CN115868412A - Davidia involucrata somatic embryogenesis regeneration plant culture method - Google Patents
Davidia involucrata somatic embryogenesis regeneration plant culture method Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention provides a method for culturing a davidia involucrata somatic embryogenesis regeneration plant, which comprises the following steps: (1) explant selection and disinfection: selecting an immature zygotic embryo in the optimal period of Davidia involucrata as an explant, and sterilizing the explant by using a disinfectant to obtain a material for inducing embryogenic callus; (2) embryonic callus induction culture: placing the explant treated in the step (1) in an embryonic callus induction culture medium for culture to obtain an embryonic callus; and (3) somatic embryo development induction culture: placing the embryogenic callus processed in the step (2) in a somatic embryo development induction culture medium for culturing to obtain a mature somatic embryo; (4) germinating the somatic embryo and culturing the transformed plant: and (4) placing the mature somatic embryo obtained by culturing in the step (3) into a germination culture medium for culturing, placing the mature somatic embryo into a transformation plant culture medium for culturing after the culture is finished, and obtaining a davidia involucrata regeneration plant after the culture is finished. The method for regenerating plants by somatic embryogenesis has the characteristics of high embryogenic callus induction rate, good somatic embryo induction effect, and strong growth of the induced somatic embryos which can normally germinate into complete plants.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a method for culturing a regenerated plant by utilizing a somatic embryogenesis way and a Davidia involucrata plant obtained by culturing the regenerated plant.
Background
Chinese dive tree (Davidia involucrataBaill.) is a deciduous tree species native to the rare or endangered species in china, the first national focus being on protecting wild plants. Because of its special white bract and beautiful tree posture, it is called world famous ornamental species. As an ancient wriggling tree species surviving in the fourth glacier movement period, the Davidia involucrata has extremely strong scientific research value in the aspects of ancient botany and ancient geography. In addition, the davidia involucrata is heavy in wood material and is a first-class material for building and manufacturing furniture. In recent years, researches show that the natural compound in the fruit and leaf of Davidia involucrata has pharmacological effects of resisting tumor, cancer and the like. Therefore, it is considered as a valuable biological resource integrating ornamental value, scientific research value, economic value and medicinal value. Davidia involucrata is usually propagated by seeds, the seeds are hard and can germinate in 2~3 years under natural conditions, and the seeds have serious abortion and low fruiting rate, so that the expansion of field population and garden application are severely restricted. The method for breeding the Chinese dive tree efficiently is especially important to be researched.
Somatic embryogenesis refers to a tissue culture rapid propagation technology for generating zygotic embryo development-like somatic cells without gamete fusion, and can be used as a substitute propagation method when conventional seed propagation is difficult. In the last decades, the regeneration of plants by somatic embryogenesis has been successful in many woody plant species, and this technology can be used not only to save endangered species from extinction, but also to preserve the genetic resources of the plants. If the Davidia involucrata somatic embryogenesis technology can be applied to industrial seedling raising, the breeding process of the Davidia involucrata is accelerated, the large-scale production of the endangered plant Davidia involucrata can be realized, and the resource and population expansion of rare species can be protected.
Disclosure of Invention
The invention aims to provide a method for culturing a davidia involucrata somatic embryogenic regeneration plant, which has the characteristics of high propagation coefficient and short period.
The specific scheme is as follows:
a method for culturing Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo in the optimal period of Davidia involucrata as an explant, and sterilizing the explant by using a disinfectant to obtain a material for inducing embryogenic callus;
(2) Embryogenic callus induction culture
Removing endosperm of the explant material treated in the step (1), and placing the immature zygotic embryo in an embryogenic callus induction culture medium for culture to obtain an embryogenic callus;
(3) Induced culture of somatic embryo development
Placing the embryogenic callus processed in the step (2) in a somatic embryo development induction culture medium for culturing to obtain a mature somatic embryo;
(4) Somatic embryo germination and transformed plant culture
And (3) placing the mature somatic embryo obtained by culturing in the step (3) into a germination culture medium for culturing, placing the mature somatic embryo into a transformation plant culture medium for culturing after the culturing is finished, and obtaining a Davidia involucrata regenerated plant after the culturing is finished. The somatic embryogenesis regeneration plant method has the characteristics of high embryogenic callus induction rate, good somatic embryo induction effect, capability of leading the induced somatic embryo to normally germinate into a complete plant and grow robustly and the like.
In a specific embodiment of the invention, (1) the explant is selected from Chinese dive tree 10 to 11 weeks after flower blooming,
i.e., immature zygotic embryos at the early cotyledon stage.
In a specific embodiment of the invention, (1) the disinfection treatment comprises multi-stage disinfection, namely soaking the standby explant in washing powder for 15-30 min, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in a clean bench, taking out immature zygotic embryo wrapped by endosperm, placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80, soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use.
In one embodiment of the present invention, (2) the induction medium is an MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5 to 2.0mg/L and 0.2 to 2.0mg/L, respectively.
In the specific embodiment of the present invention, (2), the culture temperature for induction culture is 25 ± 2 ℃, and dark culture is performed.
In a specific embodiment of the present invention, (3) the induction medium is an MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, NAA, and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, NAA, and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5 to 2.0mg/L, and 0.2 to 2.0mg/L, respectively.
In the specific embodiment of the present invention, (3), the culture temperature for induction culture is 25 ± 2 ℃, and dark culture is performed.
In a specific embodiment of the invention, (4) the germination medium is a 1/2MS medium containing sucrose, plant gel, 6-BA and IBA, wherein the concentrations of the sucrose, the plant gel, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.25mg/L;
the somatic embryo transformation plant culture medium is a 1/2MS culture medium containing sucrose, plant gel, activated carbon, 2,4-D and 6-BA, wherein the concentrations of the sucrose, the plant gel, the activated carbon, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1g/L, 0.5mg/L and 0.25mg/L.
In the specific embodiment of the invention, (4), the culture temperature of germination culture is 25 +/-2 ℃, and the illumination intensity is 40 to 50 umol.m -2 ·s -1 The total illumination time per day is 14 to 169h.
The invention also provides a davidia involucrata regenerated plant which is prepared by adopting the cultivation method of the davidia involucrata somatic embryogenesis regenerated plant.
The MS culture medium and the 1/2MS culture medium are commercially available culture mediums, for example, the MS culture medium can be selected from Beijing Solebao scientific and technology Co., ltd (the product number is M8520), and the 1/2MS culture medium can be selected from Beijing Solebao scientific and technology Co., ltd (the product number is M8521).
The sucrose, the plant gel, the L-glutamine, the hydrolyzed casein and the activated carbon are all commercially available products, for example, the sucrose can be selected from Beijing Solebao technology Co., ltd (product number S8271), the plant gel can be selected from Beijing Solebao technology Co., ltd (product number P8170), the L-glutamine can be selected from Beijing Solebao technology Co., ltd (product number G8230), the hydrolyzed casein can be selected from Beijing Solebao technology Co., ltd (product number C8221), and the activated carbon can be selected from Beijing Solebao technology Co., ltd (product number C7261).
The plant growth regulators 2,4-D, NAA, 6-BA and IBA are commonly used growth regulators in plant tissue culture and are all products sold in the market. 2,4-D is an auxin for inducing callus formation, and can maintain dedifferentiated state of cells, and is selected from Beijing Soilebao science and technology Co., ltd (product number D8100); NAA is a synthetic plant growth regulator with auxin activity, can promote longitudinal elongation of plant cells, promote dedifferentiation of the plant cells and promote growth of roots, and can be selected from Beijing Soilebao science and technology Co., ltd (the product number is N8010); 6-BA is artificially synthesized cytokinin capable of promoting cell division and differentiation of non-differentiated tissues, and is selected from Beijing Solebao science and technology Co., ltd (product number is A8170); IBA is also called indolebutyric acid, is an auxin phytohormone, can promote plant cutting rooting, improve germination rate and survival rate, and can be selected from Beijing Sorley
BaoTech Co Ltd (product number I8030).
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional seed sowing and propagation method, the method has the characteristics of material saving, higher propagation speed and higher propagation efficiency, and can be suitable for the immature zygotic embryo of the Davidia involucrata.
According to the invention, through multi-level disinfection treatment, the influence of interference substances such as germs carried by explants on the culture of somatic embryogenesis regeneration plants is avoided, and the pollution rate of Davidia involucrata callus in the induction process is further reduced. According to the invention, different plant growth regulators and other substances are added at each stage, and are matched with corresponding culture media in a synergistic manner, so that the browning of the callus is further reduced or avoided.
The method is beneficial to industrial production, and greatly saves manpower and material resources; has great popularization value in the field of industrial seedling culture of Davidia involucrata.
The method for culturing the somatic embryogenesis regenerated plant has the characteristics of high callus induction rate, good embryogenic callus induction and somatic embryo development effects, normal germination and transformation of plants of somatic embryos and the like.
Drawings
FIG. 1 is a diagram of the basic condition of explants
FIG. 2 is the embryogenic callus induction chart
FIG. 3 is a diagram of somatic embryo development induction
FIG. 4 is a diagram of somatic embryo germination and transformed plant
In the figure 1, A is a Davidia involucrata seed wrapped by fleshy mesocarp, B is a Davidia involucrata seed wrapped by woody endocarp, C is a transverse section and a longitudinal section of the Davidia involucrata seed, D is an immature zygotic embryo wrapped by endosperm, E in the figure 2 is an immature zygotic embryo inoculated with 0D, F is an immature zygotic embryo inoculated with 3D, G is an immature zygotic embryo inoculated with 5D, H is an immature zygotic embryo inoculated with 7D, I is an immature zygotic embryo inoculated with 14D, J is an immature zygotic embryo inoculated with 15D, K is a granular embryogenic callus, L is a nodular non-embryogenic callus, M in the figure 3 is a globular stage somatic embryo, N is a heart stage somatic embryo, O is a torpedo stage somatic embryo, P is a somatic embryo in the stage, Q in the figure 4 is a mature somatic embryo and a heart stage embryo, R is a cotyledon embryo germination, S is a transformation of a somatic embryo into a small plant, and T is a transformed small plant
Detailed description of the preferred embodiments
The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present application, the raw materials and auxiliary materials may be commercially available without specific claims.
Example 1
The cultivation method of Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected from Davidia involucrata Baill in 7 months and 5 days as an explant (shown in figure 1A), soaking the explant for later use in washing powder for 15 to 30min, taking out and washing with running water for 20 to 30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning shears in clean bench (shown in FIGS. 1B and C), taking out immature zygotic embryo wrapped by endosperm (shown in FIG. 1D), and adding dropwise 2~3
Dripping 10% (v/v) sodium hypochlorite of Tween 80, soaking and sterilizing for 2.5min, taking out, washing with sterile water for several times, and drying surface water with filter paper to obtain material for inducing embryo callus of Davidia involucrata Baill;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1.0mg/L and 0.2mg/L, respectively; culturing under dark conditions at 25 + -2 deg.C; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the vein at the joint of the hypocotyl and the cotyledon gradually turns yellow from milk white (figure 2F), the hypocotyl expands for about 5d and germinates, the cotyledon basically turns yellow and starts to stretch and roll over (figure 2G), the zygotic embryo obviously calluses after continuous culture to 7d, the calluses gradually form (figure 2H), the calluses basically and completely grow into white or light yellow fluffy calluses at 14d (figure 2I or J), and the calluses become light yellow, granular and glossy embryogenic calluses after induced culture for 30d (figure 2K);
(3) Induced culture of somatic embryo development
Culturing the embryogenic callus treated in (2) in a somatic embryo development induction culture medium which is an MS culture medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of the sucrose, the plant gel, the L-glutamine, the hydrolyzed casein, 2,4-D and the 6-BA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1.0mg/L and 0.2mg/L; the temperature of the induction culture is 25 +/-2 ℃, and the dark culture is carried out; the embryos are inoculated 10d to 20d and observed to generate more embryos with smooth surfaces, and grow into mature embryos (figure 4Q) after a globular period (figure 3M), a heart-shaped period (figure 3N), a torpedo period (figure 3O) and a cotyledon period (figure 3P);
(4) Somatic embryo germination and transformation plant
Culturing the mature somatic embryos cultured in (3) in a germination medium (FIG. 4Q), which is a 1/2MS medium containing sucrose, plant gel, 6-BA and IBA at concentrations of 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.25mg/L, respectively; the transformed plant culture medium is a 1/2MS culture medium containing sucrose, plant gel, active carbon, 6-BA and IBA, wherein the concentrations of the sucrose, the plant gel, the active carbon, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1g/L, 0.5mg/L and 0.25mg/L; the culture temperature of the induction culture is 25 +/-2 ℃, and the illumination intensity is 40 to 50 umol.m -2 ·s -1 The total illumination time per day is 14-16h; the whole germination culture duration is 10d (figure 4R), the transformed plant culture duration is 20d, and the cultivation is completedObtaining the regeneration plant of Davidia involucrata (figure 4S).
Example 2
The cultivation method of Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryo wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80 for soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the material for inducing embryo callus of Davidia involucrata;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein sucrose, plant gel, L-glutamine
The concentrations of amide, hydrolyzed casein, 2,4-D and 6-BA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.5mg/L; culturing under dark conditions at 25 + -2 deg.C; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the vein at the joint of the hypocotyl and the cotyledon gradually turns yellow from milk white (figure 2F), the hypocotyl expands for about 5d and germinates, the cotyledon basically turns yellow and starts to stretch and roll over (figure 2G), the zygotic embryo obviously calluses after continuous culture to 7d, the calluses gradually form (figure 2H), the calluses basically and completely grow into white or light yellow fluffy calluses at 14d (figure 2I or J), and the calluses become light yellow, granular and glossy embryogenic calluses after induced culture for 30d (figure 2K);
(3) Somatic embryo development induction culture
Culturing the embryogenic callus treated in (2) in a somatic embryo development induction culture medium, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, NAA and 6-BA, and the concentrations of the sucrose, the plant gel, the L-glutamine, the hydrolyzed casein, the NAA and the 6-BA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 1.0mg/L; the temperature of the induction culture is 25 +/-2 ℃, and the dark culture is carried out; inoculating 10d to 20d to observe to generate more embryos with smooth surfaces, and finally growing into mature embryos (figure 4Q) through a globular stage (figure 3M), a heart-shaped stage (figure 3N), a torpedo stage (figure 3O) and a cotyledon stage (figure 3P);
(4) Somatic embryo germination and transformation plant
Culturing the mature somatic embryos cultured in (3) in a germination medium (FIG. 4Q), which is a 1/2MS medium containing sucrose, plant gel, 6-BA and IBA at concentrations of 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.25mg/L, respectively; the transformed plant culture medium is a 1/2MS culture medium containing sucrose, plant gel, activated carbon, 6-BA and IBA, wherein the concentrations of the sucrose, the plant gel, the activated carbon, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1g/L, 0.5mg/L and 0.25mg/L; the temperature of the induction culture is 25 +/-2 ℃, and the illumination intensity is 40 to 50 umol.m -2 ·s -1 The total illumination time per day is 14 to 169h; the whole germination culture duration is 10d (fig. 4R), the transformed plant culture duration is 20d, and a davidia involucrata regenerated plant can be obtained after the culture is completed (fig. 4S).
Example 3
The method for culturing Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryo wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80 for soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the explant for Davidia involucrata embryo callus induction;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in an embryogenic callus induction medium (FIG. 2E), which is MS medium containing sucrose, plant gel, L-glutamine, casein hydrolysate, NAA and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, casein hydrolysate, 2,4-D and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1mg/L, 1.0mg/L and 1.0mg/L, respectively; culturing at 25 + -2 deg.C in dark; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the vein at the joint of the hypocotyl and the cotyledon gradually turns yellow from milk white (figure 2F), the hypocotyl expands for about 5d and sprouts, the cotyledon basically turns yellow and starts to stretch and roll over (figure 2G), the zygotic embryo obviously calluses after continuing to culture for 7d, the calluses gradually form (figure 2H), and the calluses basically and completely grow into white or light yellow fluffy calluses at 14d (figure 2I or J), and the induction is carried out to induce
After 30d of culture, the callus turned into yellowish, granular, glossy embryogenic callus (FIG. 2K);
(3) Induced culture of somatic embryo development
Culturing the embryogenic callus treated in the step (2) in a somatic embryo development induction culture medium, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine, casein hydrolysate, NAA and 6-BA, and the concentrations of the sucrose, the plant gel, the L-glutamine, the casein hydrolysate, the NAA and the 6-BA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1.0mg/L and 1.0mg/L; culturing at 25 + -2 deg.C in dark; the embryos are inoculated 10d to 20d and observed to generate more embryos with smooth surfaces, and grow into mature embryos (figure 4Q) after a globular period (figure 3M), a heart-shaped period (figure 3N), a torpedo period (figure 3O) and a cotyledon period (figure 3P);
(4) Somatic embryo germination and transformation plant
Placing the mature somatic embryos cultured in the step (3) in a germination culture medium for culturing, wherein the germination culture medium is a 1/2MS culture medium containing sucrose, plant gel, 6-BA and IBA, and the concentrations of the sucrose, the plant gel, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.25mg/L; the transformed plant culture medium is a 1/2MS culture medium containing sucrose, plant gel, active carbon, 6-BA and IBA, wherein the concentrations of the sucrose, the plant gel, the active carbon, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1g/L, 0.5mg/L and 0.25mg/L; the culture temperature of the induction culture is 25 +/-2 ℃, and the illumination intensity is 40 to 50 umol.m -2 ·s -1 The total illumination time per day is 14 to 169h; the whole germination culture duration is 10d (fig. 4R), the transformed plant culture duration is 20d, and a davidia involucrata regenerated plant can be obtained after the culture is completed (fig. 4S).
Comparative example 1
The method for culturing Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryo wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80 for soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the material for inducing embryo callus of Davidia involucrata;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is WPM medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA at concentrations of 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1mg/L, 1.0mg/L and 0.2mg/L, respectively; culturing at 25 + -2 deg.C in dark; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the veins at the joint of the hypocotyls and the cotyledons gradually turn yellow from milk white (figure 2F), the hypocotyls expand to sprout after 5d, the cotyledons basically turn yellow and start to stretch and roll over (figure 2G), the zygotic embryo obviously calluses after continuing to culture for 7d, the calluses gradually form (figure 2H), the calluses basically and completely grow into sauce-shaped or flocculent calluses at 14d (figure 2L), and the calluses become brown, tumor-shaped and lusterless non-embryogenic calluses after induced culture for 30d (figure 2L);
(3) Induced culture of somatic embryo development
Placing the embryogenic callus treated in the step (2) into a somatic embryo development induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine and hydrolyzed casein, and no plant growth regulator is added, and the concentrations of the sucrose, the plant gel, the L-glutamine and the hydrolyzed casein are respectively 30g/L, 3g/L, 0.4g/L and 0.8g/L; culturing under dark conditions at 25 + -2 deg.C;
no somatic embryos were observed after inoculation at 10d to 20d.
Comparative example 2
The method for culturing Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning shears on an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryos wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 dropwise tween 80 for soaking and disinfection for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the material for inducing embryonic callus of Davidia involucrata;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is 1/2MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1.0mg/L and 0.2mg/L, respectively; culturing under dark conditions at 25 + -2 deg.C; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the veins at the joint of the hypocotyls and the cotyledons gradually turn yellow from milk white (figure 2F), the hypocotyls expand to sprout after 5d, the cotyledons basically turn yellow and start to stretch and turn over (figure 2G), the zygotic embryo obviously calluses after continuing to culture until 7d, the calluses gradually form (figure 2H), the calluses basically and completely grow into white or transparent fluffy calluses (figure 2I or J) after 14d, and the calluses become white or transparent small-bulge and obviously water-stained embryogenic calluses after 30d of induced culture (figure 2K);
(3) Induced culture of somatic embryo development
Placing the embryogenic callus treated in the step (2) into a somatic embryo development induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine and hydrolyzed casein, and no plant growth regulator is added, and the concentrations of the sucrose, the plant gel, the L-glutamine and the hydrolyzed casein are respectively 30g/L, 3g/L, 0.4g/L and 0.8g/L; culturing at 25 + -2 deg.C in dark; no somatic embryos were observed after inoculation at 10d to 20d.
Comparative example 3
The cultivation method of Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryo wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80 for soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the material for inducing embryo callus of Davidia involucrata;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, NAA, and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D, and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L, and 0.2mg/L, respectively; culturing under dark conditions at 25 + -2 deg.C; 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, and the vein at the joint of the embryo axis and the cotyledon gradually turns yellow from milk white
(fig. 2F), the embryonic axis expands and sprouts about 5d, the cotyledon basically turns yellow and begins to stretch and roll over (fig. 2G), the zygotic embryo obviously callies after being continuously cultured for 7d, the callus is gradually formed (fig. 2H), the callus basically and completely grows into a light yellow fluffy shape (fig. 2I or J) at 14d, and the callus turns into a small amount of light yellow, granular and glossy embryogenic callus after being induced and cultured for 30d (fig. 2K);
(3) Somatic embryo development induction culture
Placing the embryogenic callus treated in the step (2) into a somatic embryo development induction culture medium for culture, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine and casein hydrolysate, and no plant growth regulator is added, and the concentrations of the sucrose, the plant gel, the L-glutamine and the casein hydrolysate are respectively 30g/L, 3g/L, 0.4g/L and 0.8g/L; the temperature of the induction culture is 25 +/-2 ℃, and the dark culture is carried out; no somatic embryos were observed after inoculation at 10d to 20d.
Comparative example 4
The cultivation method of Davidia involucrata somatic embryogenesis regeneration plants comprises the following steps:
(1) Explant selection and disinfection
Selecting an immature zygotic embryo collected in 10 months of Davidia involucrata as an explant (shown in figure 1A), soaking the explant for standby 15-30min by washing powder, taking out and washing with running water for 20-30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning shears on an ultraclean workbench (shown in figures 1B and C), taking out immature zygotic embryos wrapped by endosperm (shown in figure 1D), placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 dropwise tween 80 for soaking and disinfection for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use, namely the explant for Davidia involucrata embryonic callus induction;
(2) Embryogenic callus induction culture
Removing endosperm from the explant material treated in (1), and culturing the immature zygotic embryos in embryogenic callus induction medium (FIG. 2E), which is MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 2.0mg/L and 0.2mg/L, respectively; culturing under dark conditions at 25 + -2 deg.C; after 3d, the zygotic embryo contacted with the culture medium absorbs nutrition, the vein at the joint of the hypocotyl and the cotyledon gradually turns yellow from milk white (figure 2F), the hypocotyl expands for about 5d and germinates, the cotyledon basically turns yellow and starts to stretch and roll over (figure 2G), the zygotic embryo obviously calluses after continuous culture for 7d, the calluses gradually form (figure 2H), the calluses basically and completely grow into a light yellow fluffy shape (figure 2I or J) at 14d, and the calluses become light yellow, granular and glossy embryogenic calluses after induced culture for 30d (figure 2K);
(3) Induced culture of somatic embryo development
Placing the embryogenic callus treated in the step (2) into a somatic embryo development induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing sucrose, plant gel, L-glutamine and hydrolyzed casein, and no plant growth regulator is added, and the concentrations of the sucrose, the plant gel, the L-glutamine and the hydrolyzed casein are respectively 30g/L, 3g/L, 0.4g/L and 0.8g/L; culturing under dark conditions at 25 + -2 deg.C; inoculation at 10d to 20d produced a small number of somatic embryos (FIG. 3M).
The applicant experiments show that the Davidia involucrata somatic embryogenesis regeneration plant method can be applied to immature zygotic embryos of different genotypes, and other experimental results which are not listed are similar to or the same as the above-listed results of the application, and can promote the embryogenic callus induction rate to be higher, the somatic embryo induction effect is good, and the induced somatic embryo can normally germinate into a complete plant and grow robustly.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Effect of different basal Medium types and plant growth regulators on callus, embryogenic callus and somatic embryos
Claims (10)
1. The method for culturing Davidia involucrata somatic embryogenesis regeneration plants is characterized by comprising the following steps of:
(1) Explant sterilization
Selecting immature zygotic embryo at the optimum period of Davidia involucrata Baill as explant, sterilizing the explant with disinfectant, and inducing embryonic callus as material
(2) Embryogenic callus induction culture
Removing endosperm of the explant material treated in the step (1), and placing the immature zygotic embryo in an embryogenic callus induction culture medium for culture to obtain an embryogenic callus;
(3) Induced culture of somatic embryo development
Placing the embryogenic callus processed in the step (2) in a somatic embryo development induction culture medium for culturing to obtain a mature somatic embryo;
(4) Somatic embryo germination and transformed plant culture
And (4) placing the mature somatic embryo obtained by culturing in the step (3) into a germination culture medium for culturing, placing the mature somatic embryo into a transformation plant culture medium for culturing after the culture is finished, and obtaining a davidia involucrata regeneration plant after the culture is finished.
2. The method for culturing the transgenic plant with the somatic embryogenesis of Davidia involucrata according to claim 1, wherein in (1), the explant is selected from immature zygotic embryo at early cotyledon stage, which is 10 to 11 weeks (1 to 15 days 7 months) after the flower of Davidia involucrata.
3. The method for culturing the transgenic plant of davidia involucrata somatic embryogenesis according to claim 1, characterized in that in (1), the disinfection treatment comprises multi-stage disinfection, wherein the explant is soaked in washing powder for standby 15 to 30min, and then taken out and washed with running water for 20 to 30min; soaking the washed explants in 75% alcohol for 10 to 15min; removing exocarp and fleshy mesocarp of fruit with pruning scissors in a clean bench, taking out immature zygotic embryo wrapped by endosperm, placing in 10% (v/v) sodium hypochlorite dropwise added with 2~3 drops of tween 80, soaking and sterilizing for 2.5min, taking out, washing with sterile water for multiple times, and sucking surface water with filter paper for later use.
4. The method for culturing the involucra somatic embryogenesis regenerated plant of claim 1, wherein in (2), the induction medium is an MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, 2,4-D and 6-BA, wherein the concentrations of the sucrose, the plant gel, the L-glutamine, the hydrolyzed casein, 2,4-D and the 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5 to 2.0mg/L and 0.2 to 2.0mg/L, respectively.
5. The method for culturing the davidia involucrata body cell embryogenesis regeneration plant according to claim 1, wherein in the step (2), the culture temperature for induction culture is 25 ± 2 ℃, and dark shading culture is performed.
6. The method for culturing the involucra somatic embryogenesis regenerated plant of claim 1, characterized in that in (3), the induction medium is an MS medium containing sucrose, plant gel, L-glutamine, hydrolyzed casein, NAA and 6-BA, wherein the concentrations of the sucrose, the plant gel, the L-glutamine, the hydrolyzed casein, the NAA and the 6-BA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5 to 2.0mg/L and 0.2 to 2.0mg/L, respectively.
7. The method for culturing the davidia involucrata body cell embryogenesis regeneration plant according to claim 1, wherein in the step (3), the culture temperature for induction culture is 25 ± 2 ℃, and dark shading culture is performed.
8. The method for culturing the involucra cell embryogenic regeneration plant of claim 1, wherein in (4), the germination medium is a 1/2MS medium containing sucrose, plant gel, 6-BA and IBA, wherein the concentrations of sucrose, plant gel, 6-BA and IBA are 30g/L, 3g/L, 0.4g/L, 0.8g/L, 0.5mg/L and 0.25mg/L, respectively; the somatic embryo transformation plant culture medium is a 1/2MS culture medium containing sucrose, plant gel, activated carbon, 2,4-D and 6-BA, wherein the concentrations of the sucrose, the plant gel, the activated carbon, the 6-BA and the IBA are respectively 30g/L, 3g/L, 0.4g/L, 0.8g/L, 1g/L, 0.5mg/L and 0.25mg/L.
9. The method for culturing the transgenic plant with the somatic embryogenesis of Davidia involucrata according to claim 1, wherein in the step (4), the culture temperature for induction culture is 25 +/-2 ℃, and the illumination intensity is 40 to 50 umol-m -2 ·s -1 The total illumination time per day is 14 to 169h.
10. The regenerated plant of Davidia involucrata as described in claim 1~9, which is prepared by the method for culturing a transgenic Davidia involucrata somatic embryogenesis regenerated plant.
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| CN102919129A (en) * | 2012-11-14 | 2013-02-13 | 四川大学 | Method for acquiring regeneration seedlings of dove trees through tissue culture by taking dove tree leaves as explants |
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