Patent Application
20210076578
The present disclosure provides a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait and methods for obtaining same.
Improvement of agricultural methods and productivity is seen as one of the greatest challenges of the 21st century. According to the OECD-FAO report of 2012, agricultural production needs to be increased by 60% over the next 40 years, to meet the rising demand for food. Globally, the scope of area expansion is limited, i.e. the total arable land is projected to be increased by less than 5%. Thus, additional technologies will need to be developed in order to increase food production.
Traditionally, improved crops have been obtained by farmer experimentation with new varieties, plant breeding, purposeful selection, growth and cross pollination.
The first genetically modified crop plant was produced in 1982. Genetically modified crops (GM or GMC) are plants whose DNA was modified using genetic engineering techniques with a view to introduce in the plants a non-natural trait.
Transgenic (GM) plants possess a particular trait not native to the plant, which (inherited) trait is transmitted through the seeds.
While the GM technology has been widely accepted in the US and other countries, the regulatory status of the GM foods varies by country, with some countries banning or restricting them, or permitting them within limiting regulations.
Up until now, the efforts to increase the crop's intrinsic yield potential have mainly focused on exploiting the genetic variability within the crops. New combinations of plants have been mainly produced by traditional breeding techniques and molecular techniques, allowing the exchange of genetic material across species.
The role of epigenetic control mechanisms was much less studied and utilized. Epigenetics refers to the study of changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence.
Epigenetic mechanisms include functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications include DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence. In such mechanisms, “non-genetic” factors cause the organism's genes to be expressed differently.
The role of epigenetic control components in plants was demonstrated in some studies and patent documents. US patent application 2013/0117877 A1 relates to methods of finding a DNA methylation profile for a plant with high energy use efficiency. The invention enables the artisan to correlate the DNA methylation profile of a plant with potentially high energy use efficiency. US patent application 2012/0117678 A1 as well as M. Hauben et al. (Proceedings of the National Academy of Sciences of the United States of America, 106, no. 47, 20109-20114, 2009) publication, disclose methods for selecting plants with high energy use efficiency, by monitoring their cellular respiration rate. The cellular respiration rate is determined by measuring NADPH content, ascorbic acid content and/or respiratory chain complex I activity. It is emphasized that the cellular respiration rate measurements taught by US patent application 2012/0117678 A1 and Hauben et al (2009), are performed in vitro, on explants or tissue samples isolated from individual plants. Furthermore, the prior art remains silent on the ability to induce the production of plants with high yield and/or inheritable properties.
Attempts at manipulating the yield of plants and identifying yield genes have been made, with emphasis on the modification of the flowering time in plants (U.S. Pat. No. 8,935,880 and U.S. Patent Application No. 2014/0259905).
The International Patent Application Serial No. WO2017/077539, filed on Nov. 1, 2016 by Shenfeld A., incorporated in its entirety by reference, discloses methods of obtaining various non-GM improved crops with inheritable improved traits.
There is an unmet and long felt need for non-GM tomato crops with improved traits and moreover, for non-GM tomato crops with inheritable improved traits.
This disclosure provides a method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait. Also provided is a non-GM tomato plant and/or a harvestable part thereof with improved inheritable traits, produced by the method of this disclosure.
It is an object of the present disclosure to provide a method for producing a non-GM tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait as compared to a control population of untreated tomato plants, said method comprising the steps of: (a) providing a population of a cultivar or line of tomato seeds or plants; (b) exposing said population (a) of a cultivar or line of tomato plants to a predetermined light treatment by irradiating with artificial light, partly in the presence of ambient daylight, every day for a duration of 2 to 6 weeks from sowing, preferably for 30 days from sowing, wherein the predetermined light treatment consists of irradiating with artificial red light of wavelength in the range of from about 600 nm to about 700 nm for a period of about 60 minutes, starting about 30 min. before sunset in the presence of daylight, and continuing about 30 minutes after sunset. (c) growing the tomato plants in greenhouses for 2-3 months after the end of stage (b), then collecting, counting and weighing the tomatoes and collecting their seeds. d) monitoring at least one improved trait of the tomato plants at the end of step (b) and step (c) and comparing to a control population; (e) selecting at the end of the treatment of step (b) the plants exhibiting at least one improved trait as compared to a control population and selecting again at the end of the growing stage of step (c) the plants exhibiting at least one improved trait as compared to a control population; wherein the number of plants selected in the first generation is from about 1% to about 4%, preferably from about 2% to about 3% of the initial plant population provided (a); (f) propagating by sowing the tomato seeds at least one subsequent generation of the plants exhibiting at least one improved trait as compared to a control population; and (g) repeating steps (b) to (f) on 1-7 subsequent generations, preferably at least two subsequent generations; wherein the number of plants selected in step (e) increases from generation to generation, thereby obtaining a tomato crop plant and/or harvestable part thereof exhibiting at least one improved trait, wherein the improved trait is inheritable for at least one more generation, preferably for two or more generations, more preferably for 3 to 5 generations, and even more preferably for 6 to 8 generations.
It is a further object of the present disclosure to provide the method as defined above, wherein steps (b) to (g) are applied to tomato plants or tomato plant parts selected from the group consisting of tomatoes, seeds, seedlings, tissue cultures, calluses, meristems, regenerable cells, protoplasts, potted seedlings, adult tomato plants and any combination thereof.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of selecting the control population from the group consisting of untreated tomato plants, untreated tomato plants of the same generation, treated tomato plants of the same generation and any combination thereof.
It is a further object of the present disclosure to disclose the method as defined in any of the above, wherein the ambient daylight is characterized by luminous flux units of between about 1.5 to about 3000 lux, particularly from about 100 to about 2000 lux.
It is a further object of the present disclosure to provide the method as defined in any of the above, wherein the step of irradiating the tomato plants with artificial light is applied at a periodicity selected from the group consisting of: at the beginning of sunrise, during dawn, during sunrise, before sunset, during sunset, after sunset or at any combination thereof.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of irradiating with artificial light the tomato plants of step (a) with red light wavelengths, particularly in the range of about 600 nm to about 700 nm.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of irradiating with artificial light the tomato plants of step (a) with red light wavelengths every day for a duration of 2 to 6 weeks from sowing, preferably for 30 days, beginning on the sowing day.
It is a further object of the present disclosure to provide the method as defined in any of the above comprising steps of irradiating with artificial light the tomato plants of step (a) with wavelengths selected from the group consisting of red light, blue light, white light, preferably red light and any combination thereof.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of irradiating with artificial light the tomato plants of step (a) with red light wavelengths, blue light wavelengths, white light wavelengths and any combination thereof, consecutively, simultaneously or interchangeably.
Table 4 in Example 6 discloses the selection of the optimal irradiation wavelength range out of red, blue and white light irradiation, based on best average tomatoes per plant weight at each wavelength.
In some embodiments, there is provided the method of this disclosure, wherein the tomato plants are optimally irradiated with red light wavelength.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of exposing the tomato crop plants to a predetermined light treatment for about one month under greenhouse conditions and then transferring the selected best plants exhibiting at least one improved trait to field growth conditions or commercial greenhouse growth conditions. At the end of the growing cycle, the best performing plants, based on the selected traits are selected for an additional round of treatment.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of exposing the plants to a predetermined light treatment regime for about one month under field conditions and then selecting the plants exhibiting at least one improved trait responding best to the treatment. At the end of the growing cycle, the best performing plants, based on the selected traits, are selected for an additional round of treatment.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of monitoring at least one trait or parameter selected from the group consisting of tomatoes number, tomatoes weight, average seed number, average total seed weight, average single seed weight, plant height, main stem width, stem thickness, plant biomass, number of stems, number of secondary stems, photosynthesis efficiency, photosynthesis rate, nitrogen concentration in leaves and any combination thereof.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of producing a commercial tomato crop plant exhibiting an increased yield of at least 2% and up to 800% or more of the at least one yield characteristic, as compared to the control population of same.
It is a further object of the present disclosure to provide the method as defined in any of the above, wherein steps (a) to (g) are applied to tomato cultivars, plant lines or plant types selected from the group consisting of M82, Adoration, Alicante, Azoychka, Beefsteak, Better Boy, Big Beef, Big Rainbow, Blaby Special, Black Krim, Brandy wine, Campari, Celebrity, Cherokee Purple, Canario, Early Girl, Enchantment, Ferris Wheel, Flamenco, Fourth of July, Garden Peach, Gardener's delight, German Johnson, Giulietta F1, Granadero, Great White, Green Zebra, Hillbilly, Japanese, Black Trifele, Jersey Boy, Jubilee, Juliet, Kumato, Lilian's Yellow, McDreamy, Matt's Wild Cherry, Micro Tom, Moneymaker, Monterosa, Montserrat, Mortgage Lifter, Mr. Stripey, Pantano Romanesco, Paul Robeson, Plum Tomato, Pumpkin Tomato, Raf Tomato, Rebellion, Red Currant, Roma, Rosa de Barbastro, Rutgers Tomato, San Marzano, Sasha Altai, Stupice, Tiny Tim, Traveller, Arkansas Traveler, Cherry Bambelo, Cherrry Nebula, Santorini, Super Sweet, Tomaccio, Yellow Pear, White Queen.
The experiments described in this disclosure have been carried out on the tomato cultivar M82.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of producing a commercial tomato crop plant exhibiting an increased average seed number per plant of between about 10% and about 200%, preferably between about 50% and about 80%, as compared to the control population of same. It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of producing a commercial tomato crop plant exhibiting improved traits such as an increased average single tomato weight of between about 10% and about 50%, preferably between about 20% and about 40% or from about 40% to about 100% increase as compared to the control population of same.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of producing a commercial tomato crop plant exhibiting an improved trait such as an increased average total tomato weight per plant of between about 50% and about 200%, preferably between about 80% and about 150% as compared to the control population of same.
It is a further object of the present disclosure to provide the method as defined in any of the above, comprising steps of producing a commercial tomato exhibiting an improved trait such as improved room temperature (17-27° C.) shelf life of at least 45 days after harvesting, as compared to about 10 days for an untreated control tomato.
It is another further object of the present disclosure to provide the tomato obtained as described in this disclosure, wherein exhibiting an improved trait such as improved room temperature (17-27° C.) shelf life of at least 45 days after harvesting, as compared to about 10 days for an untreated control tomato.
It is a further object of the present disclosure to provide a tomato plant part or a harvestable part thereof produced by the method as defined in any of the above.
It is a further object of the present disclosure to provide the method of this disclosure wherein applied to a non-GM tomato plant.
It is a further object of the present disclosure to provide the method of this disclosure wherein applied to a genetically modified (GM) tomato plant.
It is a further object of the present disclosure to provide a plant part or a product thereof derived from the commercial plant as defined in any of the above.
It is further within the scope of this disclosure to provide a method for manipulating the plant's genome expression to increase its energy production efficiency. This may result in a dramatic increase in the number of tomatoes, seeds and biomass of the commercial tomato plant crop. Using the methods of the present disclosure, it is demonstrated that a tomato crop exhibiting increased yield capacity of up to 800% or more, between F1 (generation 1) and F2 (generation 2) or subsequent generation F3, can be produced.
It is a further object of the present disclosure to provide a tomato plant or a harvestable part thereof such as a tomato or a tomato seed, produced by the method as defined in any of the above.
Some embodiments of the disclosure are herein described, by way of example only, with reference to the accompanying Figures. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the disclosure. In this regard, the description taken with the Figures makes apparent to those skilled in the art how embodiments of the disclosure may be practiced.
In order to better understand the disclosure and its implementation in practice, a plurality of embodiments will now be described, by way of non-limiting example only, with reference to the accompanying Figures, wherein
The following description is provided, alongside all chapters of the present disclosure, so as to enable any person skilled in the art to make use of said disclosure and sets forth the best modes contemplated by the inventor of carrying out this invention. Various modifications, however, will remain apparent to those skilled in the art, since the generic principles of the present disclosure have been defined specifically to provide methods and products thereof for producing and screening for a commercial non-GM tomato plant exhibiting at least one improved trait compared to a control population of same.
It is an object of the present disclosure to provide a method for producing a non-GM tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait as compared to a control population of untreated tomato plants, said method comprising the steps of: (a) providing a population of a cultivar or line of tomato seeds or plants; (b) exposing said population (a) of a cultivar or line of tomato plants to a predetermined light treatment by irradiating with artificial light, partly in the presence of ambient daylight, every day for a duration of 2 to 6 weeks from sowing, preferably for 30 days from sowing, wherein the predetermined light treatment consists of irradiating with artificial red light of wavelength in the range of from about 600 nm to about 700 nm for a period of about 60 minutes, starting about 30 min. before sunset in the presence of daylight, and continuing about 30 minutes after sunset. (c) growing the tomato plants in greenhouses for 2-3 months after the end of stage (b), then collecting, counting and weighing the tomatoes and collecting their seeds; (d) monitoring at least one improved trait of the tomato plants at the end of step (b) and step (c) and comparing to a control population; (e) selecting at the end of the treatment of step (b) the plants exhibiting at least one improved trait as compared to a control population and selecting again at the end of the growing stage of step (c) the plants exhibiting at least one improved trait as compared to a control population; wherein the number of plants selected in the first generation is from about 1% to about 4%, preferably from about 2% to about 3% of the initial plant population provided (a); (f) propagating by sowing the tomato seeds at least one subsequent generation of the plants exhibiting at least one improved trait as compared to a control population; and (g) repeating steps (b) to (f) on 1-7 subsequent generations, preferably at least two subsequent generations; wherein the number of plants selected in step (e) increases from generation to generation, thereby obtaining a tomato crop plant and/or harvestable part thereof exhibiting at least one improved trait, wherein the improved trait is inheritable for at least one more generation, preferably for two or more generations, more preferably for 3 to 5 generations, and even more preferably for 6 to 8 generations
Examples 1-6 are provided, in which the tomato plants are M82 tomato cultivar plants.
In this way a non-GM tomato plant is produced, exhibiting inheritable improved traits compared to a control population of same.
It is further within the scope of the disclosure to disclose a method for improving tomato plant traits by manipulating environmental factors affecting the plant's growth such as artificial light irradiation.
The technology provided by the present disclosure is suitable for use on genetically modified (GM) crops as well as on non-GM crops (i.e. produced by other techniques such as breeding, grafting etc.), but the method itself is non-GM and does not lead to GM modifications by itself.
It is further within the scope of the disclosure to provide various treatment protocols for affecting tomato crop plants, preferably crop yield, resulting in different extent of trait improvements (such as tomato yield increase, biomass increase, etc.) of the treated tomato plants versus control plants.
It is further within the scope of the disclosure to provide a method for manipulating the various tomato plant inheritable traits by the method detailed in this disclosure.
According to certain aspects, the phenotypic changes in the treated plants are preserved in the next generations.
It is further demonstrated in the present disclosure that by increasing the efficiency of photosynthesis, tomato crop lines characterized by improved traits such as high yield and better growth potential were produced and selected.
According to one embodiment, the present disclosure provides methods to increase the expression of genes which accelerate photosynthesis, by manipulating environmental factors. As a result, improved tomato plant traits (such as an increase in tomato weight, tomato number, plant growth, biomass increase, inheritable phenotypic changes) is achieved.
Without wishing to be bound by theory, the environmental effects on gene expression may be associated with epigenetic mechanisms. It is noted that such genes may be responsive to environmental influence. The improved traits acquired are inheritable over a number of generations (at least one more generation and up to 5-8 generations), without the need for genetical modification.
According to a further embodiment, it is herein demonstrated that the ability to increase photosynthesis rate is associated with increased plant growth and with improved traits such as average number of tomatoes per plant, tomato weight per plant, seed weight per plant, average seed number per plant, average seed weight per plant, plant height, main stem width, stem thickness, average weight of a single seed, plant biomass, number of stems, number of secondary stems, stress resistance, pest resistance, virus resistance, drought tolerance, herbicide tolerance, delayed senescence, modified color, photosynthesis efficiency or rate, nitrogen concentration in the leaves, or any combination thereof.
According to a further embodiment, the improved trait plants of the present disclosure may be non-GM (Non-Genetically Modified) or GM plants. It is noted that modifications in the treatment protocol of the present disclosure may be adapted to each tomato cultivar type.
It is further within the scope of this disclosure that the treatment procedures (i.e. exposure to artificial environmental conditions) as well as the monitoring and evaluation of tomato yield characteristics disclosed in the present disclosure are performed during the tomato plant growth without disrupting the normal growth cycle of the plant. It is herein emphasized that the treatment procedures are external, and in preferred embodiments, are directed to the whole plant. In any case, even when the treatment is done on seeds or plant parts, the treatment is non-invasive. Furthermore, the monitoring or selection or identification of high performing plants does not interrupt or interfere with tomato plant growth.
The traits subject to improvement are selected from the group consisting of average tomato number per plant, weight per plant, average seed number per plant, average seed weight per plant, plant height, main stem width, stem thickness, tomato weight per plant, average weight of a single seed, plant biomass, number of stems, number of secondary stems, stress resistance, pest resistance, virus resistance, drought tolerance, herbicide tolerance, delayed senescence, modified color, photosynthesis efficiency or rate, nitrogen concentration in the leaves or any combination thereof.
In specific embodiments, tomato plants were exposed to irradiation with artificial light of wavelengths selected from red light, blue light, white light and any combination thereof, administered at predetermined protocols, periodicities, cycles and sessions.
In a preferred aspect, the tomato plant irradiation treatment is carried out with red light irradiation, for one hour per day, out of which half an hour before sunset and another half an hour after sunset.
The selection of the tomato plants exhibiting the best improved traits is an important part of the methods of this disclosure.
The treatment method of this disclosure uses in each generation of plant treatment (F1, F2, F3 and F4) two selection stages that differ from each other:
1. The first selection takes place at the end of the irradiation period (b), about one month from the time of sowing. The plants exhibiting at least one improved trait, best responding to the treatment, are selected and transferred to greenhouse growth conditions.
2. The second selection takes place at the end of the growing period (c) in greenhouses (about 2-3 months, depending on the season). This selection is based on the best performing chosen trait (e.g. tomato yield). The plants exhibiting at least one improved trait are selected, tomatoes are collected, counted, weighed and their seeds are collected for the next round of treatment
It was found that three rounds of treatment (F1-F3) lead to the best heredity results.
According to certain aspects of the disclosure, the following conclusions are herein demonstrated: The treated plants of the present disclosure have a significantly increased average yield of up to several hundred percent (i.e. up to 800% or more) greater than the average yield of control plants, as exemplified herein.
The increase in yield and growth of the treated tomato plants appeared not only in the treated generation but continued to the next generation and was maintained without additional treatment. It is noted that the high yield plant may have adapted to the aforementioned major phenotypic changes, over time. The demand for energy is significantly higher in the high yield tomato lines and the tomato plants increase their energy supply to adjust to the new demands but the adaptation continues vertically through several generations.
According to a further embodiment, the method as defined in any of the above, comprises steps of producing a commercial tomato plant exhibiting an increased tomato yield of at least 2% and up to 800%, or more particularly 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more, of said at least one characteristic, as compared to the control population of same.
According to a further embodiment, the method as defined in any of the above, comprises steps of producing a commercial tomato plant exhibiting an increased average total tomato weight per plant of between about 50% and about 200%, preferably between about 80% and about 150% higher than the control population of same.
It is further within the scope of this disclosure to provide the method as defined in any of the above, wherein the method is applied to a non-genetically modified (non-GM) commercial tomato crop plant or to a GM tomato crop plant.
It is further within the scope of this disclosure to provide a plant part such as a tomato or seeds thereof produced by the method as defined in any of the above.
It is further within the scope of this disclosure to disclose the tomato plant as defined in any of the above, wherein the control population is selected from the group consisting of: untreated plants, untreated plants of the same generation, treated plants of the same generation and any combination thereof.
It is further within the scope of this disclosure to disclose the method as defined in any of the above, wherein the optional ambient daylight is characterized by luminous flux units between about 100 to about 2000 lux.
It is further within the scope of this disclosure to disclose the method as defined in any of the above, wherein the step of irradiating with artificial light is applied at a periodicity selected from the group consisting of at the beginning of sunrise, during dawn, during sunrise, before sunset, during sunset, after sunset or any combination thereof.
It is further within the scope of this disclosure to disclose the tomato plant as defined in any of the above, wherein the plant exhibits an increased tomato yield of between about at least 2% and about 800%, or more of the at least one yield characteristic, as compared to the control population of same cultivar.
It is further within the scope of this invention to disclose the tomato plant as defined in any of the above wherein the plant exhibits an increased tomato yield of between about 10% and about 200% as the at least one yield characteristic, as compared to the control population of same cultivar.
It is further within the scope of this disclosure to disclose the tomato plant as defined in any of the above wherein the plant exhibits an increased average tomato seed number per plant of between about 10% and about 100%, preferably between about 50% and about 80% higher than the control population of same cultivar.
It is further within the scope of this disclosure to disclose the tomato plant as defined in any of the above wherein the plant exhibits an increased average single seed weight of between about 10% and about 50%, preferably between about 20% and about 40% higher than the control population of same cultivar.
It is further within the scope of this invention to disclose the tomato plant as defined in any of the above wherein the plant exhibits an increased average total seed weight per plant of between about 50% and about 200%, preferably between about 80% and about 150% higher than the control population of same cultivar.
It is further within the scope of this disclosure to provide a method of screening for tomato plants exhibiting improved traits such as improved tomato yield compared to a control population of same, the method comprising the steps of: (a) providing a population of a cultivar or line of tomato seeds or plants; (b) exposing said population (a) of a cultivar or line of tomato plants to a predetermined light treatment by irradiating with artificial light, partly in the presence of ambient daylight, every day for a duration of 2 to 6 weeks from sowing, preferably for 30 days from sowing, wherein the predetermined light treatment consists of irradiating with artificial red light of wavelength in the range of from about 600 nm to about 700 nm for a period of about 60 minutes, starting about 30 min. before sunset in the presence of daylight, and continuing about 30 minutes after sunset. (c) growing the tomato plants in greenhouses for 2-3 months after the end of stage (b), then collecting, counting and weighing the tomatoes and collecting their seeds. d) monitoring at least one improved trait of the tomato plants at the end of step (b) and step (c) and comparing to a control population; (e) selecting at the end of the treatment of step (b) the plants exhibiting at least one improved trait as compared to a control population and selecting again at the end of the growing stage of step (c) the plants exhibiting at least one improved trait as compared to a control population; wherein the number of plants selected in the first generation is from about 1% to about 4%, preferably from about 2% to about 3% of the initial plant population provided (a); (f) propagating by sowing the tomato seeds at least one subsequent generation of the plants exhibiting at least one improved trait as compared to a control population; and (g) repeating steps (b) to (f) on 1-7 subsequent generations, preferably at least two subsequent generations; wherein the number of plants selected in step (e) increases from generation to generation, thereby obtaining a tomato crop plant and/or harvestable part thereof exhibiting at least one improved trait, wherein the improved trait is inheritable for at least one more generation, preferably for two or more generations, more preferably for 3 to 5 generations, and even more preferably for 6 to 8 generations.
This way the best plants are screened and selected for use in further propagations.
According to a further aspect, the method of screening for commercial tomato plants exhibiting increased yield compared to a control population of same as defined in any of the above, comprises steps of applying the steps as defined above to tomato plants or plant parts selected from the group consisting of tomatoes, seeds, seedlings, tissue cultures, calluses, meristems, regenerable cells, protoplasts, potted seedlings, adult plants and any combination thereof.
It is further within the scope of this disclosure to provide the method of screening for a commercial tomato plant exhibiting increased yield compared to a control population of same as defined in any of the above, wherein the control population is selected from the group consisting of: untreated plants, untreated plants of the same generation, treated plants of the same generation and any combination thereof.
It is further within the scope of this disclosure to provide the method of screening for a commercial tomato plant exhibiting increased tomato yield compared to a control population of same as defined in any of the above, wherein the step of irradiating with artificial light is applied at a periodicity selected from the group consisting of: at the beginning of sunrise, during dawn, during sunrise, before sunset, during sunset, after sunset or any combination thereof.
According to a further aspect, there is provided a method of screening for a commercial tomato plant exhibiting at least one inheritable improved trait (such as improved tomato yield) compared to a control population of same as defined in any of the above comprises the steps (a) to (f) of the method of this disclosure, in several consecutive rounds of treatment and selection. The best non-GM tomato plants exhibiting improved traits compared to a control population or line of plants of the same plant cultivar are screened and selected by this process for use in further propagation.
It is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
As used herein, the term ‘about’ refers to a value being ±25% of the defined measure.
The term “commercial” or “commercial tomato cultivar” or “commercial crop” or “commercial crop plant” as used herein refers to a tomato cultivar or a line. In specific embodiments such tomato cultivars or lines have been selected for characteristics such as improved yield, flavor, and resistance to disease or environmental stress. In further aspects they were produced by repeated self-fertilization or inbreeding. The term “improved trait” as used herein refers to beneficial new traits acquired as a result of the treatment of the present disclosure by the treated tomato plants as compared to untreated plants of the same.
The term “control population” as used herein refers to crop plants used as reference or in comparison to crop plants of the same variety, cultivar or commercial line that were exposed to the treatment of the present disclosure. Such control population may include untreated plants, untreated plants of the same generation, treated plants of the same generation and any combination thereof. In some embodiments yield parameters have been compared between treated crop plants and a control population.
According to further embodiments, the term ‘control plant’ or ‘control population’ as used in the context of the present disclosure refers to an untreated tomato plant or a plant population of the same cultivar or variety or line of the corresponding treated plant. In one aspect, the controlled plant did not receive additional light radiation treatment on top of normal (ambient) sunlight. In another aspect, ‘control tomato plant’ or ‘control population’ used in the present disclosure refers to a corresponding plant of the same variety or line of the treated plant, exposed to the same number of treatment cycles as the treated plant, but propagated for one less generation than the treated plant. In another aspect, ‘control plant’ or ‘control population’ used in the present disclosure refers to a corresponding plant of the same cultivar, species or variety or line of the treated plant, and of the same generation of the treated plant, exposed to less treatment cycles.
The term ‘monocrop population’ as used herein refers to a population composed of genetically singular tomato crop plants. In other aspects, a monocrop population refers to crop plants of the same variety, particularly to the cultivation of a single crop cultivar.
The term ‘conditioning’ as used herein refers to the application of at least one treatment regime or protocol as defined herein below, to a predetermined tomato cultivar, to achieve improved yield characteristics or parameters.
The term ‘artificial light’ as used herein refers to any lighting that is not sunlight. In general, artificial light refers to lighting with man-made sources, such as fluorescent, tungsten, mercury vapor, sodium vapor, halogen, compact fluorescent, etc. It can be turned on and off. It is within the scope of the present disclosure that irradiation with artificial light includes exposing the plants to visible light wavelengths, corresponding to 380-750 nm and/or 450-495 nm. The following ranges can be used to distinguish between various light sources: violet (about 380-450 nm), blue (about 450-495 nm), green (about 495-570 nm), yellow (about 570-590 nm), orange (about 590-620 nm) and preferably red (620-750 nm).
The term ‘ambient light’ or ‘ambient daylight’ or ‘ambient sunlight’ as used herein refers to available or normal light or sources of light that are available naturally (e.g. the sun or moon). The term usually refers to the combination of all direct and indirect sunlight outdoors during the daytime. This includes direct sunlight, diffuse sky radiation that may be reflected from the Earth and terrestrial objects. It is within the scope of the disclosure that the outdoor illuminance can vary from 120,000 lux to less than 5 lux (even less than 1 lux for extreme cases). More specifically, the radiation treatment of the present disclosure is optionally applied in the presence of ambient light characterized by luminous flux units of between about 1.5 to about 3000 lux, particularly between about 100 to about 2000 lux, more particularly in the range of about 800 lux to about 1800 lux.
The term ‘dawn’ as used herein generally refers to the time that marks the beginning of the twilight before sunrise. In other aspects, the term dawn, include sunrise, particularly a period of about one hour at the beginning of sunrise. The exact dawn time depends on day length.
The term ‘sunset’ as used herein generally refers to the time of sunset defined in astronomy as the moment when the trailing edge of the Sun's disk disappears below the horizon. The exact time of sunset depends on day length. In some aspects it may refer to a period of about 30 minutes to about 75 minutes before sunset.
The term “lux” as used herein refers to the measuring of luminous flux per unit area, i.e. one lux is equal to one lumen per square meter.
The terms ‘dawn’ or ‘sunrise’ and ‘sunset’ are herein defined by light intensity or units of illuminance or luminous emittance or luminous flux per unit area of between about 1.5 to about 75 lux. It is noted that at sunset and sunrise (on a clear sky), ambient outdoor light may reach between about 400 lux and about 1800 lux.
The term ‘treatment’ as used herein refers to implementation of protocol comprising irradiation cycles with artificial light of wavelength ranges between 380 nm to about 750 nm, for duration of between 5 minutes to 2.5 hours per cycle, preferably one hour. A predetermined session may be defined in terms of cycles, for example, there may be sessions of 5 minutes to 2.5 hours per day at a periodicity of 24 hours apart, for duration of 2 to 6 weeks, preferably one month. A treatment cycle is directed to one generation. According to certain aspects, the aforementioned irradiation treatment is optionally applied in the presence of or in addition to normal or ambient daylight. The aforementioned treatment is applied to at least one tomato crop plant or a crop plant part. In another aspect, the irradiation treatment is applied about one hour at the beginning of sunrise, during dawn or before, during or after sunset or a combination thereof (dependent on day length).
It is noted that different combinations of light radiation treatments were applied to be suitable for the genomic structure and expression of each crop. Examples of treatment cycles used in the present disclosure may include the following:
The term ‘protocol A’ as used herein refers to a treatment protocol where the crop plants or part thereof, were irradiated with white light, for one hour at dawn or during sunset, for about one month from the time of sowing. In specific embodiments, applying protocol A treatment resulted in a decreased yield or lower magnitude of yield increase of the treated crops relative to the control crops.
The term “protocol B” or ‘treatment protocol’ as used herein refers to a treatment protocol where the crops were irradiated with red light (wavelength in the range of 600-700 nm), for about one hour before and after sunset, for about one month from the time of sowing. In specific embodiments, applying protocol B, the aforementioned treatment protocol resulted in an increased yield of the treated crops relative to the control crops.
As used herein, the term ‘progeny’ refers to the descendant(s) of a particular plant. Progeny may result from selfing (i.e., the same plant acts as the donor of both male and female gametes) or from crossing of two different plants. The descendant(s) can be, for example, of the F1, the F2, or any subsequent generation. The terms ‘increased yield’ or ‘high yield’ as used herein refers to epigenetically enhanced lines or cultivars of tomato crops that have an increased tomato crop production or increased percentage of usable plant parts, preferably tomatoes, seeds or biomass.
The ‘yield characteristic’ or ‘yield parameter’ includes at least one phenotypic parameter or characteristic selected from the group comprising average tomato number per plant, average tomato weight per plant, average seed number per plant, average seed weight per plant, plant height, main stem width, stem thickness, tomato weight per plant, average weight of a single seed, plant biomass, number of stems, number of secondary stems, photosynthesis efficiency or rate, nitrogen concentration in the leaves or any combination thereof.
The tomato plants produced by the methods provided in this disclosure exhibit enhanced or improved or improved traits such as increased tomato yield or improved measures of at least one of the parameters or characteristics listed above by at least 2% and up to 800% or more relative to a control plant, or control population.
As used herein, the term ‘population’ means a genetically homogeneous or heterogeneous collection of plants sharing a common genetic derivation.
As used herein, the term ‘variety’ or ‘cultivar’ means a group of similar plants that by structural features and performance can be identified from other varieties within the same species. The term ‘variety’ as used herein has identical meaning to the corresponding definition in the International Convention for the Protection of New Varieties of Plants (UPOV treaty), of Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991.
The term ‘plant cell culture’ or ‘tissue culture’ as used herein means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, tomatoes, seeds, seed coat or any combination thereof.
The term ‘plant material’ or ‘plant part’ used herein refers to tomatoes, leaves, stems, roots, root tips, flowers or flower parts, seeds, kernels, pollen, egg cells, zygotes, seed coat, cuttings, explant or any sample derived from a plant, cell or tissue cultures, or any other part or product derived from a plant or plant part and any combination thereof.
In other embodiments, the term ‘plant material’ or ‘plant part’ also refers to tissue culture of regenerable cells or protoplasts obtained from a plant or from a plant part selected from the group consisting of leaves, pollen, embryos, roots, root tips, anthers, flowers, tomatoes and seeds.
In some embodiments, there is provided a method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait as compared to a control population of plants of the same plant cultivar or line, the method comprising the steps of:
(a) providing a population of a cultivar or line of tomato seeds or plants;
(b) exposing said population (a) of a cultivar or line of tomato plants to a predetermined light treatment by irradiating with artificial light, partly in the presence of ambient daylight, every day for a duration of 2 to 6 weeks from sowing, preferably for 30 days from sowing, wherein the predetermined light treatment consists of irradiating with artificial red light of wavelength in the range of from about 600 nm to about 700 nm for a period of about 60 minutes, starting about 30 min. before sunset in the presence of daylight, and continuing about 30 minutes after sunset.
(c) growing the tomato plants in greenhouses for 2-3 months after the end of stage (b), then collecting, counting and weighing the tomatoes and collecting their seeds.
(d) monitoring at least one improved trait of the tomato plants at the end of step (b) and step (c) and comparing to a control population;
(e) selecting at the end of the treatment of step (b) the plants exhibiting at least one improved trait as compared to a control population and selecting again at the end of the growing stage of step (c) the plants exhibiting at least one improved trait as compared to a control population; wherein the number of plants selected in the first generation is from about 1% to about 4%, preferably from about 2% to about 3% of the initial plant population provided (a);
(f) propagating by sowing the tomato seeds at least one subsequent generation of the plants exhibiting at least one improved trait as compared to a control population; and
(g) repeating steps (b) to (f) on 1-7 subsequent generations, preferably at least two subsequent generations; wherein the number of plants selected in step (e) increases from generation to generation,
thereby obtaining a tomato crop plant and/or harvestable part thereof exhibiting at least one improved trait, wherein the improved trait is inheritable for at least one more generation, preferably for two or more generations, more preferably for 3 to 5 generations, and even more preferably for 6 to 8 generations.
In some embodiments, there is provided the above method of this disclosure for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, exhibiting at least one inheritable improved trait as compared to a control population of plants of the same plant cultivar or line, by monitoring at least one improved trait of said tomato plants, wherein the at least one improved inheritable trait being monitored is selected from the group consisting of tomato yield, tomato number, tomato weight, tomato shelf life, seed number, seed weight, plant height, main stem width, stem thickness, plant biomass, number of stems, number of secondary stems, stress resistance, pest resistance, virus resistance, drought tolerance, herbicide tolerance, delayed senescence, modified color, photosynthesis efficiency, nitrogen concentration in leaves and any combination thereof.
In some other embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved trait of said tomato plants, wherein the at least one improved inheritable trait being monitored is inheritable improved tomato yield.
According to some embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein the at least one improved inheritable trait is an increased tomato yield of at least 2% and up to 800%, or more particularly 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or higher, as compared to the control population of the same tomato cultivar or line.
According to some other embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein the at least one inheritable improved trait is the result of an epigenetic effect.
In some embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein applied to plants or harvestable plant parts selected from the group consisting of tomatoes, seeds, seedlings, tissue cultures, calluses, meristems, regenerable cells, protoplasts, potted seedlings, adult plants and any combination thereof.
In some other embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein said the step of irradiating with artificial light is applied at a periodicity selected from the group consisting of before sunset, during sunset, after sunset and any combination thereof.
According to some embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein applied to tomato cultivars, plant lines or plant types selected from the group consisting of M82, Adoration, Alicante, Azoychka, Beefsteak, Better Boy, Big Beef, Big Rainbow, Blaby Special, Black Krim, Brandy wine, Campari, Celebrity, Cherokee Purple, Canario, Early Girl, Enchantment, Ferris Wheel, Flamenco, Fourth of July, Garden Peach, Gardener's delight, German Johnson, Giulietta F1, Granadero, Great White, Green Zebra, Hillbilly, Japanese, Black Trifele, Jersey Boy, Jubilee, Juliet, Kumato, Lilian's Yellow, McDreamy, Matt's Wild Cherry, Micro Tom, Moneymaker, Monterosa, Montserrat, Mortgage Lifter, Mr. Stripey, Pantano Romanesco, Paul Robeson, Plum Tomato, Pumpkin Tomato, Raf Tomato, Rebellion, Red Currant, Roma, Rosa de Barbastro, Rutgers Tomato, San Marzano, Sasha Altai, Stupice, Tiny Tim, Traveller, Arkansas Traveler, Cherry Bambelo, Cherrry Nebula, Santorini, Super Sweet, Tomaccio, Yellow Pear, White Queen and any combination thereof.
According to some other embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein applied to a non-genetically modified (non-GM) plant.
According to some embodiments, there is provided the above method for producing a non-genetically modified (non-GM) tomato plant and/or a harvestable part thereof, by monitoring at least one improved inheritable trait of said tomato plants, wherein applied to a genetically modified (GM) plant.
In some embodiments, there is provided a tomato plant or a harvestable tomato plant part, preferably a tomato, having at least one improved inheritable trait, wherein obtained by the method of this disclosure.
In some other embodiments, there is provided a tomato plant exhibiting at least one improved trait compared to a control population of tomato plants of the same cultivar or line, wherein produced by the method of this disclosure and wherein said tomato plant preserves its at least one improved trait for at least one subsequent generation, preferably for two or more generations, more preferably for 3 to 5 generations, and even more preferably for 6 to 8 generations, without exposure to additional light treatment.
According to some embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein selected from the group consisting of tomatoes, seeds, seedlings, tissue cultures, calluses, meristems, regenerable cells, protoplasts, potted seedlings, adult plants and any combination thereof.
According to some other embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein produced by irradiating with artificial light of red wavelengths.
In some embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein the at least one improved inheritable trait is an yield characteristic selected from the group consisting of, tomato number, tomato weight, seeds number, seeds weight, plant height, main stem width, stem thickness, plant biomass, number of stems, number of secondary stems, photosynthesis efficiency, nitrogen concentration in leaves and any combination thereof.
In some other embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein exhibiting as inheritable improved trait an increased tomato yield from about 2% to about 800% or more, as compared to a control population of the same cultivar.
In some other embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein the population of a cultivar or line of tomato seeds or plants provided is a non-genetically modified (non-GM) plant population.
In some embodiments, there is provided a tomato plant or harvestable plant part of this disclosure, wherein the population of a cultivar or line of tomato seeds or plants provided is is a genetically modified (GM) plant population.
According to some embodiments, there is provided a harvestable part of this disclosure, wherein it is a tomato exhibiting as improved trait an improved room temperature (17-27° C.) shelf life of at least 45 days after harvesting, as compared to about 10 days for an untreated control tomato.
According to some other embodiments, there is provided a method of screening for a tomato plant cultivar or line amenable to exhibiting at least one inheritable improved trait by applying the method of this disclosure and selecting the best plants exhibiting at least one inheritable improved trait.
The following examples illustrate certain embodiments of the invention but are not meant to limit the scope of the claims in any way. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, percentages are weight per weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
The irradiation treatment in the Examples 1-8 below used red fluorescent lamps of wavelength between 620-750 nm (see
Reference is made to the following non-limiting examples:
Treatment procedure and results of stage F1 (first generation)
Summing up, in the first treatment F1, there was not much difference between the average height of the control plants and the average height of the treated plants. However, the few individual responsive plants in F1 selected for further treatment in F2 were significantly higher than the control plants.
Treatment of 150 plants grown from seeds of F1 treatment began on the day of sowing.
The treatment included red irradiation in the evening, for half an hour before sunset and half an hour into the night. Red fluorescent light lamps of wavelength between 620-750 nm (see
The 150 plants were irrigated 5 minutes twice a day, altogether 400 mm per growing cycle. Each pot was irrigated separately.
The treated group of plants included 89 plants (59% of the initial population provided of 150 plants) and the control group included 50 plants. The selected plants were grown in 12 cm pots arranged on two benches, four pots in a row on each bench.
18-20 treated plants exhibiting an improved trait (in respect of the yield) (20-22% of the selected 89 plants or 12-13% of the initial 150 plants) were selected and taken to the commercial greenhouse for the next round of treatment. The control plants were taken to the commercial greenhouse as is, without selection.
The treated group of plants showed significant trait improvements in comparison to the control group.
Growing conditions in the tomato greenhouses:
Measurement of Phenotypes
Summary of F2 Results:
Tomatoes from F2 treated tomato plants which were stored openly at room temperature (between 17-27° C.) in the shade, but without any cooling or addition of preservatives, exhibited an extended shelf life of at least 48 days (see
The third round of treatment (F3) was performed on seeds and seedlings which received previously two rounds of treatment. The average weight of tomatoes from the two best treated sub-lines TRT 154 and TRT213 (see
The effect of the F3 treatment can be seen in
The seed germination rate is the percentage of seeds out of a certain seed population which on sowing, germinate and turn into plants.
Germination rate is an indicator for the fertility of the seeds from treated plants versus the control seeds an indication if our treatment effect in fertility of the treated seeds. The results in Table 3 show that the treatment of this disclosure significantly improved the fertility of the seeds obtained from F3-F4 treatments.
150 F2 seedlings were divided into 4 groups and were irradiated with light different wavelengths as follows:
The average tomato weights obtained by irradiation at red, blue and white wavelength range are detailed in Table 4 below.
In order to determine if the irradiation method disclosed in the present application (one hour of red irradiation {between 620-750 nm} for half an hour before sunset and half an hour into the night, for one month) results in transcriptomic changes, RNA sequencing assay was carried out on tomato plants treated with said irradiation protocol and control plants. The RNA sequencing assay was performed on F3 tomato plants (treated and control plants).
RNA was extracted from a 50-100 mg of tomato (line M82) leaves using Sigma Spectrum Plant Total RNA Kit (Cat #SLCD4163) according to the manufacturer's guidelines. The resultant RNA was eluted in Nuclease free water (Cat #AM9938, Ambion, USA), quantified and analyzed for integrity using standard protocols.
RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 500 ng of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using JetSeq Beads (Bioline, Cat #BIO-68031). Purified cDNA was end-repaired, adenylated and ligated to Illumina adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37° C. for 15 minutes. Adapter ligated cDNA was purified using JetSeq Beads and was subjected to 11 cycles for Indexing-(98° C. for 30 sec, cycling (98° C. for 10 sec, 65° C. for 75 sec) and 65° C. for 5 min) and enriching the adapter-ligated fragments. Final PCR products (sequencing libraries) were purified with JetSeq Beads, followed by library quality control check. Illumina-compatible sequencing libraries were quantified by Qubitfluorometer (Thermo Fisher Scientific, MA, USA) and fragment size distribution was analyzed on Agilent 2200 TapeStation.
The libraries were paired-end sequenced on Illumina HiSeq X Ten sequencer for 150 cycles (Illumina, San Diego, USA) following manufacturer's instructions.
Raw data pre-processing was done to remove low quality data and adaptor sequences using the Trim Galore tool (www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The high-quality reads were considered for alignment with reference genome [Solanum lycopersicum (solgenomics.net/organism/Solanum_lycopersicum/genome)] using a splice aligner (see Kim, D., Langmead, B., & Salzberg, S. L. hisat2. Nature methods, 2015). Expression calculation was performed at gene level using HTSeq tool (see “HTSeq—a Python framework to work with high-throughput sequencing data”, Anders, S., Pyl, P. T., & Huber, W., Bioinformatics, 31(2), 166-169, 2015). DESeq was used to calculate the differentially expressed genes (see “Differential expression analysis for sequence count data. Genome biology”, Anders, S., & Huber, W., Genome biology, 11(10), R106, 2010). Genes were categorized into upregulated, downregulated and neutrally regulated based on the log 2fold change cutoff of 1 value. This experiment was carried out in triplicates, and was subjected to rigorous and stringent statistical tests and evaluations.
The following tables (Table 5-8) present the genes which are differentially expressed between the treated F3 tomato plants and control plants. Table 5 depicts genes whose expression was upregulated by a 2-4 fold increase in treated plants compared to control plants.
Table 6 depicts genes whose expression was upregulated by more than a 4-fold increase in treated plants compared to control plants.
Table 7 depicts genes whose expression was downregulated by a 2-4 fold decrease in treated plants compared to control plants.
Table 8 depicts genes whose expression was downregulated by more than a 4-fold decrease in treated plants compared to control plants.
In order to determine if the irradiation method disclosed in the present application (one hour of red irradiation {between 620-750 nm} for half an hour before sunset and half an hour into the night, for one month) results in methylation alternations, DNA was extracted from F3 tomato plants (line M82) treated with said irradiation protocol and from control plants, and analyzed as follows:
Genomic DNA was extracted from Tomato leaf samples using standard CTAB extraction protocol. The quantification and quality of the genomic DNA was assessed using Nanodrop2000 (Thermo Scientific, USA), Qubit (Thermo Scientific, USA) and agarose gel electrophoresis.
The immunoprecipitation of DNA was performed using MagMeDIP DNA Immunoprecipitation kit (MagMeDIP, Cat. No.: CO2010020 mc-magme-A10) as per manufacturer's instructions and quantified using Qubit fluorometric assays.
MeDIP-Seq libraries were prepared using ThruPLEXPlasma-Seq Kit (Takara Bio, U.S.A.) at Genotypic Technology Pvt. Ltd., Bangalore, India. Briefly, ing of immunoprecipitated or input DNA processed using the MagMeDIP method for immunoprecipitation and purification (Diagenode, Cat #CO2010020) and quantified using Qubitfluorometric assays, was used as template for end-repair and stem-loop adapter ligation, whereby the 5′ ends of the DNA were only ligated with the adapters, while the 3′ ends were left with a nick. The free 3′ ends of the adapter-ligated DNA fragments were subsequently extended for library synthesis, and Illumina-compatible indexes were added through high-fidelity library amplification. Libraries were purified using 1× JetSeq Beads (Bioline, Cat #BIO-68031), followed by library quality control check. Illumina-compatible sequencing libraries were quantified by Qubitfluorometer (Thermo Fisher Scientific, MA, USA) and fragment size distribution was analyzed on Agilent 2200 Tapestation.
The libraries were paired-end sequenced on Illumina HiSeq X Ten sequencer for 150 cycles (Illumina, San Diego, USA) following manufacturer's instructions.
The MeDIP-Seq analysis was performed by processing the raw data for removal of low-quality data and adaptor sequences. The high-quality reads were considered for alignment with reference genome. Aligned reads were used for identification of enriched regions or methylated regions as peaks and further motifs were identified in the peak regions. Methylated regions were annotated further to identify their genomic locations. This experiment was carried out in triplicates, and was subjected to rigorous and stringent statistical tests and evaluations.
Based on the annotation from the genomic alignments, exons of genes exhibiting methylation patterns were identified. Table 9 shows methylation patterns detected solely in the exons of treated samples (tomato plants exposed to the irradiation protocol disclosed in the present application), which are not detected in the control samples.
Table 10 shows methylation patterns detected solely in the exons of control samples. Since these specific methylation fingerprints are not detected in the same exons from treated F3 tomato plants, it is highly plausible that the irradiation treatment regulates or leads to demethylation events in the same genes of treated tomato plants.
The above examples, tables (Tables 1-10) and experimental results clearly indicate that tomato plants exposed to the irradiation treatment of the present disclosure (one hour of irradiation for half an hour before sunset and half an hour into the night, for one month) exhibit improved inheritable yield-related traits, such as plant height, inflorescence number, plant weight and germination percentage, as well as transcriptomic and methylation differences, which are highly distinguishable from the control plant population.
This application is a Continuation in Part of National Stage Entry of International Application No: PCT/M2019/053516, filed on 30 Apr. 2019, which claims priority to U.S. Provisional Patent Application Ser. No. 62/665,847, filed on 30 Apr. 2019 May 2, 2018, the entire contents of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62665847 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2019/053516 | Apr 2019 | US |
| Child | 17086437 | US |
