecting polynucleotide sequences effective in gene silencing. In some embodiments the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
While there is no upper limit on the concentrations and dosages of polynucleotide molecules that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency. The concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using 25-mer polynucleotide molecules is about 1 nanomole (nmol) of polynucleotide molecules per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a ssDNA polynucleotide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of a dsRNA is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 mg/mL, or about 0.14 mg/mL of dsRNA or ssDNA (21-mer) is applied. In certain embodiments, a composition of about 0.5 to about 1.5 mg/mL of a long dsRNA polynucleotide (i.e., about 50 to about 200 or more nucleotides) is applied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNA is applied to a plant. In certain embodiments, the polynucleotide composition as topically applied to the plant contains the at least one polynucleotide at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines may require correspondingly larger amounts of polynucleotides. When using long dsRNA molecules that can be processed into multiple oligonucleotides, lower concentrations can be used. To illustrate embodiments of the invention, the factor 1×, when applied to oligonucleotide molecules is arbitrarily used to denote a treatment of 0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmol of polynucleotide molecule per plant; and 100×, 80 nmol of polynucleotide molecule per plant.
The polynucleotide compositions of this invention are useful in compositions, such as liquids that comprise polynucleotide molecules, alone or in combination with other components either in the same liquid or in separately applied liquids that provide a transfer agent. As used herein, a transfer agent is an agent that, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enables the polynucleotide to enter a plant cell. In certain embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e.g., seeds, leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotide molecules into plant cells. The transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a polynucleotide-transferring agent to the plant tissue. In some embodiments the transferring agent is applied subsequent to the application of the polynucleotide composition. The polynucleotide transfer agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and/or cell wall or membrane barriers into plant cells. Suitable transfer agents to facilitate transfer of the polynucleotide into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include emulsions, reverse emulsions, liposomes, and other micellar-like compositions. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include counter-ions or other molecules that are known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e.g., plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on the worldwide web (internet) at herbicide.adjuvants.com can be used, e.g., paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. Transfer agents include, but are not limited to, organosilicone preparations.
In certain embodiments, an organosilicone preparation that is commercially available as Silwet® L-77 surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, N.Y. can be used to prepare a polynucleotide composition. In certain embodiments where a Silwet L-77 organosilicone preparation is used as a pre-spray treatment of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%. by weight (wt percent) is used or provided.
In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%. by weight (wt percent) is used or provided. In certain embodiments, any of the commercially available organosilicone preparations provided in the following Table 1 can be used as transfer agents in a polynucleotide composition. In certain embodiments where an organosilicone preparation of the Table is used as a pre-spray treatment of plant leaves or other surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation of Table 1 in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 (wt percent) is used or provided.
Organosilicone preparations used in the methods and compositions provided herein can comprise one or more effective organosilicone compounds. As used herein, the phrase “effective organosilicone compound” is used to describe any organosilicone compound that is found in an organosilicone preparation that enables a polynucleotide to enter a plant cell. In certain embodiments, an effective organosilicone compound can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of target gene expression in the plant cell. In general, effective organosilicone compounds include, but are not limited to, compounds that can comprise: i) a trisiloxane head group that is covalently linked to, ii) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, iii) a poly glycol chain, that is covalently linked to, iv) a terminal group. Trisiloxane head groups of such effective organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Poly glycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Poly glycol chains can comprise a mixture that provides an average chain length “n” of about “7.5”. In certain embodiments, the average chain length “n” can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Effective organosilicone compounds are believed to include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.
(Compound I: polyalkyleneoxide heptamethyltrisiloxane, average n=7.5).
One organosilicone compound believed to be ineffective comprises the formula:
In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a trisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a heptamethyltrisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and one or more effective organosilicone compound in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.
In certain embodiments, the polynucleotide compositions that comprise an organosilicone preparation can comprise a salt such as ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate. Ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate can be provided in the polynucleotide composition at a concentration of about 0.5% to about 5% (w/v). An ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate concentration of about 1% to about 3%, or about 2% (w/v) can also be used in the polynucleotide compositions that comprise an organosilicone preparation. In certain embodiments, the polynucleotide compositions can comprise an ammonium salt at a concentration greater or equal to 300 millimolar. In certain embodiments, the polynucleotide compositions that comprise an organosilicone preparation can comprise ammonium sulfate at concentrations from about 80 to about 1200 mM or about 150 mM to about 600 mM.
In certain embodiments, the polynucleotide compositions can also comprise a phosphate salt. Phosphate salts used in the compositions include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, the polynucleotide compositions can comprise a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the polynucleotide compositions will comprise a phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the polynucleotide compositions can comprise sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the polynucleotide compositions can comprise sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the polynucleotide compositions will comprise a sodium phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the polynucleotide compositions will comprise a sodium phosphate salt in a range of about 10 mM to about 160 mM or in a range of about 20 mM to about 40 mM. In certain embodiments, the polynucleotide compositions can comprise a sodium phosphate buffer at a pH of about 6.8.
In certain embodiments, other useful transfer agents or adjuvants to transfer agents that can be used in polynucleotide compositions provided herein include surfactants and/or effective molecules contained therein. Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. In certain embodiments, the polynucleotide compositions that comprise a transfer agent are formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Illustrative examples include tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, the polynucleotide compositions are formulated with a non-polynucleotide herbicide. Non-polynucleotide herbicidal molecules include, but are not limited to, glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides.
In certain embodiments, the polynucleotides used in the compositions that are essentially identical or essentially complementary to the target gene or transcript will comprise the predominant nucleic acid in the composition. Thus in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript will comprise at least about 50%, 75%, 95%, 98%, or 100% of the nucleic acids provided in the composition by either mass or molar concentration. However, in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to about 50%, about 10% to about 50%, about 20% to about 50%, or about 30% to about 50% of the nucleic acids provided in the composition by either mass or molar concentration. Also provided are compositions where the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to 100%, about 10% to 100%, about 20% to about 100%, about 30% to about 50%, or about 50% to 100% of the nucleic acids provided in the composition by either mass or molar concentration.
Polynucleotides comprising ssDNA, dsDNA, ssRNA, dsRNA, or RNA/DNA hybrids that are essentially identical or complementary to certain plant target genes or transcripts and that can be used in compositions containing transfer agents that include, but are not limited to, organosilicone preparations, to suppress those target genes when topically applied to plants are disclosed in co-assigned U.S. patent application Ser. No. 13/042,856. Various polynucleotide herbicidal molecules, compositions comprising those polynucleotide herbicidal molecules and transfer agents that include, but are not limited to, organosilicone preparations, and methods whereby herbicidal effects are obtained by the topical application of such compositions to plants are also disclosed in co-assigned U.S. patent application Ser. No. 13/042,856, and those polynucleotide herbicidal molecules, compositions, and methods are incorporated herein by reference in their entireties. Genes encoding proteins that can provide tolerance to an herbicide and/or that are targets of a herbicide are collectively referred to herein as “herbicide target genes”. Herbicide target genes include, but are not limited to, a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a glyphosate oxidoreductase (GOX), a glyphosate decarboxylase, a glyphosate-N-acetyl transferase (GAT), a dicamba monooxygenase, a phosphinothricin acetyltransferase, a 2,2-dichloropropionic acid dehalogenase, an acetohydroxyacid synthase, an acetolactate synthase, a haloarylnitrilase, an acetyl-coenzyme A carboxylase (ACCase), a dihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrin IX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), a para-aminobenzoate synthase, a glutamine synthase, a cellulose synthase, a beta tubulin, and a serine hydroxymethyltransferase gene. The effects of applying certain compositions comprising polynucleotides that are essentially identical or complementary to certain herbicide target genes and transfer agents on plants containing the herbicide target genes was shown to be potentiated or enhanced by subsequent application of an herbicide that targets the same gene as the polynucleotide in co-assigned U.S. patent application Ser. No. 13/042,856. For example, compositions comprising polynucleotides targeting the EPSPS herbicide target gene were potentiated by glyphosate in experiments disclosed in co-assigned U.S. patent application Ser. No. 13/042,856.
In certain embodiments of the compositions and methods disclosed herein, the composition comprising a polynucleotide and a transfer agent can thus further comprise a second polynucleotide comprising at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a transcript to a protein that confers resistance to a herbicide. In certain embodiments, the second polynucleotide does not comprise a polynucleotide that is essentially identical or essentially complementary to a transcript encoding a protein of a target plant that confers resistance to said herbicidal molecule. Thus, in an exemplary and non-limiting embodiment, the second polynucleotide could be essentially identical or essentially complementary to a transcript encoding a protein that confers resistance to a herbicide in a weed (such as an EPSPS encoding transcript) but would not be essentially identical or essentially complementary to a transcript encoding a protein that confers resistance to that same herbicide in a crop plant.
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can comprise glycerin. Glycerin can be provided in the composition at a concentration of about 0.1% to about 1% (w/v or v/v). A glycerin concentration of about 0.4% to about 0.6%, or about 0.5% (w/v or v/v) can also be used in the polynucleotide compositions that comprise a transfer agent.
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can further comprise organic solvents. Such organic solvents include, but are not limited to, DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions).
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can further comprise naturally derived or synthetic oils with or without surfactants or emulsifiers. Such oils include, but are not limited to, plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on line at www.herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.
In aspects of the invention, methods include one or more applications of the composition comprising a polynucleotide and a transfer agent or one or more effective components contained therein. In certain embodiments of the methods, one or more applications of a transfer agent or one or more effective components contained therein can precede one or more applications of the composition comprising a polynucleotide and a transfer agent. In embodiments where a transfer agent and/or one or more effective molecules contained therein is used either by itself as a pre-treatment or as part of a composition that includes a polynucleotide, embodiments of the polynucleotide molecules are double-stranded RNA oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA polynucleotides, single-stranded RNA polynucleotides, double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides, double-stranded DNA polynucleotides, single-stranded DNA polynucleotides, chemically modified RNA or DNA oligonucleotides or polynucleotides or mixtures thereof.
Compositions and methods of the invention are useful for modulating or suppressing the expression of an endogenous target gene or transgenic target gene in a plant cell or plant. In certain embodiments of the methods and compositions provided herein, expression of Polyphenol oxidase (PPO) target genes can be suppressed completely, partially and/or transiently to result in improved shelf life and reduced postharvest losses. In various embodiments, a target gene includes coding (protein-coding or translatable) sequence, non-coding (non-translatable) sequence, or both coding and non-coding sequence. Compositions of the invention can include polynucleotides and oligonucleotides designed to target multiple genes, or multiple segments of one or more genes. The target gene can include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species. Examples of target genes of the present invention include endogenous Polyphenol oxidase (PPO) genes and Polyphenol oxidase (PPO) transgenes.
Target Polyphenol oxidase (PPO) genes and plants containing those target Polyphenol oxidase (PPO) genes can be obtained from: i) row crop plants including, but are not limited to, corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, potato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; or, vi) an ornamental plant (e.g., an ornamental flowering plant or shrub or turf grass). The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i.e., a plant not grown from a seed) include fruit trees and plants that include, but are not limited to, citrus, apples, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants. Such row crop, vegetable, culinary, fruit, tree, or ornamental plants exhibiting improvements in that result from suppressing expression of Polyphenol oxidase (PPO) gene are provided herein. Such row crop, vegetable, culinary, fruit, tree, or ornamental plant parts or processed plant products exhibiting improved shelf life and reduced postharvest losses that result from suppressing expression of Polyphenol oxidase (PPO) gene are also provided herein. Such plant parts can include, but are not limited to, flowers, stems, tubers, fruit, anthers, meristems, ovules, pollen, leaves, or seeds. Such processed plant products obtained from the plant parts can include, but are not limited to, a meal, a pulp, a feed, or a food product.
An aspect of the invention provides a method for modulating expression of an Polyphenol oxidase (PPO) gene in a plant including (a) conditioning of a plant to permeation by polynucleotides and (b) treatment of the plant with the polynucleotide molecules, wherein the polynucleotide molecules include at least one segment of 18 or more contiguous nucleotides cloned from or otherwise identified from the target Polyphenol oxidase (PPO) gene in either anti-sense or sense orientation, whereby the polynucleotide molecules permeate the interior of the plant and induce modulation of the target Polyphenol oxidase (PPO) gene. The conditioning and polynucleotide application can be performed separately or in a single step. When the conditioning and polynucleotide application are performed in separate steps, the conditioning can precede or can follow the polynucleotide application within minutes, hours, or days. In some embodiments more than one conditioning step or more than one polynucleotide molecule application can be performed on the same plant. In embodiments of the method, the segment can be cloned or identified from (a) coding (protein-encoding), (b) non-coding (promoter and other gene related molecules), or (c) both coding and non-coding parts of the target Polyphenol oxidase (PPO) gene. Non-coding parts include DNA, such as promoter regions or the RNA transcribed by the DNA that provide RNA regulatory molecules, including but not limited to: introns, 5′ or 3′ untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, and riboswitches. In certain embodiments where the polynucleotide used in the composition comprises a promoter sequence essentially identical to, or essentially complementary to at least 18 contiguous nucleotides of the promoter of the endogenous target Polyphenol oxidase (PPO) gene, the promoter sequence of the polynucleotide is not operably linked to another sequence that is transcribed from the promoter sequence.
Compositions comprising a polynucleotide and a transfer agent provided herein can be topically applied to a plant or plant part by any convenient method, e.g., spraying or coating with a powder, or with a liquid composition comprising any of an emulsion, suspension, or solution. Such topically applied sprays or coatings can be of either all or of any a portion of the surface of the plant or plant part. Similarly, the compositions comprising a transfer agent or other pre-treatment can in certain embodiments be applied to the plant or plant part by any convenient method, e.g., spraying or wiping a solution, emulsion, or suspension. Compositions comprising a polynucleotide and a transfer agent provided herein can be topically applied to plant parts that include, but are not limited to, flowers, stems, tubers, meristems, ovules, fruit, anthers, pollen, leaves, roots, or seeds.
Application of compositions comprising a polynucleotide and a transfer agent to seeds is specifically provided herein. Seeds can be contacted with such compositions by spraying, misting, immersion, and the like.
In certain embodiments, application of compositions comprising a polynucleotide and a transfer agent to plants, plant parts, or seeds in particular can provide for the improved shelf life and reduced postharvest losses in progeny plants, plant parts, or seeds derived from those treated plants, plant parts, or seeds. In certain embodiments, progeny plants, plant parts, or seeds derived from those treated plants, plant parts, or seeds will exhibit improved shelf life and reduced postharvest losses that result from suppressing expression of Polyphenol oxidase (PPO) gene. In certain embodiments, the methods and compositions provided herein can provide for improved shelf life and reduced postharvest losses in progeny plants or seeds as a result of epigenetically inherited suppression of Polyphenol oxidase (PPO) gene expression. In certain embodiments, such progeny plants exhibit improved shelf life and reduced postharvest losses from epigenetically inherited suppression of Polyphenol oxidase (PPO) gene expression that is not caused by a transgene where the polynucleotide is operably linked to a promoter, a viral vector, or a copy of the polynucleotide that is integrated into a non-native location in the chromosomal DNA of the plant. Without seeking to be limited by theory, progeny plants or seeds derived from those treated plants, plant parts, or seeds can exhibit an improvement in improved shelf life and reduced postharvest losses through an epigenetic mechanism that provides for propagation of an epigenetic condition where suppression of Polyphenol oxidase (PPO) gene expression occurs in the progeny plants, plant parts, or plant seeds. In certain embodiments, progeny plants or seeds exhibiting improved shelf life and reduced postharvest losses as a result of epigenetically inherited suppression of Polyphenol oxidase (PPO) gene expression can also exhibit increased methylation, and in particular, increased methylation of cytosine residues, in the endogenous Polyphenol oxidase (PPO) gene of the plant. Plant parts, including seeds, of the progeny plants that exhibit improved shelf life and reduced postharvest losses as a result of epigenetically inherited suppression of Polyphenol oxidase (PPO) gene expression, can also in certain embodiments exhibit increased methylation, and in particular, increased methylation of cytosine residues, in the endogenous Polyphenol oxidase (PPO) gene. In certain embodiments, DNA methylation levels in DNA encoding the endogenous Polyphenol oxidase (PPO) gene can be compared in plants that exhibit the improved shelf life and reduced postharvest losses and control plants that do not exhibit the improved shelf life and reduced postharvest losses to correlate the presence of the improved shelf life and reduced postharvest losses to epigenetically inherited suppression of Polyphenol oxidase (PPO) gene expression and to identify plants that comprise the epigenetically inherited improved shelf life and reduced postharvest losses.
Various methods of spraying compositions on plants or plant parts can be used to topically apply to a plant surface a composition comprising a polynucleotide that comprises a transfer agent. In the field, a composition can be applied with a boom that extends over the crops and delivers the composition to the surface of the plants or with a boomless sprayer that distributes a composition across a wide area. Agricultural sprayers adapted for directional, broadcast, or banded spraying can also be used in certain embodiments. Sprayers adapted for spraying particular parts of plants including, but not limited to, leaves, the undersides of leaves, flowers, stems, male reproductive organs such as tassels, meristems, pollen, ovules, and the like can also be used. Compositions can also be delivered aerially, such as by a crop dusting airplane. In certain embodiments, the spray can be delivered with a pressurized backpack sprayer calibrated to deliver the appropriate rate of the composition. In certain embodiments, such a backpack sprayer is a carbon dioxide pressurized sprayer with a 11015 flat fan or equivalent spray nozzle with a customized single nozzle assembly (to minimize waste) at a spray pressure of about 0.25 MPa and/or any single nozzle sprayer providing an effective spray swath of 60 cm above the canopy of 3 to 12 inch tall growing plants can be used. Plants in a greenhouse or growth chamber can be treated using a track sprayer or laboratory sprayer with a 11001XR or equivalent spray nozzle to deliver the sample solution at a determined rate. An exemplary and non-limiting rate is about 140 L/ha at about 0.25 MPa pressure.
In certain embodiments, it is also contemplated that a plant part can be sprayed with the composition comprising a polynucleotide that comprises a transfer agent. Such plant parts can be sprayed either pre- or post-harvest to provide improved shelf life and reduced postharvest losses in the plant part that results from suppression of Polyphenol oxidase (PPO) gene expression. Compositions can be topically applied to plant parts attached to a plant by a spray as previously described. Compositions can be topically applied to plant parts that are detached from a plant by a spray as previously described or by an alternative method. Alternative methods for applying compositions to detached parts include, but are not limited to, passing the plant parts through a spray by a conveyor belt or trough, or immersing the plant parts in the composition.
Compositions comprising polynucleotides and transfer agents can be applied to plants or plant parts at one or more developmental stages as desired and/or as needed. Application of compositions to pre-germination seeds and/or to post-germination seedlings is provided in certain embodiments. Seeds can be treated with polynucleotide compositions provided herein by methods including, but not limited to, spraying, immersion, or any process that provides for coating, imbibition, and/or uptake of the polynucleotide composition by the seed. Seeds can be treated with polynucleotide compositions using seed batch treatment systems or continuous flow treatment systems. Seed coating systems are at least described in U.S. Pat. Nos. 6,582,516, 5,891,246, 4,079,696, and 4,023,525. Seed treatment can also be effected in laboratory or commercial scale treatment equipment such as a tumbler, a mixer, or a pan granulator. A polynucleotide composition used to treat seeds can contain one or more other desirable components including, but not limited to liquid diluents, binders to serve as a matrix for the polynucleotide, fillers for protecting the seeds during stress conditions, and plasticizers to improve flexibility, adhesion and/or spreadability of the coating. In addition, for oily polynucleotide compositions containing little or no filler, drying agents such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth or any other adsorbent material can be added. Use of such components in seed treatments is described in U.S. Pat. No. 5,876,739. Additional ingredients can be incorporated into the polynucleotide compositions used in seed treatments. Such ingredients include but are not limited to: conventional sticking agents, dispersing agents such as methylcellulose (Methocel A15LV or Methocel A15C, for example, serve as combined dispersant/sticking agents for use in seed treatments), polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P), polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPNA S-630), thickeners (e.g., clay thickeners such as Van Gel B to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers, surfactants, antifreeze compounds (e.g., urea), dyes, colorants, and the like that can be combined with compositions comprising a polynucleotide and a transfer agent. Further ingredients used in compositions that can be applied to seeds can be found in McCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996 and in McCutcheon's, vol. 2, “Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996. Methods of applying compositions to seeds and pesticidal compositions that can be used to treat seeds are described in U.S. Patent Application Publication No. 20080092256, which is incorporated herein by reference in its entirety.
Application of the compositions in early, mid-, and late vegetative stages of plant development is provided in certain embodiments. Application of the compositions in early, mid, and late reproductive stages is also provided in certain embodiments. Application of the compositions to plant parts at different stages of maturation is also provided.
The following examples are included to demonstrate examples of certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES Example 1 Polyphenol Oxidase (PPO) Target Gene SequencesThe Polyphenol oxidase (PPO) genes provided in Table 2 (SEQ ID NO:1-23), or their corresponding transcripts, can be used as targets of polynucleotide compositions comprising a polynucleotide that of at least 18 contiguous nucleotides that are essentially identical or essentially complementary to those genes or transcripts. The genes provided in Table 2, protein sequences encoded by those genes, or sequences contained within those genes can also be used to obtain orthologous Polyphenol oxidase (PPO) genes from plants not listed in Table 2. Such orthologous genes and their transcripts can then serve as targets of polynucleotides provided herein or as a source of polynucleotides that are specifically designed to target the orthologous genes or transcripts.
SEQ ID NO:1-23 contain the target DNA sequences from various plant species for Polyphenol oxidase (PPO) genes. For each gene having a DNA sequence provided in SEQ ID NO:1-23, single stranded or double stranded DNA or RNA fragments in sense or antisense orientation or both are mixed with an organosilicone preparation that comprises the compositions of the topical application method. This composition is topically applied to plants to effect expression of the target genes in the specified plant to obtain the desired increases in shelf life or reductions in post-harvest losses.
An exemplary set of polynucleotides that can be used to suppress expression of Polyphenol oxidase (PPO) genes in various plants is provided herewith in SEQ ID NO:24-295. The SEQ ID NOS:24-295 describe polynucleotide sequences from a variety of dicot and monocot plants as indicated that are useful for downregulating Polyphenol oxidase (PPO) expression using methods described here. The SEQ ID NO:24-295 SEQ ID NOS: describe polynucleotide sequences that can be applied to plants as ssDNA, ssRNA, dsDNA, dsRNA, and/or as DNA/RNA hybrids to provide for improved shelf life and reduced post-harvest losses. Subfragments of at least 18 contiguous nucleotides of the SEQ ID NOS:61-334 polynucleotide sequences can also be applied to plants as ssDNA, ssRNA, dsDNA, dsRNA, and/or as DNA/RNA hybrids to provide for improved shelf life and reduced postharvest losses. Other regions of Polyphenol oxidase (PPO) genes can also be targeted to modify expression including the use of antisense DNA oligonucleotides against coding regions and/or targeting promoter regions using sense/antisense dsRNA, sense or antisense ssDNA as well as sense/antisense double stranded DNA. For example, a polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any of the SEQ ID NO:1-23 PPO genes can be used to downregulate expression of those Polyphenol oxidase (PPO) genes.
Example 3 Method for Identifying Preferred PolynucleotidesA method for testing the entire sequence of each gene for selecting effective trigger molecules is described. Polynucleotides fragments are designed to cover the full length coding and untranslated regions of the gene in SEQ ID NO:1-23 as full-length sequences or as contiguous overlapping fragments of 200-300 bases length. These fragments are applied topically as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA to determine the relative effectiveness in providing the trait phenotype. Fragments providing the desired activity are further subdivided into 50-60 polynucleotide fragments which are evaluated for providing the trait phenotype. The 50-60 base fragments with the desired activity are subdivided into 19-30 base fragments which are evaluated for providing the trait phenotype. Fragments are tested singly, or in combination in one or more pools to determine effective trigger formulations for generation of the trait phenotype. Exemplary triggers developed in this manner are provided herewith in SEQ ID NO:24-295.
Example 4 Method for Identifying Conserved Preferred PolynucleotidesTriggers can also be developed to simultaneously suppress multiple Polyphenol oxidase (PPO) gene family members by alignment of coding and/or non-coding sequences of gene families in the crop of interest (SEQ ID NO:1-23), and choosing 200-300 base fragments from the most similar regions of the aligned sequences for evaluation in the topical application method (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the trait phenotype. The effective segments are subdivided into 50-60 base fragments, prioritized by greatest similarity, and re-evaluated in a topical application method. The effective 50-60 base fragments are subdivided into 19-30 base fragments, prioritized by greatest similarity, and again evaluated for induction of the trait/benefit phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger formulation for providing the trait phenotype. SEQ ID NO:296-303 provides some conserved PPO trigger sequences.
Example 5 Methods for Topical Application of Polynucleotide Molecules that Suppress PPOTomato plants at the 2-leaf stage grown in a peat moss, composted bark and perlite soil mix are spotted with polynucleotides, either ssDNA and/or dsRNA oligos or long dsRNAs directed to the promoter and/or targeting the coding region of the PPO gene or gene family. The nucleotide solution applied consists of 40-50 nmoles of each ssDNA oligonucleotide or 0.5-2 nmoles dsRNA, 0.3% Silwet L77, 5 mM Na2HPO4 and 2% ammonium sulfate in a final volume of 40 μL. Two mature leaves are spotted with 20 μL of the nucleotide solution for a total of 40 μL per plant.
Corn plants (LH244 or B73 inbred or LH244+80IDM2 hybrid) are germinated in potting medium and grown in the greenhouse for approximately 10 days. ssDNA and/or dsRNA polynucleotides directed to the promoter and/or targeting the coding region of the PPO gene or gene family are spotted onto the first and second leaves. The nucleotide solution applied consists of 40-50 nmoles of each ssDNA oligonucleotide or 0.5-2 nmoles dsRNA, 0.5% Silwet L77, 20 mM Na2HPO4 and 2% ammonium sulfate in a final volume of 50 μL. Two mature leaves are spotted with 25 μL each of the nucleotide solution for a total of 50 μL per plant.
Alternatively, corn plants (LH244 or B73 inbred or LH244+80IDM2 hybrid) grown in the greenhouse are treated between V1 and V6 stages of development by spraying leaves with a solution containing 0.14 mg/mL of dsRNA or ssDNA (21-mer) or 0.5 to 1.5 mg/mL long dsRNA polynucleotides targeted to the PPO gene or gene family with 0.5% Silwet L77, 20 mM Na2HPO4 and 2% ammonium sulfate.
Example 6 Methods Used in the Invention Related to Measuring Methyl-Jasmonate (MeJa) Induced BrowningThe following procedure is used for all assays described in this example. Lettuce heads cv SVR3606 L4 harvested at 10 days post-trigger treatment and then every 5 days after will be immersed into a container of cool water. The petioles will be cut away from the stem underwater and the cut ends will be kept in water until they are transferred. Petioles will be transferred into a solution of either 5% EtOH or MeJA+5% EtOH to ˜¾″ depth. The cups/baggies will be kept at RT on the laboratory bench and the induction of browning is followed visually. Photos taken of at 96 and 120 hrs show a clear reduction of central rib browning in trigger treated samples compared to buffer treated controls.
Example 7 Exemplary Methods Useful for Measuring Shelf LifeLettuce cv SVR3606 L4 will be grown in a greenhouse. Lettuce heads will be harvested and immersed into a container of cool water. Petioles are cut away from the stem underwater. The petioles from polynucleotide treated or untreated samples are respectively pooled and then placed onto a cutting board in ambient air and then cut perpendicular to the central mid rib into ¾″ pieces. The minimally process lettuce is then dried in a manual salad spinner to remove excess water. These are then stored inside “clam shells” or in deep well petri dishes at 4° C. The petri dishes will be stacked inside of a large Tupperware container to limit airflow and prevent drying out. Leaves are visually scored for appearance of brown/pink pigment appearance. Comparisons of treated vs. untreated minimally processed lettuce showed an improvement of 5 days after polynucleotide treatment.
Inducible PPO Enzyme AssayLettuce heads are immersed into a container of cool water. The petioles are cut away from the stem underwater and the cut ends are kept in water until they are transferred. Petioles are then inserted based base end into a solution of either 5% EtOH or MeJA+5% EtOH to ˜¾″ depth. The cups/baggies are then kept at RT on the laboratory bench and the central midribs are excised for enzymatic analysis at 48 hrs.
Enzyme analysis will be done using catechol as substrate. This analysis will show a reduction in the levels of PPO activity across biological and technical replicates of lettuce treated with polynucleotides as catechol is converted to benzoquinone.
Example 8 Methods Used in the Invention Related to Treating Plants or Plant Parts with a Topical Mixture of the Trigger MoleculesSingle stranded or double stranded DNA or RNA fragments in sense or antisense orientation are or both are identified and mixed with a transfer agent and other components in the composition of the invention. This composition is topically applied to plants to effect expression of the target PPO genes in the specified plant to obtain the desired effect on growth or development.
In this example, growing lettuce (Lactuca sativa, c.v. SVR3606) are treated with a topically applied composition for inducing suppression of a target gene in a plant including (a) an agent for conditioning of a plant to permeation by polynucleotides and (b) polynucleotides including at least one polynucleotide strand including at least one segment of 17-25 contiguous nucleotides of the target gene in either anti-sense (AS) or sense (S) orientation. Lettuce plants are treated with a topically applied adjuvant solution comprising dsRNA, ssDNA, essentially homologous or essentially complementary to the Lettuce PPO coding sequence. Plants were grown and treated in growth chamber 5 [22° C., 8 hour light (˜50 μmol, 16 hour dark cycles].
The following procedure is used for all assays described in this example. Lettuce plants germinated for approximately 20 days are used in these assays. The polynucleotide solution is spotted on the first or second leaf. Plants are pre-treated with 0.1% (vol/vol) Silwet L-77 applied to the leaf surface with a paint brush and allowed to dry for approximately 10 minutes. Final concentration for each oligonucleotide was 0.8-1 nmol ssDNA and 2-4 nMoles for dsRNA (in 0.01% Silwet L-77, 2% ammonium phosphate, 5 mM sodium phosphate buffer, pH6.8). Twenty microliters of the solution was applied to the top surface of the two leaves to provide for a total of 40 microliters for each plant.
For “one-step” application method the final concentration for each oligonucleotide or polynucleotide was 40 nMoles for ssDNA and 2-4 nMoles for dsRNA (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8) unless otherwise stated. Twenty microliters of the solution was applied to the top surface of each of the two leaves to provide a total of 40 microliters for each plant.
Spray solutions are prepared the same day as spraying. Lettuce plants are treated at 20 days after germination. Final concentration of each oligonucleotide or polynucleotide was 40 nMoles for ssDNA and 2-4 nMoles for dsRNA (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8) unless otherwise stated. The spray solution was applied to the plant to provide a total of 200-300 microliter volume.
Example 9 Effect of Inhibiting PPO Expression on Methyl-Jasmonate (MeJa) Induced BrowningTriggers targeting PPO 1-11 (SEQ ID NOs: 310-317 and 335-348) or GFP (SEQ ID NO 308) were diluted to a concentration of 2 nM ssDNA in buffer (0.01% (vol/vol) Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate) and applied to lettuce leaves cv SVR3606 L4 using a paint brush. Leaves were incubated in a plastic bag for 10 days, each treatment consisted of 2 lettuce leaves per plastic bag. Untreated (null (0)) lettuce leaves and lettuce leaves treated with trigger or buffer alone were harvested at 10 days post-treatment and immersed into a container of cool water. The petioles were cut away from the stem underwater and the cut ends were kept in water until transferred. Petioles were transferred into a solution of either 5% EtOH or MeJA+5% EtOH to ˜¾″ depth. The plastic bags were be kept at RT on the laboratory bench and the induction of browning was followed visually. Photos taken of at 96 and 120 hrs show a reduction of central rib browning in trigger treated samples compared to buffer treated controls (
The effect of trigger treatment on target mRNA levels was analyzed by TaqMan assay. Romaine lettuce leaves were removed from lettuce heads by slicing underwater. The leaves were placed in a plastic bag and incubated in the presence of dsRNA polynucleotide triggers targeting PPO11 (SEQ ID NO 307,
A significant difference in relative transcript abundance relative to buffer control was observed at a p-value of 0.0363 (
In
The effect of compositions comprising triggers targeting PPO8 or pooled PPO9, PPO10 and PPO11 triggers on browning of field-grown Romaine lettuce (Lactuca sativa, SVR3606) was assayed.
An eight by eight plot of SVR3606 was over seeded and then thinned to two rows of 10 plants in a single bed so that each plot contained 20 plants. The experiment design was a randomized complete block (RCB) design (9 blocks and 5 plots per block) with a single factor (Treatment) tested per block. See
-
- Treatment 1 (Null): Untreated control. The null plants, included as a negative control, were not marked or treated.
- Treatment 2 (Buffer treatment): 20 μl of buffer (25 mM NaPO4 (pH6.8), 1% (NH4)2SO4 and 0.01% (v/v) Silwet L-77) was applied to the pre-treated leaves using a pipette.
- Treatment 3 (PPO8 treatment): 20 μl of a solution containing 2 single-stranded antisense DNA oligonucleotides targeting PPO8 (10 nmoles each of SEQ ID NO 310 (HH162) and SEQ ID 311 (HH163)) in buffer (25 mM NaPO4 (pH6.8), 1% (NH4)2SO4 and 0.01% (v/v) Silwet L-77) was applied to the pre-treated leaves using a pipette.
- Treatment 4 (PPO 9, 10, 11 ssDNA treatment): 20 μl of a solution containing 6 single-stranded antisense DNA oligonucleotides targeting PPO 9, 10, and 11 (3.33 nmol each of SEQ ID 312-317 (HH164-169) DNA oligonucleotides) in buffer (25 mM NaPO4 (pH6.8), 1% (NH4)2SO4 and 0.01% (v/v) Silwet L-77) was applied to the pre-treated leaves using a pipette.
- Treatment 5 (PPO 9, 10, 11 dsRNA treatment): 20 μl of a solution containing 6 double-stranded RNA oligonucleotides targeting PPO 9, 10, and 11 (0.833 nmol each of SEQ ID NO 318-323 (rHH164-169) RNA oligonucleotides) in buffer (25 mM NaPO4 (pH6.8), 1% (NH4)2SO4 and 0.01% (v/v) Silwet L-77) was applied to the pre-treated leaves using a pipette.
The plants were grown to maturity, at which time; eight (8) market mature heads were harvested from each block by trained field workers by cutting at the base. Heads affected by big-vein virus or other defects were not be sampled. Lettuce heads were pooled within a single designated block and were placed into white bags (three heads/bag) for transport to the locations where the heads were processed and evaluated for shelf-life by (1) visual inspection and (2) image analysis. The total number of lettuce heads delivered in this study: 8 heads/treatment×5 treatments/block×9 blocks=360 heads. Transportation was kept to within 4 hours from time of harvest and the samples were transferred to a cold room kept at 4-7° C. for 12-24 hours prior to processing.
Shelf Life EvaluationThe lettuce heads were removed from their bags and any exterior leaves that showed damage were removed before processing (usually 5-7 leaves/head). Individual leaves were then removed by slicing from the bottom of the plant until the inner heart section was reached. The color of the leaves changed from a dark green on the outside of the head transitioning to a light lime green color for interior leaves. Leaves with a light green color were not included in the shelf-life evaluation. The total number of leaves collected ranged from 18-20 leaves per head. Excess leafy material was removed around the midrib leaving midrib pieces that were 1 inch in length and 2-3 inches wide. The chopped leaves were washed in ice cold de-ionized water for approximately 5-10 seconds and dried in a salad spinner. For each designated plot, 200-250 grams of dried leaves were placed into a 32 oz. clear plastic deli container, commonly referred to as a clamshell (Genpak, Glen Falls, N.Y. cat# AD32F). Processed lettuce samples were stored at 4° C. for 5-7 days and monitored daily for signs of discoloration. When approximately 50% of the clamshells showed browning, the lettuce leaves were scored for discoloration. See
For the visual assay, browning was quantified by dividing the number of discolored lettuce pieces by total number of counted pieces to give the percentage of discolored leaves. The two positive test applications (asDNA PPO 9,10, 11 and dsRNA PPO 9,10,11) were compared to the 3 negative controls (null, buffer, and PPO8). After the clamshells were scored visually, the lettuce was spread out in a black tray and photographed using an imaging station at three different time points, Day 5, 7 and 9. Image analysis software quantified the number of discolored vs green pixels in the imaged lettuce pieces. Statistical analyses of the image data was performed using established methods. The mean component values of each treatment were compared to the performance of the control and null treatments for statistically significant differences (p<0.05).
The ANOVA results of the shelf-life evaluation of individual heads processed by chopping indicate a trend in decreased discoloration for the plants treated with ssDNA oligonucleotides to PPO9,10 and 11 (Table 5 and
Analysis by imaging of chopped heads at Day 5 (
Claims
1. A method for producing a plant exhibiting improved shelf life and reduced postharvest losses comprising topically applying to a plant surface a composition that comprises:
- a. at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or to a transcript of said gene; and
- b. a transfer agent, wherein said plant exhibits improved shelf life and reduced postharvest losses that results from suppression of said Polyphenol oxidase (PPO) gene.
2. The method of claim 1, wherein said polynucleotide molecule comprises sense ssDNA, sense ssRNA, dsRNA, dsDNA, a double stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA.
3. The method of claim 1, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23.
4. The method of claim 1, wherein:
- said plant is a potato, apple, spinach, and lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO:24-302, and 303, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23.
5. The method of claim 1, wherein said composition comprises any combination of two or more polynucleotide molecules.
6-8. (canceled)
9. The method of claim 1, wherein said transfer agent comprises an organosilicone preparation.
10. The method of claim 1, wherein said polynucleotide is not operably linked to a viral vector.
11. The method of claim 1, wherein said polynucleotide is not integrated into the plant chromosome.
12. A plant obtained by the method of claim 1.
13. The plant obtained by the method of claim 12, wherein a progeny plant or a plant part derived therefrom exhibits improved shelf life and reduced postharvest losses.
14-15. (canceled)
16. A processed product of said plant of claim 12, wherein said processed product exhibits said improved shelf life and reduced postharvest losses.
17-18. (canceled)
19. A composition comprising a polynucleotide molecule that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene, wherein said polynucleotide is not operably linked to a promoter; and a transfer agent.
20. The composition of claim 19, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23.
21-22. (canceled)
23. The composition of claim 19, wherein said composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof.
24. The composition of claim 19, wherein said transfer agent is an organosilicone preparation.
25. The composition of claim 19, wherein said polynucleotide is not physically bound to a biolistic particle.
26-29. (canceled)
30. A method of identifying a polynucleotide for improving shelf life and reducing postharvest losses in a plant comprising:
- a) selecting a population of polynucleotides that are essentially identical or essentially complementary to a Polyphenol oxidase (PPO) gene or transcript of said gene;
- b) topically applying to a surface of at least one of said plants a composition comprising at least one polynucleotide from said population and a transfer agent to obtain a treated plant; and,
- c) identifying a treated plant that exhibits suppression of the Polyphenol oxidase (PPO) gene or exhibits improved shelf life and reduced postharvest losses, thereby identifying a polynucleotide that improves shelf life and reduces postharvest losses in a plant.
31. The method of claim 30, wherein said polynucleotide is selected from the group consisting of SEQ ID NO:24-302, and 303, or wherein said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a plant gene of SEQ ID NO:1-23.
32. The method of claim 30, wherein:
- said plant is a potato, apple, spinach, and lettuce plant, said gene or transcript is from the corresponding plant, and said polynucleotide molecule is selected from the group consisting of SEQ ID NO:24-302, and 303, or said polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a corresponding gene of SEQ ID NO:1-23.
33-46. (canceled)
Type: Application
Filed: Sep 24, 2013
Publication Date: Sep 3, 2015
Inventors: JoAnne Fillatti (St. Louis, MO), Byron Froman (St. Louis, MO), Graeme S. Garvey (St. Louis, MO), Johannes C. Hemmes (St. Louis, MO)
Application Number: 14/430,703
