Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
  • A01H1/08—Methods for producing changes in chromosome number
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/008—Methods for regeneration to complete plants

Definitions

• This invention relates to methods for generating doubled haploid plants from microspores, and to doubled haploid plants produced by the methods disclosed herein.
• FI generation the resulting progeny
• FI plant a plant identified that possesses a desirable combination of phenotypic traits.
• the FI plant is then self-fertilized to yield a population of progeny plants (termed F2 plants) that must be individually analyzed to determine which F2 plants possess the desired combination of phenotypic traits originally introduced in the FI plant. If, as is often the case, the desired phenotypic traits derive from the combined effect of several genes, then the number of F2 progeny plants that must be screened depends on the number of genetic differences between the parents of the FI plant. Thus, the greater the number of genetically-controlled differences between parents of the FI plant, the larger the number of F2 progeny that must be grown and evaluated, and the lower the probability of obtaining progeny with all the desired traits.
• haploid plants the chromosomes of which can be doubled using colchicine or other means to achieve instantly homozygous, doubled-haploid plants.
• doubled haploids can be produced from the microspores which normally give rise to pollen grains.
• the life cycle of flowering plants exhibits an alteration of generations between a sporophytic (diploid) phase and a gametophytic (haploid) phase.
• Meiosis produces the first cells of the haploid generation which are either microspores (male) or megaspores (female).
• Microspores divide and develop within anthers to become mature male gametophytes (pollen).
• polyspores are genetically programmed for terminal differentiation to form mature pollen through two cell divisions.
• microspores can be induced to initiate sporophytic development which leads to the formation of haploid or doubled haploid "embryoids".
• Androgenesis is of significant interest for developmental genetic research as well as plant breeding and biotechnology, since it is a means to produce genetically true-breeding, doubled haploid plants.
• Table 1 by producing doubled-haploid (also termed polyhaploid) progeny, the number of possible gene combinations for any number of inherited traits is more manageable.
• the present invention provides methods for generating doubled haploid and/or haploid plants from microspores.
• the methods of the present invention for producing plants from microspores include the steps of: purifying microspores at a developmental stage amenable to androgenic induction (preferably from mid uninucleate to early binucleate); subjecting the microspores to nutrient stress (and optionally to temperature stress) to obtain stressed microspores; contacting the microspores with an amount of a sporophytic development inducer effective to induce sporophytic development and chromosome doubling, the contacting step occurring before, during, after, or overlapping with any portion of the nutrient stress step; and culturing the isolated, stressed microspores with at least one live plant ovary and/or with an aliquot of plant ovary conditioned medium.
• microspores are contacted with an effective amount of an auxin and/or a cell spindle inhibiting agent before, during, after, or overlapping with any portion of the nutrient stress step.
• microspores are contacted with an effective amount of a gibberrellin and/or cytokinin before, during, after, or overlapping with any portion of the nutrient stress step.
• plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction.
• the selected plant material is tillers or branches bearing spikes or flowers that contain microspores in the mid uninucleate stage of development through the early binucleate of development.
• the microspores at a developmental stage amenable to androgenic induction are purified by blending, macerating, or otherwise breaking down, the surrounding tissue (such as a wheat plant spike), filtering the ground material through a screen having a mesh size large enough to permit the passage of the microspores, but small enough to prevent the passage of most of the unwanted material, then applying the filtrate, containing the microspores, to a screen having a mesh size that is small enough to prevent the passage of the microspores but which permits the passage of smaller particles and liquid.
• Presently preferred media in which to blend, macerate, or otherwise break down tissue, including microspores at a developmental stage amenable to androgenic induction are 0.3M mannitol and 0.25 M maltose.
• the purified microspores are preferably subjected to nutrient stress for a period of at least 0.5 hours, preferably from about 0.5 hours to about 120 hours, more preferably for a period of from about 1 hour to about 96 hours, most preferably for a period of from about 24 hours to about 48 hours.
• a presently preferred method for subjecting the purified microspores to nutrient stress is to incubate the purified microspores in NPB-A medium having the composition described in Table 9 herein.
• the purified microspores are subjected to temperature stress during all or part of the period during which they are subjected to nutrient stress.
• temperature stress is effected by incubating the purified microspores at a temperature of from about 4°C to about 40°C, more preferably at a temperature of from about 28°C to about 35°C, most preferably at a temperature of about 33°C, for a period of from about 0 minutes to about 96 hours, more preferably for a period of from about half an hour to about 24 hours, most preferably for a period of from about one hour to about six hours.
• the stressed microspores are then incubated at 27°C in the dark, preferably for up to forty eight hours, before they are transferred to culture medium for embryo development.
• the microspores are also contacted with an effective amount of at least one sporophytic development inducer (as further described herein), such as 2- hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel.
• sporophytic development inducer such as 2- hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel.
• the microspores are contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin (preferably 2,4-dichlorophenoxyacetic acid) and/or an effective amount of a cell spindle inhibiting agent (such as pronamide, amiprophos methyl, dinitroaniline, phosphoric amide and related analogs).
• auxin preferably 2,4-dichlorophenoxyacetic acid
• a cell spindle inhibiting agent such as pronamide, amiprophos methyl, dinitroaniline, phosphoric amide and related analog
• the presently preferred concentration range for auxin is from about 0.1 mg/1 to about 25 mg/1, more preferably from about 0.2 mg/1 to about 10.0 mg/1, most preferably from about 0.5 mg/1 to about 4.0 mg/1.
• the presently preferred concentration range for sporophytic development inducer is from about 0.001 mg/1 to about 1000 mg/1.
• the presently most preferred concentration range for sporophytic development inducer is from about 1 mg/1 to about 500 mg/1.
• the presently preferred concentration range for cell spindle inhibiting agent is from about 1.0 ⁇ M to about 200 ⁇ M.
• the microspores are contacted with an effective amount of a cytokinin, preferably kinetin or BAP, and/or an effective amount of a gibberellin.
• a cytokinin preferably kinetin or BAP
• gibberellin an effective amount of a gibberellin.
• the preferred concentration range for cytokinin is from about 0.1 mg/1 to about 10 mg/1, more preferably from about 0.2 mg/1 to about 4.0 mg/1, most preferably from about 0.5 mg/1 to about 2.0 mg/1.
• the presently preferred concentration range for gibberellin is from about 0.01 mg/1 to about 20 mg/1, most preferably from about 0.2 mg/1 to about 4.0 mg/1.
• microspores are contacted with some or all of the foregoing chemical agents (sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin) before, during, after, or overlapping with any portion of the nutrient stress treatment, preferably during the nutrient stress treatment.
• the isolated, treated microspores are then cultured in a liquid nutrient suspension medium, such as medium NPB98 or NPB 99, preferably NPB 99, supplemented with at least one live plant ovary or an aliquot of plant ovary conditioned medium, until the microspores develop into embryoids.
• a liquid nutrient suspension medium such as medium NPB98 or NPB 99, preferably NPB 99
• the plant ovaries are obtained from wheat varieties “Chris” or “Pavon 76", but ovaries from a wide range of genotypes, and other cereal plant species, including Igri barley and NPB 96001 oats, are effective.
• the embryoids are transferred to a regeneration medium and incubated therein until the embryoids develop into plants.
• the resulting plants may be doubled haploids, or they may be haploids which can be converted to doubled haploids by treatment with a chromosome doubling agent such as colchicine.
• the methods of the present invention for producing plants from microspores may optionally include the step of genetically transforming the microspores.
• Microspores preferably uninucleate microspores
• the presently preferred methods of genetically transforming microspores are biolistic gene transfer utilizing a particle gun or electroporation of plasmolyzed microspores.
• the present invention provides genetically transformed plants regenerated from microspores.
• the present invention provides methods of initiating microspore embryogenesis including the steps of: purifying microspores at a developmental stage amenable to androgenic induction (preferably the mid uninucleate to early binucleate stages of development); subjecting the microspores to nutrient stress (and optionally to temperature stress) to obtain stressed microspores; and contacting the microspores with an amount of a sporophytic development inducer effective to induce sporophytic development and chromosome doubling, the contacting step occurring before, during, after, or overlapping with any portion of the nutrient stress step.
• microspores are contacted with an effective amount of an auxin and/or a cell spindle inhibiting agent before, during, after, or overlapping with any portion of the nutrient stress step.
• microspores are contacted with an effective amount of a gibberrellin and/or cytokinin before, during, after, or overlapping with any portion of the nutrient stress step.
• plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction.
• the selected plant material is tillers or branches bearing spikes or flowers that contain microspores in the mid uninucleate to early binucleate stage of development.
• the microspores at a developmental stage amenable to androgenic induction are purified by blending, macerating, or otherwise breaking down, the surrounding tissue (such as a wheat plant spike), filtering the ground material through a screen having a mesh size large enough to permit the passage of the microspores, but small enough to prevent the passage of most of the unwanted, material, then applying the filtrate, containing the microspores, to a screen having a mesh size that is small enough to prevent the passage of the microspores but big enough to permit the passage of smaller particles and liquid.
• the surrounding tissue such as a wheat plant spike
• Presently preferred media in which to blend, macerate, or otherwise break down tissue including microspores at a developmental stage amenable to androgenic induction are 0.3M mannitol and 0.25 M maltose.
• the purified microspores are preferably subjected to nutrient stress for a period of at least 0.5 hours, preferably from about 0.5 hours to about 120 hours, more preferably for a period of from about 1 hour to about 96 hours, most preferably for a period of from about 24 hours to about 48 hours.
• a presently preferred method for subjecting the purified microspores to nutrient stress is to incubate the purified microspores in NPB-A medium having the composition described in Table 9 herein.
• the purified microspores are subjected to temperature stress during all or part of the period during which they are subjected to nutrient stress.
• temperature stress is effected by incubating the purified microspores at a temperature of from about 4°C to about 40°C, more preferably at a temperature of from about 28°C to about 35°C, most preferably at a temperature of about 33°C, for a period of from about 0 minutes to about 96 hours, more preferably for a period of from about half an hour to about 24 hours, most preferably for a period of from about one hour to about six hours.
• the temperature stressed microspores are then incubated at 27°C in the dark, preferably for up to forty eight hours, before they are transferred to culture medium for embryo development.
• the microspores are also contacted with an effective amount of at least one sporophytic development inducer (as further described herein), such as 2- hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel.
• sporophytic development inducer such as 2- hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel.
• the microspores are contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin (preferably 2,4-dichlorophenoxyacetic acid) and/or an effective amount of a cell spindle inhibiting agent (such as pronamide).
• auxin preferably 2,4-dichlorophenoxyacetic acid
• a cell spindle inhibiting agent such as pronamide.
• concentration range for auxin is from about 0.1 mg/1 to about 25 mg/1, more preferably from about 0.2 mg/1 to about 10.0 mg/1, most preferably from about
• the presently preferred concentration range for sporophytic development inducer is from about 0.001 mg/1 to about 1000 mg/1.
• the presently most preferred concentration range for sporophytic development inducer is from about 1 mg/1 to about 500 mg/1.
• the presently preferred concentration range for cell spindle inhibiting agent is from about 1.0 ⁇ M to about 200 ⁇ M.
• the microspores are contacted with an effective amount of a cytokinin, preferably kinetin or BAP, and/or an effective amount of a gibberellin.
• the preferred concentration range for cytokinin is from about 0.1 mg/1 to about 10 mg/1, more preferably from about 0.2 mg/1 to about 4.0 mg/1, most preferably from about 0.5 mg/1 to about 2.0 mg/1.
• the presently preferred concentration range for gibberellin is from about 0.01 mg/1 to about 20 mg/1, most preferably from about 0.2 mg/1 to about 4.0 mg/1.
• the microspores are contacted with some or all of the foregoing chemical agents (sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin) before, during, after, or overlapping with any portion of the nutrient stress treatment, preferably during the nutrient stress treatment.
• the methods of the present invention for initiating microspore embryogenesis may optionally include the step of genetically transforming the microspores.
• Microspores preferably uninucleate microspores
• the presently preferred methods of genetically transforming microspores are biolistic gene transfer utilizing a particle gun or electroporation of plasmolyzed microspores.
• the present invention provides genetically transformed plants regenerated from microspores.
• doubled haploid and/or haploid plants are provided that are produced according to the methods of the present invention. Detailed Description of the Preferred Embodiment
• doubled haploid (abbreviated as DH) is used herein to refer to plants produced by doubling the chromosome number of a gamete-derived haploid plant which is produced via male gamete sporophytic divisions.
• the chromosome doubling can occur spontaneously at any stage in the process of converting a microspore to a whole plant, or can be induced, for example, by treatment with a cell spindle inhibitor, or after embryogenesis, for example by treating haploid plantlets with colchicine.
• microspore refers herein to the male gametophyte of a plant, including all stages of development from meiosis through formation of the mature pollen grain.
• androgenic induction means induction of androgenesis, i.e., the process by which pseudo-embryos or embryoids, which can be regenerated into plants, are produced from microspores.
• the abbreviation mg/1 means milligrams per liter.
• the methods of the present invention are applicable to a broad range of plant species, including dicotyledonous plants and monocotyledonous plants.
• Representative examples of plants which can be treated in accordance with the methods of the present invention include, but are not limited to: wheat, barley, rice, corn, triticale, rye, millet, flax, wheat grasses (for example, Agropyron, Elytrigia), pasture grasses, rye grass, orchard and brome grasses (e.g., Lolium, Phleum, Bromus spp.), turf grasses (e.g., Poa pratensis), forage and pasture legumes (e.g., alfalfa, clovers), grain legumes, soybeans, peas, lentils, peanuts, ornamentals including garden and commercial flower and bulb species, fruit trees and nut trees, various vegetable species (e.g., cucurbits, onions
• the methods of the present invention can be used, for example, to produce inbred lines for use in hybrid seed production, especially to generate vigorous inbred lines from hybrids of crop plants that are susceptible to inbreeding depression. Additionally, the methods of the present invention can be used, for example, to obtain genetically and phenotypically variable progeny from pollen-producing apomictic species, such as blue grasses (Poa spp.) and buffalo grasses (Buchloe spp.). The methods of the present invention may permit recovery of viable progeny from semisterile plants, such as garlic, Burbank potato, and various self-incompatible species such as cherries, apples, clovers and broccoli. Preferably, the methods of the present invention utilize wheat spikes as starting material.
• the methods of the present invention have been successfully applied to spikes from a range of wheat genotypes including, but not limited to: the spring wheats, Calorwa, Chris, Pavon 76, Penawawa, Spillman, Waldron, WED 202-16-2, Wawawai, Red Bobs, SWSW96005, and winter wheats Claire, Eltan, Platte, Enola, Soisson, BonPain, Madsen and Svilena.
• the presently preferred wheat genotypes are Chris, WED 202-16-2 and Pavon 76.
• Spring wheat cv Spring wheat cv.
• Pavon 76 is generally considered to be a highly androgenic genotype based upon results from anther culture, although it produces a high proportion of albino plants, and a relatively low frequency of spontaneously doubled haploid plants.
• the androgenic microspores of both Chris and WED 202-16-2 produce high numbers of green progeny, many of which are spontaneously DH.
• the presently most preferred wheat cultivar is Chris.
• Chris is a public variety and Pavon 76 is available from the USD A Cereals Collection, 169 IS 2700W, Aberdeen ID 83210.
• WED 202-16-2 is a T. dicoccoides/Favon 16 derivative from the Volcani Institute, Bet Degan, Israel.
• the methods of the present invention permit the production of doubled haploid plants from wheat varieties and cultivars that have long been considered recalcitrant or non-responsive to anther or microspore culture.
• Waldron is a spring wheat considered recalcitrant to established anther culture methods. Nonetheless, Waldron is responsive to the methods of the present invention.
• wheat cultivars that are recalcitrant to other methods such as
• Waldron and WPB926, can be induced to begin sporophytic divisions at a high frequency utilizing the methods of the present invention.
• Microspore cell divisions of some of the less responsive genotypes are arrested prior to the emergence of a multi- nucleate pro-embryoid from a common microspore cell wall.
• a solution to this problem provided by the present invention is to treat plant material containing microspores with novel culture media NPB 98 or NPB 99, the compositions of which are set forth in Table 9.
• the recalcitrant genotypes respond especially well to culture in NPB 98 or NPB 99 that is conditioned by the prior growth of plant ovaries at a density of 3 to 4 ovaries per milliliter of medium for 7 to 14 days prior to use in microspore culture. Media so conditioned have been shown to accelerate cell divisions and embryogenic development of cultured micrspores. Typically, mature embryoids can be obtained one week earlier than in cultures that utilize whole ovaries instead of ovary- conditioned medium.
• An alternative solution to the problem of arrested microspore cell divisions is to make crosses between the recalcitrant cultivars, such as Waldron or WPB 926, and cultivars that efficiently produce green plants from embryoids, such as Chris and WED 202-16-2.
• the methods of the present invention can be incorporated into a more general plant breeding program in which genotypes that are amenable to culture according to the methods of the present invention are crossed with less amenable genotypes which have other, desirable characteristics.
• Pavon 76 which produces many embryoids, but relatively few green plants when treated in accordance with the methods of the present invention, can be crossed with WED 202- 16-2, Chris, or any other wheat genotype which produces a high frequency of green plants from embryoids when treated in accordance with the methods of the present invention.
• the resulting doubled haploid progeny can be screened for those genotypes that produce many embryoids and many green plants.
• crossing Chris, Pavon 76 or WED 202-16-2 with a recalcitrant genotype will result in doubled haploid progeny that are amenable to culture according to the methods of the present invention, and which also possess at least some of the desirable traits of the recalcitrant parent.
• the strategy of crossing a genotype that is amenable to the production of green, double haploid plants with a more recalcitrant plant species, having some other desirable trait(s), is generally applicable to any plant species.
• Wheat plants that are used to provide the microspore starting material (referred to as donor plants) in the practice of the presently preferred embodiment of the present invention may be cultivated in the field, but preferably are cultivated in an artificial environment that is less contaminated with microorganisms, such as a greenhouse.
• Field-grown wheat plants are often heavily infested with microorganisms that contaminate all stages of the microspore embryogenic process.
• Disinfectant treatment can be used to ameliorate the contamination problem.
• the starting plant material used in the methods of the present invention can be treated with a 20% (v/v) solution of commercial hypochlorite or chlorine bleach. Any standard growth regime that is known to one of ordinary skill in the art for growing wheat, preferably in a greenhouse, can be utilized in the practice of the present invention.
• microspores that have at least completed meiosis are useful in the practice of the present invention.
• microspores enclosed within the anthers in the middle section of a spike should be in the mid uninucleate to early binucleate stage of development, more preferably in the late uninucleate to early binucleate stage of development.
• Microspores that are to be subjected to genetic transformation should preferably be uninucleate. Morphological features of tillers containing microspores at these stages can easily be established for each plant variety by comparing the morphology of the tiller with the microspore developmental stage as determined by microscopic examination with acetocarmine stain. The stages of microspore development are set forth in Bennett, M.D.
• the microspores at a developmental stage amenable to androgenic induction are purified by blending, macerating, or otherwise breaking down, the surrounding tissue (such as the spikelets of a wheat plant), filtering the ground material through a screen having a mesh size large enough to permit the passage of the microspores, but small enough to prevent the passage of most of the ground material, then applying the filtrate, containing the microspores, to a screen having a mesh size that is small enough to prevent the passage of the microspores but which allows passage of the finer particles and liquid.
• the surrounding tissue such as the spikelets of a wheat plant
• presently preferred media in which to blend, macerate, or otherwise break down tissue including microspores at a developmental stage amenable to androgenic induction are 0.3M mannitol and 0.25 M maltose.
• a presently preferred method for isolating microspores is set forth in Example 1. Isolated microspores are subjected to stress treatment, such as nutrient and/or temperature stress, treatment with sporophytic development inducers, and optionally to treatment with auxins, cell spindle inhibiting agents, cytokinins and gibberellins as is hereinafter further described.
• Stress treatment such as nutrient and/or temperature stress, treatment with sporophytic development inducers, and optionally to treatment with auxins, cell spindle inhibiting agents, cytokinins and gibberellins as is hereinafter further described.
• Temperature stress is carried out preferably from between about 4°C to about 40°C. The optimum period of stress treatment varies with the genotype.
• microwave radiation having an energy of 106 eV to 103 eV
• the absence or reduction of all nutrients, or selected nutrients, for example the absence or reduction of minerals and/or nitrogen, from the water in which the microspores are immersed provides the nutrient stress when desired.
• Nutrient stress is one way in which to promote the induction of sporophytic development from microspores and can be used, for example, when dealing with microspores from plant genotypes that are resistant to androgenic induction.
• the microspores are contacted with an effective amount of a sporophytic development inducer.
• the microspores are contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin and/or a cell spindle inhibiting agent.
• the microspores are contacted with an effective amount of a cytokinin and/or a gibberellin.
• the microspores are contacted with the foregoing chemical agents before, during, after, or overlapping with any portion of the nutrient stress treatment, preferably during the nutrient stress step.
• Sporophytic development inducers useful in the practice of the present invention induce plant microspores to switch from gametophytic development to sporophytic development.
• sporophytic development inducers useful in the practice of the present invention may cause the development of inviable pollen grains, multicellular or multinucleate pollen grains, arrest starch formation in developing microspores and cause physical deformation of mature pollen grains that develop from microspores treated with a sporophytic development inducer.
• Some sporophytic development inducers useful in the practice of the present invention are chemical hybridizing agents. Chemical hybridizing agents (abbreviated as CHAs) are chemicals which when applied to plants cause the plants to produce inviable pollen.
• CHAs Chemical hybridizing agents
• sporophytic development inducers useful in the present invention include, but are not limited to: amiprophos methyl, 2-aminonicotinic acid; 2-chloronicotinic acid; 6-chloronicotinic acid; 2-hydroxynicotinic acid; 6-hydroxynicotinic acid; 3-hydroxypicolinic acid; Benzotriazole; 2,2'-dipyridil; 2,4-pyridine dicarboxylic acid monohydrate; 2-hydroxypyridine; 2,3-dihydroxypyridine; 2,4-dihydroxypyrimidine-5- carboxylic acid; 2,4-dihydroxypyrimidine-5-carboxylic acid hydrate; dinitroaniline, phosphoric amide, 2-hydroxypirimidine hydrate; 2,4,5-trihydroxypyrimidine; 2,4,6- trichloropyrimidine; 2-hydroxy-4-methyl pyrimidine hydrochloride; 4- hydroxypyrazolo-3,4,d-pyrimidine; quinaldic acid; violuric acid monohydrate; thymine; xant
• the presently preferred sporophytic development inducers are: 2-hydroxynicotinic acid (2-HNA); 2-chloroethyl-phosphonic acid (having the commercial name of Ethrel) available from Sigma Chemical Co., PO Box 14508, St. Louis, Mo. 63178-9916; violuric acid monohydrate, 2-chloronicotinic acid and 2- hydroxyproline.
• the presently most preferred sporophytic development inducers are 2- hydroxynicotinic acid (2-HNA) and 2-chloroethyl-phosphonic acid.
• a sufficient amount of the sporophytic development inducer is employed to effect a measurable induction of sporophytic development.
• the presently preferred concentration range of sporophytic development inducer is from about 0.001 mg/1 to about 1000 mg/1.
• representative sporophytic development inducers While not wishing to be bound to a particular theory explaining the method of action of the sporophytic development inducers useful in the practice of the present invention, certain of the presently preferred, representative sporophytic development inducers have some metal chelation ability.
• representative sporophytic development inducers can chelate Cu, Mg, Fe and Zn ions. Copper is essential to pollen fertility (Scharrer, K., and Schaumlaufel, E., Z Graph. Dung.
• the microspores are also contacted with an amount of an auxin effective to maintain callus development.
• auxins useful in the practice of the present invention include, but are not limited to: 2,4- dichlorophenoxyacetic acid (2,4-D), as well as related auxins (e.g., indoleacetic acid (IAA), indolebutyric acid (IBA), naphthalene acetic acid (NAA), analogues and/or salts of 2,4-D).
• the presently preferred concentration range for auxin is from about 0.1 mg/1 to about 25 mg/1, more preferably from about 0.2 mg/1 to about 10 mg/1, most preferably from about 0.5 mg/1 to about 4.0 mg/1.
• the microspores are also preferably contacted with an amount of a cell spindle doubling agent effective to double the chromosome number in a measurable number of microspores.
• Cell spindle doubling agents tubulin inhibitors
• the presently preferred concentration range for cell spindle inhibiting agents is from about 1.0 ⁇ M to about 200 ⁇ M.
• microspores treated in accordance with the present invention can be contacted with an amount of a cytokinin effective to improve callus quality, in particular to enhance the ability of callus tissue to grow and to increase the size to which callus tissue develops.
• cytokinins useful in the practice of the present invention include, but are not limited to: kinetin, benzaminopurine (BAP) and zeatin. Kinetin is the presently preferred cytokinin.
• water in which peeled Solanum tuberosum potatoes have been boiled contains significant amounts of cytokinin(s) which can be utilized in the practice of the present invention.
• the presently preferred concentration range for kinetin, zeatin and BAP is from about 0.1 mg/1 to about 10 mg/1, more preferably from about 0.2 mg/1 to about 4.0 mg/1, most preferably from about 0.5 mg/1 to about 2.0 mg/1.
• the microspores can be contacted with an amount of a gibberellin effective to enhance cell expansion.
• a gibberellin effective to enhance cell expansion.
• concentration range for gibberellin is from about 0.01 mg/1 to about 20 mg/1, more preferably from about 0.2 mg/1 to about 4.0 mg/1.
• the sporophytic development inducer and auxin interact with the aforedescribed temperature and/or nutrient stress treatments to enhance the induction of androgenic microspores.
• the sporophytic development inducer and auxin treatments contribute to the completion of androgenesis leading to the eventual formation of mature embryoids which, upon transfer to semi-solid medium, regenerate into green and/or doubled haploid plants.
• Obtaining enlarged microspores with a fibrillar cytoplasmic structure is a pre-requisite for embryogenesis, but only those that proceed to proembryoids will eventually develop into mature embryoids, which are then able to regenerate to produce plants.
• sporophytic development inducers act synergistically to produce embryoids from microspores which can be regenerated into whole plants.
• the isolated, treated microspores are cultured in the presence of at least one live plant ovary and/or in the presence of an aliquot of plant ovary conditioned medium.
• Presently preferred sources of ovaries are wheat cultivars Pavon 76 and Chris. It has been found that the plant ovaries do not have to be from the same variety (genotype) or species as the plant from which the embryoids are generated. Thus, for example, ovaries from Igri winter barley, or NPB96001 oats, will stimulate the development of wheat embryoids.
• embryoid culture medium typically 3 to 6 live plant ovaries will be added to a 60 mm diameter Petri dish containing approximately 5 ml of embryoid culture medium.
• the presently preferred embryoid culture medium is NPB99, the composition of which is set forth in Table 9 herein.
• the embryoid culture medium osmolarity should be around 300 mOsmo.
• Embryoids develop from treated microspores, in the presence of at least one plant ovary and/or in the presence of an aliquot of ovary conditioned medium, usually after 3-4 weeks.
• the developed embryoids are transferred to a regeneration medium, such as media 190-2 and 190-2(b) set forth in Table 9, and the remaining, less- developed embryoids are further incubated with fresh ovaries, and/or ovary- conditioned medium, until mature embryoids form.
• a regeneration medium such as media 190-2 and 190-2(b) set forth in Table 9, and the remaining, less- developed embryoids are further incubated with fresh ovaries, and/or ovary- conditioned medium, until mature embryoids form.
• the presently most preferred regeneration medium is 190-2(b).
• a population that consists almost entirely of live, androgenic microspores can be obtained by the use of a mesh filter with a pore size of 50 ⁇ m to filter out dead microspores. This filtration step is typically done about 7 to 10 days after the beginning of coculture of microspores with one or more plant ovaries (and/or culture of microspores in the presence of an aliquot of ovary conditioned medium). The medium containing the microspores is pipetted into an autoclaved 50 ⁇ m mesh filter.
• the 2 to 16 celled, multi-cellular, dividing microspores (and plant ovaries, if present) are retained on the surface of the filter, because their diameters are larger than 50 ⁇ m, while the dead microspores pass through the filter.
• the dividing microspores in the filter are rinsed 3 times with NPB99 medium, and collected by washing the filter, from the reverse side, twice with NPB99 medium (5 ml per wash). These dividing microspores are re-suspended in NPB99 medium, and plated over several plates to accommodate the large number of developing embryos. Nearly all of these microspores are androgenic and will continue their development into embryoids and/or calli.
• microspores obtained from one wheat spike will yield about 5000 to 8000 embryos.
• a highly purified, androgenic, microspore population is desirable if the microspores are to be genetically transformed, as described below.
• Microspores treated in accordance with the methods of the present invention can optionally be genetically transformed by any art-recognized means, for example to produce plants that express one or more desirable traits.
• techniques for introducing a gene, cDNA, or other nucleic acid molecule into microspores include: transformation by means of Agrobacterium tumifaciens; electroporation-facilitated DNA uptake in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA, by microspores; microinjection of nucleic acid molecules directly into microspores; treatment of microspores with polyethylene glycol; and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the microspore and enter the cell nucleus.
• microspore transformation technique that utilizes Agrobacterium tumifaciens and is broadly applicable to numerous plant species is disclosed in European Patent Application EP 0 737 748 Al.
• Isolated microspores are cocultivated with Agrobacterium containing a Ti plasmid including a transgene (within the transfer DNA of the Ti plasmid) that is to be transferred and stably integrated into the microspore genome.
• Cellulytic enzymes (such as cellulase, hemicellulase and pectinase) are added during the cocultivation step and serve to permeabilize the microspore cell wall.
• the transfer DNA (T DNA) is transferred from the Agrobacterium cells to the microspores where it is inserted into the microspore genome thereby generating stably genetically transformed microspores. Thereafter, the treated microspores are washed with a mucolytic enzyme (such as lysozyme). Whole plants can then be regenerated from the genetically transformed microspores in accordance with the present invention.
• a mucolytic enzyme such as lysozyme.
• Whole plants can then be regenerated from the genetically transformed microspores in accordance with the present invention.
• Other workers have reported the use of Agrobacterium to successfully transform microspores from Brassica (Pechan P.M., Plant Cell Rep. 8: 387-390 (1989); Swanson E.B. and Erickson L.R., Theor. Appl. Genet. 78: 831-835 (1989)).
• Joersbo et al. Plant Cell, Tissue and Organ Culture 23: 125-129 (1990).
• Joersbo et al. report the transient electropermeabilization of barley microspores to the dye propidium iodide by delivering rectangular electrical pulses to microspores in a chamber with cylindrical coaxial electrodes at a distance of 1 mm.
• the electroporation treatment had limited deleterious effect on the microspores which could be cultured to produce green plants.
• a presently preferred method for stably genetically transforming microspores is biolistic transformation whereby microspores are bombarded with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the microspore.
• Yao et al. (Genome 40(4): 570-581 (1997)) report the production of transgenic barley plants by direct delivery of plasmid DNA into isolated microspores using high velocity microprojectiles.
• the plasmid used to transform the microspores contained a bar gene, under the control of a maize ubiquitin promoter, that conferred resistance to the herbicide bialaphos.
• genetically transformed microspores or embryoids could be selected based on their resistance to bialaphos present in the culture medium.
• Jahne et al. (Theor. Appl. Genet. 89: 525-533 (1994)) also report the production of transgenic barley plants by direct delivery of plasmid DNA into isolated microspores using high velocity gold microprojectiles. Again, genetically transformed microspores or microspore-derived calli were selected based on their resistance to bialaphos present in the culture medium. Fukuoka et al. (Plant Cell Reports 17: 323-328 (1998)) report the production of transgenic rapeseed plants by direct delivery of plasmid DNA into isolated microspores using high velocity microprojectiles.
• Transformed embryos derived from the microprojectile bombarded microspores were identified by expression of a firefly luciferase gene.
• Harwood et al. (Euphytica 85: 113-118 (1995))disclose the use of the PDS1000 He particle delivery system to genetically transform barley microspores. The gus reporter gene was used to demonstrate both transient and stable transformation events. Additional examples of microspore transformation techniques are set forth in In Vitro Haploid Production in Higher Plants, Chapt. 2, Jain et al. (eds), Kluwer Academic Publishers (1996). The aforementioned publications disclosing microspore transformation techniques are incorporated herein by reference, and minor variations make these technologies applicable to a broad range of plant species.
• DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes.
• a selectable marker gene is used to select only those microspores that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit).
• the screenable gene provides another check for the successful culturing of only those microspores or microspore-derived embryoids carrying the gene(s) of interest.
• a commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the microspores grow.
• Plant cells are normally susceptible to kanamycin and, as a result, die.
• the presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable.
• another selectable marker gene is a gene, such as the bar gene, which confers resistance to a herbicide.
• a screenable gene commonly used is the -glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue.
• the plasmid will contain both selectable and screenable marker genes.
• Plants produced in accordance with the methods of the present invention can be doubled haploids, the chromosome number of which doubled during the androgenesis induction phase of development of whole plants from microspores. Additionally, plants produced in accordance with the methods of the present invention can be haploids, the chromosome number of which can subsequently be doubled by treatment with an agent such as colchicine.
• the methods of the present invention are at least approximately 250-fold more efficient than other methods for producing plants from microspores, efficiency being measured as the percentage of microspores that yield green plants.
• Fresh tillers that contain microspores at an appropriate developmental stage are cut at a position two nodes below the top of the tiller, and immediately placed in a clean container with distilled water. All leaves are removed by cutting the leaf blades at their bases.
• the time between the collection of tillers and their treatment is preferably minimized to reduce the possibility of contamination by microorganisms.
• Microspores enclosed within the anthers in the central section of a spike should preferably be in the mid- to late uninucleate stage of development. Morphological features of tillers containing microspores at these stages can easily be established for each genotype via microscopic examination with acetocarmine stain. Fresh tillers so collected are then ready for microspore isolation.
• the microspores are then washed three times with 5ml NPB-A each, then washed off the filter into a petri dish (60 x 15 mm) using lOmls of water.
• the total volume here may range from 5 to 10 ml, depending upon the number of spikes blended.
• the suspension of microspores is then transferred to Petri dishes (60 x 15 mm) at 4 ml each. In each petri dish, one ml of chemical formula stock solution is added.
• the chemical stock solutions i.e., containing one or more of sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin
• the chemical stock solutions i.e., containing one or more of sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin
• all dishes with microspore suspension are sealed with parafilm and then placed in a 33°C incubator for four to 24 hours followed by an incubation at 27oC for 0 to 48 hours.
• the suspension of microspores may be incubated only at 27oC for 24 to 72 hours.
• microspores are recovered by passing the treated suspension through a 50 ⁇ filter. The microspores are then washed three times with 5ml of solution NPB 98 (having the composition set forth in Table 9). The microspores are washed off into a Petri dish with two 3ml aliquots of solution NPB 98. The microspore density is determined using a hemocytometer, then adjusted to 10,000 microspores per ml of solution NPB 98. The tubes are incubated at 27°C for embryoid induction. The percent microspore survival can be evaluated by adding fluorescein diacetate to the tubes and counting the microspores using a haemacytometer.
• Isolated microspores are cultured in liquid NPB 98-1 medium or liquid NPB-99 medium, preferably liquid NPB-99 medium, as a suspension culture.
• the compositions of NPB 98-1 medium and NPB-99 medium are set forth in Table 6. An aliquot of 2 ml media per 35 mm x 10 mm Petri dish, or 5 ml media per 60 mm x 15 mm Petri dish, at a density of approximately 1.5 x 10 4 microspores per ml is effective. Immature ovaries are added to the culture at a density of one per ml of medium immediately preceding the incubation. Ovaries are aseptically picked out from fresh and disinfected spikes.
• Ovaries of all genotypes are effective for the present invention, but the ovaries from varieties Chris or Pavon 76 are preferred for embryogenesis of all wheat varieties tested.
• medium NPB 98 or medium NPB 9 conditioned by ovaries at a density of three ovaries per milliliter (ml) of medium for 7 to 14 days is mixed directly with microspores without the addition of any fresh ovaries. All Petri dishes are sealed with parafilm and incubated in the dark at 27°C until embryoids of 1-2 mm diameter develop. The embryoids can then be transferred to regeneration medium.
• Embryogenic microspores begin their first cell division after approximately 12 hr in culture.
• Multi-cellular proembryoids still enclosed within the microspore wall or exine, are formed in approximately one week. In approximately one more week, the exine wall ruptures and immature embryoids emerge, which grow into mature embryoids within about 10 to 14 days.
• Obtaining enlarged microspores with a fibrillar cytoplasmic structure is a pre-requisite for embryogenesis, but only those that proceed to proembryoids will eventually develop into mature embryoids, which are then able to regenerate to produce plants.
• the use of some ovary-conditioned medium appears to accelerate embryoid development and influence the uniformity of embryoid development from induced microspores.
• embryoids When embryoids reach the size of 1 to 2 mm, they are transferred aseptically to solid 190-2 or 190-2(b) medium, preferably 190-2(b) medium, at a density of 25-30 embryoids in each 100 x 15 mm Petri dish.
• the Petri dishes with embryoids are placed under continuous fluorescent light at room temperature (22°C) for plantlet development.
• green plantlets develop and are ready for transfer to soil. Green plants are raised in the greenhouse, much like plants grown from seeds. If plants appear to be haploid, colchicine can be applied to induce chromosome doubling. Seeds produced on any plants are instantly homozygous, and so can immediately be used for rapid evaluation and selection in breeding, for analyses in genetics research, or for selection and evaluation of transformants in biotechnology.
• microspores were isolated and treated as described in Example 1 except that the density of the microspores in solution NPB-A was 7700 per ml. The microspores were counted in microtitre wells. The results of the experiment are shown in Table 3.
• the following experiment was conducted to determine if the presence of 2,4 dihydroxy pyrimidine 5 carboxylic acid (2,4 DP) or violuric acid monohydrate (VAM) increases the number of microspores that survive treatment in accordance with the methods of the present invention.
• Microspores were isolated and treated as described in Example 1 except that eleven spikes were blended, using two blenders. The 33°C treatment in water was for 5.5 hours.
• the microspore density in ⁇ PB-A was 5100 microspores per ml, and there were 2ml per treatment. The viability was determined one day after transfer to ⁇ PB98. The results of the experiment are shown in Table 6.
• the 2,2 Dipyridyl had a very beneficial effect on the cell survival, particularly at a concentration of 0.18mM.
• media set forth in Table 9 are useful in the practice of the present invention.
• media NPB98 and NPB-99 are novel media of the present invention and are preferably used to coculture treated microspores with plant ovaries.
• Medium 190-2(b) is preferably used to culture embryoids during their development into green plants.
• Other media, except for MB-97, described in Table 9 are those commonly reported in the literature, but which have been found to be comparatively ineffective for production of embryoids.
• MB-97 is a novel medium of the present invention and is nearly as effective as NPB98 when used to coculture treated microspores with plant ovaries.
• the values for the amount of each medium component are milligrams per liter.
• stressed microspores are cocultured with either ovary-conditioned medium or at least one live plant ovary (although it is within the scope of the present invention to coculture stressed microspores with both ovary- conditioned medium and at least one live plant ovary).
• ovaries were excised and placed into Petri dishes supplemented with NPB-98 medium.
• the density of ovaries in the Petri dishes was initially set at ten ovaries per millilitre of medium. After being sealed with parafilm, all Petri dishes were incubated at 27°C for a period of 2 through 45 days, during which the conditioned medium was removed from various dishes at various time points. The removed medium was then mixed with NPB-98 (in a 1:3 ratio) and microspores were isolated.
• the microspore cultures were maintained at 27°C and examined with an inverted microscope periodically to monitor cell divisions and embryogenic development. Data on the numbers of initial embryogenic microspores, percentage of dividing cells, percentage of proembryoids and of embryoids were collected. The rate of embryoid development was also measured at various time points during culture.
• ovaries isolated from genotype Pavon 76 were cultured at a density of ten ovaries/ml of NPB-98. After 7, 14, 35 and 40 days in culture, 0.5, 1.0 or 2.0 ml of ovary conditioned medium was added to microspore cultures to a final volume of 3 ml medium. The dilution of aged (35 or 40 d) ovary conditioned media did not show improved microspore embryogenesis, whereas the same dilutions of younger ovary conditioned media led to decreases in frequencies of proembryoids. These results suggest that the functional ingredients released by ovaries are stimulatory rather than inhibitory in nature (Table 11).
• ovary densities were tested to determine the number of ovaries in the conditioning medium that is most effective for stimulating microspore embryogenesis once the conditioning medium is diluted to a final working concentration in microspore cultures.
• Microspores were isolated from Pavon 76 as described herein. Five to 30 ovaries per ml of original conditioning medium were all effective, 4 ovaries/ml or below were sub-effective (Table 13). On this basis, the ovary conditioned medium is as effective as the same number of fresh ovaries placed directly in the culture medium at the time cultures are initiated. Twenty ovaries per ml of medium seemed to be most effective, saturating the demand for active ingredients released by cultured ovaries.
• ovaries were excised from various genotypes and were tested for their effectiveness in stimulating microspore embryogenesis.
• Microspores were isolated from wheat variety Chris. The available data do not show genotype differences.
• Media conditioned with ovaries from all genotypes, ranging from early uninucleate through mature pollen were all effective for promoting the production of proembryoids from microspores.
• the density of ovaries and the time they were cultured in the conditioning medium were more critical than the genotype and the developmental stage of ovaries at the time of excision.
• Ten ovaries per ml of medium conditioned for a period of 7-14 days, and subsequently diluted three fold for microspore culture were the most beneficial for embryoid development.
• embryoids from some plant genotypes yield a high percentage of albino plants.
• Albino plant percentage can be reduced by lower temperature treatment during the temperature stress period, but the total number of embryoids produced is sharply reduced and the total number of green plant production is consequently low.
• the level of nutrients available to the microspores during the nutrient stress step of the methods of the present invention was assessed for its effect on the number of green plants produced.
• Three spikes of spring wheat line WED 202-16-2 were treated in a flask containing 50 ml of 100 mg/1 2 HNA, 10 mg/1 2,4-D, 2 mg/1 BAP, 3 mg/1 GA with or without 10% NPB98 induction medium at 33°C for 69 hours.
• Microspores were released as described herein. Microspores were cultured in 5 ml of NPB98 induction media in 6 cm Petri dishes at a density of 30,000 microspores per ml induction medium.
• Four fresh ovaries of WED 202-16-2 were added to each of the Petri dishes. There were 2 replications for each treatment. The results are shown in Table 14.

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