179 results about "Microspore" patented technology

Disclosed are methods for transformation of an androgenic-derived, haploid cell line with a site-specific nuclease. In some embodiments, the androgenic-derived, haploid cell line is a maize microspore-derived plant tissue culture. In addition, the disclosure provides a method for modifying, e.g., by mutating or targeting and integrating donor DNA into, a specific locus of a haploid or dihaploid tissue genome. The disclosure further provides methods for regenerating a whole plant from the haploid or dihaploid tissue that contains either the mutation at a specific genomic locus or a donor DNA integrated within a specific genomic locus may be obtained from the subject disclosure.

The invention provides a rapid multi-target property polymerization breeding method for rape, belonging to a plant breeding method, and comprising the following steps of: selecting excellent breeding target properties requiring to be polymerized, preparing single-cross combinations at room temperature in winter locally, preparing multi-parent complicated cross combinations in summer at different places, and assembling and polymerizing the multiple parents with excellent target properties based on the breeding requirements; performing microspore seedling cultivation on the complex filial generation for which multi-target property breeding restructuring polymerization is already realized, constructing a dihaploid (DH) breeding group, and realizing rapid fixation and homozygosis of the target genotypes of the generation of the multi-target property polymerization; and performing effective field and indoor identification and screening on the target genotypes of the generation for which the multi-target property polymerization is realized based on the property characteristics, performing molecular marker assistant selection on important target properties, and breeding new materials conforming to the breeding targets.

The invention discloses an SSR molecular marker method of a brassica allohexaploid and primers thereof. The SSR molecular marker method of the brassica allohexaploid comprises the steps: hybridizing a brassica allohexaploid parent, and performing microspore culture on the obtained filial generation to obtain a DH colony; extracting genome DNAs of the brassica allohexaploid parent and the DH colony, and performing PCR amplification with all genome DNAs as a template by using the primers; constructing a genetic map of the brassica allohexaploid parent according to a PCR amplification result, wherein 217 pairs of primers are included, with base sequences respectively shown in SEQ ID No. 1/2-433/434. According to the SSR molecular marker method, a first genetic map of the brassica allohexaploid is constructed, and the basis is provided for further performing QTL location and molecular marker assistant breeding.

The invention provides a method for obtaining doubled haploid (DH) plants of sweet peppers, which comprises the following steps of: (1) selecting buds of a donor plant, the microspores of which are in a development state between nonokaryotic stage with the nucleus located aside and early dikaryotic stage, and pretreating at 4 DEG C for 1-3 days; (2) stripping the calyces off, soaking in alcohol, disinfecting through oscillation with sodium hypochlorite, and cleaning with sterile water; (3) stripping the anthers, and inoculating the anthers on an N4-3 solid-liquid double-layer culture medium by the density that 12-18 anthers are inoculated to a culture dish with diameter of 60 mm; (4) firstly, culturing the anthers for 1-10 days in the dark at 28-35 DEG C, and then, transferring to the condition of 25-28 DEG C to continue culturing in the dark; and (5) culturing for 4-7 weeks, when a large quantity of embryoids occur, transferring leaf type embryoids to an MS basic culture medium free of hormones, and cultivating the seedlings. According to the cultivating method provided by the invention, the occurrence rate of the embryoids breakthrough the limitations of genotype, the culturing efficiency of sweet peppers is greatly increased, and meanwhile, the ratio of the DH plants is also increased.

The invention discloses a breeding method of rape with high-density siliques on a main inflorescence and application. The breeding method comprises the following steps: adopting double-2 in the variety of cabbage type rape and nepeta oil-2 in the variety of cabbage type rape as parents for hybridization to obtain F1, and conducting bagged selfing propagation to obtain a segregated population; selecting single plants in the segregated population to conduct identification on phenotypic characteristics in the field continuously for 4 years, selecting single plants with high-density siliques on the main inflorescence (number of effective siliques on the main influorescence/main-inflorescence length being greater than 2 pieces/cm); in a flowering period, selecting flower buds 2.5-3.5mm in length on the main inflorescence, using colchicine to double chromosomes, using microspores for culture to obtain a double-haploid population, and selecting single plants to breed into strain with high-density siliques on the main inflorescence and stable characters; using the strain and double 11 in the variety of cabbage type rape (the density of siliques on the main inflorescence is about 1.7 pieces/cm) to conduct hybridization, utilizing F2:3 populations to conduct genetic segregation analysis and finding that the characters of the high-density siliques are controlled by 2 pairs of additive-dominant-epistatic main genes. The breeding method and the application disclosed by the invention have the advantages that the variety of rape with high-density siliques on the main inflorescence can be bred by utilizing hybridization of the high-density silique materials on the main inflorscence and common rape, and the yield per unit of the rape can be greatly increased.

The invention relates to an anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana. An Arabidopsis thaliana male sterile mutant ahl16 is screened out from T-DNA-inserted mutant population, a gene AHL16 controlling the fertility of Arabidopsis thaliana is cloned and identified, the nucleotide sequence of the gene AHL16 is represented by SEQ ID N0.1, and genetic complement experiments prove that the AHL16 gene in a wild type can restore the male sterile phenotype. Amino acid sequence analysis and subcellular localization experiments indicate that the AHL16 gene codes proteins of an AT-hookmotif nuclear localized protein family and is located in the nucleus. In Arabidopsis thaliana, the mutation of the gene leads to complete male sterility. The gene has a very important application value in aspects of explanation of influences of the growth of the inner layers of the outer walls of microspores and the structures of the inner walls on the fertility of plants and improvement on yield by hybrid seed production.

The invention relates to a method for continuously culturing high frequency regeneration plants through cereal crop single plant source microspores, particularly to a culture method of the cereal crop single plant source microspores and the method for culturing and obtaining the regeneration plants through the cereal crop single plant source microspores. The method comprises the following steps: separating the microspores from young spikes of a single plant of the cereal crop; carrying out callus induction and differentiation steps on the separated microspores, wherein the step of separating the microspores from the young spikes of the cereal crop comprises the steps of continuously obtaining young spikes of which the middle floret microspores are in the mononuclear early-middle period from the same cereal crop plant which is planted indoors and continuously tillers and ears; carrying out the separation, callus induction and differentiation steps on the young spike obtained each time.

The invention discloses a culture method for a brassica oleracea L. var. acephala microspore regeneration plant. The method comprises the following steps: taking inflorescences of brassica oleracea L. var. acephala and rape, directly taking or taking after induction alabastrum of the inflorescences, adding the alabastrum into NLN-13 induced medium after disinfection to form fluid suspension, carrying out filtering, and centrifuging the filtrate to obtain a precipitate; adding NLN-13 induced medium and active carbon mixed liquor in order so as to obtain a microspore fluid suspension; carrying out a heat shock treatment, followed by culturing so as to obtain embryoids in cotyledon stage; inoculating the embryoids to embryoid differential medium until the embryoids are differentiated and regenerates into buds; cutting the regenerated buds to a rooting medium for rooting culture, and hardening and transplanting seedlings to obtain regenerated plants; taking young leaves of the regenerated plants for the detection of ploidy of corresponding regenerated plants and determining regenerated seedlings of rape and brassica oleracea L. var. acephala in the regenerated plants. According to the method provided in the invention, rape which is easy to generate embryos and brassica oleracea L. var. acephala which is difficult to generate embryos are mixedly cultured; the material which is easy to generate embryos is used to spur the material which is difficult to generate embryos; therefore the ratio of embryos of brassica oleracea L. var. acephala is improved.

The invention relates to an oryza sativa pollen germination aperture development and pollen fertility gene OsAOM, a mutant gene, and a recombinant expression vector and application thereof. The gene encoded protein is protein with the amino acid sequence shown as SEQ ID NO.1, or protein which is obtained by substituting, missing or adding at least one amino acid and has the same function as the OsAOM; the protein formed by the amino acid sequence shown as SEQ ID NO.1 has the nucleotide sequence shown as SEQ ID NO.2. The invention also discloses the recombinant expression vector of the OsAOM gene. The OsAOM gene disclosed by the invention encodes one piece of GDSL lipase; the functions of regulating and controlling the pollen germination aperture growth, the microspore cell membrane expansion and the pollen inner wall and cytoplasm development are realized; the gene is a necessary gene for developing microspores into fertile pollen; the gene function deficiency can cause germination aperture structure disorder, microspore cell membrane and cytoplasm degradation, pollen inner wall formation incapability and oryza sativa male sterility.

The invention discloses a method for efficiently obtaining a regeneration plant by cultivating isolated microspores of brassica oleracea L.var.capitata L.. The method comprises the following steps: (1) performing embryogenesis; and (2) performing embryo germination, thereby obtaining the regeneration plant, wherein the separated microspores are subjected to dark culture in a culture box at the temperature of 18+/-0.5 DEG C until an embryoid visible to the naked eyes is generated in the step (1) of performing embryogenesis; and then the small embryoid is transferred to a dark shaking table at a speed of 50-55 revolutions per minute at the temperature of 25+/-0.5 DEG C to perform shake culture until the cotyledonous embryoid is formed. According to the method, the genetically homogenous double-haploid plant materials can be obtained in batches, the germplasm resource innovation is accelerated, and the breeding progress is shortened. The method disclosed by the invention can be popularized and applied in the research field of molecular marker assisted breeding, mutant screening and cell totipotency mode systems of the brassica oleracea L.var.capitata L..

The invention discloses a cultivation method for barley microspores. The cultivation method comprises the following steps of selecting young spikes, performing pretreatment, taking florets on the young spikes, performing disinfection, washing the florets with sterile water, performing inoculation to an extracting solution, and performing treatment for 1-2 d at 1-10 DEG C; performing rotary cuttingon the florets after inoculation, performing filtering and centrifugation, and collecting microspores; putting the collected microspores in the extracting solution for 1-3 d in the dark at 24-26 DEGC, and purifying the microspores with maltose; and washing the purified microspores with an induction medium, performing suspension, regulating the density of the microspores, performing inoculation to a culture dish, performing sealing, and performing cultivation for 15-24 d in the dark at 24-26 DEG C. In the cultivation method, by performing low temperature treatment on the florets in vitro, thecallus yield and the seedling formation amount in cultivation of the barley microspores are increased, the regeneration capacity is greatly improved, the problem of less amount of a donor material issolved, the operation is simple and feasible, and the cost is saved.

The invention belongs to the field of rapeseed breeding, and particularly relates to a cultivating method for a high-oleic acid rapeseed variety. The cultivating method is characterized by comprising the following steps: performing hybridization by taking first Brassica napus L. A177 with the oleic acid content of 65 percent or below as a female parent and taking first high-oleic acid Brassica napus L. H003 which has the preservation number of CCTCC NO:P201604 and has the oleic acid content of at least 78 percent as a male parent, and obtaining F1 through artificial emasculation; planting the F1, obtaining a separated DH line through microspores culture; performing high-oleic acid molecular marker identification on strain seedling stage of a DH line group, and determining the genotype of each strain; measuring the content of oleic acid of each single plant by utilizing a gas chromatograph, and determining the content of the oleic acid of the each primarily selected line; selecting the parent strains of which the characters, such as the whole plant pod number, the seed number per pod, the thousand seed weight and the yield per plant, are superior to the parent strain with the oleic acid content lower than 69 percent from the DH strain with the oleic acid content of greater than 75 percent to obtain the high-oleic acid rapeseed selected line and variety of which the oleic acid is greater than 78 percent.

The invention provides a culture medium formulation for regenerated plantlets of Brassica isolated microspores, which relates to improvement of a culture medium formulation for regenerated plantlets of Brassica microspores as horticultural crops, and belongs to the field of bioengineering. The invention provides the culture medium formulation for regenerated plantlets of Brassica isolated microspores, which is high in induced embryo formation rate and differentiation rate. A culture medium of the invention comprises an embryoid induction culture medium, a differentiation culture medium and a rooting culture medium, wherein the embryoid induction culture medium, the differentiation culture medium and the rooting culture medium all comprise macroelements, trace elements and organic elements. The formulation has the advantages of providing a large number of new genotype pure lines for breeding and greatly enriching breeding resources of Brassica.

The invention provides a method for cultivating actinidia arguta anther into haplobiont. The method comprises the following steps: (A) collecting flower buds developed by actinidia arguta microspores, carrying out low-temperature refrigeration treatment for 1-10 days, and peeling anther; and (B) putting the anther in a callus tissue induction medium for carrying out dark culture for 20 days or more, culturing the anther for 40 days or more in a plant differentiation medium, and finally transferring the anther into a rooting medium for culturing and rooting to obtain the haplobiont. Through the method for cultivating actinidia arguta anther into haplobiont of the embodiment, multiple homozygous breeding materials are provided for breeding of actinidia arguta; selection and breeding materials are provided; meanwhile, an ideal receptor material is provided for the basic theoretical research; compared with the manners such as an in-vitro microspore culturing manner and a somatic chromosome eliminating manner, the anther culturing method is simple and convenient to operate; a method of artificially culturing in-vitro anther is an effective method for obtaining haplobiont of actinidia arguta; the method is extremely worthy of extensive popularization and application.

The invention relates to a method for separating and purifying a plant tapetum tissue, and belongs to the fields of developmental biology and cell biology. The method comprises the steps of collection of anthers, preparation of a cleaning liquid, preparation of an enzymolysis liquid, pretreatment of the anthers, removal of anther microspores, and separation and collection of tapetum cells. Suitable enzyme species and concentration are used for enzymolysis separation of a tapetum tissue, and then the tapetum is collected through filtration, centrifugation and purification. The method has the advantages of simple steps, convenient operation, shorter consumed time and the like, the separation efficiency is good, and the obtained tapetum tissue can be used for subsequent experiments.

The invention belongs to the technical field of plant cytology, and particularly relates to a method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly in batch in the early stage in a method of chloroplast counting. The method is characterized by comprising the following steps of: inoculating an embryoid cultured by in-vitro microspores onto a B5 culture medium; when the embryoid is grown into a complete plant, cutting the fifth even leaf sterilely; dyeing the lower epidermis tissue in the middle of the leaf by using iodine-potassium iodide; observing chloroplasts of guard cells of air vents by using an optical microscope; and recording the number of the chloroplasts of 10 air vents on each prepared leaf (namely the same leaf), wherein more than 7 air vents have 6 to 8 chloroplasts, and the plants of the chloroplasts are the haploid. By the method, the problem of long-time subculture of the haploid is solved, and in ten thousands of plants, the cost of reagents, lands and labors can be saved by 19,083 RMB. The method has a simple process, is easy to operate and low in cost and can be used for identifying the ploidy of a large number of plants, and one plant of seedling is identified only for 5 minutes, so the application efficiency of the double haploid (DH) technology is improved.

The invention provides a complex breeding method of barley with high chlorophyll content and/or large tiller number, a method for preparing barley green products and a method for improving the chlorophyll content and/or the tiller number of the barley. The method provided by the invention comprises the following steps: selecting spikelets with barley microspores in development single-nuclear early stage and/or middle stage, and performing treatment at the temperature of 2-10 DEG C for 10-30 days; harvesting the microspores, culturing the microspores and regenerating to obtain a homozygous doubled haploid; culturing the homozygous doubled haploid; and further screening to obtain the barley with the high chlorophyll content and/or the large tiller number, and preparing the barley into the required barley green products. According to the method, a plurality of excellent traits are polymerized through a hybridization technology, a microspore culture technology is utilized to perform fast homozygous stabilization on target traits, multi-index screening is performed under an artificial controllable environment, and a barley germplasm material with high chlorophyll content and multiple tillers is finally obtained. The material can be used as the excellent germplasm material for producing the barley green and other nutritional and health care products in a field planting or water culture way.

The invention discloses a method for restoring a new germplasm by rapidly creating male sterility of processing type hot pepper. The method comprises the following steps: placing a processing type hot pepper donor parent plant at the temperature of 26.4+/-0.4 DEG C; performing anther culture and selecting small microspores which are in a mid-late uninucleate stage for inducing the generation of embryoid bodies; taking flower buds before 9 am every day, and performing low-temperature pre-treament on the flower buds for 1 to 3 days in a refrigerator at 4 DEG C; soaking in alcohol under an aseptic condition, then sterilizing, washing with sterile water, and taking anther out from the flower buds and inoculating to an induced culture medium by using tweezers; inoculating weight flower buds in a culture vessel; after the anther is taken out from the flower buds under the sterile condition, uniformly dispersing the anther on the induced culture medium; transferring the induced embryoid bodies to an MS culture medium; when the root length reaches 4 to 5 cm, transplanting, wherein seedling hardening is required to be performed before transplanting. According to the method, the anther culture work is carried out more effectively and conveniently; morphological indexes of sampling flower buds are mastered; operation links are reduced; the anther culture induction efficiency is ensured.

The invention discloses a method for culturing non-heading Chinese cabbage microspore plantlets. The method comprises the following specific steps of (1) flower bud selection, wherein flower buds withthe number ratio of 1:1 between petals and anthers are selected; (2) culture of microspore derived embryos, wherein the flower buds collected in the step (1) are disinfected by a sodium hypochloritesolution for 10-15 minutes, washed with sterilization water and rinsed by an NLN-13 culture medium; the rinsed flower buds are completely ground, filtered, centrifuged, diluted by the NLN-13 culture medium containing 10-30 mg/L of a boron substance and subpackaged in culture dishes, activated carbon is added, heat shock treatment is conducted, dark culture is conducted at 24-26 DEG C for 14-16 days, and the embryoids are formed; (3) germination into the plantlets from mature embryos, wherein the embryoids are subjected to shake culture, mature embryos turning to green are transferred into a solid MS culture medium for culture, and after 38-42 days of culture, acclimatization and transplant can be directly conducted. According to the method, on the premise of ensuring the embryo formation rate, the operation steps are simplified, the operation time is greatly shortened, and the plantlet production efficiency is improved.