WO2001001761A1 - Procede de regeneration d'une plante appartenant au genre lilium - Google Patents

Procede de regeneration d'une plante appartenant au genre lilium Download PDF

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Publication number
WO2001001761A1
WO2001001761A1 PCT/JP2000/004297 JP0004297W WO0101761A1 WO 2001001761 A1 WO2001001761 A1 WO 2001001761A1 JP 0004297 W JP0004297 W JP 0004297W WO 0101761 A1 WO0101761 A1 WO 0101761A1
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Prior art keywords
culture
protoplast
nurse
callus
regenerating
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PCT/JP2000/004297
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English (en)
Japanese (ja)
Inventor
Noriko Tabayashi
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Sankyo Company, Limited
Hokkai Sankyo Co. Ltd.
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Publication date
Application filed by Sankyo Company, Limited, Hokkai Sankyo Co. Ltd. filed Critical Sankyo Company, Limited
Priority to AU57059/00A priority Critical patent/AU5705900A/en
Publication of WO2001001761A1 publication Critical patent/WO2001001761A1/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
    • A01H4/008—Methods for regeneration to complete plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells

Definitions

  • the present invention relates to a method for regenerating a plant of the genus Juli, which comprises subjecting a protoplast obtained by dedifferentiation of a plant of the genus Juli to nurse culture and then redifferentiating the plant.
  • the present inventors have conducted intensive studies on the dedifferentiation method, protoplast culture method, and individual plant regeneration method of lily plants, and as a result, the protoplast colonies obtained by nurse culture of aprotoplasts embedded in agarose were used.
  • the present invention was completed by regenerating the individual.
  • the present invention provides a universal individual regeneration method for lily plants, comprising subjecting a protoplast obtained by dedifferentiation of a lily plant to nurse culture and then redifferentiating the protoplasts. It is an object.
  • the present invention is a.
  • the nurse cells used in the nurse culture are cells obtained by subculturing callus derived from shoot apex culture in a liquid medium and cells having an active dividing ability.
  • the solid medium in which the protoplasts are embedded is an agarose-containing medium.
  • Protoplast is a fusion protoplast obtained by cell fusion
  • a wild species of a lily plant is referred to as an original species.
  • the original species of the lily genus plant include a lily of the valley and a wild mushroom.
  • a species that has been crossed and raised based on the original species is referred to as a cultivated species.
  • cultivated species of the lily genus plant include Oriental 'hybrid type (scientific name: Lilium speciorybel), and Asiatic' hybrid type (Lilium Xelegans).
  • a species is called a strain.
  • the heterogeneous line of the genus Lily means a heterologous species of the genus Lily.
  • a horticultural variety is simply referred to as a variety.
  • the method for regenerating a lily plant includes: [i] induction of callus by dedifferentiation of a lily plant tissue, [ii] purification of protoplasts from the callus culture,
  • the tissue of the Curie plant is not particularly limited as long as it is a tissue usually used for callus induction, and examples thereof include a shoot apex, a scallop, and a flower vase. is there.
  • the separated tissue is placed on a medium containing 1 mg / m 1 or more of a hormone having a dedifferentiating effect, and cultured under ⁇ conditions to induce callus and proliferate.
  • Culture temperature The lower limit is 18 to 23 ° (:, the upper limit is 26 to 28 ° C, and the preferable range is 23 to 26 ° C.
  • the plant hormone having a dedifferentiating action is not particularly limited as long as it can dedifferentiate a plant of the genus Juli, and is preferably an auxin-based compound.
  • the auxin-based compound is not particularly limited as long as it is commonly used, and examples thereof include Picloram.
  • the medium used for callus growth is not particularly limited as long as it is a medium usually used for callus culture.
  • MS medium Murashige & Skoog Medium
  • Reference Mrashige
  • T. & Skoog F., Physiol. Plant., 15, 473-497 (1962)
  • Linsmeier-Stag medium Linsmaier
  • the properties of the medium may be either liquid or solid.
  • the lower limit of the shaking speed is 90 to 100 revolutions per minute (hereinafter referred to as "rp mj"), and the upper limit is 110 to 120 rpm. 0 to 110 rpm
  • the solid medium is prepared by adding a solidifying agent to the liquid medium, and is not particularly limited as long as it is one that is usually used for solidifying the medium. For example, Rinsu Loin, Gelam gum and the like can be mentioned.
  • the callus grown in [i] is immersed in a cell wall degrading enzyme solution, and incubated at room temperature for 2 to 16 hours to dissolve the cell wall and connective tissue in the callus.
  • the cell wall degrading enzyme is not particularly limited as long as it is usually used for decomposing the cell wall of a plant, and examples thereof include cellulase and pectinase.
  • the decomposition enzyme reaction is performed in a buffer solution containing an inorganic salt.
  • the inorganic salt to be added to the buffer is not particularly limited as long as it is essential for the degrading enzyme reaction or has a favorable effect on the degrading enzyme reaction. Examples thereof include 10 mM calcium chloride. Is mentioned.
  • the decomposing enzyme reaction is performed in an isotonic solution.
  • an isotonic solution For example, 0.6 M sorbitol or the like is added to the buffer solution. Is added.
  • the reaction product is filtered with a nylon mesh to remove undigested substances and the like.
  • the collected filtrate is subjected to fractionation by density gradient centrifugation to purify protoplasts.
  • - Reagents used for density gradient centrifugation are those generally used in the method and are used for protoplasts. There is no particular limitation as long as it does not impart tress, and examples thereof include saccharides and synthetic colloids. As saccharides, sucrose and dextran are preferable, and as synthetic colloid, percoll (Percoll: manufactured by Pharmacia: polyvinyl pyrrolide conjugate of silicic acid) is preferable.
  • the protoplast fraction obtained by the density gradient centrifugation has few contaminants such as a solid culture medium for callus culture, has a dense cytoplasm, and has high division activity.
  • the protoplast fraction is embedded in a solid medium.
  • the basic medium is not particularly limited as long as it is a liquid medium usually used for culturing protoplasts, and examples include an IS medium.
  • the protoplast fraction obtained in [ii] is suspended in the above liquid medium, an equal amount of a solution obtained by dissolving a solidifying agent in the liquid medium is added, mixed, poured into a petri dish, and left at room temperature to solidify.
  • the solidifying agent is not particularly limited as long as it is generally used for solidifying a medium, and examples thereof include agarose and gelangam, and agarose is preferred.
  • nurse culture refers to culturing a protoplast derived from a lily plant embedded in a gel in a liquid medium in the presence of nurse cells.
  • a nurse cell is a cultured cell derived from a plant of the genus Uri that is actively dividing. Nurse cells can be obtained by inducing calli by dedifferentiating lily plant tissues and culturing the callus. Induction of callus and its culture are described in [i] Repeat in the same way. The cultured cells are subcultured every 14 days. The culture rate of the cultured cells becomes stable 4 to 6 months after the callus induction. Cultured cells with a stable growth rate are removed 10 to 12 days after the last subculture and used immediately for nurse culture.
  • Nurse culture of gel with embedded protoplasts Divide the gel with embedded protoplasts into several equal parts and immerse them in liquid medium. Add vigorous growing nurse cells to the liquid medium in which the gel is soaked, and culture under shaking conditions. The range of the shaking speed is a lower limit of 10 to 20 rpm, an upper limit of 30 to 50 rpm, and a preferable range is 20 to 30 rpm. The lower limit of the culture temperature is 18 to 23 ° C. and the upper limit is 26 to 28 ° C. The preferred range is 23 to 26 ° C.
  • a preferred medium for nurse culture is a liquid medium used for embedding protoplasts, and more preferably an osmotic pressure regulator is added.
  • the osmotic pressure regulator is not particularly limited as long as it is commonly used, and examples thereof include glucose having a final concentration of 0.5 M.
  • the first division starts 1 to 2 weeks after the start of nurse culture, and a colony with a cell number of 10 or more is formed about 3 weeks later. Nurse culture is continued while changing the medium periodically until the diameter of the colony becomes 100 ⁇ m or more. The medium is preferably changed about once a week.
  • the protoplasts to be used for nurse culture may be fusion protoplasts obtained by cell fusion.
  • the cell fusion method is not particularly limited as long as it is a known method. For example, an electric pulse method (see Akagi, H., et al., Mol. Ge. Genet, 325, 501-506 (1989)) And the like.
  • the fusion protoplast may be a fusion protoplast obtained by cell fusion between different strains of the genus Lily.
  • the cell fusion method and the individual regeneration method of the present invention it is possible to cross between different strains of the genus Juli, which could not be crossed by the conventional method.
  • examples of cell fusion between different strains by combining cultivated species and cultivated species include, for example, a combination of an oriental hybrid type and an Asiantic / hybrid type.
  • the colonies obtained in [iii] are placed on a growth medium to form calli.
  • the lower limit of the culture temperature is 18 to 23 ° C
  • the upper limit is 26 to 28 ° C
  • the preferable range is 23 to 26 ° C.
  • the regeneration medium is not particularly limited as long as it is a normal medium that does not contain plant hormones, and examples thereof include an MS medium.
  • Example 1 Protoplast culture and plant regeneration of lily cultivars
  • Stain was collected from a sterile plant of Oriental hybrid (cultivar: Hakumyo), which is a lily cultivar.
  • Picloram (Picloram: manufactured by Ridel der De Heen) lmg / L and gellan gum (Gellan) Gum: Kanto Chemical Co., Ltd.) Callus was induced by placing the plate on an MS medium containing 2 g / L.
  • the resulting callus was cultured at 24 ° C. in an MS liquid medium containing 1 mg of Picloram (Riddel 'D') under shaking at 110 rpm. It was replanted weekly. Approximately 4 months after the start of the culture, calli with stable growth were used as protoplast preparation materials.
  • MS culture medium containing modified protoplasts as described above modified MS medium (206. SSmg ZL ammonium nitrate, lmg ZL picloram (Picloram: Ridel der der Hahn), 60 g / L glucose) ).
  • modified MS medium 206. SSmg ZL ammonium nitrate, lmg ZL picloram (Picloram: Ridel der der Hahn), 60 g / L glucose
  • modified MS medium containing 2.5% agarose
  • the solidified gel was divided into eight equal parts in a fan shape, immersed in 5 ml of a modified MS medium in a plastic dish having a diameter of 60 mm, added with 100 mg of the following cultured cells, and rotated at 30 rpm. Nurse culture was performed under shaking at 25 C with shaking. The medium was changed every two weeks.
  • Nurse cells used for nurse culture are derived from Oriental 'Hipride (variety: Red' Ruby '), which is a cultivar used for nursery. Calli induced from the shoot apex of the cultivar by the method described above were cultured in MS medium at 24 ° C. for 16 hours while shaking at 110 rpm. Subculture was carried out every 14 days, and after 5 to 6 months, the growth rate became stable. After 10 to 12 days from the last subculture, the culture was taken out and used immediately for nurse culture.
  • Oriental 'Hipride variety: Red' Ruby '
  • the first division started 1 to 2 weeks after the start of the nurse culture, and a colony having a cell number of 10 to 20 or more was formed about 1 month later. After about 2 months, it grows into callus with a diameter of 1 to 3 mm, and this is converted to MS solids containing 1 mg / L picloram (Picloram: Riedel de Heen) and 60 g / L glucose.
  • the cells were subcultured, cultured for 24 hours at a temperature of 24 ° C. for 16 hours, and grown to a callus having a diameter of about 5 mm.
  • the grown calli are transferred to a regeneration medium (MS medium containing 3% sucrose and no plant hormones), cultured at 24 ° C for 16 hours with a photoperiod of 1 to 3 months to regenerate the plant did.
  • the resulting callus was cultured in an MS liquid medium containing Picloram (Ricdel 'de' Hahn) lm gZL at 24 ° C ⁇ with shaking at 110 rpm. It was replanted weekly. Approximately 4 months after the start of the culture, calli with stable growth were used as protoplast preparation materials.
  • Cellulase Onozuka RS manufactured by Yakult Honsha
  • Maserozym R—10 manufactured by Yakult Honsha
  • 0.05% Cellulose Y—23 A solution consisting of 10 mM calcium chloride, 5 mM MES, and 0.6 M sorbitol (hereinafter referred to as “enzyme solution”) was adjusted to ⁇ ⁇ 5.8 and the pore size was adjusted to 0.
  • the solution was sterilized by filtration through a 45 / zm filter (Sterlie Acrodisc: manufactured by Germanic).
  • the protoplasts purified as described above were placed in a modified MS medium (206. SS mg / L ammonium nitrate, lmg / L picloram (Picloram: MS medium containing 60 g ZL glucose). Suspended. 0.5 ml of the suspension was mixed with an equal volume of a modified MS medium containing 2.5% agarose, and solidified by pouring into a plastic dish having a diameter of 35 mm. The gel was divided into eight equal parts in a fan shape, immersed in 5 ml of a modified MS medium placed in a 60 mm diameter plastic dish, and 100 mg of the cultured cells was added, and nurse culture was performed under a dark condition of 25 ° C. while shaking at 30 rpm. The medium was changed every two weeks.
  • the nurse cells used for nurse culture are derived from Oriental 'hybrid (cultivar: Red Ruby), which is a cultivar used for nursery. Calli induced from the shoot apex of the cultivar by the method described above were cultured in MS medium at 24 ° C. for 16 hours while shaking at 110 rpm. Subculture was carried out every 14 days, and after 5 to 6 months the growth rate was stabilized, the culture on the 10th to 12th day from the last subculture was taken out and used immediately for nurse culture.
  • the first division started 10 to 14 days after the start of the nurse culture, and 4 to 6 weeks later, colonies having 10 to 20 or more cells were formed. After about 2 months, they grow into calli of 1-3 mm in diameter, which are then transferred to MS solid medium containing 1 mg ZL picloram (Ricdel de Heen) and 60 g ZL glucose. The cells were cultured for 16 hours at 24 ° C for 16 hours, and grown to a callus having a diameter of about 5 mm.
  • the grown calli were subcultured on a regeneration medium (MS medium containing 3% sucrose and no plant hormone), cultured for 24 to 16 hours with a photoperiod of 1 to 3 months, and the plants were regenerated. .
  • a regeneration medium MS medium containing 3% sucrose and no plant hormone
  • Spices were collected from lily cultivars, Asiatic hybrid (variety: Mona) and Orienta Nore 'hybrid (variety: Casablanca), and Picloram (Picloram: Ridel del de Callus was induced by placing on an MS medium containing 1 mg ZL and 2 g L of dielan gum (Ge 11 an Gum : manufactured by Kanto Chemical Co., Ltd.).
  • the resulting callus picloram shaking at MS liquid medium containing (picloram Riedel 'de' ⁇ emissions Co.) lmgZ L at 1 1 0 rpm and cultured in 2 4 a C dark conditions, every 2 weeks Planted. Calli with stable growth were used as protoplast preparation materials.
  • the callus on the 12th day after the subculture was collected, suspended in an enzyme solution, and subjected to an enzyme treatment at 25 ° C for 3 hours.
  • the mixture was filtered through a cell sorting mesh (manufactured by Kyojin Riko Co., Ltd.), and a 2-fold volume solution of 10 mM calcium chloride, 5 mM MES, 0.6 M sorbitol was added to the filtrate, and mixed. Centrifugation was performed at 100 XG for 3 minutes. The protoplast fraction that settled at the bottom of the centrifuge tube was collected. This fraction was washed twice with 0.6 M sorbitol.
  • IOA iodoacetamide
  • Cell fusion was performed by the electric pulse fusion method (Akagi, H., et al., Mol. Gen. Genet., 215, 501-506, (1989)), a cell fusion device EFS-100 (IWAKI CORPORATION). Manufactured).
  • a buffer for cell fusion (10 mM calcium chloride, 5 mM MES, 0.6 M sorbitol), and high frequency (intensity: 400 Vp-p / cm
  • An electric field applied once, applied for 50 seconds was applied to bring the protoplasts into a continuous chain.
  • a pulse (voltage 1.5 to 2.5 KVZcm, width 50 / z sec) was applied to both ends of the cell chain under microscopic observation, and a hole was formed where the cell membranes were in contact. Opened and cells were fused. After confirming the fusion of the protoplasts under microscopic observation, the suspension was centrifuged at 100 ⁇ g for 5 minutes, and the protoplast fraction was recovered in the precipitate.
  • Method for selecting fusion protoplasts of different varieties The protoplasts derived from Casablanca, which had retained the fission activity, were treated with IOA according to the above-mentioned method to lose the fission ability. On the other hand, protoplasts derived from mona did not retain division activity.
  • the fusion protoplasts purified as described above were modified with an MS medium (206.25 mg / L ammonium nitrate, lmgZL picloram (Picloram: Ridel der der), and 60 g / L glucose. Containing MS medium).
  • MS medium 206.25 mg / L ammonium nitrate, lmgZL picloram (Picloram: Ridel der der), and 60 g / L glucose. Containing MS medium.
  • 0.5 ml of the suspension was mixed with an equal volume of a modified MS medium containing 2.5% agarose, and solidified by pouring into a 35 mm-diameter plastic petri dish.
  • the solidified gel is divided into eight equal parts in a fan shape, immersed in 5 ml of modified MS medium in a plastic dish with a diameter of 6 mm, added with 100 mg of the following cultured cells, and shaken at 30 rpm. Nurse culture was performed under 25 ° C conditions. The medium was
  • Nurse cells used for nurse culture are derived from Oriental 'hybrid (cultivar: red, ruby), which is a cultivar of lily. Calli induced from the shoot apex of the cultivar by the method described above were cultured in MS medium at 24 ° C. for 16 hours while shaking at 110 rpm. Subculture was carried out every 14 days, and after 5 to 6 months the growth rate was stabilized, the culture on the 10th to 12th day from the last subculture was taken out and used immediately for nurse culture.
  • Oriental 'hybrid cultivar: red, ruby
  • the callus grown to a diameter of 1 to 3 mm was subcultured on an MS solid medium containing 1 mg ZL picloram (Picloram: Riedel-Den) and 60 gZL glucose, and allowed to grow for 24 hours at 24 C for 16 hours.
  • the cells were cultured and grown to calli about 5 mm in diameter.
  • the grown calli are transferred to a regeneration medium (MS medium containing 3% sucrose and no phytohormone), and cultured at 24 ° C for 16 hours with a photoperiod of 1 to 3 months to regenerate the plant did.
  • a regeneration medium MS medium containing 3% sucrose and no phytohormone
  • Example 1 The method described in Example 1 was used for Oriental Hybrid (cultivar: Casablanca, Shiromyo), Asiatic Hybrid (cultivar: Mona), which is a lily cultivar, and Sasuri, which is a native lily species. Then, callus was induced from each scaly piece, and protoplasts were purified and cultured.
  • Each of the obtained protoplasts is referred to as (1) conventional dielan gum embedding method (hereinafter, referred to as “conventional method”) and (2) combination method of agarose embedding and nurse culture of the present invention (hereinafter, “present invention method”). ) of cultured in two ways, c and compared their efficiency 1 literature (Godo, T., et al ., Plant Cell Report, 15,401-404 (1996) in), 2 the actual Example 1 was followed.
  • conventional method conventional dielan gum embedding method
  • present invention method combination method of agarose embedding and nurse culture of the present invention
  • the method of the present invention shows a variation according to the experimental number compared to the conventional method, but the splitting rate is about 2 to 8 times higher, and the agar is more aggressive than the conventional method by embedding dielan gum.
  • the superiority of the method of the present invention by oral embedding and nurse culture was demonstrated.
  • the method for regenerating lily plants of the present invention can be widely applied to both original species and cultivated species, and is useful as a universal individual regenerating method for lily plants.
  • the method of the present invention for regenerating a plant of the genus Lily can be applied to a fusion port toplast obtained by a cell fusion method, the method differs from that obtained by a conventional crossing method. Crossing between strains is also possible.

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Abstract

Cette invention concerne un procédé de régénération d'une plante du genre Lilium qui comprend les opérations suivantes : (1) obtention d'un cal par dé-différentiation d'un tissu de plante appartenant au genre Lilium); (ii) purification d'un protoplaste à partir de la culture du cal susmentionné ; (iii) culture du protoplaste ainsi purifié ; (iv)obtention d'un cal à partir d'une colonie du protoplaste obtenu par culture ; et (v) re-différentiation de la culture du cal. Ce procédé permet de régénérer efficacement un individu à partir d'un protoplaste de plante appartenant au genre Lilium de sujets de souche ou d'une espèce cultivée. Ce procédé de régénération peut également s'appliquer à un protoplaste hybridé obtenu par fusion cellulaire.
PCT/JP2000/004297 1999-07-01 2000-06-29 Procede de regeneration d'une plante appartenant au genre lilium WO2001001761A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57059/00A AU5705900A (en) 1999-07-01 2000-06-29 Method of regenerating individual of plant belonging to the genus lilium

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JP11/187806 1999-07-01
JP18780699 1999-07-01

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WO2001001761A1 true WO2001001761A1 (fr) 2001-01-11

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102696483A (zh) * 2012-06-19 2012-10-03 西安文理学院 绿花百合快速繁殖方法
CN102907313A (zh) * 2012-11-03 2013-02-06 云南省农业科学院花卉研究所 一种构建百合杂交后代无性系群体的方法
CN108949665A (zh) * 2018-07-12 2018-12-07 北京林业大学 百合花瓣原生质体的制备方法
CN114303956A (zh) * 2022-03-02 2022-04-12 鲁东大学 一种重瓣百合离体快繁育苗方法

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JPS62232382A (ja) * 1986-04-02 1987-10-12 Mitsubishi Corp イネプロトプラストの培養方法
JPH02222626A (ja) * 1989-02-27 1990-09-05 Pias Arise Kk タマネギとニンニクの雑種植物の生産方法

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
JPS62232382A (ja) * 1986-04-02 1987-10-12 Mitsubishi Corp イネプロトプラストの培養方法
JPH02222626A (ja) * 1989-02-27 1990-09-05 Pias Arise Kk タマネギとニンニクの雑種植物の生産方法

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"Supplement/Engei shokubutsu no kikan to soshiki no baiyou", SEIBUNDOU SHINKOUSHA, 28 February 1985 (1985-02-28), pages 54 - 75, XP002932684 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102696483A (zh) * 2012-06-19 2012-10-03 西安文理学院 绿花百合快速繁殖方法
CN102907313A (zh) * 2012-11-03 2013-02-06 云南省农业科学院花卉研究所 一种构建百合杂交后代无性系群体的方法
CN108949665A (zh) * 2018-07-12 2018-12-07 北京林业大学 百合花瓣原生质体的制备方法
CN114303956A (zh) * 2022-03-02 2022-04-12 鲁东大学 一种重瓣百合离体快繁育苗方法

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