Production of double haploid plants

Abstract

Provided herein are methods for producing double haploid plants. The methods include obtaining a haploid plant tissue, contacting the haploid plant tissue with a safener compound, contacting the haploid plant tissue with a chromosome doubling agent, and regenerating a double haploid plant. The safener compound (e.g., mecarbafen) and the chromosome doubling agent (e.g., colchicine) are administered sequentially or simultaneously. In addition, the methods include further contacting the haploid plant tissue with a viability-responsive compound (e.g., thiamethoxam) wherein the viability-responsive compound and a chromosome doubling agent are administered either sequentially or simultaneously.

Description

Technical Field

[0001] The present invention relates to efficient methods for plant breeding and production of doubled haploid plants. Background Art

[0002] The production of double haploid (DH) plants is important and useful for breeders. Once haploid induction line plants are used to produce haploid plants or embryos, the haploid plants or embryos must be converted into double haploids afterwards. The typical method for producing DH plants requires a large amount of resources. The method for improvement needs to reduce the burden and improve the efficiency of DH production. A kind of mode of obtaining DH plants is by processing haploid plants with doubling agents (for example, colchicine). Colchicine and other chemicals are formed by suppressing spindle assembly and blocking cell wall formation during cell division and haploid plants are converted into DH plants. However, the use of colchicine and many doubling agents has stress, toxicity, and may be lethal to embryos and seedlings.

[0003] Therefore, improving haploid stress tolerance can improve the production efficiency of the DH method. Improving stress tolerance will lead to higher survival rates throughout the process.

[0004] Here, we present novel strategies demonstrating that selected chemicals can be used to induce protective mechanisms in plants to better protect haploids from stress during the DH production process, leading to increased haploid survival and haploid-to-doubled haploid conversion rates, and thereby to increased DH production efficiency. Summary of the Invention

[0005] In order to reduce the burden and improve the efficiency of producing double haploid plants, a method is disclosed herein, which includes obtaining haploid plant tissue, contacting the haploid plant tissue with a safener compound, contacting the haploid plant tissue with a chromosome doubling agent, and regenerating double haploid (DH) plants. In this method, the safener compound and the chromosome doubling agent are applied sequentially or simultaneously. The haploid plant tissue is further contacted with a vigor response compound, which can be applied sequentially or simultaneously with the chromosome doubling agent. In addition, in this method, the safener compound and the vigor response compound can be applied sequentially or simultaneously. The vigor response compound is selected from the group consisting of thiamethoxam and saponin. The safener compound is selected from the group consisting of metcamifen, benoxacor, dichlormid, isoxadifen, cloquintocet-mexyl, fenclorim, and cyprosulfamide. The chromosome doubling agent is selected from the group consisting of colchicine, trifluralin, natron, dithiopyr, nitrous oxide, and amosulfuron. The haploid plant tissue in the method can be maize, wheat, rice, barley, sunflower, soybean, watermelon, cucumber, tomato, pepper, or Brassica plant. When the haploid plant tissue is from maize, the haploid plant tissue can be an ear, microspore, callus, or embryo.

[0006] Another method of the present invention includes producing a doubled haploid plant, the method comprising obtaining haploid plant tissue, contacting the haploid plant tissue with a vigor-responsive compound, contacting the haploid plant tissue with a chromosome doubling agent, and regenerating the doubled haploid plant. The contacting with the vigor-responsive compound and the contacting with the chromosome doubling agent can occur sequentially or simultaneously.

[0007] definition

[0008] While it is believed that the following terms are well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0009] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. References to the techniques employed herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and / or alternatives to equivalent techniques that are well known to those of ordinary skill in the art. Although it is believed that the following terms may be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0010] In accordance with long-standing patent law conventions, the terms "a," "an," and "the" as used in this application, including the claims, mean "one or more." For example, the phrase "a cell" refers to one or more cells, and in some embodiments may refer to tissues and / or organs. Similarly, the phrase "at least one," when used herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more of the entity, including but not limited to all integer values ​​between 1 and 100 and integers greater than 100.

[0011] Unless otherwise indicated, all numbers used in the specification and claims expressing amounts of ingredients, reaction conditions, and the like should be understood as being modified in all instances by the term "about." As used herein, the term "about," when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of ±20% from the stated amount in some embodiments, ±10% from the stated amount in some embodiments, ±5% from the stated amount in some embodiments, ±1% from the stated amount in some embodiments, ±0.5% from the stated amount in some embodiments, and ±0.1% from the stated amount in some embodiments, as such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, polypeptides, and the like. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter of the present disclosure.

[0012] As used herein, the term "allele" refers to a variant or alternative sequence form at a genetic locus. In a diploid, a single allele at each locus is inherited by the progeny of each parent. Although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all the alleles present in the species, the two alleles present in a given locus in a diploid organism occupy corresponding positions on a pair of homologous chromosomes.

[0013] As used herein, the term "and / or," when used in the context of listing entities, refers to the entities individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, the one or more elements to which "and / or" refers may also be present individually in single or multiple occurrences in one or more combinations and / or in one or more subcombinations.

[0014] The term "comprising" is synonymous with "including," "containing," and "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that means that the specified elements and / or steps are present, but other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0015] As used herein, the phrase "consisting of excludes any elements, steps, or ingredients not specifically recited. When the phrase "consisting of" appears in a clause of the body of a claim, rather than directly following a preamble, it limits only the elements recited in that clause; other elements are not excluded from the claim as a whole.

[0016] As used herein, the phrase "consisting essentially of limits the scope of the associated disclosure or claim to the specified materials and / or steps, plus those that do not materially affect one or more basic and novel characteristics of the disclosed and / or claimed subject matter.

[0017] As used herein, the term "biomarker" refers to a measurable indicator of various biological states or conditions, including stress tolerance. These biomarkers may include, for example, GST27, glycosyltransferases, and cytochrome P450.

[0018] As used herein, the term "chromosome doubling agent" means a chemical that doubles the number of chromosomes in a cell (e.g., from haploid to diploid or from diploid to tetraploid, etc.) by blocking normal cell cycle division. Such agents are typically anti-microtubule agents, such as colchicine, nafopyl, dithiopyr, trifluralin, or another known anti-microtubule agent. Compounds that can cause chromosome doubling are well known to those skilled in the art.

[0019] As used herein, the term "doubled haploid embryo" refers to an embryo having one or more cells containing two sets of homozygous chromosomes. The related term "doubled haploid" means a plant cell, tissue or plant derived from a haploid. A diploid has two sets of chromosomes (2n) and is typically homozygous. A "doubled haploid" is formed by doubling the haploid set of chromosomes using a "chromosome doubling agent."

[0020] As used herein, the term "embryo" refers to an embryo formed after a sperm nucleus from a pollen grain fuses with an egg cell to produce a diploid (2N) embryo. A diploid (2N) embryo can be converted into a haploid (1N) embryo by genome elimination. A haploid (1N) embryo can also be formed after sperm fertilization or sperm-egg fusion fails, when the egg is stimulated to develop directly into a haploid embryo via parthenogenesis.

[0021] As used herein, the term "endosperm" refers to the tissue formed after one sperm nucleus from a pollen grain fuses with the polar nuclear sac to produce triploid (3N) endosperm.

[0022] As used herein, the term "escape rate" refers to the percentage of pseudohaploids that result from the non-marker expression of diploids in all colorless embryos (true haploids plus pseudohaploids). As used herein, "escape" refers to that diploids are not detected throughout the haploid selection process.

[0023] As used herein, the term "gene" refers to a unit of inheritance comprising a DNA sequence that occupies a specific location on a chromosome and contains the genetic instructions for a specific characteristic or trait in an organism.

[0024] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. As is known in the art, a genotype can relate to a single locus or multiple loci, whether these loci are correlated or uncorrelated, and / or linked or unlinked. In some embodiments, a genotype is represented by a haplotype (defined below).

[0025] As used herein, the term "germination" refers to the germination of an embryo, typically after a dormant period. Germination depends on appropriate environmental conditions, such as temperature, water, and oxygen. The embryo can exist intact as part of the seed, or it can be isolated and cultured on an artificial medium under sterile conditions.

[0026] As used herein, the term "germplasm" refers to the totality of the genotypes of a population or another group of individuals (e.g., species). The term "germplasm" may also refer to plant material; for example, a group of plants that serves as a repository for various alleles. The phrase "adapted germplasm" refers to plant material that has been shown to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "exotic germplasm" refer to plant material of unknown or unconfirmed genetic value; for example, for a given environment or geographic region; therefore, the phrase "unadapted germplasm" in some embodiments refers to plant material that is not part of an established breeding population and has no known relationship to a member of an established breeding population.

[0027] As used herein, the term "haploid" means a plant cell, tissue or plant that has one set (n) of chromosomes. In a haploid organism, only half the normal number of chromosomes is present.

[0028] As used herein, haploid induction rate ("HIR") means the ratio of the number of surviving haploid kernels or embryos to the total number of kernels or embryos after pollination of an ear with haploid induction line pollen or after pollination of an ear of a haploid induction line with wild-type pollen.

[0029] As used herein, term " heterotic group " refers to when with the genotype of different germplasm groups from other genes or the hybridization of inbred strains, show a group of genotype or the inbred strain of similar hybrid vigor response.With respect to the strain of the more remote degree genetic relationship compared between the heterotic group, the strain that is included in the heterotic group has a closer degree genetic relationship.Generally speaking, the hybrid that two inbred strains in the same heterotic group hybridize together shows much less heterosis than the hybrid from the inbred strain of a heterotic group and the hybrid from the inbred strains of different heterotic groups.Specific heterotic group can comprise a plurality of strains with different heredities.

[0030] As used herein, the term "hybrid" refers to a plant that is the offspring of genetically different parents produced by crossing plants of different lines or varieties or species, including but not limited to a cross between two inbred lines (e.g., genetically heterozygous or mostly heterozygous individuals).

[0031] As used herein, the term "locus" refers to a location on a chromosome in a given species (eg, the location of a gene, genetic marker, etc.).

[0032] As used herein, a "maternal haploid inducer line" refers to a line that produces pollen and, when used as a male in a hybrid, results in the development of gynogenetic haploid seeds. A "paternal haploid inducer line" refers to a line that, when used as a female in a hybrid, results in the development of androgenetic haploid seeds. Haploid inducer plants can be derived haploid using either of these maternal or paternal mechanisms and, without specifying the mechanism of a particular line, may be collectively referred to as an "inducer line" or "haploid inducer line."

[0033] As used herein, the term "plant" can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise indicated, the term "plant" can refer to any of the following: a whole plant, a plant component or organ (e.g., leaves, stems, roots, etc.), a plant tissue, a seed, and / or a plant cell.

[0034] A plant cell is a plant cell obtained from a plant, or a plant cell derived from a cell taken from a plant by culture. Thus, the term "plant cell" includes, but is not limited to, cells within seeds, suspension cultures, embryos, meristems, callus, leaves, buds, gametophytes, sporophytes, pollen, and microspores. The phrase "plant part" refers to a part of a plant, including single cells and cell tissues (such as intact plant cells in a plant), cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, and seeds; as well as scions, rhizomes, protoplasts, callus, and the like.

[0035] As used herein, the terms "phenotype," "phenotypic trait," or "trait" refer to one or more traits of a plant or plant cell. A phenotype is observable by the naked eye or by any other evaluation method known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponding to a "single gene trait"). For example, in the case of haploid plants being evaluated for SCD, the phenotype may refer to fertile pollen shed and / or seeds obtained via pollination with that pollen. In other cases, a phenotype is the result of interactions between several genes, and in some embodiments, is also caused by the interaction of a plant and / or plant cell with its environment.

[0036] As used herein, the term "transplant survival rate" refers to the percentage of germinated embryos successfully transferred to soil from the total treated haploid embryos. "Transplant survival rate" is used as an indicator of plant health to monitor the success of recovery and growth after haploid treatment. As used herein, the term "transplant rate" refers to the survival rate of healthy plants after embryo germination and transfer to soil.

[0037] As used herein, term " filial generation " and " filial generation plant " refer to the plant produced by asexual or sexual reproduction from one or more parental plants.In maternal haploid induction, the seed desired on maternal parent is haploid, is therefore not the filial generation of inducing haploid strain.The filial generation of haploid seed is not unique filial generation.Can also have hybrid (diploid) seed and by its subsequent plant of growth, and the further filial generation of hybrid and female plant backcross.In addition, filial generation can comprise aneuploid filial generation (comprising the chromosome uneven mixture from parental plant), and these filial generations can be eliminated conventionally.Haploid seed and hybrid seed both can be filial generation.Filial generation plant can be by cloning single parental plant or making single parental plant self-pollination or by making two or more parental plants hybridize and obtain.For example, filial generation plant can be by cloning or self-pollination of a parental plant or by the hybridization of two parental plants and obtain, and comprise self-pollinated body and F1 or F2 or even more distant generation. An F1 is the first generation of progeny produced from two parents (at least one of the two parents is the first time used as a donor for a trait), while progeny of the second (F2) or subsequent (F3, F4, etc.) generations are specimens produced from self-crosses, reciprocal crosses, backcrosses, and / or other crosses of the F1, F2, etc. Thus, an F1 can be (and in some embodiments is) a hybrid produced from a cross between two breeding parents, while an F2 can be (and in some embodiments is) a progeny produced from self-pollination of an F1 hybrid.

[0038] As used herein, the term "regeneration" and its grammatical variations refer to the generation of plants from tissue culture.

[0039] As used herein, the term "safener" refers to a chemical compound that is used in combination with an herbicide to reduce damage to crop plants caused by the herbicide.

[0040] As used herein, the term "trait" refers to a phenotype of interest, a gene that contributes to a phenotype of interest, and a nucleic acid sequence associated with a gene that contributes to a phenotype of interest. DETAILED DESCRIPTION

[0041] The following description and examples illustrate various aspects and embodiments of the compositions and methods of the present invention. The specific examples are not intended to limit the scope of the compositions and methods. Rather, the examples merely provide non-limiting examples of various compositions and methods that are at least within the scope of the disclosed compositions and methods. The descriptions should be read from the perspective of one of ordinary skill in the art; therefore, they do not necessarily include information that would be familiar to a skilled artisan.

[0042] One aspect of the invention provided herein is a method for producing doubled haploid plants, the method comprising obtaining haploid plant tissue, contacting the haploid plant tissue with a safener compound, contacting the haploid plant tissue with a chromosome doubling agent, and regenerating doubled haploid plants. In one embodiment, the safener compound and the chromosome doubling agent are applied sequentially or simultaneously. In another embodiment, the haploid plant tissue is further contacted with a vigor-responsive compound. In another embodiment, the vigor-responsive compound and the chromosome doubling agent are applied sequentially or simultaneously.

[0043] In another embodiment, the safener compound is contacted with the haploid plant tissue prior to contacting the haploid plant tissue with the chromosome doubling agent. In another embodiment, the safener compound is contacted with the haploid plant tissue 24 to 168 hours prior to contact with the chromosome doubling agent. In another embodiment, the safener compound is at a concentration of 5-50 mg / L. In another embodiment, the safener compound contact occurs at 4°C to 8°C. In yet another embodiment, the safener compound contact occurs at 6°C.

[0044] In one embodiment, the safener compound is contacted with the haploid plant tissue and the chromosome doubling agent is contacted simultaneously. In another embodiment, the safener compound is contacted with the haploid plant tissue and the chromosome doubling agent is contacted simultaneously for 24 to 48 hours. In yet another embodiment, the safener compound is at a concentration of 400-800 mg / L. In another embodiment, the safener compound is contacted with the chromosome doubling agent at 24°C to 32°C. In yet another embodiment, the safener compound is contacted with the chromosome doubling agent at 28°C.

[0045] In one embodiment, the safener compound is contacted with the haploid plant tissue after the haploid plant tissue is contacted with the chromosome doubling agent. In an embodiment, the safener compound is contacted with the haploid plant tissue 8-504 hours after the haploid plant tissue is contacted with the chromosome doubling agent, and the safener compound concentration is 0-20 mg / L. The safener compound contact can occur at 24°C-32°C, and in another embodiment, the contact occurs at 28°C.

[0046] In an embodiment, the safener compound and the vigor response compound of the method are applied sequentially or simultaneously. The haploid plant tissue of the method can be from maize, wheat, rice, barley, sunflower, soybean, watermelon, cucumber, tomato, pepper or Brassica plant. In an embodiment, the haploid plant tissue is maize, and can be ear, microspore, callus or embryo. In an embodiment, the maize plant tissue is an embryo.

[0047] The safener compound of the method is selected from the group consisting of: mecarphen, fenthiocarb, dichlorvos, isoxadiazine, cloquintocet-mexyl, fenthiocarb, and cyproconazole. In an embodiment, the safener compound is mecarphen. In an embodiment, the chromosome doubling agent is selected from the group consisting of: colchicine, trifluralin, nafop-butyl, dithiopyr, nitrous oxide, and amoxicillin. In another embodiment, the chromosome doubling agent is colchicine. In one embodiment, the vitality response compound is selected from the group consisting of: thiamethoxam and saponin. In another embodiment, the vitality response compound is thiamethoxam.

[0048] In one embodiment, before colchicine treatment, haploid plant tissue is contacted with thiamethoxam. In another embodiment, thiamethoxam is applied at 10-1000mg / L. In yet another embodiment, thiamethoxam is applied for 24 to 48 hours. In another embodiment, during colchicine treatment, haploid plant tissue is contacted with thiamethoxam. In yet another embodiment, thiamethoxam is applied at 100 to 8000mg / L. In another embodiment, thiamethoxam is applied for 24 to 48 hours. In another embodiment, after colchicine treatment, haploid plant tissue is contacted with thiamethoxam. In yet another embodiment, thiamethoxam is applied at 0.5-12g / L. In another embodiment, thiamethoxam is applied for 24-504 hours. In another embodiment, all previously mentioned contacts are carried out in solid culture medium or liquid culture medium.

[0049] Another aspect of the present invention is a method for producing doubled haploid plants, the method comprising obtaining haploid plant tissue, contacting the haploid plant tissue with a vigor-responsive compound, contacting the haploid plant tissue with a chromosome doubling agent, and regenerating the doubled haploid plant. In one embodiment, the vigor-responsive compound is selected from the group consisting of thiamethoxam and saponins. In another embodiment, the vigor-responsive compound is thiamethoxam.

[0050] In another embodiment, contact with thiamethoxam occurs before contact with the chromosome doubling agent. In another embodiment, thiamethoxam is applied at 10-1000 mg / L. In yet another embodiment, thiamethoxam is applied for 0.5-24 hours. In another embodiment, contact with thiamethoxam and contact with the chromosome doubling agent occur simultaneously. In another embodiment, thiamethoxam is applied at 100-8000 mg / L. In yet another embodiment, thiamethoxam is applied for 24-48 hours. In another embodiment, contact with thiamethoxam occurs after contact with the chromosome doubling agent. In yet another embodiment, thiamethoxam is applied at 0.5-12 g / L. In yet another embodiment, thiamethoxam is applied for 24-504 hours.

[0051] Examples

[0052] Example 1: Metabolism of colchicine in maize embryos

[0053] An experiment was completed to evaluate the uptake of methamphetamine in corn embryos, whether colchicine is metabolized in the embryos, and whether methamphetamine increases the rate of colchicine metabolism. Corn embryos were co-treated with 500 ppm colchicine for 24 or 48 hours in the presence of 200 ppm methamphetamine, 1000 ppm methamphetamine, or without methamphetamine (control) and frozen. One biological replicate was represented by five embryos, and three biological replicates were used for each co-treatment.

[0054] Prior to extraction, embryos were washed in 80:20 (v / v) acetonitrile:H2O for 20 seconds to remove any surface colchicine and mecarfen. Each replicate was extracted in 0.5 ml of methanol using a rapid preparative immersion method. The samples were then diluted 1:10 before analysis by liquid chromatography mass spectrometry (LCMS). The samples were analyzed with matrix-matched standards for colchicine and mecarfen. Metabolite studies were performed to identify any metabolites.

[0055] In the co-treated samples, methamphetamine was detectable at both 24 and 48 hours. The amount of colchicine recovered after 48 hours was twice the amount recovered at 24 hours. In addition, the addition of methamphetamine did not reduce the amount of colchicine recovered. Duration and methamphetamine co-treatment increased the recovery of a colchicine metabolite (-14) that undergoes O-demethylation, however, this metabolism did not lead to a decrease in colchicine recovery, indicating that this metabolite is a minor component. Therefore, the use of methamphetamine does not significantly interact or reduce the availability of colchicine to double haploid chromosomes.

[0056] For the following examples, GST27 was used as a quick indicator of treatment effectiveness.

[0057] Example 2: Pretreatment of corn ears with mecarfen

[0058] The effect of temperature was independently tested by comparing 150 PAR light at 6°C and 28°C for 3 and 5 days. The 28°C temperature caused embryo bleaching and death, so this temperature was discarded. The 6°C temperature resulted in a normal, non-bleached phenotype and was selected for all subsequent tests of mikaphen pretreatment of maize ears.

[0059] Maize ears were pretreated with methamphetamine before embryo isolation and before embryo transfer to doubling medium containing colchicine. In all experiments, doubling medium was semi-solid, thickened with Gelzan, unless liquid medium was specified. Two methods of delivering safeners (at concentrations of 100 and 1000 mg / L) were tested. The first method wrapped the ear in a paper towel moistened with formulated methamphetamine. The second method used a sprayer to spray the formulated methamphetamine onto the ear surface at 6°C and 150 PAR light. GST27 was measured after methamphetamine pretreatment and again at the end of colchicine treatment. Low levels of GST27 (<10 ng / mg total soluble protein (TSP)) were induced at the end of ear pretreatment. After ear pretreatment, embryos were isolated and treated with colchicine for 48 hours. At the end of colchicine treatment, GST27 induction results increased but were not significant (see Table 1).

[0060] Table 1. GST27 induction results from ears pretreated with 100 and 1000 mg / L carfenol at 6°C for 3 and 5 days

[0061]

[0062] The addition of 0.5% Tween 20 to both 100 and 1000 mg / L of mecarfen was tested and sprayed onto maize ears as a pretreatment before embryo isolation. This addition did not improve the resulting GST27 expression levels over 4 days.

[0063] Example 3: Mekafen pretreatment of isolated embryos

[0064] Before the colchicine doubling treatment, embryos from maize ears were isolated and treated with methamphetamine. The isolated embryos placed on filter paper were pretreated with methamphetamine formulations at a concentration of 0, 10, 100, and 1000 mg / L at 6°C and 150 PAR light for a duration of 1, 3, 5, or 7 days. After pretreatment, GST27 induction was measured at each time point and again at the end of the colchicine treatment. The resulting embryos were germinated and the percentage of embryos successfully transferred to the soil (transplant survival percentage) was measured as an indicator of plant health and pretreatment effectiveness. GST27 was induced to a moderate level (<100 ng / ml TSP) by methamphetamine, but higher than ear pretreatment (less than 10 ng / ml TSP). Pretreatment with methamphetamine (10 mg / L) for 1 day at 6°C produced the highest transplant survival percentage (see Table 2).

[0065] Table 2. GST27 induction and transplant survival results from embryos pretreated with mecarfen in MS medium at 6°C for different durations

[0066]

[0067] The addition of 10% DMSO to 10, 100, and 1000 mg / L mekafen formulations in MS medium was tested to potentially increase mekafen uptake. The test was conducted at 6°C and 20°C over a period of 0 to 4 days. Addition of 10% DMSO did not result in increased GST27 expression at either temperature. Administration of 10 mg / L mekafen continued to provide optimal GST27 induction at all time points, regardless of temperature, compared to the control and other doses.

[0068] Embryos were pretreated with 10 mg / L of methamphetamine for 1 day at 6°C and 20°C to measure the effect of temperature on embryo health. The 6°C pretreatment had a significantly higher transplant survival rate than the control. The 20°C pretreatment caused low embryo survival during the colchicine treatment, resulting in a 2% transplant survival rate (see Table 3). Overall, the addition of methamphetamine to the culture medium for pretreatment of isolated embryos resulted in a higher induction of GST27 than the co-treatment of the ear performed in Example 1 (see Tables 1 to 3).

[0069] Table 3. GST27 induction and transplant survival results of embryos pretreated with 10 mg / L carfenol in MS medium for 1 day at 6°C and 20°C

[0070]

[0071] To optimize the concentration of methamphetamine, additional experiments were performed to pre-treat embryos with methamphetamine for 24 hours. Concentrations ranging from 5 mg / L to 20 mg / L were tested. Following pre-treatment, the pre-treated embryos were then treated with colchicine for 24 and 48 hours.

[0072] Example 4: Co-treatment of isolated embryos with colchicine during doubling period

[0073] During the colchicine doubling (also known as co-treatment) period, isolated embryos were treated with methamphetamine. Methamyl was added as a formulation to the colchicine co-treatment medium (also known as doubling medium) at concentrations of 0, 200, 400, 800, and 1200 mg / L. The methamphetamine concentration range tested over a 24-hour duration was 0-1000 mg / L. The methamphetamine concentration range tested over a 48-hour condition was 0-1200 mg / L. Three to four genotypes representing two heterotic groups were tested.

[0074] Initial testing of the three genotypes involved co-treatment of 0, 200 and 1000 mg / L of methamphetamine and colchicine in Gelzan medium at 28°C and 150 PAR light to identify a range of working concentrations. Concentrations of 200 and 1000 mg / L induced similar levels of GST27 expression for both 24 and 48 hour durations. The resulting induction was higher than that recorded for pre-treatments of ears or isolated embryos (400 ng / mg TSP GST27). The results of the 48 hour co-treatment showed that 200 mg / L methamphetamine increased the percentage survival to transplant and provided better plant health and germination rates compared to 1000 mg / L methamphetamine and the control. In the 24 hour co-treatment, both concentrations of methamphetamine (200 and 1000 mg / L) increased the percentage survival to transplant relative to the control, and the production of double haploid ears also increased. Optimization of the co-treatment of isolated embryos with methamphetamine was continued by testing levels of 0, 400, 600, and 800 mg / L for 24 and 48 hours, encompassing four genotypes from two heterotic groups. GST induction (ng / mg TSP) was measured after methamphetamine co-treatment. Although GST27 induction was observed and higher than the control in all co-treatments, the highest induction was measured at 800 mg / L in the 48 hour co-treatment. At 800 mg / L for 48 hours, one genotype produced significantly higher GST27 induction than the remaining genotypes. When categorized by genotype or heterotic group, the transplant survival percentages after methamphetamine co-treatment were similar over both durations.

[0075] Both durations tested provided benefits over the control, with the 24-hour co-treatment (800 mg / L) yielding the best percentage transplant survival and the 48-hour co-treatment (1200 mg / L) yielding the highest average GST27 induction. Mecarfen levels at 200, 400, and 800 mg / L for 24 hours and 800 and 1200 mg / L for 48 hours showed evidence of GST27 induction and better transplant survival than the colchicine-only control (see Table 4).

[0076] Table 4: GST27 induction and transplant survival results of embryos co-treated with colchicine and 0-1200 mg / L carfentanil for 24-48 hours.

[0077]

[0078] Example 5: Post-treatment with Mecarfen during germination

[0079] Double haploid embryos were treated with methamphetamine during germination. 24 or 48 hours after the colchicine treatment was completed, methamphetamine was tested at 0, 5, and 10 mg / L in the germination medium. The germination medium contained Gelzan at a concentration of 3.5 g / L. Four genotypes representing two heterotic groups were tested. GST27 expression was higher at 24 and 48 hours than in the control group. The percentage of transplant survival did not increase due to these post-treatments. It was observed that methamphetamine induced rapid root growth, and the root tips were unable to penetrate the medium. This resulted in root curling. Gelzan concentrations were tested from 3.5 g / L to 2.5 g / L (comparative data not presented here). The experiment was then conducted with methamphetamine post-treatment at 2.5 g / L Gelzan, while the control (0 methamphetamine) contained 3.5 g / L Gelzan. 24 and 48 hours after the colchicine treatment was completed, methamphetamine was tested at 0, 5, 10, and 20 mg / L. Gelzan reduction after mecarfen treatment resulted in a higher percentage of transplant survival (see Table 5).

[0080] Table 5: Effects of 0-20 mg / L Mecarfen on GST27 induction and transplant survival after 24-48 hours of embryo treatment

[0081]

[0082] Example 6: Thiamethoxam co-treatment during the colchicine doubling period

[0083] In addition to testing safeners such as mecarfen, the effects of other compounds, including thiamethoxam (TMX), were also tested. Isolated embryos were co-treated with TMX during the colchicine doubling period in doubling medium. Co-treatments were incubated for 24 or 48 hours. Four genotypes representing two heterotic groups were tested in duplicate. The effect of in vitro culture on transplant survival was evaluated.

[0084] Initial testing measured the 24 or 48 hour effects of 0, 150, or 750 mg / L TMX concentrations. At 24 hours, the effect of co-treatment with TMX on transplant survival showed an overall higher transplant survival rate (80%+) compared to the control. The strongest effect of TMX co-treatment observed over a 48 hour period was 750 mg / L TMX compared to the control. Seedling height from the same experiment was used as a measure of plant health and showed an overall increase in plant height after co-treatment with 150 or 750 mg / L TMX at both the 24 and 48 hour periods.

[0085] Additional tests were performed and TMX was applied to isolated embryos at 0, 750, 1500, and 2000 mg / L levels during the colchicine co-treatment. The co-treatments were incubated for 24 or 48 hours. Co-treatments of 1500 and 2000 mg / L for 24 hours resulted in the highest overall survival to transplant percentages and the highest percentages of transplantation rates (see Table 6).

[0086] Table 6: Co-treatment of embryos with colchicine and 0-2000 mg / L TMX for 24-48 hours: Effects on transplant survival and engraftment rates.

[0087]

[0088] During the colchicine co-treatment, TMX was applied to isolated embryos at levels of 0, 2000, 4000, and 8000 mg / L. The co-treatments were incubated for a duration of 24 or 48 hours. The 4000 mg / L TMX co-treatment for 24 hours produced the highest overall transplant survival percentage and transplantation rate (see Table 7).

[0089] Table 7: Effects of co-treatment of embryos with colchicine and 0-8000 mg / L TMX for 24-48 hours on transplant survival and engraftment rates

[0090]

[0091] During the colchicine co-treatment period, TMX was applied to the isolated embryos at a level of 0, 2000, 4000 or 6000 mg / L. The co-treatments were incubated for a duration of 24 or 48 hours. The highest transplant survival percentages of 90% and 89% were achieved for the 4000 mg / L co-treatment for 24 hours and the 6000 mg / L co-treatment for 48 hours. For the 4000 mg / L co-treatment for 24 hours and the 6000 mg / L co-treatment for 48 hours, the highest transplant survival percentages were 86% and 83% (see Table 8).

[0092] Table 8: Effects of co-treatment of embryos with colchicine and 0-6000 mg / L TMX for 24-48 hours on transplant survival and engraftment rates

[0093]

[0094] Higher escape rates were observed with co-treatment of 6000 or 8000 mg / L TMX for 24 hours. TMX levels in the range of 2000-6000 mg / L produced the highest transplant survival rates while providing reliable marker visibility for haploid selection due to low escape rates (see Table 9). Table 9: Co-treatment of embryos with colchicine and 0-8000 mg / L TMX for 24-48 hours: Effect on escape rates

[0095] TMX (mg / L) 48-hour escape rate 24-hour escape rate comparison 0％ 0％ 2000 0％ 0％ 4000 0％ 8％ 6000 0％ 60％ 8000 33％ 50％

[0096] Example 7. Pretreatment of Thiamethoxam Liquid Culture Medium Before Doubling Colchicine

[0097] It is to carry out and set repetition in 2 kinds of genotypes that the embryo of separation is carried out TMX pretreatment before colchicine doubling.Separated embryo is placed on filter paper, and is used in the TMX formulation pretreatment of 10-1000mg / L in liquid culture medium concentration range.Pretreatment thing is hatched 0.5 to 24 hour.After pretreatment, with colchicine treatment embryo, be used for chromosome doubling and plant regeneration.Assessed the effect (referring to Table 10) on transplant survival rate and transplant rate.All liquid pretreatments all do not produce transplant survival rate or transplant rate higher than solid culture medium control, but the 58% transplant survival rate and 55% transplant rate that 1000mg / L pretreatment reaches in 1 hour are closest to solid culture medium control (65% and 64%) respectively.Liquid culture medium itself may not provide improvement, but using TMX to improve embryo transplant success rate and transplant rate in liquid culture medium will be useful in production environment, and wherein liquid treatment provides other advantages aspect throughput or lower cost of goods.

[0098] Table 10: Effects of embryo pretreatment with 0-1000 mg / L TMX for 1-24 hours on transplant survival and engraftment rates

[0099]

[0100] In subsequent experiments, liquid pretreatment was tested at 30 minutes and 1 hour durations to determine whether shorter durations would result in better transplant survival and transplantation rates than those observed in the 1 hour treatment shown in Table 10. These were performed in triplicate in the same manner as described above. The results showed that 30 minutes of liquid medium pretreatment was not superior to 1 hour (see Table 11).

[0101] Table 11: Effects of embryo pretreatment with 0-1000 mg / L TMX for 30 minutes to 1 hour on transplant survival and engraftment rates

[0102]

[0103] Example 8. Thiamethoxam post-treatment after doubling colchicine (during germination)

[0104] TMX was applied to doubled haploids by adding it to the germination medium during the germination process. TMX was tested in triplicate with three genotypes at concentrations ranging from 0 to 12 g / L, and the post-treatments were incubated for 14 to 21 days (the normal duration of the regeneration phase before transplanting). The effect on transplant survival was assessed (see Table 12); due to poor germination, no plants were evaluated for transplant survival in advance.

[0105] Table 12: Effect of embryo treatment with 0-12 g / L TMX 14-21 days after germination on transplant survival

[0106]

[0107] Example 9. Co-treatment of isolated embryos with methamphetamine and thiamethoxam, including growth to maturity, self-pollination, and DH1 fruit set assessment

[0108] In order to evaluate the impact of safeners and vigor-enhancing compounds on the complete DH production process, experiments were designed and conducted that encompassed haploid embryo separation, doubling treatment, germination, transplanting, growth to maturity, selfing, and fruiting assessment. Embryos from two genotypes of maize ears were separated and co-treated with methamphetamine and thiamethoxam during the colchicine doubling treatment in triplicate. A combination of methamphetamine (400 to 800 mg / L) and TMX (4000 to 6000 mg / L) was added to Murashige and Skoog culture medium (MS culture medium). Co-treatments were run in parallel with a control of separate methamphetamine, separate TMX, or colchicine alone. Concentrations for co-treatment were selected based on the best results from the experiments in Example 1-8. The co-treatments were incubated at 28°C and 150 PAR light for 24 and 48 hours. The resulting embryos were germinated, and the percentage of embryos successfully transferred to the soil was measured as the transplant survival rate. The survival rate of healthy plants was measured as the transplant rate.

[0109] Transplanted seedlings were grown to maturity and self-pollinated to assess the rate of fertile DH plants producing ears with greater than 4 and greater than 49 kernels per ear (ear>4 and ear>49 ratios). The highest transplant survival (96%) and transplantation rate (95%) were achieved in the 24-hour co-treatment with a combination of 400 mg / L of methamphetamine and 4000 mg / L TMX, but were not statistically significantly different from the rates observed with 4000 mg / L TMX without methamphetamine (p<0.0001). At both time points, transplant survival and transplantation rates for all co-treatments were significantly better (p<0.0001) than the colchicine control. In all co-treatments and colchicine-only controls, the 48-hour experiments resulted in lower overall transplant survival and transplantation rates (12%-69% and 10%-64%, respectively) than those observed in the corresponding 24-hour experiments (58%-96% and 48%-95%, respectively) (see Table 13). This may be due to the increased toxicity of colchicine with longer exposure durations.

[0110] The proportion of ears bearing more than 4 kernels (ratio of ears > 4) was significantly increased in all co-treatments at both 24 and 48 hours compared to the colchicine-only control (p < 0.005), but no significant differences were observed between the co-treatments (see Table 13). For the proportion of ears bearing more than 49 kernels (ratio of ears > 49), there was no statistically significant difference between any co-treatment and the colchicine-only control at the 24 hour time point. For the 48 hour co-treatment, the combination of mecarphen and TMX was more likely to produce an ear > 49 kernels compared to either mecarphen alone or TMX alone.

[0111] Table 13: Effects of co-treatment of embryos with colchicine and 0-6000 mg / L TMX, and / or 0-800 mg / L micarfen for 24 or 48 hours on transplant survival, transplantation rate, and kernel set rate

[0112]

[0113] Escapes were recorded in two steps in the experiment. The first recording was performed in the laboratory when the embryos changed color after being transferred from the doubling medium to the germination medium. The second recording was performed in the greenhouse when the plants showed purple or other obvious diploid characteristics (tall, wide leaves, a large amount of fertile pollen, and purple kernels when seeds matured). As observed in the experiment in Example 6 (see Table 9), co-treatment with TMX produced a higher number of escapes. This was also the case when TMX was combined with methamphetamine for embryo co-treatment, although 24% of escapes were detected in the greenhouse in the absence of TMX in the 24-hour methamphetamine co-treatment group. Of the two genotypes tested, one strain had a much higher escape rate than the other strain (see Table 14).

[0114] Table 14: Co-treatment of embryos with colchicine and 0-6000 mg / L TMX, and 0-800 mg / L micarfen for 24-48 hours: Effect on escape rate

[0115]

[0116] A way to measure the success of DH production is the ratio of output to input, defined as "DH yield", where a larger ratio is generally desired for a more efficient production system. In this study, we measured in two ways: DH ear / total haploid embryo and DH ear / transplant (see Table 15). For these data, in the 24-hour co-treatment groups, the DH yield (based on each haploid) of ears with >4 grains in the co-treatment with mekaphen, TMX, and mekaphen+TMX was significantly higher than that of the colchicine control alone. In the 24-hour co-treatment, no significant changes in DH yield were observed for ears with >4 grains (based on each transplant) or for ears with >49 grains (based on each haploid), but there was a trend of increase in DH yield (based on each haploid) for >49 grains compared to the co-treatment with mekaphen. Interestingly, there was a significant negative trend in DH yield of ears with >49 kernels (per transplant basis) in the co-treatments including TMX, which may indicate that TMX co-treatment increased transplant survival and transplantation rates, but not all of these transplants produced ears with higher kernel numbers.

[0117] For these data, we generally saw positive trends associated with all treatments within the 48-hour co-treatment, but the data are difficult to interpret quantitatively because none of the control plants successfully produced ears with kernels. This is likely due to the stress caused by the additional time spent on colchicine-containing media during the 48-hour duration of the co-treatment; excessive colchicine exposure is known to cause sterility in both male and female reproductive systems in plants.

[0118] Table 15: Co-treatment of embryos with colchicine and 0-6000 mg / L TMX, and 0-800 mg / L micarfen for 24-48 hours: Effect on DH yield ratio (compared to ear ratio) on total haploids and transplants

[0119]

[0120] Example 10. Co-treatment of Mecarfen and Thiamethoxam during Doubling of Colchicine in Liquid Culture Medium Compared to Culture Medium Containing Gelzan

[0121] The isolated embryos were placed on filter paper in a Petri dish moistened with liquid Murashige and Skoog medium (MS medium) containing colchicine and 800 mg / L mikafen, 4000 mg / L TMX, or a combination of 800 mg / L mikafen and 4000 mg / L TMX. The colchicine treatment control used the same conditions as the liquid medium without Gelzan, except that the medium contained 3.5 g / L Gelzan. The co-treatments were incubated at 28° C. and 150 PAR light for 24 or 48 hours and were performed on two genotypes in 2 replicates. The effects on GST expression, transplant survival, and transplantation rates were reported (see Table 16). The observed diploid escape rate increased to 6.4%-8.5% in the 24-hour co-treatment with 4000 mg / L TMX and 4000 mg / L TMX in combination with 800 mg / L mikafen. In all other co-processings, escape rate was 0%.The transplant survival rate that the co-processing that all 24 hours durations produce is compared higher (91.2%-92.5%) with contrast (78%), and is higher than 48 hours co-processings (22%-82%).The best transplanting rate observed is 4000mg / L TMX co-processings (90%) at 24 hours durations, is 72% compared to contrast.

[0122] Table 16: Co-treatment of embryos with 0-800 mg / L mecarfen and 0-4000 mg / L TMX for 24-48 hours in liquid culture: Effects on GST expression, transplant survival, and engraftment rate

[0123]

[0124] In separate experiments, additional concentrations of methamphetamine and TMX co-treatments were tested in liquid co-treatments. The experimental setup was identical to the data in Table 16 described above, but the co-treatment concentrations were varied. The colchicine-only control remained unchanged, methamphetamine co-treatment was maintained at 800 mg / L, TMX co-treatment was increased to 6000 mg / L in the 48-hour co-treatment and maintained at 4000 mg / L in the 24-hour co-treatment, and methamphetamine and TMX co-treatments reduced methamphetamine concentrations to 400 mg / L, with TMX at 4000 mg / L. The reduced concentrations were associated with lower escape rates in the 24-hour co-treatment, with only the methamphetamine and TMX co-treatment producing escapes (1.8%). Transplant survival and engraftment rates remained high, with the best performance in the 24-hour methamphetamine and TMX co-treatment (96% engraftment and 87% engraftment) compared to the colchicine-only control (60% engraftment and 47% engraftment) (see Table 17). High rates of bacterial and / or fungal contamination were observed in all liquid media co-treatments. A total of 35% of pilot experiments were abandoned due to contamination issues and were excluded from data analysis.

[0125] Table 17: Co-treatment of embryos with 0-800 mg / L mecarfen and 0-6000 mg / L TMX for 24-48 hours in liquid culture: Effects on GST expression, transplant survival, and engraftment rate

[0126] Example 11. Co-treatment of isolated embryos with the safeners benoxacor or dichloropropane during colchicine treatment

[0127] Benthiocarb is applied to the embryo of the separation of 2 kinds of genotypes, during the colchicine treatment in doubling medium, carries out 2 repetitions with the level of 0,20,50,100 or 200mg / L.The co-processed thing was hatched for 24 or 48 hours.Under 50mg / L, co-processed and produced 90% the highest percentage transplanting survival rate in 24 hours.The performance of all 48 hours co-processings was not all better than 24 hours co-processings (referring to Table 18) of equals.In these experiments, do not directly measure escape rate, but in 48 hours, co-processed, along with the increase of benthiocarb concentration, color expression (promptly presenting the part of the diploid embryo of purple pigment) slightly declined.

[0128] Table 18: Effects of co-treatment of embryos with colchicine and 0-200 mg / L fenthiocarb for 24-48 hours on transplant survival

[0129]

[0130]

[0131] Dichloropropene is applied to the embryo of separation from 2 kinds of genotypes, during colchicine treatment in doubling medium, with the level of 0,20,50,100,200 and 300mg / L, carry out 2 repetitions.Co-processed thing is hatched the duration of 24 or 48 hours.Under 100mg / L and 50mg / L, co-processed 24 hours and produced the highest transplanting survival rate percentage ratio of 96% and 90% respectively.For under 100mg / L and 50mg / L, co-processed 24 hours, the highest transplanting rate percentage ratio is respectively 92% and 88%.The performance of all 48 hours co-processings is not better than the 24 hours co-processings of equals (referring to Table 19).In these experiments, do not directly measure escape rate, but when concentration is greater than 100mg / L, color expression (i.e., the part that diploid embryo color is purple) is sharply declined; This may cause the risk of escape to increase under the co-processing of higher ratio. Table 19. Effects of co-treatment of embryos with colchicine and 0-300 mg / L dichloropropane for 24-48 hours on transplant survival and transplantation rates

[0132]

[0133] Example 12. Hohenheim corn DH method (Deimling et al., 1997; Prigge and Melchinger, 2012) for application of methamphetamine and TMX on R1-nj mature seeds.

[0134] During the colchicine doubling treatment, the germinated haploid seedlings can be co-treated with methamphetamine and TMX. A combination of methamphetamine (at a concentration range of 80 to 800 mg / L) and TMX (at a concentration range of 400 to 4000 mg / L) is added to the colchicine treatment solution. The co-treatment is incubated for 6-12 hours. The co-treated seedlings are rinsed with water, then transplanted into soil and grown to maturity to produce doubled haploid seeds.

[0135] References

[0136] Ian Jepson, et al. 1994. Cloning and characterization of maizeherbicidesafener-induced cDNAs encoding subunits of glutathione S-transferaseisoforms I, II and IV. Plant Molecular Biology 26:1855-1866. David Holt,Ian Jepson,et al.1995.Characterization of the safener-inducedglutathione S-transferase isoform II from maize.Planta 196:295-302 Riechers DE,Kreuz K,Zhang Q.2010.Detoxification without intoxication:herbicide safeners activate plant defense gene expression.PlantPhysiology153,3-13.

[0137] Melissa Brazier-Hicks,Gavin Hall et al.2020.Chemically inducedherbicidetolerance in rice by the safener Metcamifen is associated with aphasedstress response.Journal of ExperimentalBotany,Vol.71,No.1pp.411-421.

[0138] Deimling S, F,Geiger HH.Methodik und Genetik der in vivo-Haploideninduktion bei Mais. für Pflanzenzüchtung.1997;38:203-224.

[0139] Prigge V,Melchinger AE(2012)Production of haploids anddoubledhaploids in maize.In:Loyola-Vargas VM,Ochoa-Alejo N(eds)Plantcellculture protocols.Springer,Berlin,pp 161-172.

Claims

1. A method for producing doubled haploid plants, the method comprising a) obtaining haploid plant tissue, b) contacting said haploid plant tissue with a safener compound, c) contacting the haploid plant tissue with a chromosome doubling agent, and d) Regeneration of doubled haploid plants.

2. The method of claim 1, wherein the safener compound and the chromosome doubling agent are administered sequentially or simultaneously.

3. The method of claim 1, wherein the haploid plant tissue is further contacted with a vigor-responsive compound.

4. The method of claim 3, wherein the viability-responsive compound and the chromosome doubling agent are administered sequentially or simultaneously.

5. The method of claim 1, wherein prior to step c), the safener compound is contacted with the haploid plant tissue.

6. The method of claim 5, wherein prior to step c), the safener compound is contacted with the haploid plant tissue for a period of 24 hours to 168 hours.

7. The method of claim 6, wherein the safener compound is contacted with the haploid plant tissue at a concentration of 5-50 mg / L.

8. The method of claim 7, wherein contacting the safener compound with the haploid plant tissue occurs at 4°C to 8°C.

9. The method of claim 8, wherein contacting the safener compound with the haploid plant tissue occurs at 6°C.

10. The method of claim 1, wherein the contacting of the safener compound with the haploid plant tissue is performed simultaneously with step c).

11. The method of claim 10, wherein the contacting of the safener compound with the haploid plant tissue is performed simultaneously with step c) and lasts for 24 to 48 hours.

12. The method of claim 11, wherein the safener compound is contacted with the haploid plant tissue at a concentration of 400-800 mg / L.

13. The method of claim 12, wherein contacting the safener compound with the haploid plant tissue occurs at 24°C-32°C.

14. The method of claim 13, wherein contacting the safener compound with the haploid plant tissue occurs at 28°C.

15. The method of claim 1, wherein after step c), the safener compound is contacted with the haploid plant tissue.

16. The method of claim 15, wherein after step c), the safener compound is contacted with the haploid plant tissue for 8 to 50 hours.

17. The method of claim 16, wherein the safener compound is contacted with the haploid plant tissue at a concentration of 0-20 mg / L.

18. The method of claim 17, wherein contacting the safener compound with the haploid plant tissue occurs at 24°C to 32°C.

19. The method of claim 18, wherein contacting the safener compound with the haploid plant tissue occurs at 28°C.

20. The method of claim 3, wherein the safener compound and the viability response compound are administered sequentially or simultaneously.

21. The method of claim 1, wherein the haploid plant tissue is from maize, wheat, rice, barley, sunflower, soybean, watermelon, cucumber, tomato, pepper, or Brassica.

22. The method of claim 21, wherein the haploid plant tissue is from maize.

23. The method of claim 22, wherein the maize haploid plant tissue is an ear, microspore, callus, or embryo.

24. The method of claim 23, wherein the maize haploid plant tissue is an embryo.

25. The method of claim 1, wherein the safener compound is selected from the group consisting of: mecarphen, benoxacor, dichlorvos, isoxadiazine, cloquintocet-mexyl, fenclorac, and cyprosulfamide.

26. The method of claim 25, wherein the safener compound is mecarphen.

27. The method of claim 1, wherein the chromosome doubling agent is selected from the group consisting of colchicine, trifluralin, nafopyl, dithiopyr, nitrous oxide, and amosulfuron.

28. The method of claim 27, wherein the chromosome doubling agent is colchicine.

29. The method of claim 3, wherein the vitality-responsive compound is selected from the group consisting of thiamethoxam and saponin.

30. The method of claim 29, wherein the vitality-responsive compound is thiamethoxam.

31. The method of claim 30, wherein the haploid plant tissue is contacted with thiamethoxam prior to the colchicine treatment.

32. The method of claim 31, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 10-1000 mg / L.

33. The method of claim 32, wherein the thiamethoxam is applied to the haploid tissue for 24 to 48 hours.

34. The method of claim 30, wherein the haploid plant tissue is contacted with the thiamethoxam during the colchicine treatment.

35. The method of claim 34, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 100 to 8000 mg / L.

36. The method of claim 35, wherein the thiamethoxam is applied to the haploid plant tissue for 24 to 48 hours.

37. The method of claim 30, wherein the haploid plant tissue is contacted with the thiamethoxam after colchicine treatment.

38. The method of claim 37, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 0.5-12 g / L.

39. The method of claim 38, wherein the thiamethoxam is applied to the haploid plant tissue for 24-504 hours.

40. The method of any one of claims 1-39, wherein the contacting is performed in a solid culture medium or a liquid culture medium.

41. A method for producing a doubled haploid plant, the method comprising e) obtaining haploid plant tissue, f) contacting the haploid plant tissue with a vigor-responsive compound, g) contacting the haploid plant tissue with a chromosome doubling agent, and h) Regeneration of doubled haploid plants.

42. The method of claim 41, wherein the vitality-responsive compound is selected from the group consisting of thiamethoxam and saponin.

43. The method of claim 42, wherein the vitality-responsive compound is thiamethoxam.

44. The method of claim 43, wherein the contacting with thiamethoxam occurs before the contacting with the chromosome doubling agent.

45. The method of claim 44, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 10-1000 mg / L.

46. ​​The method of claim 45, wherein the thiamethoxam is applied to the haploid plant tissue for 0.5-24 hours.

47. The method of claim 43, wherein the contacting with thiamethoxam and the contacting with the chromosome doubling agent occur simultaneously.

48. The method of claim 47, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 100-8000 mg / L.

49. The method of claim 48, wherein the thiamethoxam is applied to the haploid plant tissue for 24-48 hours.

50. The method of claim 43, wherein the contacting with thiamethoxam occurs after the contacting with the chromosome doubling agent.

51. The method of claim 50, wherein the thiamethoxam is applied to the haploid plant tissue at a concentration of 0.5-12 g / L.

52. The method of claim 51, wherein the thiamethoxam is applied to the haploid plant tissue for 24-504 hours.

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