Patent Application
20190150383
The invention relates to the field of manufacturing methods and manufactured compositions. More specifically, it relates to methods for hybrid corn seed production and hybrid corn compositions produced through such methods.
Commercial hybrid corn seed production involves planting male and female inbred lines in separate rows or blocks in an isolated field where possibility of foreign pollen contamination is rare. The female inbred is normally detasselled before pollen shed to ensure cross-pollination by the male inbred. Male inbreds are destroyed following pollination to prevent seed mixing during harvest. Ears from the cross-pollinated female inbred are harvested, processed, and sold to farmers for planting as hybrid seed. Production of hybrid corn seed is an expensive process due to manual or mechanical detasseling and input costs of herbicides, insecticides, fungicides, nutrients, and irrigation. The yield of hybrid seed tends to be lower resulting in lower revenues. Lower revenues and increasing cost of production result in lower profitability for manufacturers. Manufacturers of hybrid corn seed, thus, are always interested in enhancing profitability of a hybrid corn seed production system.
It is also known in the art that the cost of producing homozygous inbreds with multiple traits for creating hybrids is quite high and becomes higher with the introduction of each additional trait as breeders need to ensure a right combination of traits, right position of these traits in the genome, and integration of these traits in the right germplasm. The probability of obtaining the right homozygous output is 1:4 in case of producing an inbred with one trait and is 1:65,536 in case of producing an inbred with eight traits. These probabilities exponentially increase the difficulty in identifying a homozygous inbred with multiple traits and consequently raising the breeding costs substantially. It is important then to identify and combine suitable traits that will result in enhanced profitability of a corn seed production system.
The cost of breeding an uneven number of traits into a hybrid is similar to the breeding cost of even number of traits. Also, in certain situations, a trait may have adverse effect on a parent. There is also a need to balance the number of traits on the male and the female inbreds to reduce the cost of breeding these traits into a hybrid per se and to provide the trait causing agronomic adverse effect in one parent to another parent.
Further, due to evolving market and environmental conditions, there is increasing demand for well-adapted germplasm by end users, such as farmers and processors, comprising traits that enhance yield, promote stress resistance, and enhance grain quality.
Although, hybrid corn seeds with up to three traits are known in the market place and different methods of introducing the multiple traits are known, the current state of the art lacks a systematic method for delivering at least four transgenic traits in hybrid corn seed, such as ones described herein. That is, the art lacks a systematic means for identification and selection of cost decreasing traits, deciding which trait or traits should be provided on which parent, and then selecting a combination of traits from each parent to enhance the profitability of a hybrid corn seed production system.
A method for hybrid corn seed production system comprising: identifying at least one transgenic trait having a high impact on decreasing cost of production, wherein the trait is introduced into germplasm of a female parent; identifying at least three transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of a male parent and/or the female parent; planting and crossing the male and the female parent; and harvesting a hybrid seed, wherein the production of the hybrid seed results in enhanced profitability over a hybrid seed not produced by providing the transgenic traits on the female and the male parent.
The method further comprising balancing the transgenic traits on the male parent and the female parent, wherein the production of the hybrid seed results in an enhanced profitability as compared with the profitability of a hybrid seed produced by not balancing the transgenic traits.
In one embodiment, the method comprises identifying at least four transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of the male parent and/or the female parent.
In another embodiment, the method identifying at least five transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of the male parent and/or the female parent.
In another embodiment, the trait providing the high impact on decreasing cost of production is selected from the group consisting of herbicide tolerance, male sterility system, enhanced yield, and nutrient use efficiency, and a combination thereof. Examples of these and other traits having the high impact on decreasing cost of production are also given in one or more of Tables 2A-9.
In another embodiment, the trait providing the low impact on decreasing cost of production is selected from the group consisting of cold tolerance, drought tolerance, diseases resistance, insect resistance, and a combination thereof. Examples of these and other traits having the low impact on decreasing cost of production are also given in one or more of Tables 2A-9.
In another embodiment, the enhanced profitability is achieved by increasing yield of the hybrid corn seed.
In another embodiment, the method of present invention further comprises providing an end user transgenic trait on the female and/or the male parent. The end user trait is selected from the group consisting of enhanced amino acid content, enhanced protein content, modified or enhanced fatty acid composition, enhanced oil content, enhanced carbohydrate content, and a combination thereof. Examples of these and other end users traits are also given in Tables 10A and 10B.
The present invention also discloses a hybrid corn seed produced by the method of present invention.
In one embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits and three or more of the insect resistance traits.
In another embodiment the traits in the hybrid seed consists of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, and the drought tolerance trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the drought tolerance trait, and the male sterility system trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the drought tolerance trait, the male sterility system trait, and the intrinsic yield trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, one or more of the drought tolerance traits, the male sterility system trait, the intrinsic yield trait, and the nutrient use efficiency trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance trait, two or more of the insect resistance traits, one or more of drought tolerance traits, the male sterility system trait, the intrinsic yield trait, the nutrient use efficiency trait, and the cold tolerance trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, three or more of the insect resistance traits, and the enhanced amino acid content.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the enhanced amino acid content trait, and the drought tolerance trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the enhanced amino acid content trait, the drought tolerance trait, and the male sterility system trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, one two or more of the insect resistance traits, the enhanced amino acid content trait, the drought tolerance trait, the male sterility system trait, and the intrinsic yield trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the enhanced amino acid content trait, the drought tolerance trait, the male sterility system trait, the intrinsic yield trait, and the enhanced oil content trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, one two or more of the insect resistance traits, one or more of the enhanced amino acid content trait, one or more of the drought tolerance trait, the male sterility system trait, the intrinsic yield trait, nutrient use efficiency trait, the enhanced oil content trait, the enhanced protein content trait, and the cold tolerance trait.
In another embodiment the traits in the hybrid seed consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, one or more of the enhanced amino acid content trait, one or more of the drought tolerance trait, the male sterility system trait, the intrinsic yield trait, the nutrient use efficiency trait, the enhanced oil content trait, the enhanced protein content trait, and the cold tolerance trait.
The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.
The word “comprising” means including but not limited to.
The present invention provides a method for enhancing profitability of a hybrid corn seed production system. This is achieved by identifying and providing traits having high impact on decreasing cost of production or low impact on decreasing cost of production. Some of the cost decreasing traits may also influence yield directly or indirectly thereby enhancing revenues and profitability. For example, an intrinsic yield trait can decrease costs associated with production land by enhancing yield i.e., more units of hybrid corn seed can be produced on less land. Although, hybrid corn seeds with up to three traits are known in the market place, there does not appear to be a systematic method such as the one described herein available for identifying, selecting which cost decreasing traits should be provided on which parent and then selecting a combination of traits on each parent to enhance profitability of the hybrid corn seed production system.
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Traits having high impact on decreasing cost of production may be balanced by inclusion of traits having a low impact on decreasing cost of production, by selecting certain traits having high impact and certain traits having low impact from a gradient of traits such that the profitability of the hybrid corn seed production system is enhanced. For example, if the goal is to combine 7 traits in a hybrid, one may select, for example from Table 1, herbicide resistance, male sterility system, an intrinsic yield trait, and nitrogen use efficiency as high impact traits and introduce them into the female parent. One may also select cold tolerance, disease resistance, and drought resistance traits as low impact traits, and introduce them into the female or the male parent. In some embodiments, the traits may be balanced as±one or two or three traits on the female or the male parent to enhance profitability. In other embodiments, the traits may be balanced as±one or two or three traits on the female or the male parent to enhance profitability.
In some instances, provision of a trait on female parent may cause a yield penalty, e.g., due to its insertion into an important endogenous gene. In such situations, the trait may be provided on the male parent.
Thus, one aspect of the present invention provides a method for hybrid corn seed production comprising: identifying at least one transgenic trait having a high impact on decreasing cost of production, wherein the trait is introduced into germplasm of a female parent; identifying at least three transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of a male parent and/or the female parent; planting and crossing the male and the female parent; and harvesting a hybrid seed, wherein the production of the hybrid seed results in enhanced profitability over a hybrid seed not produced by providing the transgenic traits on the female and the male parent.
The method facilitates crop breeding decisions, for instance by allowing for balancing of transgenic traits on the male parent and the female parent, wherein production of the resulting hybrid seed results in an enhanced profitability over a hybrid seed produced by not balancing the transgenic traits.
The method may comprise identifying at least four transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of the male parent and/or the female parent. Alternatively, the method allows for identifying at least five transgenic traits having a low impact on decreasing cost of production, wherein the traits are introduced into germplasm of the male parent and/or the female parent.
In another embodiment of the method, a trait or traits providing a high impact on decreasing cost of production may be selected from the group consisting of herbicide tolerance, male sterility system, enhanced yield, and nutrient use efficiency, and a combination thereof. Examples of traits having the high impact on decreasing cost of production are also given in one or more of Tables 2A-9.
In another embodiment, a trait providing a low impact on decreasing cost of production may be selected from the group consisting of cold tolerance, drought tolerance, disease resistance, insect resistance, and a combination thereof. Examples of traits having the low impact on decreasing cost of production are also given in one or more of Tables 2A-9.
Enhanced profitability may also be achieved by increasing the yield of a hybrid corn seed production system.
The present invention also discloses hybrid corn seed produced by the method of the present invention. In one embodiment, traits in the hybrid seed may consist of one or more herbicide tolerance traits and three or more insect resistance traits. In another embodiment traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, and a drought tolerance trait. In yet another embodiment, traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, a drought tolerance trait, and a male sterility system trait. In another embodiment the traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, a drought tolerance trait, a male sterility system trait, and an intrinsic yield trait. In a further embodiment, traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, one or more drought tolerance traits, a male sterility system trait, an intrinsic yield trait, and a nutrient use efficiency trait. In yet another embodiment the traits in the hybrid seed may consist of one or more herbicide tolerance trait, two or more insect resistance traits, one or more drought tolerance traits, a male sterility system trait, an intrinsic yield trait, a nutrient use efficiency trait, and a cold tolerance trait. Examples of such traits may be found in one or more of Tables 2A-9.
A trait specifying a phenotype (e.g. an agronomic trait such as herbicide tolerance) may be encoded by one gene or by more than one gene specifying the same or a different mode of action or mechanism.
Nucleic acids encoding for a trait or traits specifying abiotic stress resistance, such as drought, cold, salt, or nutrient stress resistance, may also provide tolerance to more than one stress.
Cost decreasing traits may include traits that provide increased herbicide tolerance, male sterility system, increased intrinsic yield, increased nutrient use efficiency e.g., nitrogen use efficiency, increased cold tolerance, increased disease resistance, increased drought tolerance, and increased insect resistance. These traits may decrease cost of production by any where from 14.2% to 0.1% or more (see Table 1).
Provision of an herbicide tolerance trait in a parent in combination with use of a corresponding herbicide can be used to manage weeds, thereby reducing the utilization of resources by weeds and decreasing the need for inputs such as nutrients and water. This trait can also enhance yield as more resources will be available for growing the hybrid parents. Further, it has been found that providing an herbicide tolerance trait, for example a glyphosate tolerance trait, on a female parent may reduce the production failure rate, i.e. the estimated chance of not producing pure seed, to 0.06% to 10% as measured by trait purity in a seed lot, for example by a measured glyphosate susceptibility rate equal to or more than 2%. This compares to a possible failure rate of 36.1% if the glyphosate tolerant trait is provided on the male parent. An increase in production failure rate means an incremental rise in production acres to reproduce units not meeting the susceptibility tolerance criteria thereby increasing cost of hybrid seed production. By providing an herbicide tolerance trait, such as glyphosate tolerance, on a female parent, a decrease in cost of about 14.2% can be realized resulting in enhanced profitability (Table 1).
One or more herbicide tolerance traits can be provided to control weeds more effectively and to reduce the risk of developing herbicide resistance weeds in a field. For example, herbicide tolerance traits providing tolerance to glyphosate, glufosinate, dicamba, or 2,4-D can be provided. When more than one herbicide tolerant trait is provided then it may be provided on the male and/or the female parent. When a herbicide tolerance trait is provided as a part of a male sterility system (described below), the herbicide tolerance trait may also be provided on the male parent, since the application of the herbicide may render the pollen on a female parent non-viable. In order to provide viable pollen from a male parent, the male parent preferably has a corresponding herbicide tolerance trait to survive the application of the herbicide in the production system. Examples of proteins responsible for herbicide tolerance are exhibited in Tables 2A and 2B.
Various methods can be used to prevent self pollination of the female parent. In conventional plant breeding schemes, at the time of flowering, the tassels of all female parents are typically removed. The detasseling can be achieved manually or by machine. This technique, while effective, is extremely labor intensive and greatly increases the overall cost of hybrid seed production. Alternatively, conventional nuclear or cytoplasmic male sterility systems may be used. The present invention uses biotechnological and chemical male sterility systems (MSS) as a cost decreasing trait. By using such methods a decrease in cost of about 10.8% can be realized resulting in enhanced profitability (Table 1). Examples of such methods can be found in documents listed in Tables 2A and 2B which are also incorporated herein by reference.
By providing a yield trait on a female parent, the number of usable units harvested per acre can be increased. An increase in usable units/per acre means a proportional decrease in the number of acres needed to realize a given unit yield target. For example, a 10% intrinsic yield gain can result in a 10% decrease in the number of acres required to produce hybrid corn. By providing an intrinsic yield trait on a female parent, a decrease in cost of about 3.7% can be realized resulting in enhanced profitability (Table 1). A yield trait may increase yield by improving biomass, grain yield, number of seeds, germination, and high density growth of plants. Examples of proteins responsible for yield traits are exhibited in Tables 2A and 2B to 9.
Some traits that are able to provide tolerance to abiotic stresses such as nutrient deficiency are also cost decreasing traits by allowing reduced use of nutrient inputs, such as nitrogen, or by increasing yield when a given level of nutrient inputs (e.g. fertilizer) is applied. This may subsequently reduce the requirement for production acreage. By providing a trait for nitrogen use efficiency on a female parent a decrease in cost of about 3.7% can be realized resulting in enhanced profitability (Table 1). Examples of proteins responsible for nutrient use efficiency traits are exhibited in Tables 2A and 2B to 9.
Other stress tolerance traits such as a cold tolerance trait, for instance a cold germination tolerance trait, can result in cold tolerance. Assuming a 1″ improvement in standard deviation of seed spacing in final stand of female parents, a 7% increase in yield can be realized, thereby enabling reduced requirement of production acreage and a decrease in cost of about 2.6% resulting in enhanced profitability (Table 1). The cold tolerance trait can be provided on the male and/or the female to optimize the production cost decrease. Examples of proteins responsible for cold stress tolerance are exhibited in Tables 2A and 2B to 9.
A disease resistance trait may be provided on a male parent because the impact of a disease resistance trait on decreasing production cost is typically low. Assuming 99% of total acres is sprayed with fungicides with one application per acre per year at an application cost of $15.10, providing a trait for disease resistance can result in a cost decrease of about 0.7% resulting in enhanced profitability (Table 1). The disease resistance trait may be provided on the female parent in certain situations, for example, if the female is made male sterile by detasseling. Detasseling may further cause wounding. Thus, it may be beneficial to protect the female from any infection through wounds left after detasseling by providing a resistance trait against, for example, fungal diseases such as gray leaf spot or rust diseases, that can seriously harm a corn plant. Disease resistance traits, such as those effective against Helminthosporium carbonum or common rust, may be of further benefit in decreasing cost in a production system. Examples of proteins responsible for disease resistance are exhibited in Tables 2A and 2B to 9
Provision of another trait for combating abiotic stresses such as lack of water can also decrease cost of producing hybrid seed by allowing reduced use of water on irrigated land or increasing yield on dry land. For example, in a 6,000,000 unit production plan at 86 usable units/acre, assuming use of 70% irrigated acres with an irrigation cost of $8.00/acre/inch and the water requirement of 23″/year, with a 10% reduction in irrigation cost and 5% increase in yield on dry land, a producer could realize a decrease in cost of 0.5% in hybrid seed production. This trait can be provided on the male or the female or on both given its low impact on decreasing cost of production (see Table 1). Examples of proteins responsible for drought tolerance are exhibited in Tables 2A and 2B to 9.
With the use of an insect resistance trait in a parent, no foliar or soil insecticidal treatments may be required. For example, in a 6,000,000 unit production plan at 86 usable units/acre, assuming 26.4% of the acres will be sprayed for 1st generation European corn borer, with the female parent representing 77.3% of the total acres, a custom application cost of $5.35/acre, a chemical cost of $6.56/acre, and an application rate of 4 oz/acre, provision of a trait against protection for European corn borer could reduce chemical applications by 80-90% resulting in decrease in total production cost of about 0.1% (see Table 1), as well as environmental benefits. Provision of traits providing protection against several insects such as root worms, spider mites, grasshoppers, Western bean cutworm or other cutworms, or earworms could decrease cost of production by 0.75%. Such protection against several insects can be obtained by combining novel and chimeric genes and/or RNAi methods. One or more insect resistance traits can be provided on the female or the male parent or both. Examples of proteins responsible for insect resistance are exhibited in Tables 2A and 2B to 9.
The nucleic acids encoding proteins that confer insect resistance can be derived from a number of organisms that include, but are not limited to, Bacillus thuringiensis, Xenorhabdus sp., or Photorhabdus sp. For example, transgenic plants which express one or more B. thuringiensis proteins toxic to the same insect species or multiple insect species can be produced in order to allow for resistance management, which may delay the onset of resistance in a population of an otherwise susceptible insect species to one or more of the insecticidal nucleic acids expressed within the transgenic plant. Alternatively, expression of a B. thuringiensis insecticidal protein toxic to a particular target insect pest along with a different proteinaceous agent toxic to the same insect pest but which confers toxicity by a means different from that exhibited by the B. thuringiensis toxin is desirable. Such other different proteinaceous agents may comprise any of Cry insecticidal proteins, Cyt insecticidal proteins, insecticidal proteins from Xenorhabdus sp. or Photorhabdus sp., B. thuringiensis vegetative insecticidal proteins, and the like. Examples of such proteins encoded by insect toxin genes includes, but are not limited to, ET29, TIC809, TIC810, TIC127, TIC128, TIC812 and ET37 (WO 07/027776), TIC807, AXMI-027, AXMI-036, and AXMI-038 (WO 06/107761), AXMI-018, AXMI-020, and AXMI-021 (WO 06/083891), AXMI-010 (WO 05/038032), AXMI-003 (WO 05/021585), AXMI-008 (US 2004/0250311), AXMI-006 (US 2004/0216186), AXMI-007 (US 2004/0210965), AXMI-009 (US 2004/0210964), AXMI-014 (US 2004/0197917), AXMI-004 (US 2004/0197916), AXMI-028 and AXMI-029 (WO 06/119457) and AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 (WO 04/074462). All of the foregoing references are incorporated herein in their entirety.
Proteins conferring insect resistance are preferably toxic against coleopteran insect pests that comprises of coleopteran families consisting of Chrysomelidae, Cucujidae, Scarabaeidae, Trogositidae, Tenebrionidae, Curculionidae, Elateridae and Bruchidae. The exemplary coleopteran insects in the family Chrysomelidae may include those that are from the genus Diabrotica including D. virgifera (WCR), D. undecimpunctata (SCR), D. barberi (NCR), D. virgifera zeae (MCR), D. balteata (BZR), and Brazilian Corn Rootworm complex (BCR) consisting of D. viridula and D. speciosa.
A protein conferring insect resistance may also be toxic against hemipteran insect pests that may be selected from the group of hemipteran suborders consisting of Auchenorrhyncha (e.g., cicadas, spittlebugs, hoppers), Sternorrhyncha (e.g., aphids, whiteflies, scales), Heteroptera (e.g., true bugs including Lygus) and Coleorrhyncha. The hemipteran insects can be from the suborder Heteroptera. Exemplary hemipteran insects in the suborder Heteroptera may include those that are from the genus Lygus including Lygus hesperus (western tarnished plant bug), Lygus lineoloris (tarnished plant bug) and Lygus elisus (pale western legume bug).
A protein conferring insect resistance may also be toxic against a Lepidopteran insect pest such as European corn borer (Ostrinia nubilalis), Southwestern corn borer (Diatraea grandiosella), Sugarcane borer (Diatraea saccharalis), Corn earworm (Helicoverpa zea), Fall armyworm (Spodoptera frugiperda), Black cutworm (Agrotis ipsilon) and Western bean cutworm (Loxagrotis albiocosta).
A protein conferring insect resistance can be encoded by one or more genes encoding toxins to nematodes which attack crops. Some exemplary nematode species affecting corn are the corn cyst nematode (Heterodera zeae), the Root knot nematode (Meloidogyne spp.), and the sting nematode (Belonolaimus longicaudatus).
The decrease in cost of production associated with provision of certain traits described above could even be higher for traits where reduced material inputs such as nutrient, fungicide, water, and insecticide would also results in reduced costs related to equipment, labor, fuel, and personnel safety.
In addition to cost decreasing transgenic traits, end user transgenic traits may be added to the female and/or male parent. These traits are considered neutral in terms of enhancing profitability of a hybrid corn production system to a producer. However, these traits will be of benefit to the end users, such as farmers and processors, of hybrid seed. Such end users traits include feed quality, food quality, processing, pharmaceutical, and industrial traits. Example of proteins responsible for end user traits are exhibited in Tables 10A and 10B.
In one embodiment, the method of the present invention further comprises providing an end user transgenic trait on the female and/or the male parent. An end user trait may be defined for this purpose as a trait that requires identity preservation by the end users. Examples of these traits are also given in Tables 10A and 10B. The end user trait may be selected from the group consisting of enhanced amino acid content, enhanced protein content, modified or enhanced fatty acid composition, enhanced oil content, enhanced carbohydrate content, and a combination thereof. Examples of these and other end users traits are also given in Tables 10A and 10B.
The present invention also discloses a hybrid corn seed produced by the method of the present invention.
In another embodiment the traits in the hybrid seed may consist of one or more herbicide tolerance traits, three or more insect resistance traits, and an enhanced amino acid content trait.
The traits in the hybrid seed may consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the enhanced amino acid content trait, and the drought tolerance trait. The traits in the hybrid seed may also consist of one or more of the herbicide tolerance traits, two or more of the insect resistance traits, the enhanced amino acid content trait, the drought tolerance trait, and the male sterility system trait. Alternatively, the traits in the hybrid seed may consist of one or more herbicide tolerance traits, one two or more insect resistance traits, an enhanced amino acid content trait, a drought tolerance trait, a male sterility system trait, and an intrinsic yield trait.
Nucleic acid sequences encoding proteins that confer cost decreasing traits or end-user traits are operably linked to various expression elements to create one or more expression units. These expression units generally comprise in 5′ to 3′ direction: a promoter (usually with one or more enhancers), a nucleic acid encoding a trait of interest, and a 3′ untranslated region. Other expression elements such as a 5′UTRs, organelle transit peptide sequences, and introns may be added to facilitate expression of the trait. Also, instead of using a nucleic acid encoding a trait, one may alternatively provide a nucleic acid sequence for transcription of an RNA molecule for instance via an RNAi-mediated approach in order to manipulate the expression of an endogenous or heterologous gene. Such methods are well in the art.
In another embodiment the traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, an enhanced amino acid content trait, a drought tolerance trait, a male sterility system trait, an intrinsic yield trait, and an enhanced oil content trait.
In another embodiment the traits in the hybrid seed consist of one or more herbicide tolerance traits, one two or more insect resistance traits, one or more enhanced amino acid content trait, one or more drought tolerance traits, a male sterility system trait, an intrinsic yield trait, a nutrient use efficiency trait, an enhanced oil content trait, an enhanced protein content trait, and a cold tolerance trait.
In another embodiment the traits in the hybrid seed may consist of one or more herbicide tolerance traits, two or more insect resistance traits, one or more enhanced amino acid content trait, one or more drought tolerance trait, a male sterility system trait, an intrinsic yield trait, a nutrient use efficiency trait, an enhanced oil content trait, an enhanced protein content trait, and a cold tolerance trait.
Nucleic acids for proteins disclosed in the present invention can be expressed in plant cells by operably linking them to a promoter functional in plants, preferably in monocots, such as corn. Tissue specific and/or inducible promoters may be utilized for appropriate expression of a nucleic acid for a particular trait in a specific tissue or under a particular condition. Examples describing such promoters include U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (light inducible promoters), U.S. Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent application publication US 2004-0216189 (maize chloroplast aldolase promoter). Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert, et al., 1987, Proc. Natl. Acad. Sci. USA, 84:5745-5749), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton, et al., 1987 Plant Mol. Biol. 9: 315-324), the CaMV 35S promoter (Odell, et al., 1985, Nature, 313:810-812), the figwort mosaic virus 35S-promoter (Walker, et al., 1987, Proc. Natl. Acad. Sci. USA, 84:6624), the sucrose synthase promoter (Yang, et al., 1990, Proc. Natl. Acad. Sci. USA, 87:4144-4148), the R gene complex promoter (Chandler, et al., 1989 Plant Cell, 1:1175-1183), and the chlorophyll a/b binding protein gene promoter, etc. In the present invention, CaMV35S with enhancer sequences (U.S. Pat. Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Pat. Nos. 6,051,753; 5,378,619), PC1SV (U.S. Pat. No. 5,850,019), Os.Act1 (U.S. Pat. No. 5,641,876), maize globulin 1 promoter (U.S. Pat. No. 6,329,574; US20050132437), maize ubiquitin promoter (US20060037095), rice cytosolic those phosphate isomerase promoter (OsTPI; U.S. Pat. No. 7,132,528), rice actin 15 gene promoter (OsAct15; US Patent Application Publication 20060162010), wheat peroxidase promoter (US2006007013), corn B-32 protein promoter (Hartings et al. 1990, Plant Mol. Biol. 14:1031-1040), wheat peroxidase promoter (GenBank Accession X53675 S54871), and AGRtu.nos promoter (GenBank Accession V00087; Depicker, et al, 1982; Bevan, et al., 1983) may be of particular benefit. In some instances, e.g., OsTPI and OsAct 15, a promoter may include a 5′UTR and/or a first intron. A chimeric promoter may be useful in some instances e.g., a chimera of actin and 35S enhancer and promoter (e.g., see US 2005-0283856).
The 3′ untranslated sequence/region (3′UTR), 3′ transcription termination region, or polyadenylation region is understood to mean a DNA molecule linked to and located downstream in the direction of transcription of a structural polynucleotide molecule responsible for a trait and includes polynucleotides that provide a polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing or gene expression. The polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3′ end of the mRNA precursor. The polyadenylation sequence can be derived from the natural gene, from a variety of plant genes, or from T-DNA genes. Examples of these include polyadenylation molecules from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi, et al., 1984, EMBO J., 3:1671-1679) and AGRtu.nos (Rojiyaa, et al., 1987, Genbank Accession E01312). In the present invention, 3′ UTR from the following genes, AGRtu.nos (Rojiyaa, et al., 1987, Genbank Accession E01312), maize globulin 1 (Belanger and Kriz, Genetics, 129:863-872, 1991; US20050132437), E6 (Accession #U30508), ORF25 from Agrobacterium tumefaciens (Barker et al., 1983, Plant Mol. Biol. 2:335-350; US20050039226), and TaHsp17 (wheat low molecular weight heat shock protein gene; GenBank Accession #X13431), and CaMV.35S may be of particular benefit.
A 5′ UTR that functions as a translation leader sequence is a genetic element located between the promoter sequence and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences include maize and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coat protein leaders, plant rubisco leaders, GmHsp leader (U.S. Pat. No. 5,659,122), PhDnaK leader (U.S. Pat. No. 5,362,865), AtAnt1 leader, TEV leader (Carrington and Freed, Journal of Virology, 64:1590-1597, 1990), and AGRtunos leader (GenBank Accession V00087; Bevan, et al., 1983, Nature, 304:184-187) among others. In the present invention, 5′ UTRs that may in particular find benefit are from the following genes, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990, J. Virology 64:1590-1597), wheat major chlorophyll a/b-binding protein (Lamppa et al., 1985, Mol. Cell. Biol. 5, 1370; Genbank accession M10144), AtAnt1 (US Patent Application 20060236420), OsAct1 (U.S. Pat. No. 5,641,876), OsTPI (U.S. Pat. No. 7,132,528), OsAct15 (US Publication No. 20060162010), and AGRtunos (GenBank Accession V00087; Bevan et al., 1983).
In some embodiments, a protein product of a nucleic acid responsible for a particular trait is targeted to an organelle for proper functioning. For example, targeting of a protein to chloroplast is achieved by using a chloroplast transit peptide sequence. These sequences can be isolated or synthesized from amino acid or nucleic acid sequences of nuclear encoded but chloroplast targeted genes such as small subunit (RbcS2) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, and thioredoxin F proteins. Other examples of chloroplast targeting sequences include the maize cab-m7 signal sequence (Becker, et al., 1992, Plant Mol. Biol. 20:49; PCT WO 97/41228), the pea glutathione reductase signal sequence (Creissen, et al., 1992, Plant J., 2(1):129-131; PCT WO 97/41228), and the CTP of the Nicotiana tabacum ribulose 1,5-bisphosphate carboxylase small subunit chloroplast transit peptide (NtSSU-CTP) (Mazur, et al., 1985, Nucleic Acids Res., 13:2373-2386). In the present invention, AtRbcS4 (CTP1; U.S. Pat. No. 5,728,925), Petunia hybrida EPSPS CTP (della-Cioppa, et al., 1986), AtShkG (CTP2; Klee, et al., 1987), AtShkGZm (CTP2 synthetic; see SEQ ID NO:14 of WO04009761), and PsRbcS (Coruzzi, et al., 1984) may be used, among others. Other examples of CTPs suitable for the present invention can also be found in SEQ ID NOs:1-22 of this application and in Behrens et al. (Science 316:1185-1189, 2007).
The nucleic acids for traits described herein can be targeted to other organelles such as mitochondria for proper functionality. This can be achieved by adding pre-sequences to a nucleic acid of interest. The nucleic acids can also be targeted to both chloroplast and mitochondria by a dual-targeting peptide to make use of organellar biochemistry more effectively. Such pre-sequence elements are known to those skilled in the art. For example, mitochondrial pre-sequences are described in Silva Filho et al., Plant Mol. Biol. 30:769-780 (1996). Nucleic acid sequences that encode dual-targeting peptide sequences can be identified from the nucleic acids coding for the following proteins which are known be targeted to both chloroplasts and mitochondria: Zn-MP (Moberg et al., Plant J. 36:616-628, 2003), gluthathione reductase (Rudhe et al., J. Mol. Biol. 324:577-585, 2002; Creissen et al., Plant J. 8:167-175, 1995) and histidyl-tRNA synthetase (Akashi et al., FEBS Lett. 431:39-44, 1998).
The term “intron” refers to a polynucleotide molecule that may be isolated or identified from the intervening sequence of a genomic copy of a gene and may be defined generally as a region spliced out during mRNA processing prior to translation. Alternately, introns may be synthetically produced. Introns may themselves contain sub-elements such as cis-elements or enhancer domains that effect the transcription of operably linked genes. A “plant intron” is a native or non-native intron that is functional in plant cells. A plant intron may be used as a regulatory element for modulating expression of an operably linked gene or genes. A polynucleotide molecule sequence in a transformation construct may comprise introns. The introns may be heterologous with respect to the transcribable polynucleotide molecule sequence. Examples of introns useful in the present invention include the corn actin intron and the corn HSP70 intron (U.S. Pat. No. 5,859,347), and rice TPI intron (OsTPI; U.S. Pat. No. 7,132,528).
Duplication of any genetic element across various expression units is avoided due to trait silencing or related effects. Duplicated elements across various expression units are used only when they do not interfere with each other or do not result into silencing of a trait.
Methods are known in the art for assembling and introducing constructs into a cell in such a manner that the nucleic acid molecule for a trait is transcribed into a functional mRNA molecule that is translated and expressed as a protein product. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press. Methods for making transformation constructs particularly suited to plant transformation include, without limitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, all of which are herein incorporated by reference in their entirety. These types of vectors have also been reviewed (Rodriguez, et al., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, 1988; Glick, et al., Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., 1993).
The expression units may be provided between one or more T-DNA borders on a transformation construct designed for Agrobacterium-mediated transformation. The transformation constructs permit the integration of the expression unit between the T-DNA borders into the genome of a plant cell. The constructs may also contain plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404, EHA101, and EHA105 carrying a plasmid having a transfer function for the expression unit. Other strains known to those skilled in the art of plant transformation can function in the present invention.
The traits of the present invention are introduced into inbreds by transformation methods known to those skilled in the art of plant tissue culture and transformation. Any of the techniques known in the art for introducing expression units into plants may be used in accordance with the invention. Examples of such methods include electroporation as illustrated in U.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; protoplast transformation as illustrated in U.S. Pat. No. 5,508,184; and Agrobacterium-mediated transformation as illustrated in U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301. Preferably, inbreds of corn are transformed by the method disclosed in U.S. Pat. Nos. 5,981,840, 7,060,876, 5,591,616, or WO9506722, US2004244075 and other methods known in the art of corn transformation.
After effecting delivery of expression units to recipient cells, the next steps generally concern identifying the transformed cells for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene with a transformation construct prepared in accordance with the invention. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait. Examples of various selectable or screenable markers are disclosed in Miki and McHugh, 2004 (“Selectable marker genes in transgenic plants: applications, alternatives and biosafety”, Journal of Biotechnology, 107:193-232).
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In an exemplary embodiment, any suitable plant tissue culture media, for example, MS and N6 media may be modified by including further substances such as growth regulators. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation had occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.
To confirm the presence of the DNA for a transgenic trait in the regenerating plants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays, such as Southern and northern blotting and PCR™; “biochemical” assays, such as for detecting the presence of a protein product, e.g., by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Once a transgene for a trait has been introduced into a plant, that gene can be introduced into any plant sexually compatible with the first plant by crossing, without the need for ever directly transforming the second plant. Therefore, as used herein the term “progeny” denotes the offspring of any generation of a parent plant prepared in accordance with the present invention. A “transgenic plant” may thus be of any generation.
In one embodiment of the present invention, the identified cost decreasing traits and/or end-user traits are introduced into the germplasm of the female or the male parent as required either by direct transformation of elite inbreds or by first transforming an easily transformable inbred and then introducing the trait to elite germplasm by breeding into an elite inbred. The traits are introduced individually in individual inbreds. The traits present in individual inbreds are then combined to obtain required traits in a particular female or male inbred.
In another embodiment of the present invention, more than one traits are introduced into an inbred by repeatedly transforming the same inbred with a new trait provided on a transformation construct.
In another embodiment of the present invention, more than one trait is introduced into an inbred by providing more than one trait on a DNA construct used for transformation. For example, two traits may be provided on the same DNA construct and inserted into one locus thereby saving one locus which can be used for inserting different one or more traits.
In another embodiment of the present invention, more than one trait is introduced into an inbred by providing more than one trait on mini-chromosomes, for example, of the type described in the U.S. Pat. No. 7,235,716, U.S. Pat. No. 7,227,057, U.S. Pat. No. 7,226,782, U.S. Pat. No. 7,193,128, U.S. Pat. No. 6,649,347, US20050268359A1, all of which are incorporated herein by reference.
In another embodiment of the present invention, a combination of methods described above is applied to introduce more than one trait in an inbred. Further, inbreds with more than one trait can be crossed with at least one other inbred. In another aspect, an inbred may be crossed with at least two inbreds, non-limiting examples of which may include three-way, four-way, or multi-way crosses known to those skilled in the art of plant breeding.
As used herein, the term “inbred” means a line that has been bred for genetic homogeneity. Without limitation, examples of breeding methods to derive inbreds include pedigree breeding, recurrent selection, single-seed descent, backcrossing, and doubled haploids.
As used herein, the term “hybrid” means a progeny of mating between at least two genetically dissimilar parents. Without limitation, examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three-way cross, and double cross, wherein at least one parent in a modified cross is the progeny of a cross between sister lines.
As used herein, the term “tester” means a line used in a test cross with another line wherein the tester and the lines tested are from different germplasm pools. A tester may be isogenic or nonisogenic.
Herein, “germplasm” includes breeding germplasm, breeding populations, collection of elite inbred lines, populations of random mating individuals, biparental crosses, heterotic groups, and apomictic germplasm.
It is known in the art that maize germplasm can be divided into a number of distinct heterotic groupings. A key goal of hybrid breeding programs is to maximize yield via complementary crosses. Crosses from distinct germplasm pools that result in a yield advantage constitute heterotic groups. The identification of heterotic groups facilitates informed crosses for a yield advantage. During inbred line development, advanced inbred lines are crossed with different tester lines in order to determine how the inbred line performs in hybrid combinations. The effect of a single cross reflects the specific combining ability (SCA) and the effect of the inbred in multiple crosses with different testers (typically in multiple locations) reflects the general combining ability (GCA). The heterotic groups may be used as female or male parents.
Apomictic germplasm can be produced by introducing certain genes such as those disclosed in these published patents and patent applications which are incorporated herein by reference: U.S. Pat. No. 5,710,367, U.S. Pat. No. 5,811,636, U.S. Pat. No. 6,750,376, U.S. Pat. No. 7,148,402, US2002069433A1, US2003082813A1, US2004016022A1, US2004098760A1, US2004103452A1, US2004148667A1, US2004168216A1, US2004168217A1, US2004216193A1, US2005155111A1, and US2005262595A1. Apomixis is a form of reproduction that produces seeds without the need for fertilization to take place. Progeny are consequently clones of the mother plant.
An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance. Examples of elite lines suitable for use in the present invention are provided in Tables 11 and 12.
Descriptions of breeding methods that are commonly used for different traits and crops can be found in one of several reference books (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98, 1960; Simmonds, “Principles of crop improvement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen, “Plant breeding perspectives,” Wageningen (ed), Center for Agricultural Publishing and Documentation, 1979; Fehr, In: Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph., 16:249, 1987; Fehr, “Principles of variety development,” Theory and Technique, (Vol 1) and Crop Species Soybean (Vol 2), Iowa State Univ., Macmillan Publ. Co., NY, 360-376, 1987). The production of new elite corn hybrids requires the development and selection of elite inbred lines, the crossing of these lines and selection of superior hybrid crosses. Crosses for the production of the seed include traditional single crosses, three-way crosses, and double crosses or four-way cross. A single cross means the first generation of a cross between two inbred lines (preferably from two different heterotic groups), an inbred line and a foundation back cross, or of two foundation back crosses. A three-way cross means the first generation of a cross between a foundation single cross as one parent and an inbred line or a foundation backcross as the other parent. A double cross or four-way cross means the first generation of a cross between two foundation single crosses. The hybrid seed can be produced by manual crosses between selected male-fertile parents or by using male sterility systems. Additional data on parental lines, as well as the phenotype of the hybrid, influence the breeder's decision whether to continue with the specific hybrid cross. Details on hybrid crop breeding can be found in Bernardo, Breeding for Quantitative Traits in Plants, Stemma Press, Woodbury, Minn., 2002.
In general, two distinct breeding stages are used for commercial development of the female and male inbred containing a transgenic trait. The first stage involves evaluating and selecting a superior transgenic event, while the second stage involves integrating the selected transgenic event in a commercial germplasm.
First a transformation construct responsible for a trait is introduced into the genome via a transformation method. Numerous independent transformants (events) are usually generated for each construct. These events are evaluated to select those with superior performance. The event evaluation process is based on several criteria including 1) transgene expression/efficacy of the trait, 2) molecular characterization of the trait, 3) segregation of the trait, 4) agronomics of the developed event, and 5) stability of the transgenic trait expression. Evaluation of large population of independent events and more thorough evaluation result in the greater chance of success.
Events showing an appropriate level of gene expression or inhibition (via RNAi-mediated approaches) that corresponds with a desired phenotype (efficacy) are selected for further use by evaluating the event for insertion site, transgene copy number, intactness of the transgene, zygosity of the transgene, level of inbreeding associated with a genotype, genetic background, and growth response in various expected environmental conditions.
Events demonstrating, for instance, a clean single intact insert are found by conducting molecular assays for copy number, insert number, insert complexity, presence of the vector backbone, and by development and use of event-specific nucleic acid detection assays and are used for further breeding and development.
Segregation of a trait may be followed to identify transgenic events that follow a single-locus segregation pattern. A direct approach is to evaluate the segregation of the trait. An indirect approach may assess selectable marker segregation (if genetically linked to the transgenic trait). Agronomic performance of a transgenic inbred may vary due to somaclonal variation introduced during tissue culture process, insertional effects, homozygosity of a transgene, level of inbreeding, and genetic background. In advanced generations, agronomic performance is evaluated in several genetic backgrounds in replicated trials to identify the best gene and germplasm combination. For hybrid crops such as corn, agronomic trials are conducted in both inbred and hybrid background.
Transgene event instability over generations may be caused by transgene inactivation due to multiple transgene copies, zygosity level, highly methylated insertion sites, or level of stress. Thus, stability of transgenic trait expression may be ascertained by testing in different generations, environments, and in different genetic backgrounds. Events that show transgenic trait silencing are discarded. Events performing appropriately in a given germplasm background are selected for further development.
Generally, events with a single intact insert that is inherited as a single dominant gene and follow Mendelian segregation ratios are used in commercial breeding strategies such as backcrossing and forward breeding.
In a preferred embodiment, backcrossing is used to recover the genotype of an elite inbred with an additional transgenic trait. In each backcross generation, plants that contain the transgene are identified and crossed to the elite recurrent parent. Several backcross generations with selection for recurrent parent phenotype are generally used by commercial breeders to recover the genotype of the elite parent with the additional transgenic trait. During backcrossing the transgene is kept in a hemizygous state. Therefore, at the end of the backcrossing, the plants are self- or sib-pollinated to fix the transgene in a homozygous state. The number of backcross generations can be reduced by marker-assisted backcrossing (MABC). The MABC method uses molecular markers to identify plants that are most similar to the recurrent parent in each backcross generation. With the use of MABC and appropriate population size, it is possible to identify plants that have recovered over 98% of the recurrent parent genome after only two or three backcross generations. By eliminating several generations of backcrossing, it is often possible to bring a commercial transgenic product to market one year earlier than a product produced by conventional backcrossing.
Forward breeding is any breeding method that has the goal of developing a transgenic variety, inbred line, or hybrid that is genotypically different, and superior, to the parents used to develop the improved genotype. When forward breeding a transgenic crop, selection pressure for the efficacy of the transgene is usually applied during each generation of the breeding program. Additionally, it is usually advantageous to fix the transgene in a homozygous state during the breeding process as soon as possible to uncover potential agronomic problems caused by unfavorable transgene x genotype interactions.
After integrating the transgenic traits into a commercial germplasm, the final inbreds and hybrids are tested in multiple locations. Testing typically includes yield trials in trait neutral environments as well as typical environments of the target markets. If the new transgenic line has been derived from backcrossing, it is usually tested for equivalency by comparing it to the non-transgenic version in all environments.
Several types of genetic markers are known to those skilled in the art and can be used to expedite breeding programs. These genetic markers may include Restriction Fragment Length Polymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR), Single Nucleotide Polymorphisms (SNP), Insertion/Deletion Polymorphisms (Indels), Variable Number Tandem Repeats (VNTR), and Random Amplified Polymorphic DNA (RAPD).
Doubled-haploid breeding technology can be used to expedite the development of parental lines for crossing as known to those skilled in the art. The development of parental lines can be further enhanced by combining doubled-haploid breeding technologies with high-throughput, non-destructive seed sampling technologies. For example, U.S. Patent Application Publication US2006 0046264 (filed Aug. 26, 2005) and U.S. Patent Application Publication US2007 0204366 (filed Mar. 2, 2007), which are incorporated herein by reference in their entirety, disclose apparatuses and systems for the automated sampling of seeds as well as methods of sampling, testing and bulking seeds.
In another aspect of the present invention, only those transgenic events are selected for further development in which the nucleic acids encoding for cost decreasing traits and/or end user traits are inserted and linked to genomic regions (defined as haplotypes) that are found to provide additional benefits to the crop plant. The transgene and the haplotype comprise a T-type genomic region. Methods for using haplotypes and T-type genomic regions for enhancing breeding are disclosed in U.S. Application Publication 2006 0282911.
The present invention also provides for parts of the plants of the present invention. Plant parts, without limitation, include seed, endosperm, ovule and pollen. In a preferred embodiment of the present invention, the plant part is a seed. The invention also includes and provides transformed plant cells which comprise a nucleic acid molecule of the present invention.
Brassica seed coat gene
Corynebacterium glutamicum
Brassica turgor gene-26
Zea mays brittle2 gene
Arabidopsis CtpA (C terminal
Papaya ringspot virus coat
Papaya ringspot virus coat
Xanthomonas hypersensitive
thaliana emb|CAA19877.1|(AL031032) protein kinase-like protein [Arabidopsis
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
Arabidopsis thaliana chromosome 3, F24B22.150~unknown protein [Oryza sativa
thaliana]
cerevisiae]
tumefaciens str. C58 (U. Washington)]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana] dbj|BAB10429.1|unnamed protein product [Arabidopsis thaliana]
thaliana]
thaliana] sp|P55034|PDS4_ARATH 26S proteasome non-ATPase regulatory subunit 4
subtilis str. 168]
campestris str. ATCC 33913]
thaliana]
Arabidopsis thaliana gb|AAD25788.1|Similar to gb|U21858 transcription initiation factor
cerevisiae] sp|P25297|PH84_YEAST Inorganic phosphate transporter PHO84
fungorum]
Arabidopsis thaliana gb|AAD25802.1|Belongs to the PF|01027 Uncharacterized protein
luminescens subsp. laumondii TTO1]
thaliana]
cerevisiae]
campestris str. ATCC 33913]
thaliana gb|AAB64035.1|stearoyl-ACP desaturase [Arabidopsis thaliana]
Arabidopsis thaliana
thaliana]
thaliana]
thaliana]
subtilis str. 168]
Arabidopsis thaliana emb|CAA64407.1|CONSTANS protein [Arabidopsis thaliana]
thaliana]
thaliana]
thaliana]
thaliana] p
cerevisiae]
thaliana]
thaliana] pir||T47821 methylenetetrahydrofolate reductase MTHFR1-
thaliana]
laumondii TTO1]
cerevisiae] sp|P32328|DBFB_YEAST Serine/threonine-protein kinase DBF20
Agrobacterium tumefaciens (strain C58, Dupont)
cerevisiae] sp|P32943|CGS6_YEAST S-PHASE ENTRY CYCLIN 6 pir||S64417 cyclin B6-
cerevisiae]
thaliana]
thaliana]
thaliana] pir||E84585 26S proteasome subunit 4 [imported] - Arabidopsis thaliana
cerevisiae] prf||2211396A SSU72 protein
thaliana]
thaliana] pir||H86427 unknown protein [imported] - Arabidopsis thaliana
melanogaster and contains a PF|00249 Myb-like DNA-binding domain.
thaliana]
thaliana] pir||D96772 probable RING zinc finger protein
thaliana]
E. coli glnB
Anabaena SPP
Synechocystis Sucrose phosphate synthase
Nostoc sp. PCC 7120 glnB
Brassica P-II
Nitrosomonas europaea dual function SBPase/FBPase-
Nostoc sp. PCC 7120 GlpX protein
Nostoc punctiforme strain ATCC 29133 GlpX protein-NOS1c0617
Anabaena SPS C154
Anabaena SPS C287
Arabidopsis SUT2
Arabidopsis SUT4
Arabidopsis sucrose export defective 1-AF302188
Nostoc sp. PCC 7120 sdx1-like - 17134979
Synechocystis sp. PCC 6803 sdx1-like - 1652844
Nostoc punctiforme sdx1-like
Arabidopsis Suc5 - AJ252133
Emericella nidulans alxA
Arabidopsis LFY
E. coli clpB
Synechocystis clpB
Xylella clpB
Synechocystis 1-deoxy-D-xylulose-5-phosphate reductoisomerase-D64000
Agrobacterium 1-deoxy-D-xylulose 5-phosphate reductoisomerase-
Agrobacterium 1-deoxy-D-xylulose-5-phosphate synthase-AAK86554
Xylella 1-deoxyxylulose-5-phosphate synthase-AAF85048
sorghum phyA with corn phyC intron 1
sorghum phyB with corn phyC intron 1
sorghum phyC with corn phyC intron 1
Synechocystis biliverdin reductase
Arabidopsis salt-tolerance protein
Aspergillis phytochrome
S. pombe ALA1-like-CAA21897
E. coli adhC - AE000142
Nostoc sp. PCC 7120 glutathione dependent formaldehyde dehydrogenase-
sorghum elF-(iso)4F
sorghum elF-4F
Synechocystis cobA w cp transit peptide
Xylella tetrapyrrole methylase with transit peptide
sorghum proline permease
Xylella citrate synthase
E. coli citrate synthase
Chlamydomonas reinhardtii envelope protein LIP-36G1
Xylella SAG13-like - E82748
Nostoc punctiforme SAG13-like
Xylella adenylate transporter - XF1738
Agrobacterium ornithine decarboxylase
Synechocystis Rieske iron-sulfur protein
Arabidopsis AGL15
Arabidopsis agl8 - Q38876
E. coli guaB - NP_417003
Arabidopsis G748
Arabidopsis NAM (no apical meristem)-like protein-
Arabidopsis CRE1b
Arabidopsis HK2
Arabidopsis HK3
Arabidopsis RAV2/G9
Arabidopsis nitrate transporter NTL1 like sequence
E. coli cytoplasmic ferritin
E. nidulans cysA - AF029885
Arabidopsis AtHAP3a
Arabidopsis CCA1
Synechocystis hypothetical sugar kinase - BAA10827
Synechocystis S-adenosylhomocysteine hydrolase-BAA18079
Aspergillus yA (laccase 1) - X52552
Synechocystis ssr3189 - BAA17701
Synechocystis ssr2315 - BAA17190
Agrobacterium aiiA-like protein [attM]- AAD43990
Xylella aiiA-like protein - XF1361
Xanthomonas aiiA - like protein
E. coli betT
Xenorhabdus BetT-like 1
Arabidopsis eskimo 1
E. coli yagD homocysteine S-methyltransferase-Q47690
sorghum 14-3-3 10
sorghum 14-3-3 10 N-terminus
sorghum TTG1-like
Arabidopsis G975
E. coli fructose-1,6-bisphosphatase
Synechocystis fructose-1,6-bisphosphatase F-I
Synechocystis fructose-1,6-bisphosphatase F-II
Solanum lycopersicum (tomato) wound
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
sativa (japonica cultivar-group)]
Nostoc punctiformethaliana]
thaliana]
thaliana]
thaliana] pir||T51812 phosphoribosyl-
thaliana]
thaliana]
cerevisiae] emb|CAA80789.1|YBLO515
thaliana]
halodurans C-125]
luminescens subsp. laumondii TTO1]
thaliana]
halodurans C-125]
thaliana]
thaliana]
cerevisiae] sp|P38789|SSF1_YEAST
thaliana]
thaliana]
thaliana] emb|CAB79362.1|NADPH-
Synechocystis sp.
thaliana]
thaliana]
thaliana]
sativa (japonica cultivar-group)]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
cerevisiae] gb|AAB68212.1|Lpg21p
cerevisiae] emb|CAA50895.1|
halodurans C-125]
subtilis str. 168]
laumondii TTO1]
thaliana]
thaliana]
thaliana]
thaliana] ref|NP_199325.1|aspartyl
thaliana]
meliloti (strain 1021) magaplasmid
Arabidopsis thaliana
aurantiacus]
thaliana
grisea pathogenicity protein
thaliana]
flexneri 2a str. 301] EDL933]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana]
thaliana] sp|Q93WK5|APRR7_ARATH
thaliana]
punctiforme PCC 73102]
subtilis str. 168]
thaliana]
sativa (japonica cultivar-group)]
officinarum]
thaliana]
thaliana]
glutamicum ATCC 13032]
The following examples are included to illustrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Examples 1-12 are outlined in Table 13. In these examples several trait combinations for enhancing profitability of hybrid corn seed production system are provided. In general, traits having high impact on decreasing cost of production are provided on the female parent. These traits include herbicide tolerance, male sterility system, yield, and nutrient use efficiency. Traits having low impact on decreasing cost of production are provided on the male parent and/or the female parent. These traits include cold tolerance, drought tolerance, disease resistance, and insect resistance. For example, in example 5 or 6, by providing an enhanced yield, corn borer resistance, male sterility system trait, and herbicide tolerance on the female parent, producers will be able to decrease their cost of production by an estimated 28.8% (=14.2+10.8+3.7+0.1; based on Table 1). Similarly, as shown in example 7, by providing two herbicide tolerance traits, and 2 or 3 insect resistance traits on the female parent, producers will be able to decrease their cost of production by 28.6% or 28.7%.
Because the cost of breeding uneven number of traits into a hybrid is similar to the cost of breeding even number traits, it is of benefit to balance similar number of traits on each parent. The trait combinations exemplified here have a similar or identical number of traits on each parent, wherein similar is defined as±one or two or three traits to reduce the cost of breeding these traits into a hybrid. If a trait is found to have an adverse effect on a parent then that traits can be provided on another parent to remove the adverse effect.
Also, if a herbicide tolerance trait is used as a part of an MSS on the female parent then the same herbicide tolerance trait is also provided on the male parent as shown in examples 4-6, 9-12.
Description of various abbreviations used for trait combinations is given below. The sources of trait genes or trait events are shown in parentheses, which are all incorporated herein by reference. G2: glyphosate tolerance mechanism 2 (Event NK603; U.S. Pat. No. 6,825,400); CRW2-G2: corn root worm resistance mechanism 2 (Event MON88017; WO05059103) linked to glyphosate tolerance mechanism 2 (Event NK603; U.S. Pat. No. 6,825,400); CB2: corn borer resistance mechanism 2 (Event MON89034; U.S. Application No. 60/808,834); CB3-Glu: corn borer resistance mechanism 3 and glufosinate tolerance (Event 1507; US20060037095, US 20050039226); CRW3-Glu: corn root worm resistance mechanism 3 and glufosinate tolerance (Event 59122; US20060070139); D1: drought tolerance mechanism 1 (Tables 2A and 2B to 9); MSS: male sterility system (Tables 2A and 2B); IY1: intrinsic yield mechanism 1 (Tables 2A and 2B to 9); CRW4: corn root worm resistance mechanism 4 (US20060021087); NUE1: nitrogen use efficiency mechanism 1 (Tables 2A and 2B to 9); G3: glyphosate tolerance mechanism 3; D2: drought tolerance mechanism 2 (Tables 2A and 2B to 9); C1: cold tolerance mechanism 1 (Tables 2A to 2B to 9); and CB4: corn borer resistance mechanism 4.
Examples 13-28 are outlined in Table 14. In these examples several trait combinations for enhancing profitability of a hybrid corn seed production system are provided. In general, traits having high impact on decreasing cost of production are provided on the female parent. These traits include herbicide tolerance, male sterility system, yield, and nutrient use efficiency, Traits having low impact on decreasing cost of production are provided on the male parent or the female parent. These traits include cold tolerance, drought tolerance, diseases resistance, and insect resistance. In addition, end user traits, which are considered neutral in terms of enhancing profitability of a hybrid corn production system to a producer can be provided on the male and/or the female parent. These traits will be of benefit to the end users, such as farmers and processors. These traits may include, among others, enhanced amino acid, protein, fatty acid, carbohydrate, and oil content.
Because the cost of breeding an uneven number of traits into a hybrid is similar to the cost of breeding an even number traits, it is of benefit to balance similar number of traits on each parent. The traits combinations exemplified here have a similar number of traits on each parent, wherein similar means±one, two, or three traits to reduce the cost of breeding these traits into a hybrid. If a trait is found to have an adverse effect on a parent then that trait can be provided on another parent to remove the adverse effect. Also, a trait may be provided in a heterozygous state on each parent to remove the adverse effect such as the LI. Also, if a herbicide tolerance trait is used as a part of an MSS on the female parent then the same herbicide tolerance trait is also provided on the male parent as shown for instance in examples 17-20 and 24-27.
Descriptions of various abbreviations used for trait combinations are given below. The sources of trait genes or trait events are shown in parentheses, which are all incorporated herein by reference. G2: glyphosate tolerance mechanism 2 (Event NK603; U.S. Pat. No. 6,825,400); L1: enhanced lysine content mechanism 1 (Event LY038; US20050132437); CB1: corn borer resistance mechanism 1 (Event MON810; U.S. Pat. No. 6,713,259); CRW1—corn root worm resistance mechanism (Event MON863; US20060095986); CRW2-G2: corn root worm resistance mechanism 2 (Event MON88017; WO05059103) linked to glyphosate tolerance mechanism 2 (Event NK603; U.S. Pat. No. 6,825,400); CB3-Glu: corn borer resistance mechanism 3 and glufosinate tolerance (Event 1507; US20060037095, US 20050039226); CRW3-Glu: corn root worm resistance mechanism 3 and glufosinate tolerance (Event 59122; US20060070139); D1: drought tolerance mechanism 1 (Tables 2A and 2B to 9); L2: enhanced lysine content mechanism 2; MSS: male sterility system (Tables 2A and 2B); IY1: intrinsic yield mechanism 1 (Tables 2A and 2B to 9); NUE1: nitrogen use efficiency mechanism 1 (Tables 2A and 2B to 9); CRW4: corn root worm resistance mechanism 4; L2-O: enhanced lysine content mechanism 2 linked to enhanced oil content trait (U.S. Pat. No. 6,822,141); T-P: enhanced tryptophan content (US20030213010) linked to enhanced protein content; G3: glyphosate tolerance mechanism 3; D2: drought tolerance mechanism 2 (Tables 2A and 2B to 9); and C1: cold tolerance mechanism 1 (Tables 2A and 2B to 9).
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This application claims the priority of U.S. provisional application Ser. Nos. 60/848,952 (filed Oct. 3, 2006) and 60/922,013 (filed Apr. 5, 2007), the entire disclosure of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60922013 | Apr 2007 | US | |
| 60848952 | Oct 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12440160 | Jun 2010 | US |
| Child | 16266827 | US |
