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The following quote was obtained by the news editors from the background information supplied by the inventors: "Traditional plant breeding to include parental lines for desirable traits involves crossing selected parental lines to introduce those desirable traits into the progeny of the cross. In a crossing-based approach, often, not only the desirable trait is transferred to the progeny but some randomization of the genomes of both parental lines occurs. This results in a wide segregation and variation of morphology and other traits of the progeny, which are not predictable. The uncontrolled variation renders the progeny selection process very long, cumbersome and laborious especially if the desired traits are not expressed early in the progeny or if the desired trait is recessive.
"In an effort to minimize the random variation, breeders prefer homozygous parental lines (inbreds) so that the genetic make up of the F.sub.1 generation is more predictable. The inbreds with a desirable trait are generated by back-crossing a heterozygote with its parental lines, followed by segregation selection and repeated back-crossing. However, this repeated back-crossing is also very long, usually up to 6 to 7 times, depending on the plant, would produce a homozygous plant with the desired trait. Of course, the time scale involved here is dictated by the rate at which plants grow to maturity and set seed and several years can be necessary to produce the desired homozygous parent line.
"Haploid plants contain one half of the usual complement of genes. Normal plants are diploid in that they have two complete sets of chromosomes, one from each parent. Polyploid plants have more than two sets of chromosomes. Haploid plants are capable of growing to maturity but are generally sterile. There are several known methods of generating haploid plants.
"One way to obtain homozygous plants without the need to cross two parental lines followed by a long selection of the segregating progeny, and/or multiple back-crossings is to produce haploids and then double the chromosomes to form doubled haploids. The production of doubled haploid plants yields highly uniform inbred lines and is especially desirable as an alternative to sexual inbreeding of longer-generation crops. By producing doubled haploid progeny, the number of possible gene combinations for inherited traits is more manageable. Thus, an efficient doubled haploid technology can significantly reduce the time and the cost of inbred and cultivar development. Chemicals, such as colchicines are used as chromosome doubling agents. However, due to their mammalian toxicity and low efficiency, their use in plant breeding has been limited. Therefore, agents to induce chromosome doubling in plants with low mammalian toxicity are desirable."
In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "Methods for generating doubled haploid plants, seeds, cells, and cell cultures are provided. The methods disclosed herein, increase the efficiency of the doubled haploid generation by increasing the number of doubled haploids obtained, exhibit low mammalian toxicity and reduced the time required to produce the doubled haploids.
DETAILED DESCRIPTION OF THE INVENTION
"Methods to obtain doubled haploid plants or plant components using low mammalian toxicity chromosome doubling agents are disclosed. Contacting a haploid plant component, e.g., whole plant or a collection or population of plant cells with low mammalian toxicity chromosome doubling agents results in the generation of doubled haploid plants or plant components.
"Chemicals listed in the U.S. Pat. No. 5,866,513 to Michelotti et al., are used to generate doubled haploid plants. The disclosure of U.S. Pat. No. 5,866,513 is incorporated herein by reference in its entirety. For example, Table I and Ia on Cols. 3 and 4, 5 and 6, and 7 and 8 of U.S. Pat. No. 5,866,513, list about 76 specific example compounds and each of those compounds is herein incorporated by reference in its entirety. In addition, methods of making some of the specific examples as described in Cols. 11-22 are incorporated herein by reference in its entirety.
"In one embodiment a method is described for generating doubled haploid plant cells comprising contacting a population of haploid plant cells with a composition comprising a low mammalian toxicity chromosome doubling agent of following formula:
"wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are in each instance independently selected from the group consisting of hydrogen, alkoxy, nitro, halo, alkyl, and haloalkyl; A and B are each independently CH.sub.2, CHR.sup.a or CR.sup.aR.sup.b, wherein R.sup.a and R.sup.b are in each instance independently selected from the group consisting of hydrogen, halogen, cyano, alkyl, alkenyl, alkoxy, haloalkyl, haloalkoxyl, alkylcarbonyl and alkoxycarbonyl, or R.sup.a and R.sup.b together with the attached carbon foam a cycloalkylene; Z is CH.dbd.CH, CH.sub.2--CH.sub.2, CH.sub.2.dbd.CH(CH.sub.3), or CR.sup.d.dbd.CR.sup.e, where R.sup.d and R.sup.e are each independently hydrogen, alkyl or R.sup.d and R.sup.e together with the attached carbons form a cycloalkylene; X and Y are each independently selected from the group consisting of hydrogen, NO.sub.2, CN, halogen, alkylcarbonyl, alkoxycarbonyl, and CH.sub.2--NO.sub.2, provided that X and Y are not both hydrogen; and diastereomers and stereoisomers thereof.
"In a preferred embodiment a method is described for generating doubled haploid plant cells comprising contacting a population of haploid plant cells with a composition comprising a low mammalian toxicity chromosome doubling agent of following formula:
"wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are in each instance independently selected from the group consisting of hydrogen, alkoxy, nitro, halo, alkyl, and haloalkyl; Z is CH.dbd.CH, CH.sub.2--CH.sub.2, CH.sub.2.dbd.CH(CH.sub.3), or CR.sup.d.dbd.CR.sup.e, where R.sup.d and R.sup.e are each independently hydrogen, alkyl or R.sup.d and R.sup.e together with the attached carbons form a cycloalkylene; X and Y are each independently selected from the group consisting of hydrogen, NO.sub.2, CN, halogen, alkylcarbonyl, alkoxycarbonyl, and CH.sub.2--NO.sub.2, provided that X and Y are not both hydrogen; and diastereomers and stereoisomers thereof.
"In one embodiment the low mammalian toxicity chromosome doubling agent comprises a compound of the following formula and diastereomers and stereoisomers thereof:
"Suitable dosage for the low mammalian toxicity chromosome doubling agents for the seedling soak method, disclosed herein, include, for example, 0.01 .mu.M, 0.5 .mu.M, 1 .mu.M, 2 .mu.M, 3 .mu.M, 4 .mu.M, 5 .mu.M, 10 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M, 35 .mu.M, 40 .mu.M, 45 .mu.M, 50 .mu.M, 60 .mu.M, 70 .mu.M, 80 .mu.M, 90 .mu.M, 100 .mu.M, 125 .mu.M, 150 .mu.M, 200 .mu.M, 500 .mu.M, and 1000 .mu.M. Suitable ranges also include, for example, 0.1-10 .mu.M, 1-100 .mu.M, 5-125 .mu.M, 25-200 .mu.M, 50-500 .mu.M, 15-150 .mu.M and 1-10,000 .mu.M.
"The low seedling mortality of the chromosome doubling agents, disclosed herein, when compared to colchicine (e.g., at 0.06% w/v) can range, for example, from less than 10% to about 40% or less than about 5% to about 20% or less than about 15% to about 25% or less than 50% of the total number of seedlings or plant cells treated.
"Suitable dosage for the low mammalian toxicity chromosome doubling agents for the seedling foliar application method, disclosed herein, include, for example, 3.5 g ai/ha, 70 g ai/ha, 140 g ai/ha, 280 g ai/ha. Suitable application rates ranges include, for example, 5 g ai/ha to 1120 g ai/ha, and more preferably to 2,800 g ai/ha.
"The low mammalian toxicity of the chromosome doubling agents, disclosed herein, may show LD.sub.50 (rat) that is greater than or equal to 400 mg/kg or 500 mg/kg or 600 mg/kg or 700 mg/kg or 800 mg/kg or 900 mg/kg or 1000 mg/kg. Suitable LD.sub.50 (rat) range may also include 200-1000 mg/kg or 100-1000 mg/kg or greater than 100 mg/kg or greater than 1000 mg/kg.
"A haploid plant has a single set of chromosomes and the reduced number of chromosomes (n) in the haploid plant is equal to that in the gamete. For example, a haploid corn plant has 10 chromosomes, instead of 10 pairs of chromosomes as in a normal diploid corn plant. A diploid plant has two sets of chromosomes and the chromosome number (2n) is equal to that in the zygote. Each chromosome in a pair is derived from the maternal or paternal line.
"A doubled haploid ('DH') or a doubled haploid plant or cell refers to the plant or its component developed by the doubling of a haploid set of chromosomes. A doubled haploid plant is considered a homozygous plant. A plant is considered to be doubled haploid even if the entire vegetative part of the plant does not consist of the cells with the doubled set of chromosomes, as long as the double haploid plant is fertile. The haploid plant may be derived from any plant and may be derived from a heterozygous plant. Such a heterozygous plant may or may not be a member of a heterogeneous population of plants, such as from an open pollinated population of plants. The heterozygous plant may be obtained from any heterozygous materials including local varieties, composites of different genetic backgrounds and other collections of plants.
"A 'haploid immature embryo' is defined as the embryo formed after one sperm nucleus from a pollen grain fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm and before dry down.
"A 'doubled haploid embryo' is an embryo that has one or more cells that contain 2 sets of homozygous chromosomes. 'Callus' refers to an undifferentiated proliferating mass of cells or tissue.
"The phrase 'contacting' includes reference to 'direct contact' and 'indirect contact'. For example, the medium comprising a doubling agent may have direct contact with the haploid cell or the medium comprising the doubling agent may be separated from the haploid cell by a barrier, such as a filter paper, plant tissue, or other cells thus the doubling agent is transferred through the filter paper or cells or tissue to the haploid cell. Contacting is achieved in any suitable manner, e.g., hydroponic treatment of roots, spraying, injecting, infiltrating, soaking, and wetting.
"The term 'plant' includes reference to whole plants and plant components (e.g., leaves, stems, roots, pollen, etc.), seeds and plant cells and progeny of same. 'Plant cell,' as used herein, includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plants that are capable of being used in the methods, disclosed herein, include both monocotyledonous and dicotyledonous plants.
"Suitable plants include, for example, monocotyledonous plants, such as a maize, rice, wheat, barley, oats, onion, rye, millet, sorghum, sugarcane, lily or turfgrass plant.
"The methods disclosed herein, generally produce a high frequency of doubled haploid plants that are fertile.
"In an embodiment, methods to obtain a doubled haploid embryo, seed, or plant by contacting a haploid embryo with a doubling agent and obtaining a doubled haploid embryo, seed, or plant are disclosed.
"In another embodiment, methods to obtain a doubled haploid cell culture from a suitable plant component, e.g., doubled haploid tissue culture are disclosed.
"A method to obtain a haploid plant component for inducing chromosome doubling includes, for example, a) pollinating ovules, or stigmas, of a plant with pollen from an inducer line, wherein the inducer line has a marker gene that is expressed in embryos and/or endosperm tissue; and b) selecting a haploid embryo that has no marker gene expression. Pollination of the plant can be by any method, including natural cross-pollination and manual pollination.
"Somatic haploid cells, haploid embryos, haploid seeds, or haploid seedlings produced from haploid seeds can be treated with a low mammalian toxicity chromosome doubling agent. Homozygous plants can be regenerated from haploid cells by contacting the haploid cells, such as embryo cells or callus produced from such cells, with chromosome doubling agents, disclosed herein, to create homozygous doubled haploid cells. Treatment of a haploid seed or the resulting seedling or tissue generally may produce a chimeric plant or tissue, partially haploid and partially doubled haploid. It may be beneficial to nick the seedling before treatment with the doubling agent. When reproductive tissue contains doubled haploid cells, then doubled haploid seed is produced.
"Haploid embryos, haploid seeds, somatic haploid cells or tissue from haploid plants can be harvested and transformed by any known means prior to treatment with a doubling agent. Transgenic homozygous plants, regenerated from the transformed cells can be treated with a chromosome doubling agent and growing the resulting seed/tissue to produce a doubled haploid plant having homozygous seeds.
"In an aspect the inducer line may contain a scorable marker gene, for example, colored markers in the endosperm, embryo, stem or leaves. Such markers include GUS, luciferase, CRC, anthocyanin genes, such as A, C, R-nj, and others known in the art. When an inducer line with a scorable marker is crossed with the selected/desired line, the resulting haploid seeds will have, for example, a colored endosperm with colorless embryo. It may be desirable to express the marker gene in the embryo. In particular, it may be desirable to express the marker gene in the early stage of development, about 8-15 days after pollination using an appropriate promoter, such as an oleosin or a Lec1 promoter. Marker negative embryos are then selected to obtain haploid embryos. This method provides the advantage of obtaining haploid embryos without marker genes. Similarly, haploid inducer lines, such as RWS, KEMS, RWS, ZMS or KMS, can be transformed with a lethal gene that is expressed specifically in embryos of the mature seeds. After crossing the inducer line and the female parent the embryos of all the diploid seeds contain the inducible lethal gene, but the embryos of the haploid seeds do not contain the lethal gene. The diploid F1 seeds cannot germinate due to expression of the inducible lethal gene in their embryos. However, the haploid seeds can germinate normally because they do not contain the lethal gene in their embryos. The germinating seedlings are haploid.
"One or more genes of interest that are reflective of the commercial markets and interests of those involved in the development of crops can be transformed or otherwise introduced into a line of interest prior to generating a haploid seed or tissue by crossing with an inducer line. Such gene or genes of interest can also be transformed into a haploid cell or tissue prior to treatment with a chromosome doubling agent to generate double haploid plant component.
"General categories of genes of interest include, for example, include genes encoding agronomic traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting, for example, kernel size, sucrose loading, and the like.
"Another method is obtaining a doubled haploid seed that includes: a) obtaining a haploid seed by pollinating an ovule with an inducer line wherein the ovule comprises a set of maternal chromosomes and wherein the inducer line comprises a set of paternal chromosomes; b) contacting the haploid seed with a medium comprising a low mammalian toxicity chromosome doubling agent; c) selecting a doubled haploid seed wherein the doubled haploid seed comprises a triploid endosperm and a doubled haploid embryo. The doubled haploid seed produced by such a method wherein the triploid endosperm includes two sets of maternal chromosomes and one set of paternal chromosomes, and wherein the doubled haploid embryo has a first and second set of maternal chromosomes and wherein the first set of maternal chromosomes is homozygous to the second set of maternal chromosomes can be produced.
"In an embodiment, a method of obtaining a population of doubled haploid maize plants include the following steps: a) obtaining a set of haploid kernels by pollinating an ear with an inducer line wherein the ear includes a set of maternal chromosomes and wherein the inducer line has a set of paternal chromosomes; b) contacting the set of haploid kernels with a medium having a low mammalian toxicity chromosome doubling agent; c) selecting a set of doubled haploid kernels wherein each kernel of the set of doubled haploid kernels includes a triploid endosperm and a doubled haploid embryo; d) growing the doubled haploid kernels into a population of doubled haploid maize plants. In other aspects, the ear can be removed before, during, or after pollination and placed into a solution. The doubling agent can come in contact with the ear after pollination and before or after the ear is removed from the plant. The chromosomal doubling agent may come into contact with the ear directly or indirectly, for example, via filter paper or cotton or other suitable material.
"A method of inbred selection may include the following steps: a) cross-pollinating two inbred maize plants; b) growing the F1 seed; c) pollinating the F1 plant with an inducer line to produce haploid embryos; d) contacting the haploid embryos with a low mammalian toxicity chromosome doubling agent to produce doubled haploid embryos; e) generating doubled haploid plants; f) analyzing the doubled haploid plants for agronomic performance. The development of haploids step may also be done at later generations, e.g., F2, F3, F4, etc. Producing haploids from later generations allows for additional opportunities for recombination, if needed.
"If a haploid embryo is used as a source material for treatment with a low mammalian toxicity chromosome doubling agent, embryo rescue is performed. Embryo rescue is performed by contacting an embryo with a medium containing nutrients and generating a plant. Phytohormones may or may not be included in the embryo rescue medium. A method of obtaining a transgenic doubled haploid embryo may comprise isolating a haploid embryo, transforming the haploid embryo, placing the haploid embryo on a medium comprising a chromosome doubling agent and selecting a transgenic doubled haploid embryo.
"In any of these methods disclosed herein, the chromosomes can be doubled at the immature embryo stage, at the mature seed stage, or anytime between pollination of the plant and before the germination of the haploid seed.
"In any of these methods the haploid embryo that undergoes chromosomal doubling may be isolated, may be in the seed or kernel, may be in the kernel on a slice of cob, may be on the ear or spike, or the haploid embryo may be in the kernel, which is on the ear and on the plant. The doubling agent may reach the haploid embryo while the ear is on the plant and the plant is intact. For example, the doubling agent may be contacted directly or indirectly with the haploid embryo. In some cases the doubling agent can be transported by the plant. The plant may be grown hydroponically and the doubling agent can be taken up through the roots of the plant and transported to the haploid embryo. The seedlings may be initiated on soil or a growing medium and then transferred to a hydroponic solution where the doubling agent can be added. In another aspect of the method, the plant may be grown in soil or a growing medium and then the doubling agent is added to the soil or growing medium so that it can be transported to the haploid embryo.
"The methods disclosed herein, avoid time consuming selfing and crossing methods to obtain a homozygous trait of interest or a substantially homozygous plant. The methods can be used to produce doubled haploid populations that do not contain the residual heterozygosity of inbreds obtained though the traditional method of self pollination. The methods can be useful for functional genomics, such as knock-out analysis, functional analysis of recessive genes, gene replacement, homologous recombination, gene targeting, transgene stacking, and evaluating lethal versus non-lethal analysis of genes. In addition, the chromosome doubling agents, disclosed herein, are less toxic to mammalian systems compared to the traditional agent, such as colchicine.
"Haploid plant production systems have been developed and practiced for various plants to generate haploid tissues including embryo, plants and seeds. The haploid plants or plant components from any genotype are generated by crossing a selected/desirable line (as female) with an inducer line (male). Exemplary inducer lines for maize include, for example, Stock 6 (Coe, 1959, Am. Nat. 93:381 382; Sharkar and Coe, 1966, Genetics 54:453 464) RWS, KEMS (Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203 224), or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk & Chebotar, 2000, Plant Breeding 119:363 364), and indeterminate gametophyte (ig) mutation (Kermicle 1969 Science 166:1422 1424). The disclosures of which are incorporated herein by reference. Wide hybridization crosses can also be used to produce haploids. This method is sometimes referred to as the bulbosum method (Kasha and Kao, 1970, Nature 225:874-876). This method of haploid production occurs due to the elimination of the chromosomes from the pollinating parent."
URL and more information on this patent application, see: Cui,
Keywords for this news article include: Gases, Elements, Hydrogen, Chemistry, Treatment, Chromosomes, Cell Nucleus, Intranuclear Space, Cellular Structures, Inorganic Chemicals, Intracellular Space,
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