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WO2002001940A2 - Procede permettant de produire des vegetaux fertiles a partir de microspores isoles - Google Patents

Procede permettant de produire des vegetaux fertiles a partir de microspores isoles Download PDF

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Publication number
WO2002001940A2
WO2002001940A2 PCT/DK2001/000456 DK0100456W WO0201940A2 WO 2002001940 A2 WO2002001940 A2 WO 2002001940A2 DK 0100456 W DK0100456 W DK 0100456W WO 0201940 A2 WO0201940 A2 WO 0201940A2
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WIPO (PCT)
Prior art keywords
plant
microspores
plants
medium
wheat
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PCT/DK2001/000456
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English (en)
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WO2002001940A3 (fr
Inventor
Anni Jensen
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Pajbjergfonden
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Publication date
Application filed by Pajbjergfonden filed Critical Pajbjergfonden
Priority to AU2001268955A priority Critical patent/AU2001268955A1/en
Publication of WO2002001940A2 publication Critical patent/WO2002001940A2/fr
Publication of WO2002001940A3 publication Critical patent/WO2002001940A3/fr

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    • 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

  • the present invention relates to the field of plant breeding. It concerns a method for the production of wheat plants from microspores co-cultured with ovaries. In other aspects, this invention relates to the regenerated plants, and to seeds and progeny of the regenerated plants derived from cultured microspores/ovaries.
  • Crop and plant improvement is an area of great commercial interest.
  • Wheat for example is a major world-wide cereal crop with an important commercial value.
  • Un- notably current methods for improving breeding of wheat are time consuming and labour intensive.
  • seeds from those homozygous plants shown to possess the desired characteristics are selected for further breeding. They are crossed and the resulting offspring is inbred through generations of selfing (i.e. self fertilization) in order to obtain homozygous plants having new gene combinations.
  • the number of heterozygous plants is halved.
  • generations of selfing plants which are essentially homozygous may be obtained.
  • a sufficient degree of homozygosity is generally believed to be obtained after 6-8 generation. This process is time consuming and generally takes from 3 to 6 years.
  • Wheat plants displaying the desired characteristics are contin- uesly selected among the generations of selfing. For practical reasons the selection process takes place before the offspring has been inbred to 100 % homozygosity. This means that selection is carried out on the basis of both homozygous and heterozygous plants. The selection of heterozygous plants is disadvantageous since these plants are carrying recessive genes with unknown characteristics, which are passed on to the next generations.
  • the process of developing inbred parents generally takes from 3-6 years. Another 3-4 years is required for field testing which gives a total of 7-10 years.
  • the time period to produce a new homo- zygot wheat variety may be reduced from 3-6 years to about 1-2 years. Further- more, selection becomes more efficient, as it is carried out exclusively with homo- zogous plants.
  • haploid microspores have a single haploid nucleus whereas mature pollen have 3 nuclei.
  • Haploids contain only one- half of the chromosome number present in somatic cells. Somatic cells are those other than gametic cells, the latter being inherently haploid. Haploid complements may also be doubled to produce homozygous diploids. The production of doubled haploid plants is one way to produce truly homozygous lines. Douple haploid plants have both male and female sex organs and produce ears with good seed through self-pollination after chromosomes have been doubled.
  • chromosome complement may be doubled by various agents, such as colchicine, a mitotic spindle inhibitor, the use of which results in chromosome duplication without cell division.
  • chromosome doubling occurs spontaneously.
  • the plants resulting from induced or spontaneous chromosome doubling are diploid.
  • Doubling of haploid complements allows homozygous lines to be produced from heterozygous parents in a single generation.
  • Microspore culturing followed by chromosome doubling is one method of producing doubled haploids.
  • doubled haploid plants in wheat breeding requires a successful haploid production system.
  • Factors that affect the frequency of in vitro plant production include genotype, donor plant physiology, the stage of pollen development, pretreatment conditions and the concentrations and nature of ingredients in the media used for culture and regeneration. For example, sucrose levels, plant growth regulators and additives to culture media affect success.
  • mannitol and of cold temperature pretreatment have been explored separately as means for increasing the frequency of embryo formation from isolated microspores.
  • Wei et al., 1986 Wei et al. applied mannitol pretreatment to isolated barley pollen, but had limited success, possibly because the methods were applied to binucleate pollen, whereas uninucleate pollen (microspores) are believed to be the most responsive in culture. Uninucleate cells most likely lost viability during isolation from the anthers. If mannitol pretreatment was applied to whole anthers containing microspores then results were said to be better.
  • Touraev et al. (Touraev, A., Indrianto, A., Wratschko, I., Vicente, O., 1996, Sex Plant Reprod., 9, pp. 209-215) report on the use of a combination of starvation and heat shock treatments to induce the formation of embryonic microspores in wheat. It has been observed that microspore cultures have not resulted in a similar amount of embryo-like structures as reported for the anther culture systems.
  • Puolimatka et al. (Puolimatka, M., Laine, S., & Pauk, J., 1996, Cereal Research Communications, 24, 4, pp.
  • Mejza et al. (Mejza, S. J., Morgant, V., DiBona, D. E., & Wong, J. R., 1993, 12, pp. 149-153) describe how co-culturing wheat microspores with barley ovaries were critical, in addition to how pretreatment with cold temperatures resulted in less embryos than pretreatment at high temperatures. Also, HU & Kasha (Hu, T. O, Kasha, K. J., 1997, Plant Cell Reports, 16, pp. 520- 525) report on the improvement of isolated microspore culture of laboratory wheat through ovary co-culture.
  • the present inventor has developed a new and improved culture technique for general use in commercial plant breeding, wherein the period of generation of fertile plants from haploid isolated microspore cultures is shortened by 3-4 years.
  • the microspore cultures are co-cultured with ovaries.
  • the present invention discloses a method which is successful for commercially desirable plant lines, such as wheat.
  • the inventor has addressed the major problems in the culture of haploids, that is, low initial response frequency as determined by embryo-like growth, difficulties in plant regeneration, and difficulties in chromosome doubling to make diploids from haploids, the latter process leading to fertile plants.
  • the inventor has combined environmental stress conditions which divert the microspores from microsporogenesis to embryogenesis, such as combining stress factors comprising culture medium components, such as mannitol with cold pretreatments and oxygen treatment during culturing, to recover surprisingly high yields of embryos and of regenerated fertile plants.
  • environmental stress conditions which divert the microspores from microsporogenesis to embryogenesis, such as combining stress factors comprising culture medium components, such as mannitol with cold pretreatments and oxygen treatment during culturing, to recover surprisingly high yields of embryos and of regenerated fertile plants.
  • the present invention relates to plant breeding and discloses a method of generating fertile whole plants from isolated microspores of wheat, comprising the steps of:
  • This invention relates to a method for the production of plants, and in particular fertile whole plants, from isolated microspores of wheat. Further, the present invention concerns the use of a method for generating fertile whole plants from isolated microspores of wheat to be employed in the commercial plant breeding industry.
  • the scope of the present invention is intended to further include the production of cereal plants, such as maize, sorghum, barley, rye, oat or rice plants.
  • the invention presents a new and improved methodology for the production of plants which comprises subjecting plant compositions containing mi- crospores to a combination of stress factors.
  • the microspores of the invention are co-cultured with ovaries, and the method comprises the steps of:
  • microspore/ovary co-cultures - culturing said microspores in a culturing medium, removing ovaries from a second donor plant, contacting said ovaries with said microspore cultures, - obtaining microspore/ovary co-cultures,
  • a plant composition are obtained.
  • the plant composition may be plant organs, such as spikes which include microspores.
  • Microspores may be cultured to produce fertile plants.
  • elite breeding parent plants are preferred.
  • the term "elite” is defined in a commercial context as the breeding parent plants having the highest yield, good resistance properties and/or good quality properties, such as wheat having excellent baking properties.
  • a preferred method for producing fertile wheat plants from microspore co-cultures will generally include the following steps:
  • the microspores of the cultures are from spikes of a first donor plant.
  • spikes When spikes are employed as the plant donor, it is generally preferred to sterilize their surface. Following surface sterilization of the spikes, for example, with a solution of korsolin, the spikelets A are removed from spikes (small A portions of the spikes) and cut into 2-3 cm pieces and then placed in an isolation medium. In another embodiment the spikes are cut into cut into 2-3 cm pieces and then placed in an isolation medium. The amount of medium used may be dependent on the number of spikes or spikelets.
  • ovaries for the use in co-culturing including wheat are from a second donor plant of the invention.
  • the second donor plant may be selected from a group of plants different from the first donor plant.
  • the second donor plant may be selected from wheat, barley, maize or rice.
  • the ovaries may be selected from spikes when the second donor plant is wheat or barley, or the ovaries may be selected from tassels when the second donor plant is maize.
  • the second donor plant is selected from the same group of plants as the first donor plant.
  • the ovaries and the microspores may all be selected from wheat.
  • the second donor plant of wheat is identical to the first donor plant of wheat.
  • the ovaries may be selected from the same individual donor plant as the microspores.
  • the term "first" and "second" donor plant means the one and same donor plant.
  • the temperature under which the donor plant is growing is between 16-22 °C, preferably between 17-21 °C, more preferably between 18-20 °C. If the donor plant is grown in a greenhouse environment the temperature may be set to a temperature corresponding to the temperature of the season of the year. For example in the month of May a temperature of approximately 20-21 °C is preferred.
  • the length of exposure to light under which the donor plant is growing is between 16-20 hours/24hours, such as between 16.5-19.5 hours/24 hours, for example between 17-19 hours/24 hours, such as 17.5-18.5 hours/24 hours, for exam- pie 17-18 hours/24 hours.
  • light electromagnetic radiation in the wavelength range of 300-2000 nannometres.
  • mercury lamps may be used to mimic daylight and/or radiation having photosynthetic activity (400-700 nm).
  • the protocol of the present invention consists of pretreating a plant composition, for example, organs such as spikes containing microspores, under conditions which divert the microspores from gametophytic development to that of embryogenic development.
  • the pretreatment includes incubation of the plant composition if it includes spikes, at a cold temperature which is a stress factor.
  • microspore-containing plant organs such as spikes may generally be pretreated at any cold temperature below about 10 °C, a range between 1-10 °C is preferred, and a range between 2-7 °C is more preferred, particularly a temperature of between 3-6 °C is preferred. Other temperatures may yield embryos and regen- erated plants, however response rates may be less when pretreatment is outside the preferred temperature ranges.
  • response rate is defined as either the number of embryos or the number of regenerated plants per number of microspores initiated in culture.
  • the exposure of spikes to cold treatment may according to the invention be in a period of between 2-25 days, such as between 6-18 days, for example between 10- 14 days. Further, other pretreatment periods are envisioned within the scope of this invention, as long as the isolated microspores result in regenerated plants.
  • dissected spikes are further pre- treated in an environment that is capable of diverting microspores from their devel- opmental pathway.
  • the function of the preculture medium is to switch the developmental program from one of pollen development to that of embryo development.
  • the spikes are preferably selected at a stage where the microspores are uninucleate, that is, include only one, rather than 2 or 3 nuclei. Methods to determine the correct developmental stage are well known to those skilled in the art.
  • microspores are separated from the donor spikes at the mid to late uni-nucleate developmental stage.
  • the mid to late uni-nucleate microspore stage has been found to be the developmental stage most responsive to the disclosed methods of the invention.
  • the mid to late uni-nucleate stage is the developmental stage of the microspore when the nucleus is situated in the center of the cell before the formation of a vacu- ole.
  • the developmental stage is at the late uni-nucleate stage. During this stage the nucleus is migrating to a posi- tion next to the cell wall at the same time a vacuole is formed. Eventually the nucleus will have migrated to a position opposite the germination pit.
  • an isolation medium is preferred.
  • An isolation medium may help in the separation of the microspores from spike walls, whilst the viability and embryogenic potential of the microspores are maintained.
  • An illustrative embodiment of the isolation medium into which microspores are released from the disrupted spike includes mannitol, and optionally macronutrients.
  • the term "medium” is defined as any combination of nutri- ents that permit the microspores/ovaries to develop into embryos or callus.
  • the isolation medium allows for the blending and filtration of the microspores, and the culture medium is used for growing the microspores/ovaries.
  • suitable embryo/callus promoting media are well known to those skilled in the art. These media will typically comprise mineral salts, carbon sources, vitamins, growth regu- lators and water.
  • microspores may be released from the anthers into isolation medium.
  • One method of release is by disruption of the anthers, for example, by chopping the anthers into pieces with a sharp instrument, such as a razor blade, scalpel or Warring blender.
  • the resulting mixture of released microspores, spike fragments and isolation medium are then passed through a filter in order to separate the microspores from the spike wall fragments.
  • a filter is a mesh, having a mesh of about 150 ⁇ pore size.
  • the filter may consist of a nylon mesh, or of stainless steel.
  • the filtrate which results from filtering the microspore-containing solution is preferably substantially free from spike fragments, cell walls and other debris after centrifugation.
  • the microspore filtrate may be washed several times in isolation medium prior to centrifugation. For best results, washing and subsequent centrifugation is repeated approximately two times.
  • the purpose of the washing and centrifugation is to eliminate any toxic compounds which may be contained in the non-microspore part of the filtrate which are created by the release process.
  • the centrifugation may be done at a spin speed of for example 800 rpm.
  • spikes may be dissected into an isolation medium comprising a sugar alcohol, such as sorbitol, mannitol, in- ositol and/or carbohydrates, such as sucrose.
  • the medium comprises mannitol.
  • the mannitol concentration of the invention may be between 0.31-0.39 M, preferably between 0.32-0.37 M, more preferably between 0.33-0.36 M, such as 0.35 M.
  • the mannitol may be added to the plant organ during cold-treatment.
  • mannitol or other similar carbon structures or environmental stress factors induce starvation of the microspores and functions to force the microspores to focus their energies on entering developmental stages.
  • the cells are unable to use, for example, mannitol as a carbon source at this stage of development. It is believed that these treatments confuse the cells causing them to develop into embryos and plants from microspores instead of developing into gamatophytes.
  • the isolated wheat microspores may then according to the invention be exposed to ovaries from a second donor plant such as wheat.
  • the ovaries may be obtained by hand selecting the ovaries with tweezers transferring them to a dish containing culture medium.
  • the ovaries are selected for culturing on between day 5 and day 0 prior to culturing said microspore cultures.
  • the microspores may be contacted with the ovaries to establish a co-culture on day 1 of culturing the microspores.
  • the induced embryo/callus structures may be sub-cultured into a series of media which are capable of inducing tissue development, for example, roots and shoots.
  • This medium may be designated maturation and regeneration medium.
  • the plant cultures are exposed to oxygen during culturing. It is believed that oxygen supplementation may increase the rate and success of cell growth.
  • the cell cultures of the invention are exposed to oxygen by flushing a stream of oxygen through the cell culture container. This may be achieved by leading a stream of oxygen over the top of the culture medium by hand, or by having the exposure of oxygen automated, such as by placing the cell cultures in boxes or in a climate incubator and then centrally applying oxygen to the cell cultures through a valve in the lid or any other side of the box, or through a valve of any of the sides of the climate incubator.
  • the individual culture containers in the box or climate incubator have their lids on.
  • the individual culture containers in the box or climate incu- bator are without lids or sealing.
  • the lids or sealing may be oxygen permeable.
  • oxygen permeable By supplementing the cell cultures with oxygen a small over-pressure is created, which promotes the exchange of gases in the cultures.
  • the concentration of oxygen in the plant culture medium is dependent on the surface to volume ratio of the medium in the container, such as a petri dish and the concentration in the im- mediate gas phase.
  • the cultures are exposed to oxygen approximately every 2-3 days.
  • the duration of the oxygen exposure according to the invention is between 1-20 minutes, such as between 4-16 minutes, for example between 8-12 minutes. It is preferred that the oxygen is a 100 % oxygen having a pressure of 2 bar.
  • the culture medium of the invention may have a pH value of between 5.4-6.0.
  • the components of the culture medium of the invention essentially comprises components capable of maintaining good microspore/ovary co-culture viability, and is described in detail below.
  • the medium optionally comprises ascor- bic acid.
  • the culture medium of the invention may be added freshly to the cultures.
  • fresh medium is added to the cultures at least once a week.
  • fresh medium is added to the cultures every 6 days, such as every 5 days, for example every 4 days, such as 3 days, for example every 2 days, or such as every day.
  • the length of the intervals between the additions of fresh medium is dependent on the culture conditions, such as temperature, level of oxygen, density of the microspores, and growth rate of the microspores.
  • density is measured as the number of cells per predefined culture area.
  • growth rate is meant how fast the cells divide within a predefined time frame, i.e how "vital" the cells are.
  • Other factors that are important for the intervals in between addition of fresh medium are the size and volume of the culture dish.
  • the amount of fresh medium may be added considering the culture conditions, such as those mentioned above and the amount may differ from addition to addition.
  • the fresh medium is added to the culture dishes in an amount of between 0.1 tol .O ml, such as between 0.2 to 0.9 ml, for example between 0.3 to 0.8, such as between 0.4 to 0.7 ml.
  • the culture medium is a 2-layered medium.
  • a bilayer plate support for the microspores/ovaries during culturing. This allows the cells to be near the surface of the medium.
  • suitable supports are within the scope of the invention, one such support is gellan gum (Gel-Rite®).
  • the bilayer support for development of the microspore/ovary cultures consist of a 2-layer medium, wherein the medium is on top of a solid base. Culturing isolated microspores/ovaries on a solid support prevents microspores/ovaries from sinking into the medium with oxygen tension and possibly death as a consequence.
  • the solid support is a nylon mesh in the shape of a raft.
  • a "raft" is defined as a support material which is capable of floating above the bottom of a tissue culture vessel, such as a petri dish.
  • the solid support is a stainless steal mesh.
  • the solid medium allows the embryos to mature. The medium passes through the mesh while the microspores are retained at the medium-air interface. The surface tension of the liquid medium in the petri dish causes the raft to float. The medium is able to pass through the mesh and as a consequence the microspores/ovaries stay on top. The mesh raft will remain on top of the total volume of medium.
  • An advantage of the mesh raft is to permit diffusion of nutrients to the microspores/ovaries.
  • the use of a raft also permits transfer of the microspores/ovaries from dish to dish during sub-culturing.
  • the solid support is the basal part of the culture vessel (solid layer), where upon a liquid layer is situated.
  • a raft cell support enables the transferral of the raft with its associated microspores/ovaries to various media containing desired ingredients, such as subculture media, without substantial loss, disruption or disturbance of the developing embryos.
  • a liquid or semi-solid based medium which promotes embryo maturation (regeneration medium). It is desirable to obtain high quality embryos, such as embryos exhibiting organized developmental structures, for example shoots and meristems. Embryos are aggregates of multi-cellular structures each generally arising during a period of approximately 3 to 4 weeks. The embryos are preferred for subsequent steps to regenerating plants.
  • intact fertile plants may then be regenerated in a regeneration medium.
  • a regeneration medium During the regeneration process individual embryos are induced to form plantlets. It is within the scope of the invention to use a sequence of regeneration media for whole plant formation from the embryos. The number of different media in the sequence may vary depending on the specific protocol used.
  • a rooting medium may be used prior to transplanting the plantlets into soil.
  • a haploid embryonic cell culture is obtained and fur- ther the plant embryos obtained are haploid.
  • One method of chromosome doubling is by using chromosome doubling agents.
  • a widely applied chromosome doubling agent is colchicine, which disrupts the mitotic spindle during cell proliferation, and thus prevents the cell from dividing.
  • the chromosome doubling agents may be added to the isolation medium, whereby di- haploid cell cultures are obtained.
  • the chromosome doubling agent may be added to plantlets.
  • This may be performed by rinsing the soil off the roots and the placing the plantlets in a container, wherein the roots are covered with the chromosome doubling agent.
  • the plantlets are placed in a container of approx. 0.05 % colchicine and 1 % DMSO, and left for approx. 13-20 hours.
  • the plantlets when they reach a height of about 5-10 cm they are transferred to containers, i.e. pots for further growth into flowering plants for example in a greenhouse by methods well known to the skilled artisan.
  • the ratio of fertile regenerated wheat plants per embryo is at least 7:10, such as 8:10, for example 9:10.
  • the fertile regenerated whole plants are capable of producing seeds and progeny. These seeds and progeny are also within the scope of this invention.
  • the seeds from the regenerated plants are prepared.
  • An object of the invention is to obtain a plant regenerated from the embryonic cell culture as described above.
  • the plant may be haploid and in another embodiment the plant is dihaploid.
  • the invention relates to producing progeny of the regenerated plants, and therefore encompass seeds obtained from the regenerated plants, either by crossing or selfing.
  • the plant of the invention is transgenic.
  • the method of the present invention may shorten the time required for improvement of crops by integrating new genetic traits.
  • the method of regenerating plants from or microspore/ovary co-cultures may facilitate genetic manipulations directed at plant improvement.
  • nucleic acid segments may be introduced into microspores for integration in the chromosomes and expression on a whole plant level. Accordingly, it is within the scope of the present invention to transform plants, whereby transgenic plants are produced for example by the incorporation of large segments of DNA or artificial chromosomes using standard commercially available techniques.
  • microspores and ovaries from wheat are co-cultured.
  • the spike has just started to emerge. Leaves are cut off. The spikes are placed in container of 1.25 mM Hydroxyurea and then placed in a re- fridgerator. The spikes are moved to water the following morning (18 hours in hy- droxyurea).
  • the spikes are rinsed of leaves and are collected in bundles of 5 spikes per bundle. 3 bundles are collected to sterilise 15 spikes at a time. They are rinsed for a few seconds in 70% ethanol with a drop of tween added. Thereafter the spikes are placed in a container with 2.5 % korsolin while stirring for 12 min. New fresh korsolin every day. Rinse in steril water 4 x and place in an empty steril container.
  • Spikelets from 5 spikes are removed from the spikes by twisting with tweezers so that the ovaries stay on the spikes. Spikelets are placed in a steril 100 ml beaker or 9 cm petri dishes, and the ovaries are transferred to 3.5 cm petri dishes with approx. 5 ml culture medium and 20 ovaries per dish.
  • the blender is a Warring with MC3 top.
  • the stopper is put in a glas container with 96% ethanol for at least a couple of minuttes.
  • the blending is with 96 % ethanol, 2 x 60 seconds. At high speed.
  • Spikelets are blended for 2 x 3 sec. in 0.37 M mannitol. Mannitol is placed in the blender so that the bottom third of the top knives are covered. The blended sub- stance is lead through a coarse filter into a 100 ml beaker. The filtrate is transferred to 3 x 15 ml centrifuge tubes. The remains on the filter is reentered into the blender with a spoon. Mannitol is added to the blender so that the liquid reaches the top knives. Blending for 2 x 3 sec. The blended substance is lead through the same filter and the filtrate is transferred to 3 centrifuge tubes and an additional 2 centrifuge tubes. Accordingly, the filtrates from the 2 blendings are placed in 5 centrifuge tubes all together.
  • Centrifugation is carried out for 3 min at 800 rpm. The supernatant is discarded.
  • Pellets from the 5 centrifuge tubes are collected in a 15 ml centrifuge tubes with a pipette, and the centrifuge tube is filled with mannitol.
  • Centrifugation is carried out for 3 min at 800 rpm. The supernatant is discarded.
  • Pellet is resuspended in 12 ml CHB-2 medium, which is transferred from the petri dishes containing the ovaries with a disposable pipette.
  • the microspores are spread out evenly in the medium with the pipette.
  • 12 ml microspore suspension are distributed with 4 ml in each petri dish with the ovaries.
  • the dished are sealed with para- film.
  • the resuspension is in 6 ml CHB-2 medium divided by 2 ml per dish.
  • the dishes are placed in plastic boxes with valves in darkness at 28 °C. 100 % oxygen (2 bar) is added every 3 day for 5 minutes.
  • the embryos are transferred to the differentiation medium: The ovaries are removed with tweezers, and the medium is sucked up with a pipette and the embryos are transferred to containers with a spoon or spatula.

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  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

La présente invention trouve des applications dans le domaine de la sélection végétale. Elle concerne un procédé permettant de produire des végétaux fertiles entiers à partir de microspores isolés de blé. Plus spécifiquement, l'invention se rapporte à un procédé permettant de produire des plants de blé à partir de microspores cultivés conjointement avec des ovaires. Sous d'autres aspects, l'invention concerne des végétaux modifiés, et des graines ainsi que la descendance des végétaux modifiés dérivés des microspores/ovaires cultivés. Cette invention concerne également l'utilisation d'un procédé permettant de produire des végétaux fertiles entiers à partir de microspores isolés de blé.
PCT/DK2001/000456 2000-06-30 2001-06-29 Procede permettant de produire des vegetaux fertiles a partir de microspores isoles WO2002001940A2 (fr)

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CN103210843A (zh) * 2013-04-09 2013-07-24 南京农业大学 一种高频的鹅观草幼胚愈伤组织诱导和再生的培养方法
CN113950978A (zh) * 2021-11-26 2022-01-21 潍坊市农业科学院(山东省农业科学院潍坊市分院) 一种提高萝卜种质创制效率的方法
CN113950977A (zh) * 2021-11-26 2022-01-21 潍坊市农业科学院(山东省农业科学院潍坊市分院) 一种提高大白菜种质创制效率的方法

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WO2000014202A1 (fr) * 1998-09-09 2000-03-16 Northwest Plant Breeding Company Procedes servant a generer des plantes haploides doublees
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CN102599059A (zh) * 2012-03-28 2012-07-25 中国农业科学院作物科学研究所 一种提高低再生力小麦基因型幼胚组织培养再生率的方法
CN103210843A (zh) * 2013-04-09 2013-07-24 南京农业大学 一种高频的鹅观草幼胚愈伤组织诱导和再生的培养方法
CN113950978A (zh) * 2021-11-26 2022-01-21 潍坊市农业科学院(山东省农业科学院潍坊市分院) 一种提高萝卜种质创制效率的方法
CN113950977A (zh) * 2021-11-26 2022-01-21 潍坊市农业科学院(山东省农业科学院潍坊市分院) 一种提高大白菜种质创制效率的方法
CN113950978B (zh) * 2021-11-26 2022-10-04 潍坊市农业科学院(山东省农业科学院潍坊市分院) 一种提高萝卜种质创制效率的方法

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