RU2815108C2 - Production of grain products - Google Patents

Abstract

FIELD: biotechnology; chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to a method for establishing a time window within a day when the designated female plants are ready to receive the intended male pollen. Disclosed is a method of producing grain.

EFFECT: invention makes it possible to effectively increase crop capacity of an agricultural grain.

20 cl, 3 dwg, 3 tbl, 7 ex

Description

Cross references to related applications

[0001] This application claims priority under U.S. Provisional Patent Application Ser. No. 62/184596, filed June 25, 2015, entitled SEED PRODUCTION, U.S. Provisional Patent Application Ser. No. 62/269496, filed December 18, 2015, entitled SEED PRODUCTION, and U.S. Provisional Patent Application Ser. No. 62/269531, filed December 18, 2015 and entitled "GRAIN PRODUCTION", as well as US Provisional Patent Application Ser. No. 62/269514, filed December 18, 2015 and entitled “GRAIN PRODUCTION”. Contents of U.S. Provisional Patent Applications Ser. No. 62/184,596; 62/269.496; 62/269.531; and 62/269,514 is incorporated herein by reference in its entirety.

Field of technology to which the present invention relates

[0002] The present invention generally relates to new methods for the production of grain products aimed at increasing the yield of agricultural crops. In particular, the present invention relates to on-demand pollination technology applicable to cereal crops and not dependent on the active emission of pollen, neighboring plants or seed mixtures.

Prerequisites for the creation of the proposed invention

[0003] The present invention is applicable in the grain production and plant pollination industries, including (but not limited to) economically important grain crops such as corn, soybeans, wheat, rice, sunflowers, canola, sorghum, barley and switchgrass . The term “cereal production” as used in this application shall mean the production of grain products on a commercial scale using fields of at least 0.8 hectares (2 acres) for such grain production. A traditional grain field is typically occupied by first-generation (F1) hybrid plants that carry the genes of two inbred parents (although a standard bag of grain also contains a small percentage of contaminant, i.e. non-F1, seeds). In other circumstances, the topcross technique is used for the production of grain products. Unlike a traditional field occupied by F1 plants, when using the topcross technique, the following mixture of plants is grown on the field: a hybrid with male sterility (approximately 93%) and a pollen donor plant with male fertility - a carrier of idioplasm with high oil content (7%) . Commercial grain production often depends on field production of hybrid plants. Hybrid plants are the result of fertilization with male pollen from a source with one background genotype of the female reproductive organs of a plant with a different background genotype. Hybridization in grain crops generally provides an increase in yield in commercial production. Currently, corn, rice, sorghum, sunflower and canola are the primary grain crops that provide an advantage when using hybrid seeds. To a lesser extent, other grain crops are grown as hybrids, in particular soybeans, wheat, barley, millet, etc.

[0004] In the case of crops for which grain production is a common practice, existing methods for producing grain products vary somewhat from species to species, but generally, to grow plants that will produce the desired grain products at the stage of maturity of their seeds, they involve sowing fields with seeds of the same variety. In such fields, plants usually self-pollinate or are pollinated by pollen from neighboring plants. There may also be some cross-pollination with pollen from plants of the same species from other nearby fields.

[0005] For the purposes of the present invention and its applicability to the production of grain products, the term “own pollen”, which in the strict sense means only the own pollen of an individual plant, in the present description also includes the concept of “sib pollen”, which in the strict sense means pollen from sibling plants (descendants of the same parents) having the same genetics. Likewise, the term “self-pollination,” which strictly means the pollination of an individual plant by its own pollen, in the present description also includes the concept of “sib-pollination,” which strictly means pollination by the pollen of sibling plants, having the same effect as and self-pollination itself. In a hybrid seed field, selfing and sibling pollination have a selfing effect on seed production that results in reduced yield (compared to cross-pollination with pollen from a male parent from another line) and increased genetic purity in the case of food corn. For purposes of the present invention, the term “cross-pollination” as used herein refers to genetic exchange between plants of two inbred lines or hybrids in adjacent or surrounding fields, and not between plants in the same field. Thus, cross-pollination is the attraction for pollination of pollen taken from other places, which is genetically different from the pollen of plants growing in a given grain production field.

[0006] The advantage of using hybrid seeds for the production of grain products is that hybrids, as is known, tend to exhibit the effect of heterosis, which is manifested in the superior parental vitality of the hybrid; the plant - a heterotic hybrid is a more viable and climate-resistant plant. The result of heterosis is also higher grain yield. The heterosis effect is sometimes also called the “xenia effect” (Stamp et al. (2002) Crop Sci. 42: 1848-1856; Stamp et al. (2002) Maydica 47: 127-134). Heterosis also occurs when a hybrid plant (a plant obtained by crossing parent plants of two different lines) is pollinated by pollen from a plant of another line, which ensures a further increase in yield. Hybrid grain crops are significantly superior in yield to non-hybrid cultivars and also exhibit better susceptibility to fertilizing agents. However, growing season conditions vary from year to year, and environmental pressures, disease outbreaks, and insect pests can significantly impact crop yields.

[0007] Theoretically, cross-pollinating hybrids can provide a yield advantage by eliminating inbreeding depression caused by selfing or sibling pollination, or by creating new gene combinations that provide heterosis effects on grains (seeds). This effect is called the “xenia effect”. “Xenia may be defined as the action of pollen genes on the development of fruits or seeds” (Bulant et al. ((2000) Crop Sci. 40: 182-188).

[0008] Numerous studies have shown the influence of pollen source on grain (seed) development. According to Kiesselbach's earliest report (Kiesselbach, T. A. (1926) Neb. Agric. Exp. Stn. Bull. 33: 1-69; Kiesselbach, T. A. & W. H. Leonard (1932) J. Am. Soc. Agron. 24: 517-523), compared with self-pollination, with cross-pollination, the weight of the grain (seed) increases by an average of 10.1% (by 11.8% in the embryo, by 10.04% in the endosperm and by 3.2% in pericarp. Tsai and Tsai (1990) Crop Sci. 30: 804-808) showed that an early hybrid, when pollinated by late hybrid pollen, has an approximately 30% increase in grain yield and an increase in protein content using inbred corn lines, Bulan et al. (2000) Crop Sci. 40: 182-188 report a 13% weight advantage for cross-pollinated kernels (seeds). compared to those produced by self-pollination. Breeding research conducted at South Dakota State University confirms that cross-pollination of specific hybrids can increase grain size (seed) and protein content, and that cross-pollination between hybrids of similar maturity accounts for 40% up to 60% of grains produced in mixed stands (Wicks III, Z., (1994) Proc. Annual Corn and Sorghum Res. Conf. 4.

[0009] Grain (seed) development can be altered by cross-pollination (Tsai, C. L. & C. Y. Tsai (1990) Crop Sci. 30: 804-808; Poneliet, C. G. and D. B. Egli, (1983) Crop Sci. 23: 872-875 ). The cited work by Poneliet and Egli in 1983 (see Poneliet, C.G. and D.B. Egli, (1983) above) showed that the duration of the effective filling period in the case of cross-pollination was longer than in the case of self-pollination. Pollen source also influences endosperm development in terms of protein content, amino acid profile and translucency (Pixley, K.V. and M.S. Bjarnason (1994) Crop Sci. 34: 404-408; Bulant et al. (2000) Crop Sci. 40: 182- 188). On the fourteenth day after pollination, the benefit from cross-pollination was expressed in an average increase in starch content by 28.8%, an increase in the activity of ADP (adenosine diphosphoric acid)-glucose pyrophosphorylase (enzyme code EC 2.7.7.27) by 24.8% and an increase in the activity of neutral invertase (EC 3.2.1.26) by 24.1% (Bulant et al. (2000) Crop Sci. 40: 182-188). Tsai et al. (1991) J. Sci. Food Agric. 57: 163-174 modified endosperm P3732 through cross-pollination, which significantly increased grain weight, grain protein content and yield over a range of nitrogen applications. fertilizers Additional nutrients that were translocated into the developing grains of cross-pollinated P3732 plants mainly came from increased duration of dry matter production and input of nitrogen into the cenosis by vegetative plant tissues (Tsai et al. (1991) J. Sci. Food Agric. 57: 163-174 ). These well-recognized effects on grain composition are the basis for the “topcross” technique for producing high-oil corn. The "Topcross" system for producing high-oil corn is a technique used in the 1990s and early 2000s where high oil content was provided by the combination of a male-fertile hybrid (approximately 93%) and a male-fertile pollen donor plant. germplasm of high oil content (7%). The result was an increase in oil content from approximately 3-4% (as in conventional commercial corn) to approximately 6% in high-oil topcross grains. These high-oil grain products were sold at a premium price on the grain exchange (Thomison, P. R. et al. (2002) Agron. J. 94: 290-299).

[0010] The degree of manifestation of the xenia effect differs between male and female genotypes. The greater the genetic difference between the pollen source plant and the female plant, the greater the expected response to cross-pollination (Leng, E.R., (1949) Agron. J. 41: 555-558; Bulant, C. and A. Gallais, (1998) Crop Sci 38: 1517-1525). For simple hybrids, the advantage of cross-pollination was weaker than for their inbred parents, and this advantage varied depending on the male plant. For hybrids obtained by crossing inbred plants, the advantage was 13.8% and 14.5%, and for plants obtained by crossing their hybrids, the advantage was only 2.5% (Bulant, S. and A. Gallais, (1998) Crop Sci. 38: 1517-1525). The outcome of cross-pollination is influenced by both the properties of the pollen and the properties of the mother plant (Seka, D and H.Z. Cross (1995) Crop Sci. 35: 80-85; Seka, D. et al. (1995) Crop Sci. 35: 74- 79).

[0011] The results of cross-pollination between hybrids observed by Bulant and A. Gallais (1998) Crop Sci. 38: 1517-1525 indicate that cross-pollination can increase the drainage strength of the entire ear and that the advantage in terms of grain mass can be ensured in unfavorable circumstances. Positive xenia effects were interpreted in terms of source-sink relationships. If resources are limited, then an increase in the flow force leads to an increase in the average weight of grain with mixed pollination compared to the case of pure self-pollination. No relationship was found between cross-pollination advantage and the average weight of seed produced by selfing of a male or female pollen source. The advantage of cross-pollination was favorable for both small grains and large grains (Bulant, S. and A. Gallais, (1998) Crop Sci. 38: 1517-1525).

[0012] The success of pollination is critical to the yield of cereal crops. This yield is measured in the mass obtained per unit area of grain production at a given moisture content of the latter (for example, for corn the standard moisture content is set at 15.5%). A low degree of pollination leads to low grain yield. Therefore, grain producers usually rely on self-pollination and pollination by neighboring plants in this field, since they know that pollination will occur over the correct period. window when the female reproductive components of the plant are ready to accept pollen. Unfortunately, self-pollination does not guarantee maximum grain yield, and failure cannot be attributed to changing conditions and stresses that could affect the plant during the growing season.

[0013] Thus, there is a need in the industry for an invention that satisfies one or any combination of the following requirements: higher grain yield, improved seed composition, increased genetic purity of seeds, improved seed characteristics, reduced genetic contamination. The proposed invention provides for the creation of a method for increasing the yield of grain crops through targeted cross-pollination of plants producing grain products. In addition, the present invention provides a method for increasing grain size and changing grain composition through special cross-pollination using pollen with a different background genotype, a method that also provides the ability to make real-time production decisions in connection with problems caused by conditions prevailing in pollination period, or other production problems. This method also makes it possible to reduce unwanted genetic contamination of grain products collected from the field. In addition, the present invention also provides a method for maximizing the synchrony of pollination with one's own pollen or sibling pollen, thereby reducing genetic contamination from unwanted cross-pollination, this is especially applicable to the so-called organic farming industry. These two methods can be combined to simultaneously increase yield by changing grain size and composition, and reduce genetic contamination.

[0014] The invention described here would provide a 5% increase in corn yield (an admittedly low estimate), the annual income from the proposed invention, applied to 33% of most highly productive land, would be US$1.1 billion throughout the chain value creation only in the USA. This calculation is based on US corn production of 345 million metric tons (hereinafter referred to as mmt) in 2015 (U.S. Corn Growers Association (2016) World of Corn [online, retrieved June 13, 2016], taken from the Internet:

http://www.worldofcorn.com/#us-corn-production), and the price of corn on the grain exchange is 185.04 US dollars per metric ton (hereinafter referred to as “mt”) (June 13, 2016, corn price [online, retrieved June 13, 2016] taken from the Internet:

https://www.ams.usda.gov/market-news/livestock-grain-market-news-publications) (Calculation: $185.04/mt×345 mmt×5%×33%). Similarly, the proposed invention could provide a 5% increase in rice yields, which, using 10% of the available acreage, mostly on highly productive land, would generate a global annual income of US$1.5 billion. This calculation is based on a global rice production of 746 million metric tons (mmt) (GeoHive (2016) [online] World: Rice production in metric tons, retrieved June 20, 2016. Retrieved from the Internet: http://www. geohive.com/charts/ag_rice.aspx),

and Grain Exchange Rice Price 408.91 per metric ton (mt) (Grain Exchange Paddy Rice Price May 2016 [Online], retrieved from the Internet on June 23, 2016. Retrieved from the Internet:

http://www.indexmundi.com/commodities/?commodity=rice&months=60) (Calculation: $408.91/mt×746 mmt×5%×10%).

Brief description of the attached graphic materials

[0015] In FIG. 1 is a photograph of an ear of corn produced from hybrid H2 and pollinated with pollen from pollen donor P2, as described in Example 2. The photograph is in black and white. In the color version of this photo you can see that the lighter grains are yellow in color and the darker grains are purple in color. This ear demonstrates the successful cross-pollination that has occurred throughout the ear. Self-pollination produced light grains, while cross-pollination produced dark grains. This ear allows us to determine the difference in yield between these two types of pollination.

[0016] In FIG. 2 is a photograph of an ear of corn produced from hybrid H2 and pollinated with pollen from pollen donor P2, as described in Example 2. The photograph is in black and white. In the color version of this photo you can see that the lighter grains are yellow in color and the darker grains are purple in color. This ear demonstrates the successful cross-pollination that has occurred throughout the entire ear. Self-pollination produced light kernels and cross-pollination produced dark kernels, and it can be seen that the proportion of kernels resulting from cross-pollination is higher than in the ear shown in Fig. 1. This cob allows you to determine the difference in yield between these two types of pollination.

[0017] In FIG. 3 is a photograph of an ear of corn produced from hybrid H2 and pollinated with pollen from pollen donor P2, as described in Example 2. The photograph is in black and white. In the color version of this photo you can see that the lighter grains are yellow in color and the darker grains are purple in color. Self-pollination produced light grains, while cross-pollination produced dark grains. This ear shows a very high degree of cross-pollination (more than 85%).

Brief description of the proposed invention

[0018] The subject of the present invention is a method for producing commercial grain products of any plant species by targeted cross-pollination of a grain plant with pollen from a plant with a different background genotype. The proposed method ensures an increase in yield due to the heterosis effect, otherwise called the xenia effect. This method has the additional advantage of being able to change the characteristics of the grain, including its content and composition.

[0019] The subject matter of the present invention is also a method of increasing self-pollination and/or sibling-pollination levels in commercial grain production, comprising the operation of targeted application of pollen from a designated male parent plant (with the same background genotype as the background genotype of the designated female parent plant) on assigned female parenting development one or more times. During targeted pollen application, the designated female parent plant is in a state where its female reproductive components are ready to accept pollen. As for the male reproductive components of this designated female parent plant, during such targeted pollen application they may or may not be capable of releasing pollen. In the event that these male reproductive components emit pollen, some female reproductive components will receive targeted pollen from the designated male parent plant, while others may receive their own pollen or pollen from nearby offspring of the same parents, resulting in an overall increase successful pollination events, thereby reducing genetic contamination due to unwanted cross-pollination from extraneous sources.

[0020] One embodiment of the present invention is a method of increasing grain size and changing grain characteristics, including its content and composition, through special cross-pollination. For example, the oil, protein, or starch content of the grain may be changed, and the specific components of the oil, protein, or starch contained in the grain may also be changed. This method provides the ability to make real-time production decisions related to solving specific problems during pollination and provides the farmer with the ability to solve production problems in the field. During targeted pollen application, the designated female parent plant is in a state where its female reproductive components are ready to accept pollen. As for the male reproductive components of this designated female parent plant, during such targeted pollen application they may or may not be capable of releasing their own pollen. In the event that these male reproductive components emit pollen, some female reproductive components will receive targeted pollen from the designated male parent plant, while others may receive their own pollen or pollen from nearby offspring plants of the same parents. In the event that the male reproductive components of a designated female parent plant do not emit pollen, the majority of its female reproductive components will receive targeted pollen from a designated male parent plant with a different background genotype, resulting in a heterosis effect, or xenia effect, accompanied by an increase in grain size (seed). ), as well as targeted changes in grain content or composition.

[0021] Both of these methods may also be useful in reducing genetic contamination of grain due to cross-pollination from an undesirable pollen source. In addition, these methods can be combined to provide both an increase in grain yield and grain size and a change in grain characteristics.

[0022] These methods are applicable to a wide range of plants, including, but not limited to, corn, soybeans, rice, sunflowers, canola, sorghum, cotton, barley, and switchgrass. Application of the pollen of the designated male parent plant may be accomplished by automatic means, mechanical means, pressurized means, reduced pressure means, manual means, semi-automatic means, or combinations of the above. In addition, pre-canned or fresh pollen from the male parent plant designated as a source of pollen can be taken for pollination. This pollen may be taken from one or more of the following sources: in the field, in a growth chamber, in a grow house, in a greenhouse, in a shade house, in an arched greenhouse, in a vertical cropping setup, or in a hydroponic setup. Preserved pollen can be obtained by any means that allows the pollen to remain viable, such preservation can be achieved through various forms of refrigeration or freezing, including freezing, cryopreservation, freeze-drying, storage in liquid nitrogen, etc. In addition, pollen, either fresh or preserved, may be collected from a source with an altered circadian rhythm, from a source with normal circadian flowering but in which the emission of pollen by said male reproductive components of said designated female parent plant is delayed, or from such a source from which said male reproductive components of the designated female parent plant are allowed to emit pollen without delay. Pollen, fresh or preserved, may be taken from one or more sources and may be combined with pollen from other sources before application.

[0023] In addition, the proposed invention also provides for the creation of a method for increasing the production of grain products. This method involves the targeted application of specially selected pollen from a genetically unrelated source and provides the ability to make specific agronomic decisions in real time when selecting genetically unrelated sources. This method provides the ability to maximize seed performance under any specific environmental conditions or market needs for any crop of interest. For example, in one embodiment of the present invention, specifically selected pollen is obtained from sources that provide pollen application that is optimal in terms of environmental conditions, including both abiotic and biotic environmental factors. In another embodiment of the present invention, specifically selected pollen is obtained from a source that provides pollen application that is optimal for plant performance (eg, yield testing) or for exchange and market prices. In yet another embodiment of the present invention, specially selected pollen affects such characteristics of grain products collected from the female plant as the content and composition of the grain, including (but not limited to) the oil content and composition of the grain, the starch content and composition of the grain and content and composition of protein in grain.

[0024] According to one embodiment of the present invention, the designated female parent plants may be either male sterile or male fertile, and pollen application may occur before or at the same time as the designated female parent plant's own pollen emission. the time when a plant is receptive to its own pollen or the pollen of neighboring plants with the same genotype. In addition, the proposed method may involve the use of male plants - sources of specific pollen that are optimal in terms of environmental conditions or market trends and provide seeds with more valuable specific composition (i.e., starchiness, oiliness), as well as qualities such as drought resistance , tolerance to flooding, etc. In addition, pollen can be taken from a single source or from a combination of sources to ensure different combinations of traits in the seeds.

Detailed description of the proposed invention

[0025] The following is a detailed description of an embodiment of the proposed technology and methods for increasing the yield of grain crops and changing the size, content and composition of the grain (seed). Such technologies and methods can be used in relation to any plants from which it is desirable to obtain grain products. To facilitate discussion and understanding in the following detailed description of the present invention, it will be frequently illustrated by its application to corn. It should be understood that the proposed technology and methods can be applied to any plants, and corn or other specifically named plants are used for illustration purposes only and are not limited to the scope of the present invention. The proposed technology can be used in relation to any plant - a source of grain products, including hybrids, non-hybrids, plants with male sterility and plants with male fertility.

[0026] Cereal products are produced for a variety of purposes, including for human consumption, animal feeding, industrial use, new planting, and research purposes. When producing grain products, they strive, first of all, to ensure high yields and high quality grain products. Seed companies are pouring billions of dollars into research in an effort to develop plants with the best set of genes for increasing crop yields. Regardless of the end use of the grain product, its production is dependent on proper pollination of the designated female parent plant with pollen from the designated male parent plant at the appropriate time.

[0027] The synchronicity of the pollen availability of the designated male parent and the receptivity of the designated female parent is herein referred to as reproductive synchrony. Reproductive synchrony is easily achieved by self-pollination or by pollination by neighboring plants of the same species. However, it is much more difficult to achieve reproductive synchrony between different varieties, which may mature at different times or depending on different temperatures, as well as other environmental factors. A grain producer may be interested in pollinating a crop with pollen from a variety that could impart specific characteristics to the final product. This usually cannot be achieved unless the following conditions are met: (1) the grower knows at the beginning of the growing season which variety of pollen he wants to use for pollination, (2) the varieties emit pollen and are susceptible to pollen at the same time, and ( 3) varieties are planted in close physical proximity to each other. Thus, if a grower, for example, has experience of drought and would like to pollinate with pollen a variety that shows good yield even in dry periods, then he will not be able to do this, unless it is a variety that has already been selected for planting and growing and is already in his field and also releases pollen at the appropriate time. Typically, this grower does not pollinate the crop with pollen from a variety that could help overcome problems with low yields due to drought and has a different ripening period from the variety already grown. The present invention can overcome all three of these potential barriers to reproductive synchrony, thereby providing the grower with a wider choice of pollen, as well as the ability to make production decisions in real time.

[0028] Synchronous pollination within and between ears is particularly important in corn because it has been found to improve kernel set, which is a critical factor in increasing yield. Carcova et al. (2000) Crop Sci. 40: 1056-1061) showed that synchronous pollination of the opened pistils on the apical and pre-apical ears five days after the emergence of the pistillate styles results in an increase in the number of grains per plant.

[0029] Close synchrony in the flowering of male and female flowers and a minimum density of pollen per opened pistil are fundamental requirements for achieving high seed yields and genetic purity. In conditions of reduced pollen viability, in cases where the pistillate phase is delayed relative to pollen emission, or where there is a high risk of inbreeding of male and female plants, previously collected and preserved pollen or fresh pollen can be applied to susceptible pistils for a period of seven to ten days, thereby guaranteeing setting seeds. A high risk of inbreeding occurs when the development of male and female flowers is perfectly synchronous (that is, when the peak of pollen emission from male flowers coincides with the peak of pistillate emergence from female flowers), so that pollen grains will produce successful pollination, resulting in fertilization. For seed producers, this peak coincidence occurs when 50% of the male population begins to emit pollen on the same day that 50% of the female population begins to produce pistillate styles.

[0030] A real-time production decision refers to a decision that is made during the growing season and maturation of a crop, and not a decision made at the planning stage before the crop is planted. Grain producers choose which variety of grain crop to grow each production season based on characteristics that are optimal for the local climate, soil, growing season conditions and other factors. Once the decision is made and the crop is planted, growers make real-time decisions during the crop's growing season, such as when to spray to suppress weeds, treat diseases, or control insect pests. Such decisions have to be made as the crop grows in response to specific conditions and problems that arise during the growing season of the crop being grown. However, prior to the creation of the present invention, grain producers could not make real-time decisions such as pollen selection, because the pollen involved in the production of grain products comes from plants that are already in the field. Thus, the choice of pollen is usually already made at the time of choosing a variety for cultivation, even before planting, without any information about the specific conditions that may arise later during the growing season and affect the yield and safety of grain products.

[0031] The subject of the present invention is a method for the production of grain products, containing the operations of collecting, storing and delivering pollen from male plants to female plants (hereinafter, the delivery of pollen should be understood as its delivery to the reproductive components of the designated female plant and applying this pollen to them). The industry knows several methods for collecting pollen. For example, US Pat. No. 4,922,651 discloses a device for performing or increasing the efficiency of plant pollination.

[0032] In addition, several methods for delivering pollen are known in the industry, particularly from US Pat. No. 4,922,651. In addition, the industry knows various methods for storing pollen. For example, US Pat. No. 5,596,838, which describes a method and apparatus for preparing pollen for cryopreservation, states that pollen can be stored for months while remaining viable. Although pollen delivery methods are known in the industry, these methods do not directly or indirectly direct or incentivize the user to target pollen delivery in such a way that the grower benefits from the effect of heterosis while also providing the ability to make real-time decisions based on current crop yields. conditions. The result of the present invention is pollination carried out before the active period when the plant would be exposed to self-pollination and/or the risk of pollination from an undesirable pollen source, or during the active period when the plant is exposed to self-pollination and/or pollination from another pollen source, with thereby increasing the efficiency of pollination. The proposed invention increases the effect of heterosis and the ability to make decisions in real time. The present invention does not require the use of male sterility or isolation, although both may be used without departing from the scope of the present invention.

[0033] As discussed above, the present invention is applicable to the general practice of growing plants to produce seeds that will germinate and become seed plants producing grain products that can be sold to a grain elevator or other buyers, used for animal feeding, or in research programs . However, rather than relying on self-pollination or pollination by neighboring plants, as is currently the case, the present invention provides a better alternative by increasing the rate of successful self-pollination and/or sibling pollination and/or by changing the level of cross-pollination by targeted application of pollen male plants to female plants at a specific time. In the context of this application, the characteristics “targeted”, “targeted” in relation to pollination means the special application of pollen (to the female reproductive components of the plant) in a manner that does not involve natural pollination by wind, insects or other natural factors. Purposefully applied pollen is pollen applied to a plant as a result of intentional human activity, human decision, or human intervention, and may be applied by hand or other means. One example of targeted pollination is "prescriptive" pollination, which consists of using pollen to respond to specific needs or conditions in an industry. Which specific pollen should be used and when its targeted application should be carried out depends on the need or on the conditions that must be taken into account.

[0034] The acquisition of pollen from a designated male parent plant (hereinafter may sometimes be referred to simply as "male pollen" for short) required to produce seeds to be used for grain production may be accomplished through a pollen bank. A pollen bank is a source of stored pollen, previously collected from one or more pollen sources and stored under conditions such that its viability is ensured. The plants that were used as a source of pollen for such a pollen bank could be grown and brought to maturity for pollen collection in any environment, including, but not limited to, in the field, in a growth chamber, in a grow house, in a greenhouse, in a shade house, in an arched greenhouse, in a vertical crop growing installation, in a hydroponic installation, etc. Pollen from a pollen bank can have different origins. For example, in one embodiment of the invention, fresh pollen can be collected from male plants grown in a controlled environment whose circadian rhythm is ahead of female plants grown naturally in the field by a time interval of two to eight hours. This method will be described in more detail below. According to another embodiment of the present invention, the pollen that is stored in the pollen bank may be canned pollen collected days, weeks, months or years before it is released from the bank for pollination purposes. Preserved pollen can be obtained by any preservation methods that ensure the preservation of its viability, for example (but not limited to) by refrigeration or freezing, including (but not limited to) freezing, cryopreservation, freeze-drying, aging in liquid nitrogen.

[0035] According to one or more embodiments of the present invention, pollen can be obtained from an anther studio. The anther studio provides optimal growing conditions for the reproductive tissues of male plants of any species or variety. These tissues (eg corn panicles) are cut from plants grown under standard outdoor conditions, such as a field, or under controlled conditions, such as a greenhouse or growth chamber. The preferred solution seems to be to cut these tissues from the plant when it begins to emit pollen and place it in a pollen studio. This tissue can then be cultured in a nutrient medium that allows for further growth. This anther studio may cycle through at least one of the following: special lighting, temperature, and/or humidity, allowing the tissue to continue to grow. This growth can be regulated, that is, it can be accelerated or retarded, thereby regulating the duration of the pollen state. With this solution, it is possible to have pollen available on request for pollination, which can be carried out at any time of the day or night. This has practical value for the introduction of several beneficial and valuable technologies related to the production of seeds and grain products in terms of pollination. This solution also provides concentrated sources of pollen for canning. Any pollen collected in a pollen studio and preserved could be used in the same way as freshly collected pollen, but long after the fresh pollen that was not preserved has died (R.I. Grayson (1994) Maize inflorescence culture , p. set in maize. The influence of silk, pollen, and ear-learf water status and tassel heat treatment at pollination, Plant Phyiol. 83: 121-125).

[0036] Mechanical delivery of pollen from the designated male parent can be accomplished as soon as the designated female parent becomes receptive to pollen, which is always before the designated female parent's own pollen becomes viable on any given day, thereby ensuring successful cross-pollination of all female plants susceptible to pollen. In other words, the female reproductive components of the designated female plant became open to receiving pollen from the designated male plant before the male reproductive components of the same female plant became ready to produce pollen. In corn, female plants are receptive to pollen when the pistils are open to receive pollen. Pistils are receptive to pollen before the cob emerges from the leaf sheath and remains susceptible for many days after emerging from the leaf sheath. Additionally, in corn there are two possibilities: pollen can be emitted before the pistils emerge (protandry), or pollen can be emitted after the pistils emerge (protogyny). In both cases, the pistils will be receptive to pollen before releasing pollen on a given day. The present invention is applicable in both of these cases. Additionally, there are situations where pistils are receptive to pollen all day long for about seven days. Accordingly, the pollen can be purposefully applied any number of times, for example (but not limited to) once a day, twice a day, or continuously. Alternatively, mechanical delivery of the designated male parent's pollen may occur while the designated female parent plants are receptive to it, and while the designated female parent plant's own pollen becomes viable on any given day, thereby allowing both self-pollination and self-pollination. and pollination by pollen from the designated male parent plant. Unless male sterilization is resorted to, such as in corn, the female parent's own pollen usually begins to be emitted in the mid-morning hours and continues until the late morning hours or early afternoon hours, after which all of the female parent's own pollen finds receptive female reproductive components and germinates to ensure successful self-pollination, or dies within four hours or less if this fails (Luna V. et al. (2001) Crop Sci. 41 (5): 1551-1557). Thus, the female corn plant has a window during which pollen from the designated male parent plant can be delivered to it and during which the female plant's own pollen is not emitted or is not viable, or a combination of self-pollination and cross-pollination occurs. The present invention provides the ability to cross-pollinate nearly all viable female reproductive components before or after the designated female parent plant's own viable pollen has emerged, depending on the grain grower's preference and field situation.

[0037] Accordingly, one aspect of the present invention is schedule of pollen delivery to the female reproductive components of the designated female parent plants. All crops have a daily cycle of pollination window (a period of time during which the female reproductive components of a pollinated plant are receptive to pollen and during which pollination can be successful) in which female plants mature and grow relatively continuously, while male plants have a different cycle in which pollen becomes viable and is emitted, or the anthers open, which begins in mid-morning and ends in the late morning or early afternoon. In some cases, the designated female parent plants' own pollen may become viable before those plants' female reproductive components become fertile (ready to be fertilized), but there will always be a first morning on which those female reproductive components become fertile before those plants' own pollen plants will begin to emit on this day. Therefore, the female plants' own pollen, which may have been emitted the day before, is no longer alive and has become nonviable before the female reproductive components become fertile the next morning. Therefore, the period from the early morning hours to mid-morning is ideal for applying pollen from the designated male parent plants to cross-pollinate all the female plants before their own pollen is released, although it is also possible to cross-pollinate the pollen before the female plants emit self-pollen is applied over several days, or several times a day, or continuously and even under conditions of emission of self-pollen, to ensure the likelihood of increasing the incidence of heterosis and to maximize the yield of the crop. According to one embodiment of the present invention, pollen delivery can be carried out at six o'clock in the morning (6.00). But even when delivering pollen at any time of the day, the proposed invention provides a great advantage over traditional technologies.

[0038] When pollen is delivered at the proper time within During the fertile window of the plant, adequate pollination can be achieved. The present invention provides for the first time the efficient production of grain products without relying only on self-pollination or pollination by pollen of neighboring plants, which during pollination may be undesirable due to stresses caused to the crop by biotic and abiotic environmental factors. Sources of unwanted biocompatible pollen may hereinafter be referred to simply as “undesirable pollen” for brevity. The present invention enables targeted pollination so that when unwanted pollen begins to be emitted, all fertile designated female parent plants have already been pollinated from the selected pollen source, and the unwanted pollen, not finding female plants ready to fertilize, dies after a short period of time depending on the plant species and environmental conditions (Luna V. et al. (2001) Crop Sci. 41 (5): 1551-1557; Stanley, RG & Linskens, HF (1974) Pollen: Biology, Biochemistry, Management. Springer-Verlag, Heidelberg. ; Shivanna, KR (2003) Pollen Biology and Biotechnology. Science Publishers, Inc.), thereby avoiding pollination of the designated female parent plants from a source of unwanted pollen. This would apply to situations where the unwanted pollen is considered to be one's own pollen or pollen from neighboring plants. Different species have different periods of pollen viability, which can be influenced by environmental conditions (Dafni, A. & D. Firmage (2000) Plant Systemics and Evolution 222 (1): 113-132). High humidity and low temperatures can prolong the period of pollen viability. For example, rice (Oryza sativa) has been found to have only four minutes of pollen viability (Koga et al. (1971) Cytologia 36: 104-110), or 50% of the pollen to die within up to 20 minutes (Khatum, S . and T. J. Flowers (1995) J. Exp. Bot. 46: 151-154). In contrast, pollen from field-grown radish (Raphanus sativas) was found to remain viable for five days (Siddiqui, V.A. (1983) Acta Bot. Ind. 11: 150-154).

[0039] The ability to deliver viable pollen on demand to ensure timely pollination of receptive pistils removes a number of limitations common to field-scale grain production. Namely, the use of the present invention solves the problem of low levels of pollen production and eliminates problems caused by insufficient synchrony of flowering of male and female flowers.

[0040] Pollen from the designated male parent can be delivered to the female reproductive components of the designated female parent in a variety of ways, including (but not limited to) entirely manually, manually using a small hand-held device for semi-automatic pollen dispersal, using field-driven technical means equipped with mechanisms for dispersing pollen or using fully automatic dispersal of pollen using self-propelled and (or) human-guided vehicles, such as unmanned aerial vehicles (drones) equipped with a device for dispersing pollen, and the dispersal of pollen is carried out automatically means, including (but not limited to) mechanical means, means of creating increased pressure, means of creating reduced pressure, or pneumatic means. The use of drones would be a particular innovation and would seem practical from the point of view of carrying out the method according to the invention. Small drones, the use of which does not require administrative regulation, could be directed to spray pollen directly over designated female parent plants using a GPS system. It has been estimated by the present inventors that with any of these pollen delivery methods, approximately 140 g (5 oz) of male pollen delivered over three to four days is sufficient to successfully cross-pollinate one hectare (2.47 acres) of female corn parents. contract. This estimate is based on literature indicating how many corn pollen grains there are per 1 mg, and estimating that for successful pollination there should be three to five pollen grains per pistil (three to four pollen grains per pistil is the generally accepted amount among specialists necessary to ensure successful pollination (M.E. Westgate, J. Lizaso, W., Batchelor (2003) Quantitative relationships between pollen shed density and grain yield in maize, Crop Science 43: 934-942; M. Uribelarrea, J. Carcova, M.E. Otegui, M. Westgate (2002) Pollen production, pollination dynamics, and kernel set in maize, Crop Science 42: 1910-1918). See, for example, Porter (1981) Environ. Health Perspectives 37: 53-59; Miller (1982) In Maize for Biological Research. W.F. Sheridan (ed.), pp.279-293). The timing of pollen delivery, the amount of pollen required and the number of days over which pollen should be delivered can be adjusted as necessary depending on circumstances such as crop type and meteorological conditions (i.e. dominant stand height, plant density, rainfall frequency and their abundance, wind speed and direction, etc.). The proposed method can be adapted, for example, for soybeans, which have a longer pollination period compared to corn. In addition, it is expected that the amount of pollen required for pollination to produce hybrid seeds can be reduced due to the efficiency of the pollen delivery methods mentioned above. The inventors estimate that by using these more efficient methods, pollen use could be reduced to a thousandth (1/1000) or even less of the amount of pollen currently produced in the hybrid seed industry. Research conducted by the authors of the present invention indicates that to ensure a complete set of corn grains per pistil, approximately 3000 pollen grains are required, emitted by corn panicles onto the field. Hand pollination studies and open field tests have shown that three to four pollen grains per pistil are sufficient to ensure a full complement of corn kernels. (M.E. Westgate, J. Lizaso, W., Batchelor (2003) Quantitative relationships between pollen shed density and grain yield in maize, Crop Science 43: 934-942; M. Uribelarrea, J. Carcova, M.E. Otegui, M Westgate (2002) Pollen production, pollination dynamics, and kernel set in size, Crop Science 42: 1910-1918). The result of this is the conclusion that a thousandth (1/1000) of the amount of pollen currently produced in the hybrid seed industry is sufficient.

[0041] A person skilled in the art can easily modify the above options if the need arises. For example, in some circumstances it may be advantageous to conduct pollination at night. In other cases, it may be beneficial to pollinate throughout the day. In corn, pollination efficiency increases when pistil health is at optimal levels and viability is at its maximum, which often coincides with conditions where there is no heat and high humidity - meaning that the pistils will not dry out quickly. Based on these weather conditions, the corn grower has the opportunity to choose the time to pollinate the corn so that the health of the pistil is at an optimal level and its viability is maximized.

[0042] The present invention can be applied to any agricultural crop in order to increase or ensure the production of grain products. It is applicable in any environment, including (but not limited to) ideal or targeted growing environments, out-of-season growing environments, or controlled growing environments (e.g., shade house, greenhouse, grow house, arch greenhouse, in a growth chamber, in an installation for vertical cultivation of crops, in a hydroponic installation, in an aeroponic installation, etc.). Application of pollen from the designated male parent plants to the female reproductive components of the designated female parent plants can be done as soon as the latter are receptive to pollination and regardless of the viability of the female parent plants' own pollen or other unwanted pollen. The present invention can also be used to increase the efficiency of methods currently used in the production of grain products even in the presence of all normal circumstances, thus ensuring an increase in the yield of grain products in problem situations.

[0043] According to one of its embodiments, the present invention can be used to prevent or reduce losses caused by drought. This represents an advantage for everyone involved in this sector of agricultural production, including both direct producers and insurers. During drought, plants experience stress. When stressed, a plant often produces only male reproductive components. Accordingly, there is pollen, but no female reproductive components to accept it. Sometimes a plant produces female reproductive components later, but at this time the pollen is no longer viable and is not suitable for pollination. When using the method according to the invention to stressed plants, when their female reproductive components are ready for pollination, pollen can be delivered from a pollen bank or from a controlled environment, and thus, at least to some extent, the harvest of grain products can be ensured.

[0044] In addition, according to another embodiment of the present invention, with respect to pollen intended for pollination, research or production decisions can be made in real time to ensure the production of crop plants with characteristics that are desired by the manufacturer, including (but not only) increased oil content of grains, decreased oil content of grains, increased starchy grain content, reduced starchy grain content, increased drought resistance, resistance to flooding or other factors of excess moisture, increased productivity in conditions of high temperatures, increased productivity in conditions of low temperatures, increased resistance to fungal diseases, increased resistance to insects, increased productivity, increased pollination in case of non-synchrony of the reproductive cycles of male and female reproductive components. Any of these objectives is achieved by selecting a pollen source specifically optimized to maximize yield under appropriate conditions from the crop of interest. With a range of pollen sources stored in a pollen bank, the user has the opportunity to make research or production decisions of this type immediately before applying pollen to the female reproductive components of the pollinated plant. In particular, pollen from the designated male parent plant may be applied before self-pollination has begun or conditions for pollination by neighboring plants have occurred, or simultaneously with self-pollination or potential pollination by neighboring plants. The pollen bank from which the grower takes pollen may contain sources of pollen that are optimal for specific environmental conditions. In addition, any pollen used in the present invention, including the contemplated embodiment, can be taken either from a single source or from a combination of multiple sources.

[0045] One example of a real-time production decision would be the use of plant performance data to decide which male parent plant to use for hybrid or grain production. For example, in the southern hemisphere, winter grain production is often practiced outside the growing seasons of the northern hemisphere. It often happens that fields in the southern hemisphere are planted before complete data on plant productivity have been collected in the northern hemisphere. Therefore, in the southern hemisphere, decisions regarding plant production are often made on the basis of incomplete information and/or delays, resulting in errors in the choice of plant to grow or the volume to grow when space is limited. The present invention provides the ability for a grower to grow designated female parent plants and then wait approximately 45 to 75 additional days to make a more informed decision about which male plant to use to pollinate those female plants. Production solutions of this type would also be applicable in cereal production, where a cereal producer in the northern hemisphere is waiting for better information from the southern hemisphere in order to decide which male parent plant to use.

[0046] As will be appreciated by those skilled in the art, the selection of male pollen to be used in the process of the invention is of great importance in terms of the potential benefits of the invention. Pollen vigor and vitality are factors that need to be considered, along with the various genetic traits and characteristics that pollen is endowed with. There may be circumstances where using pollen with lower vigor or viability is still the right choice due to the genetic traits that will be transferred to the resulting grain product. In such circumstances, where pollen is known to have lower vigor or viability, a more modest increase in yield should be expected. Such decisions can be made by the grower based on the specific characteristics of the crop, the desired grain product, and the prevailing conditions at the expected time of pollination due to environmental influences and other biological and non-biological influences. Thus, the ability to select pollen during pollination represents an additional significant benefit from the use of the present invention. Table 1 below shows the potential benefits of using the present invention.

[0047] As should be clear to those skilled in the relevant field, the technology disclosed by the present invention can be used to modify cross-pollination by increasing the level of cross-pollination (for example, to increase the heterosis / xenia effect in corn and thereby increase field yield grain production), but it can also be used to reduce the level of cross-pollination and instead focus on self-pollination (for example, in so-called organic production or in the production of grain products that do not involve the production of genetically modified organisms, to increase the success of self-pollination and , thereby reducing the incidence of unwanted cross-pollination). In such a case, the present invention can be used specifically to prevent cross-pollination by increasing the efficiency of self-pollination, thereby helping to reduce the level of genetic contamination from sources of unwanted pollen that are genetically modified organisms.

[0048] In addition, the proposed invention provides the agricultural producer with the opportunity to use varieties for pollination of a crop that would not naturally reach maturity or would not be a suitable pollen donor in the climate in which the agricultural producer operates. Such a variety might have superior characteristics that would make it a pollen donor for cross-pollination suitable for maximizing crop yield, but it is not possible to grow it in the climate in which the grower operates, or in that climate it cannot reach the stage , on which it could emit pollen. The present invention overcomes this limitation and provides the grower with a wider range of pollen types from which to select the ideal candidate for a particular crop and the particular stresses it may be exposed to as pollination time approaches.

[0049] Another real-time production decision enabled by the present invention is deciding which pollen to use based on market and commodity price information. A grain producer might have approximately 45 to 75 days to observe market trends and decide which male parent to use for private market reasons, such as waxy corn or food corn, or No. 2 yellow dent corn ( on the commodity grain market).

[0050] In addition, real-time production decisions could be made based on environmental information. For example, a grain producer might know from experience that flowering is preceded by a dry period and might therefore choose to pollinate female parent plants with pollen from a male parent known to be drought-tolerant. This solution reduces the percentage of seed underdevelopment due to stress caused by drought, thereby ensuring increased yield compared to allowing the hybrid to self-pollinate in grain production. A similar situation would arise for an agricultural producer faced with the problem of seed-eating pests. In this case, the pollen source could be the male parent, which has a gene that confers resistance to seed-eating pests and protects the developing seeds, thereby providing increased yield of the hybrid seeds compared to the case when the pollen source the male parent that was originally planned before the problem of seed-eating pests arose, or that would have been selected from a more convenient block system, would have been used. Environmental information used in real-time production decisions may include, but is not limited to, abiotic environmental factors such as drought, nitrogen or other nutrient availability, moisture fluctuations and temperature extremes, and such (but not limited to) biotic environmental factors such as harmful insects and nematodes, weeds and diseases.

[0051] The following are examples that illustrate the present invention in more detail and explain how the proposed invention can be implemented in relation to corn. Basically, the proposed method is applicable to any crop with appropriate modifications appropriate to the specific crop. The results of Examples 1 and 2 were collected in fields in central Iowa during the summer of 2015.

[0052] Example 1

[0053] The invention described herein was field tested in a field in central Iowa using a commercially available hybrid (H1) as the female parent. Blocks of 12 rows (each row was 0.76 m (2.5 ft) wide for a total block width of 9.1 m (30 ft)) 13.7 m (45 ft) long for each hybrid were grown. One inbred pollen donor (P1), unrelated to H1, was grown more than 45.7 m (150 ft) from the hybrid block. This pollen donor was a publicly available line from the USDA Agricultural Research Service's National Plant Germplasm System and carried the gene responsible for anthocyanin in grain endosperm. When using P1 as a male parent - a source of pollen, the success of pollination can soon be verified without the help of optical instruments by the visible purple color of mature grains on the cob.

[0054] To practice the present invention, water was sprayed onto the panicles of a grain production block (H1) from approximately 8:15 a.m. to approximately 8:45 a.m. to delay the release of pollen by these designated female parent plants. After splashing water on these panicles, they immediately began to put the proposed invention into practice, for which pollen was forcibly taken from panicles P1, which was placed in bags. This designated male parent pollen was then promptly applied to the H1 pistils by hand using a well-known industry technique of gently tapping the pollen sac to release pollen grains onto the pistils. These acts of pollination took place before the hybrids emitted pollen. In the production block H1, 13 acts of pollination were carried out, directing pollen to the pistils of plants in block H1, which were located in the middle of the block. This was a simulated case where pollen could be collected from a nearby field from an unrelated corn hybrid, placed in storage for short-term storage, and then removed from storage in the early morning hours before pollen emission began in the grain field planted with the hybrid. , used as a female parent. It can be assumed that on these ears, after harvest, most, if not all, of the yellow kernels will be the result of self-pollination from the H1 plants in the grain production block, while the purple kernels on these ears are the result of cross-pollination with pollen from the P1 pollen donor. It is important to note that the manual application of pollen was very precise and measured, since only two purple grains were found on six adjacent ears on which pollen from pollen donor P1 was not directly applied (see Table 2). These ears and the resulting kernels served as a control to measure the basal percentage of selfing in the block. Therefore, from these data we can conclude that the frequency of self-pollination in the middle of this block of hybrids with known methods of grain production is close to 100%. This experiment replicated the result that would be expected from the present invention if pollen were collected from a nearby field of unrelated corn hybrid, placed in storage for short-term storage, and then removed from storage in the early morning hours before naturally occurring. hybrid plants used as female parents will begin to emit pollen.

[0055] The results shown in Table 2 indicate that the proposed invention is very effective in terms of increasing the percentage of cross-pollination in the field of grain production. The 13 ears on which the present invention was practiced had a total of 6564 kernels, of which 2034 kernels, or 31% of the total, were purple. This is a huge increase in the frequency of cross-pollination events of more than 38,000% (or 38 times) compared to the six ears that served as the control group. It is important to note that P1 and the other pollen donors mentioned below in Example 2 are not elite inbreds, these inbred lines are more than 20 years old and have never been used for commercial grain production. In addition, according to the literature (Sari Gorla, (1975) Theor. Appl. Genet. 46: 289-94), pollen from hybrids such as H1 is more resilient and stronger than pollen from inbred plants. As a result, P1 pollen had unfavorable competitive conditions in terms of viability and vigor compared to H1 pollen. This further illustrates the effectiveness of the present invention in increasing the percentage of cross-pollination.

[0056] Example 2. The present invention was put into practice using two commercial hybrids (H1 and H2) as female parents in one of the central areas of Iowa. Blocks of 12 rows (each row was 0.76 m (2.5 ft) wide for a total block width of 9.1 m (30 ft)) 13.7 m (45 ft) long for each hybrid were grown. Three inbred pollen donors (P1, P2, and P3) that were not related to the hybrids were grown more than 45.7 m (150 ft) from the hybrid blocks. These three pollen donors were publicly available lines from the USDA Agricultural Research Service's National Plant Germplasm System and carried the gene responsible for anthocyanin in grain endosperm. When using these pollen sources, the success of pollination can soon be confirmed without the aid of optical instruments by the visible purple color of the mature grains on the cob.

[0057] At the very beginning windows, the proposed invention was put into practice, for which pollen was forcibly taken from the panicles of male pollen donor plants (P1, P2, and P3), which was placed in bags. This pollen from the designated male parents was then promptly applied to the pistils of the H1 and H2 hybrids by hand using a well-known industry technique of gently tapping the pollen sac to release the pollen grains onto the pistils. The female parent of the hybrid H1 was an early maturing variety, as was the pollen donor P1, so pollen from this pollen donor P1 was used to apply to the pistils of the female parent H1. It should be noted that the portion of the ears of the female parent H1, which in this experiment consisted of 11 ears, which in Example 1 above met the criterion for inclusion in this data set and therefore were the result of "splashing water" to delay the release of the female parent H1 of its own pollen and the result pollination carried out before the H1 hybrid emitted its own pollen. The female parent hybrid H2 was a later maturing variety, as were the pollen donors P2 and P3. Therefore, pollen from donors P2 and P3 was used to pollinate the pistils of the maternal parent H2. All H2 pollinations and most H1 pollinations (with the exception of 11 H1 pollinations performed before natural pollination windows, as described in Example 1) were performed at the very beginning windows in the morning.

[0058] During the early period pollination window, in relation to the female parent plants H1 and H2, more than 75 manual pollination acts were carried out by applying pollen to the pistils of those plants H1 and H2 that grew in the middle of the block. This method implements an embodiment of the proposed invention, in which in the early part During natural pollination windows, fresh pollen can be collected and immediately applied directly to unrelated hybrid plants growing in an adjacent field. In addition, this experiment simulated, although not exactly, the case where pollen could be collected from a nearby field of unrelated plants, placed in storage for a period of time, and then removed from storage in the early morning hours before the plants used as female parents will begin to emit their own pollen, and apply to the female reproductive components of said female parent plants. Pollinations were labeled as to which pollen donor was used for each pollinated ear. The yellow grains on these ears can be assumed to be largely, if not entirely, the result of self-pollination, while the purple grains on these ears are the result of cross-pollination from pollen donors. As noted in Example 1 discussed above, it can also be assumed that in this experiment, using known methods of producing grain products in the middle of blocks with hybrids, selfing was close to 100%.

[0059] Some of the harvested ears subject to targeted pollination have a high percentage of kernels that are the result of cross-pollination, often more than 50%. These ears often have large solid blocks of purple seeds in one part of the ear, while the rest of the ear is occupied by yellow seeds. This can be seen in Fig. 3, which shows an ear in which the proportion of kernels resulting from cross-pollination (have a dark (in color image purple) color) is approximately 85%. In other cases, ears have been produced with a lower proportion of purple kernels, often between 10% and 30%. Among them there were only 49 ears, in which a mixture of separated purple and yellow grains formed, not forming solid blocks. Two cob samples are shown in Fig. 1 and fig. 2. These ears show a clear mixture of dark (purple) and light (yellow) kernels. These ears were particularly illustrative in terms of self-pollinated yield compared to cross-pollinated yield, while the ear shown in FIG. 3 is not illustrative in terms of yield, since there are not enough grains obtained as a result of self-pollination to establish a sample size sufficient for comparison. The grains in these cobs developed under almost identical environmental conditions with the only difference being the different gene pool of the male parent plants (self-pollination versus cross-pollination). These kernels were collected and, for each cob, divided into two groups: 100 yellow and 100 purple kernels from approximately the middle of the cob (sometimes when there were not enough purple kernels, a smaller number could be used - but not less than 40 kernels of each type). For each cob, measurements were taken from these sets of kernels. Table 3a and Table 3b show the differences between yellow and purple kernels from these ears in individual kernel weight and kernel weight (weight per unit volume). The data summarized in Table 3a and Table 3b illustrates several points:

[0060] Firstly, the result of the implementation of the proposed invention in practice was an increase in useful productivity in at least one parameter (grain weight or natural grain weight) of grains obtained as a result of cross-pollination, compared with grains obtained as a result of self-pollination, in all three grain types resulting from cross-pollination (Table 3a and Table 3b).

[0061] Secondly, the implementation of the present invention in practice can increase useful productivity in various ways depending on the specific combination of pollinated hybrid and pollen donor (Table 3a and Table 3b). The first cross (a set of organisms obtained as a result of cross-pollination) - hybrid H1, pollinated by the inbred pollen donor P1 - due to cross-pollination, gave an increase in grain weight by 8.7% and an increase in natural grain weight by 1.9%. The second cross-hybrid H2, pollinated by the inbred pollen donor P2, gave an increase in the natural grain weight by 2.8%, but did not increase the grain weight. The third cross - hybrid H2, pollinated by the inbred pollen donor P3 - due to cross-pollination, gave an increase in grain weight by 5.1%, but did not increase the natural grain weight.

[0062] Example 3. The embodiment of the present invention described here characterizes the general situation in commercial grain production, where large areas (fields from 20.2 hectares to 40.5 hectares (50 to 100 acres)) are planted with various unrelated hybrids. plants, which in many cases come from different sellers. These fields are largely self-pollinating and therefore produce lower yields than would otherwise be the case if cross-pollinated from unrelated pollen sources. In a pure stand of corn, on average, more than 95% of the kernels harvested are the result of selfing. Goggi et al. (2006) Field Crops Res. 99: 147-157 showed that inadvertent cross-breeding between adjacent grain fields was much lower: 0.4% at 35 m. and less than 0.05% at a distance of 100 m. Many of these large corn fields are at different stages of maturity, and therefore flowering and pollen release occur at different times in them. In this embodiment, pollen from one field, where ripening occurred earlier, is collected and preserved/stored until flowering begins in another field with unrelated hybrid plants, and then pollen from the first field is used to pollinate the second field, causing it on the female reproductive components of the plants early in the morning (for example, at five o'clock in the morning) in order to maximize cross-pollination and minimize self-pollination in this second field. This application of pollen is carried out for two or three days in a row during the period of the most vigorous flowering. This process of collecting pollen, preserving it and storing it is repeated for the second field and this pollen is used to pollinate unrelated hybrid plants starting to bloom in the third field and so on for all fields until the flowering time ends for them. With this solution, a significant part of the corn grains in a given district will be obtained as a result of cross-pollination, rather than self-pollination, which will affect the yield in the form of an increase of 5-10%. It should be noted that the process described above does not necessarily have to be carried out in the exact sequence described above. One could take pollen from the first field (the one that bloomed first) and use it to pollinate many (say, five or more) fields. With this particular solution, cross-pollination according to the present invention could very quickly spread from one field to a group of five fields, from five fields to 25 fields, and so on. As a result, cross-pollination would occur in most of the corn fields in the county.

[0063] Example 4 This embodiment of an embodiment of the invention described herein is similar to Example 2 discussed above, in which pollen from fields planted with unrelated hybrids flowering at approximately the same time in each field was collected as soon as the panicles began to emit pollen. . Some of the pollen from each field was immediately applied directly to the opened pistils of plants in the other field, and the remaining pollen was canned/stored. Flowering of hybrid plants in fields usually lasts approximately three to four days, and the stored pollen is used for pollination for the next two to three days in a row in the early morning before the natural release of its own pollen begins - in order to increase the percentage of cross-pollination. With this solution, an increase in the percentage of cross-pollination is ensured, and therefore, the yield on both fields will be increased by 5-10%.

[0064] Example 5 From the perspective of specialty grain markets, it is important that a field be left to self-pollinate so that the percentage of cross-pollination from nearby fields is very low. One example of such a specialty grain product is so-called food corn, which must be 99% or more self-fertilized to qualify (Strayer, D. (2002) Preserved Systems: A reference handbook. CRC Press .). In this example, the present invention is used to reduce the percentage of cross-pollination in order to provide a genetically pure harvest of a specialty grain product that will be sold to the end consumer at a premium price. In this case, pollen is collected as soon as the first panicles begin to emit pollen in the field for growing special grain products. Some of this pollen is immediately and purposefully applied directly to the opened pistils in this field. At this initial stage of flowering, only a small proportion of the pistils are open. The remaining pollen collected is canned/stored and used for the next two to three consecutive days for very early morning pollination before natural pollen emission begins, so as to enhance self-pollination and minimize cross-pollination. With this solution, the manufacturer of specialty grain products successfully ensures the cultivation and supply of specialty grain products that meet the requirements of the end consumer. In this embodiment, the proposed invention could also be carried out with less intensity, namely, the application of pollen could be carried out only along the outer edge of the field in a strip of approximately 12.2 m (40 ft) wide, that is, where there is the greatest risk of cross-pollination. pollination, and the inner area of the field can be left to natural self-pollination.

[0065] Example 6 The present invention can be applied to the grain corn industry in such a way that it allows the producer to make decisions based on market indicators up to the point of pollination (early to mid-July), rather than before planting, as is the case. occurs in the prior art. By collecting and preserving/storing pollen from many different sources, which are available in abundance around the world for different production situations, the selection of pollen for pollination can ensure different characteristics of the grain product that is removed from the field. The farmer has the ability to monitor the market and make decisions regarding specialty grain products, such as high-oil corn or No. 2 yellow dent corn, and can issue orders (“place an order”) to purchase pollen that will produce the desired grain product. In this example, the planted hybrid is simply a female parent plant to accept pollen and generally has the desired characteristics. The pollen contains the genes required by the market, and with this solution, the ability to satisfy market needs by modifying the crop remains until the time of pollination. There are many other similar signs that apply in this example. For example, if a drought occurs, a pollen source can be used that minimizes the percentage of underdeveloped grains during grain filling. Alternatively, if the season has not been water limited, a pollen source may be used that maximizes absorption rates and crop yield.

[0066] For the high-oil corn and No. 2 yellow dent corn mentioned above, as well as many others, the desired characteristics can be achieved using natural, non-GM pollen sources. For other traits, such as resistance to insect pests, the desired characteristics can be achieved using genetically modified organisms as pollen sources. In addition, the proposed invention makes it possible to impart the characteristics of a genetically modified organism to grain products without causing public concern about the negative effect of conventional genetically modified organisms. Thus, the proposed invention bypasses a serious barrier that manufacturers of useful genetically modified organisms are forced to overcome in order to push them to the market.

[0067] Example 7

[0068] Another example of the invention described herein concerns the rescue of set seeds that are lost under drought conditions that occur before or during flowering. Drought during this period is still the main cause of loss of grain production per year in the United States. Under favorable conditions, that is, at adequate humidity and normal temperatures, there is a close synchronicity between the release of pollen by male flowers on panicles and the appearance of pollen-sensitive reproductive components (pistils) in female flowers. However, in drought conditions, three problems arise that prevent successful pollination. First, during drought, pistil emergence lags behind pollen release (Basset, R. & M. Westgate (1993) Crop Sci. 33: 279-282). That is, it lengthens the interval between the emission of pollen by panicles and the spawning of pistillate styles in the cobs, which can lead to the absolute impossibility of pollination of late swept pistillate styles. Second, drought reduces the time that emerging pistils are receptive to pollen (Araus, JL et al. (2012) Front. Physiol. 3: 305). That is, their ability to support pollen germination and pollen tube growth is reduced for several days. Third, the low humidity and high temperatures that typically accompany drought reduce pollen viability and longevity (Schoper, J.B. et al. (1987) Plant Physiol. 83 (1): 121-125). To apply the present invention to this case, fresh pollen is collected from this field or from a nearby field in which an earlier mature corn hybrid has been planted. If necessary, the pollen is stored for some time and then applied directly to the female flowers as soon as the pistils appear. This pollen application overcomes all three of the above drought-related limitations on pollination and seed set. To enhance natural self/sib pollination, stored pollen can be applied for several days in a row. In cases where drought prolongs the interval between the emission of pollen by the panicles and the emergence of pistillate columns in the cobs is up to four or more days, the use of the present invention can increase the yield by 25% or more.

[0069] Various embodiments of the present invention have been described in some detail above, however, those skilled in the art are able to make numerous modifications to the described embodiments of the present invention without deviating from its spirit and without going beyond its scope as disclosed herein and the attached claims. In some cases, in the procedures disclosed herein, directly or indirectly, the various steps and operations are described in one possible order of operation, but those skilled in the art will appreciate that rearranging, substituting, or eliminating some of these steps and operations will not necessarily result in a departure from spirit of the proposed invention and to go beyond its scope. It is intended that all material contained in this specification or in the accompanying drawings should be interpreted as merely illustrative and not limiting. Changes in details or structure may be made without deviating from the spirit of the proposed invention and without going beyond the scope thereof, which is defined by the appended claims.

[0070] The present invention has been described with reference to the embodiments discussed above, however, those with at least average skill in the art will recognize various alternatives, modifications, variations, improvements and/or equivalents per se as are known , and those that are predicted or can be predicted. Listing the stages of the method in a certain order does not impose any restrictions on the order in which these stages of the method are performed. Thus, the embodiments of the present invention described above are to be understood as illustrative and not limiting. It should be clear to those skilled in the relevant field that changes can be made to the invention in form and detail without deviating from the spirit of the proposed invention or going beyond its scope. It is therefore intended that the scope of the present invention cover all known or previously developed alternatives, modifications, variations, improvements and/or equivalents in essence.

Claims (56)

1. A method of establishing one or more times at least one time window within a day when designated female plants are ready to accept intentionally applied designated male pollen ( ^m pollen) in a manner that does not involve natural wind pollination, insect activity, or other natural factors, for the purpose of production of grains of interest, wherein the designated female plant is any one or more of the following: corn, soybean, wheat, rice, sunflower, canola, sorghum, barley, pearl millet, wherein designated female plants include female components and are grown under conditions that may expose the female components to unwanted pollen ( ^U pollen), which is: a. own pollen produced by the male components of the designated female plants, and/or b. pollen produced by neighboring plants that is capable of pollinating designated female plants, wherein the method includes the following steps: determining that the female components of the designated female plants are receptive to pollen, and establishing one or more times at least one time window within a day for designated female plants, said time windows being or being: i. first time the time window when designated female plants first become receptive to pollen, and/or ii. a second time by a time window occurring during a period of time at a time of day when ^U pollen is not being released and/or the ^U pollen being released is no longer viable, so that when, on one or more occasions, ^pollen is deliberately applied to the female components of designated female plants, not including natural wind pollination, insect activity or other natural factors during the second period, then the selected designated female plants will already be pollinated with pollen by the time ^pollen begins to be emitted. 2. A method of producing grain for human, animal or industrial use, comprising: a. Growing designated female plants having female components under conditions that may expose the female components to undesirable pollen ( ^U pollen), wherein said ^U pollen is: own pollen produced by the male components of said female plants, and/or pollen produced by neighboring plants, which is capable of pollinating said female plants, b. selecting designated female plants as ready to receive applied designated male pollen ( ^m pollen) in a manner that does not involve natural wind pollination, insect activity, or other natural factors, for the purpose of obtaining the grain of interest in one or more times, as determined by the method of .1, and c. intentional application of ^pollen to designated female plants in one or more times selected in step b, in one or more times during the first and/or second time according to the method of claim 1, in a manner that does not involve natural wind pollination, insect activity or other natural factors, wherein the designated female plant is any one or more of the following: corn, soybean, wheat, rice, sunflower, canola, sorghum, barley, pearl millet, and the method does not include replanting the grain produced by the designated female parent plants. 3. A method of producing grain for human, animal or industrial consumption, comprising: a. Growing designated female plants having female components under conditions that may expose the female components to undesirable pollen (pollen ⁰ ), wherein pollen ⁰ is: own pollen produced by the male components of these female plants, and/or pollen produced by neighboring plants that is capable of pollinating the designated female plants, b. selecting designated female plants as ready to receive intentionally applied designated male pollen ( ^m pollen) in a manner that does not involve natural wind pollination, insect activity, or other natural factors, for the purpose of producing the grain of interest in one or more times, as determined by the method of paragraph 1, and c. intentional pollination of the designated female parent plants in one or more times, selected in step b, in one or more of the first and/or second times in accordance with paragraph 1, with ^pollen in a manner that does not involve natural wind pollination, insect activity or other natural factors, d. growing the designated female plants obtained at stage c to maturity, and e. harvesting the grain of a designated female plant, wherein the designated female plant is any one or more of the following: corn, soybeans, wheat, rice, sunflower, canola, sorghum, barley, pearl millet, and the method does not include replanting the grain produced by the designated female parent plants. 4. The method of claim 3, further comprising the step of using the harvested grain for human, animal or industrial consumption, and wherein the method is not an essentially biological plant growing process. 5. The method of any one of the preceding claims, wherein the pollen ^m is pollen from a male plant having a different genetic background than the designated female plants. 6. The method according to any of the preceding claims, wherein the ^pollen consists of one or more of the following components: A. fresh pollen, b. preserved pollen. 7. Method according to any one of paragraphs. 2 or 3, or any other claim thereunder, wherein the intentional application of the designated male plant pollen is carried out by one or more of the following means: mechanical means, pneumatic means, positive pressure means, negative pressure means, manual means, combinations thereof. 8. A method as claimed in any one of the preceding claims, wherein the pollen ^m is fresh pollen collected from one or more fields, grow room, greenhouse, greenhouse, shade house, arch greenhouse, vertical farming or hydroponic setup. 9. The method as claimed in any one of the preceding claims, wherein the pollen ^m is preserved pollen that has been previously collected and preserved by refrigeration, cryopreservation, freezing, freeze-drying or storage in liquid nitrogen. 10. Method according to any one of paragraphs. 2 or 3, in which pollen ^m is applied more than once to the same designated female plant. 11. The method of claim 10, wherein the ^pollen is applied at a time when the designated female plants first become susceptible to the pollen. 12. The method of claim 3, which results in modified grain characteristics, which include one or more of the following: grain size, grain content or grain composition. 13. The method according to any of the preceding claims, wherein the ^pollen is obtained from sources that are optimal for use under environmental conditions. 14. The method according to claim 13, wherein said environmental conditions are: i. abiotic conditions, ii. abiotic conditions including at least one of the following: drought, moisture, temperature, nitrogen availability or nutrient availability, iii. biotic conditions or iv. biotic conditions including at least one of the following: insect pest pressure, disease pressure. 15. The method according to any of the previous paragraphs, in which the pollen ^is obtained from sources that are optimal for use based on plant productivity data. 16. Method according to any one of paragraphs. 2 or 3, wherein the ^pollen influences the characteristics of grain collected from female plants, wherein said influence affects one or more of the following: i. grain oil content, ii. starch content in grain, iii. protein content in grain, iv. composition of grain oil, v. composition of grain starch, vi. protein composition of grain. 17. The method according to any of the previous paragraphs, in which the pollen ^is obtained from: (a) a single genetic source, or (b) multiple genetic sources and combined before use. 18. The method of claim 3, wherein grain yield and grain characteristics are changed as a result of pollination. 19. The method of claim 18, comprising growing a plurality of designated female plants having both female and male components, wherein at least a portion of the male components of the designated female plants are capable of releasing pollen from the designated female plants. 20. The method of claim 19, wherein the designated female plants are intentionally cross-pollinated ^{with pollen} from a different genetic background before said male components release at least a portion of the pollen from the designated female plants.