CN102124955B - Induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos - Google Patents
Induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos Download PDFInfo
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- CN102124955B CN102124955B CN 201110033701 CN201110033701A CN102124955B CN 102124955 B CN102124955 B CN 102124955B CN 201110033701 CN201110033701 CN 201110033701 CN 201110033701 A CN201110033701 A CN 201110033701A CN 102124955 B CN102124955 B CN 102124955B
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Abstract
The invention provides an induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos, comprising the following steps: (1) building a regeneration system of a Photinia fraseri asepsis test tube seedling; (2) building a high-frequency rapid-propagation system induced by an in-vitro somatic embryo and an adventitious bud; and (3) rooting, acclimatizing and transplanting for the test tube seedling. The induction rapid-propagation culture method for Photinia fraseri in-vitro leaf somatic embryos is characterized in that coordinated sets of measures of selecting a proper explant, improving a basic culture medium and ingredients, regulating culture conditions and the like are adopted, and the Photinia fraseri of which the seed is difficult to breed is subjected to path rapid propagation by the somatic embryos; the plant transplantation survival rate is higher than 95%; the germchit grows strongly to solve the problem that an excellent germchit quality degenerates, and a great quantity of purified and rejuvenated detoxification nursery stock with good quality can be provided for production; and in addition, and ideal receptor system is provided for breeding new specimens for Photinia fraseri by genetic transformation or mutation breeding.
Description
Technical field
The present invention relates to the fast numerous cultural method of a kind of photinia glabra excised leaf somatic embryo inducement, belong to the botany field.
Background technology
Photinia glabra (
Photinia ⅹ frasery) be the crossbreed of Photinia (P.glabra) and Chinese photinia (P.serrulata); Belong to rose family Photinia evergreen broad-leaved dungarunga or racemosus bush, it is distributed widely in ground such as Japan, the U.S. and Europe, the bright-coloured as fire because of its young sprout and tender leaf; Beautiful lasting; It is strong to prune back rudiment power, and strain shape is compact, in garden landscape, uses always and makees high-grade colour band; Or cultivate solely do, spherical tree crown, isolated planting in the greenery patches; Or do street tree; Or be arranged in porch or indoor after potted plant, and effect is all good, and it is the high ornamental plant of a kind of ornamental value.
Since last century, the nineties photinia glabra was introduced into China, it is just classified as had the color leaf seeds that higher exploitation is worth, but photinia glabra is compared with China many original seeds; Rare in China provenance, not seeing so far has solid report, and from the improved seeds of external introducing also because of cottage propagation for a long time; Fungal diseases such as easy infection leaf spot, spring and summer, it was subject to aphid damage again, and its virus is serious; The blade piebald is prone to come off, and sight descends, and the improved variety degree reduces; And the use of seedling inferior causes the intrinsic good characteristic of photinia glabra to be lost day by day; This has retrained the development of these seeds to a certain extent and has applied; Therefore, selecting, breed good photinia glabra strain becomes the outstanding production problem of a ten minutes, and tissue-culturing rapid propagation is a kind of effective way of photinia glabra breeding industrialization; The report of related fields is also more, but the report that success is arranged is not seen in the technical research of the direct inductor blast of relevant Photinia glabra leaves and the fast numerous system of regenerating.
Summary of the invention
In order to overcome the deficiency of prior art; The object of the present invention is to provide a kind of good photinia glabra strain convenient but the seminal propagation difficulty of drawing materials is the formation of the direct inductor embryo of blade and the fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement that obtains regeneration plant; And utilize the formation of embryoid regeneration plant blade inductor embryo and indefinite bud or the formation of direct evoking adventive bud to obtain high-frequency regeneration plant; Solving the problem that the good seed germplasm is degenerated, the detoxification photinia glabra nursery stock of, purification and rejuvenation of fine quality greatly for the amount of providing on producing;
Another object of the present invention provides and a kind ofly a desirable receptor system is provided for photinia glabra genetic transformation or mutation breeding seed selection new varieties.
In order to realize the foregoing invention purpose, the present invention realizes through following technical scheme:
The fast numerous cultural method of a kind of photinia glabra excised leaf somatic embryo inducement is characterized in that, may further comprise the steps:
(1) set up photinia glabra sterile test tube seedling regenerating system:
(1.1) selection and material processed: choose robust growth, individual plant that The Characters is good the fine morning in spring, spray the branch of choosing with 5% carbendazim, sprinkling once weekly; Sprayed one month continuously, the firm anosis worm spray of sprouting of clip comprises front end not lignification and semi-lignified part afterwards; Remove unnecessary cauline leaf, need be partly with liquid detergent solution soaking 5-10min, after cleaning with cotton balls is careful then; Again with flowing water flushing 1 hour; Use 2%NaClO solution soaking 15-20min at last, clean with aseptic water washing again, with subsequent use behind the sterilization filter paper suck dry moisture;
(1.2) selection sterilization and bud start the sprouting cultivation: the material that step (1.1) was handled places on the superclean bench, the alcohol disinfecting 15-20s with 75%, and aseptic water washing 5-6 time is again with 0.1% mercuric chloride sterilization 8-15min; Aseptic water washing 6-8 times, with sterilization filter paper suck dry moisture, clip 1-2cm is with the stem section of an axillary-bud or top-bud then; Vertically insert bud with ecological hypomere and start on the medium, 3 explants of every bottle graft kind are induced terminal bud and axillary bud sprouting; At 25 ± 2 ℃ of culturing room's temperature daytimes, 20 ± 2 ℃ of nights, intensity of illumination 1500-2000 lx; Under the condition of light application time 14 h/d, carry out the cultivation of 15-20d, stem segment with axillary buds begins to sprout; Stem apex obviously extends, and the bud point grows up to the young sprout of 2-4cm behind the 40-50d, forms the sterile test tube seedling;
(1.3) the sterile test tube seedling proliferation is cultivated: the sterile test tube seedling that step (1.2) is induced removes radical leaves; Being cut into the stem section or the stem apex that contain 1-2 axillalry bud is seeded on the test-tube plantlet proliferated culture medium; In 25-28d follow-up generation 1 time,, the NAA concentration through above-mentioned test-tube plantlet proliferated culture medium is at 0.1～0.5mgL
-1Controlled range in adjustment change, obtain a large amount of sterile test tube seedlings, set up photinia glabra sterile test tube seedling regenerating system;
(2) set up the fast traditional font of the high frequency of excised leaf body embryo and adventitious bud inducing system:
(2.1) formation of inductor embryo and indefinite bud: launching leaf with test-tube plantlet the 3rd to the 6th is explant; The short petiole of blade band; Blade paripheral zone zigzag leaf margin is cut; Do following processing afterwards: blade is cut into left and right sides two halves along master pulse, backsight blade size row dry the 1-2 cutter or with blade along master pulse crosscut 2-3 cutter; Be seeded in the first inductor embryo and/or indefinite bud and form medium; Perhaps, the second inductor embryo and/or indefinite bud form on the medium, and the medium that requires the explant blade back to be close to is separately down placed; In dark culturing 7-10d; The formation of inductor embryo, in dark culturing 15-20d, the formation of evoking adventive bud;
(2.2) growth of body embryo and indefinite bud and differentiation: with the first inductor embryo and/or indefinite bud form medium and, the second inductor embryo and/or indefinite bud form the explant of cultivating on the medium and are transferred on the first body embryo and/or indefinite bud growth and the differential medium; Put into climatic cabinate then; When temperature is 10 ± 2 ℃, secretly cultivates and change culturing room over to after the 3-5d and secretly cultivate 35-40d, or directly secretly cultivate 40-45d in culturing room; Middle subculture once; Cultivation through the above-mentioned first body embryo and/or indefinite bud growth and differential medium forms many buds of growing thickly on explant, the bud height is at 2cm～5cm;
(2.3) the fast numerous cultivation of adventitious buds proliferation: stem section or stem apex that above-mentioned steps (2.2) is induced the bud of growing thickly of formation to be cut into to contain 1-2 axillalry bud are transferred to the test-tube plantlet proliferated culture medium; Perhaps; Replace subculture on the second body embryo and/or indefinite bud growth and the differential medium; Breed fast numerous cultivation, every 18-22d subculture once, the propagation frequency is 10-15;
(3) rooting of vitro seedling acclimatization and transplants:
(3.1) strengthening seedling and rooting is cultivated:
Above-mentioned steps (2.3) the grow thickly bud of healthy and strong length behind the 3-4cm that grow is cut into individual plant and is transferred on the strengthening seedling and rooting medium, cultivate 25-35d, guarantee that cauline leaf grows normally, root growth is healthy and strong, and root grows to 1.5cm when above, carries out acclimatization and transplants;
(3.2) acclimatization and transplants:
At first the test-tube plantlet bottle cap is opened, after culturing room adapts to 1d, moved on to the shading screen plastic tunnel of obscurity 70-80%, at greenhouse experiment lower refining seedling 2-3d; With tweezers seedling is pressed from both sides out, the medium that clear water flush away base portion is residual is transplanted to and is equipped with in the cave dish that mixes seedling medium; Utilize intermittent spraying device or artificial water spray to keep relative moisture more than 80%; Behind domestication refining seedling 5-10d, after new root sprouting eruption, reducing gradually waters number of times and be transplanted to flowerpot carries out Routine Management;
Described bud starts medium: improvement MS+0.5～1.0 mgL
-1BA+0.5～1.0mgL
-1KT+0.1～0.2mgL
-1NAA+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described test-tube plantlet proliferated culture medium is: improvement MS+1.0～2.0mgL
-1BA+1.0～2.0mgL
-1KT+0.1～0.5mgL
-1NAA+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described first inductor embryo and/or indefinite bud form medium: improvement MS+0.5mgL
-12,4-D+0.5～1.0mgL
-1BA+0.5～2.0mgL
-1NAA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described second inductor embryo and/or indefinite bud form medium: improvement MS+0.1mgL
-12,4-D+0.5mgL
-1BA+10～30 mgL
-1NAA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described first body embryo and/or indefinite bud growth with differential medium are: improvement MS+2.0～4.0mg L
-1BA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described second body embryo and/or indefinite bud growth with differential medium are: MS+2.0mgL
-1BA+1.0 mgL
-1IBA+2.0 mgL
-1KT+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described strengthening seedling and rooting medium is: 1/2 improvement MS+0.2～0.5mgL
-1IBA+0～0.05mgL
-1NAA+6.0～7.0gL
-1Agar+20gL
-1Sucrose, and the pH value is 5.8～6.0,
Described improvement MS comprises grand nutrition element, micronutrient element and organic reagent,
Wherein, the component of described grand nutrition element is following with its corresponding concentration:
Potassium nitrate 1900 mg/L;
Ammonium sulfate 1650 mg/L;
Epsom salt 370mg/L;
Calcium chloride dihydrate 440 mg/L;
Anhydrous potassium dihydrogenphosphate 170 mg/L;
Disodium ethylene diamine tetraacetate 37.3 mg/L;
Ferrous sulfate heptahydrate 27.8 mg/L,
The component of described micronutrient element is following with its corresponding concentration:
Four water manganese sulphates, 22.3 mg/L;
Zinc sulphate 8.6 mg/L;
Boric acid 6.2 mg/L;
KI 0.83 mg/L;
Sodium molybdate 0.25 mg/L;
Copper sulphate 0.025 mg/L;
Cobalt chloride 0.025 mg/L,
The component of described organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 10.0 mg/L;
Nicotinic acid 1.0 mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100.0 mg/L.
And the composition of above-mentioned mixing seedling medium is counted with volume ratio: vermiculite: turfy soil: garden mould=3:5:2.
The invention has the beneficial effects as follows: the fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement of the present invention is through selecting supplementary measures such as proper explant, improvement minimal medium and composition, adjustment condition of culture; Make the plantlet of transplant survival rate can reach more than 95%; Seedling early growth is healthy and strong; Can effectively solve good seed germplasm degenerate problem; Also can be the detoxification photinia glabra nursery stock that the purification and rejuvenation of a large amount of high-qualitys are provided in the production, in addition, also can be the photinia glabra genetic transformation or mutation breeding seed selection new varieties provide a desirable acceptor systems.
Description of drawings
Fig. 1 is the test-tube plantlet that increment of the present invention is cultivated;
Fig. 2 is the somatic embryo of the blade of photinia glabra of the present invention at different times;
Fig. 3 is the indefinite bud of the blade body embryogenesis of photinia glabra of the present invention;
Fig. 4 is the indefinite bud that the blade of photinia glabra of the present invention directly forms;
Fig. 5 is the bud of growing thickly of the fast numerous formation of propagation of the present invention;
Fig. 6 is of the present invention through the test-tube plantlet behind culture of rootage and the refining seedling;
Fig. 7 is the plant that grows up to through transplanting of the present invention.
Embodiment
To combine accompanying drawing and specific embodiment below, specify embodiment of the present invention:
Fig. 1 is the test-tube plantlet that increment of the present invention is cultivated; Fig. 2 is the somatic embryo of the blade of photinia glabra of the present invention at different times; Fig. 3 is the indefinite bud of the blade body embryogenesis of photinia glabra of the present invention; Fig. 4 is the indefinite tooth that the blade of photinia glabra of the present invention directly forms; Fig. 5 is the bud of growing thickly of the fast numerous formation of propagation of the present invention; Fig. 6 is of the present invention through the test-tube plantlet behind culture of rootage and the refining seedling; Fig. 7 is the plant that grows up to through transplanting of the present invention.
Like Fig. 1-shown in Figure 7:
Embodiment 1
The fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement may further comprise the steps:
(1) set up photinia glabra sterile test tube seedling regenerating system:
(1.1) selection and material processed: choose robust growth, individual plant that The Characters is good the fine morning in spring, spray the branch of choosing with 5% carbendazim, sprinkling once weekly; Sprayed one month continuously, the firm anosis worm spray of sprouting of clip comprises front end not lignification and semi-lignified part afterwards; Remove unnecessary cauline leaf, need be partly with liquid detergent solution soaking 5min, after cleaning with cotton balls is careful then; Again with flowing water flushing 1 hour; Use 2%NaClO solution soaking 15min at last, clean with aseptic water washing again, with subsequent use behind the sterilization filter paper suck dry moisture;
(1.2) selection sterilization and bud start the sprouting cultivation: the material of the described processing of step (1.1) is placed on the superclean bench alcohol disinfecting 15s with 75%, aseptic water washing 5 times; Again with 0.1% mercuric chloride sterilization 8min; Aseptic water washing 6 times, with sterilization filter paper suck dry moisture, then about clip 1cm with the stem section of an axillary-bud or top-bud; Vertically insert bud with ecological hypomere and start on the medium, bud starts medium and is improvement MS+0.5mgL
-1BA+0.5mgL
-1KT+0.1mgL
-1NAA+6.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8,3 explants of every bottle graft kind are induced terminal bud and axillary bud sprouting; At 25 ± 2 ℃ of culturing room's temperature daytimes, 20 ± 2 ℃ of nights, intensity of illumination 1500-2000 lx; Under the condition of light application time 14 h/d, carry out the cultivation of 15d, stem segment with axillary buds begins to sprout; Stem apex obviously extends, and 40d left and right sides bud point grows up to the young sprout about 2cm, forms the sterile test tube seedling;
(1.3) the sterile test tube seedling proliferation is cultivated: the sterile test tube seedling that step (1.2) is induced removes radical leaves, is cut into the stem section or the stem apex that contain 1-2 axillalry bud and is seeded on the test-tube plantlet proliferated culture medium, and the test-tube plantlet proliferated culture medium is improvement MS+1.0mgL
-1BA+1.0mgL
-1KT+0.1～0.5mgL
-1NAA+6.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8, general basal part of stem has a small amount of callus to produce and goes out 2-3 budlet from the callus director, and subculture was 1 time in 25 days, and the NAA concentration through above-mentioned test-tube plantlet proliferated culture medium is at 0.1～0.5mgL
-1Controlled range in adjustment change, and constantly cut breeding through basal part of stem bud clump, can obtain a large amount of sterile test tube seedlings, set up photinia glabra sterile test tube seedling regenerating system;
(2) set up the fast traditional font of the high frequency of excised leaf body embryo and adventitious bud inducing system:
Launching leaf with test-tube plantlet the 3rd to the 6th is explant; The short petiole of blade band cuts blade paripheral zone zigzag leaf margin, and blade is cut into left and right sides two halves along master pulse; 1 cutter rows dry on blade; Blade back is close to the medium placement down, is seeded in the first inductor embryo and/or indefinite bud and forms in the medium, and wherein the first inductor embryo and/or indefinite bud form medium for improveing MS+0.5mgL
-12,4-D+0.5mgL
-1BA+1.0mgL
-1NAA+3.0gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8, in dark culturing 10d, and the formation of inductor embryo;
Above-mentioned explant is transferred in the first body embryo and/or indefinite bud growth and the differential medium, and the first body embryo and/or indefinite bud growth and differential medium are improvement MS+ 2.0mgL
-1BA+3.5gL
-1Phytagel+20gL
-1Sucrose, pH value 5.8 is secretly cultivated 40d in culturing room, and middle subculture once just can be seen somatic embryo about 10d under stereomicroscope, and developmental stage can be from globular embryo, heart-shape embryo to the torpedo embryo, about the high 2-3cm of 35d left and right sides bud;
Induce the bud seedling of formation to be cut into the stem section or the stem apex that contain 1-2 axillalry bud above-mentioned steps and be transferred to MS+1.0mgL
-1BA+1.0mgL
-1KT+0.1mgL
-1NAA+6.5gL
-1Agar+30gL
-1On the medium of sucrose, or MS+2.0mgL
-1BA+1.0mgL
-1IBA+2.0mgL
-1KT+6.5gL
-1Agar+30gL
-1On the medium of sucrose, pH value 5.8, two medium replace subculture and carry out, and every 20d subculture is once bred frequency averaging and reached 12, and growth coefficient uses the test-tube plantlet enrichment culture to improve more than 3 times more separately.
(3) rooting of vitro seedling acclimatization and transplants:
(3.1) strengthening seedling and rooting is cultivated:
Above-mentioned clump bud length of growing healthy and strong is cut into individual plant during to 4cm is transferred on the strengthening seedling and rooting medium, cultivate 25d, guarantee that cauline leaf grows normally, root growth is healthy and strong, when root grows to 1.5-2.5cm and 2-3 bar lateral root is arranged, gets final product acclimatization and transplants.
(3.2) acclimatization and transplants:
At first the test-tube plantlet bottle cap is opened, adapted to 1d, move on to the shading screen plastic tunnel greenhouse experiment lower refining seedling 2-3d of obscurity 70-80% afterwards in culturing room; With tweezers seedling is pressed from both sides out; The medium that clear water flush away base portion is residual is transplanted to and is equipped with in the cave dish that mixes seedling medium, utilizes intermittent spraying device or artificial water spray to keep relative moisture more than 80%; Behind domestication refining seedling 5-10d, treating that the eruption of new root sprouting reduces to water number of times and be transplanted to flowerpot gradually carries out Routine Management.
The composition of cave dish or flowerpot mixing seedling medium is counted with volume ratio: vermiculite: turfy soil: garden mould=3:5:2.
Embodiment 2
The fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement may further comprise the steps:
(1) set up photinia glabra sterile test tube seedling regenerating system:
(1.1) selection and material processed: choose robust growth, individual plant that The Characters is good the fine morning in spring, spray the branch of choosing with 5% carbendazim, sprinkling once weekly; Sprayed one month continuously, the firm anosis worm spray of sprouting of clip comprises front end not lignification and semi-lignified part afterwards; Remove unnecessary cauline leaf, need be partly with liquid detergent solution soaking 10min, after cleaning with cotton balls is careful then; Again with flowing water flushing 1 hour; Use 2%NaClO solution soaking 15min at last, clean with aseptic water washing again, with subsequent use behind the sterilization filter paper suck dry moisture;
(1.2) selection sterilization and bud start the sprouting cultivation: the material of the described processing of step (1.1) is placed on the superclean bench alcohol disinfecting 20s with 75%, aseptic water washing 6 times; Again with 0.1% mercuric chloride sterilization 15min; Aseptic water washing 8 times, with sterilization filter paper suck dry moisture, then about clip 2cm with the stem section of an axillary-bud or top-bud; Vertically insert bud with ecological hypomere and start on the medium, bud starts medium and is improvement MS+1.0mgL
-1BA+0.5mgL
-1KT+0.2mgL
-1NAA+6.5gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8,3 explants of every bottle graft kind are induced terminal bud and axillary bud sprouting; At 25 ± 2 ℃ of culturing room's temperature daytimes, 20 ± 2 ℃ of nights, intensity of illumination 1500-2000 lx; Under the condition of light application time 14 h/d, carry out the cultivation of 15d, stem segment with axillary buds begins to sprout; Stem apex obviously extends, and 45d left and right sides bud point grows up to the young sprout about 3cm, forms the sterile test tube seedling;
(1.3) the sterile test tube seedling proliferation is cultivated: the sterile test tube seedling that step (1.2) is induced removes radical leaves, is cut into the stem section or the stem apex that contain 1-2 axillalry bud and is seeded on the test-tube plantlet proliferated culture medium, and the test-tube plantlet proliferated culture medium is improvement MS+1.0mgL
-1BA+2.0mgL
-1KT+0.1～0.5mgL
-1NAA+6.5gL
-1Agar+30gL
-1Sucrose, and the pH value is 6.0, general basal part of stem has more callus to produce and goes out 3-4 budlet from the callus director, and subculture was 1 time in 28 days, and the concentration of the NAA growth hormone through above-mentioned test-tube plantlet proliferated culture medium is at 0.1～0.5mgL
-1Control range in adjustment change, and constantly cut breeding through basal part of stem bud clump, can obtain a large amount of sterile test tube seedlings, set up photinia glabra sterile test tube seedling regenerating system;
(2) set up the fast traditional font of the high frequency of excised leaf body embryo and adventitious bud inducing system:
Launching leaf with test-tube plantlet the 3rd to the 6th is explant; The short petiole of blade band; Blade paripheral zone zigzag leaf margin is cut, and blade is along master pulse crosscut 2 cuttves, and blade back is close to medium down and is placed; Be seeded in the second inductor embryo and/or indefinite bud and form in the medium, wherein the second inductor embryo and/or indefinite bud form medium for improveing MS+0.1mgL
-12,4-D+0.5mgL
-1BA+10mgL
-1NAA+3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8, in dark culturing 7d, and the formation of inductor embryo;
Above-mentioned explant is transferred to improvement MS+2.0mgL
-1BA+3.5gL
-1Phytagel+20gL
-1On the medium of sucrose, the pH value is 5.8, puts it into climatic cabinate; Temperature is 10 ℃ of dark 5d of cultivation, changes culturing room afterwards over to and secretly cultivates 35d, and middle subculture once; Just can be observed torpedo embryo or cotyledonary embryos about 20d or sprout the bud of growing thickly that forms, about the high 3-4cm of 35d left and right sides bud by it;
Induce the bud seedling of formation to be cut into the stem section or the stem apex that contain 1-2 axillalry bud above-mentioned steps and be transferred to MS+1.0mgL
-1BA+1.0mgL
-1KT+0.1mgL
-1NAA+6.5gL
-1Agar+30gL
-1On the medium of sucrose, or MS+2.0mgL
-1BA+1.0mgL
-1IBA+2.0mgL
-1KT+6.5gL
-1Agar+30gL
-1On the medium of sucrose, pH value 5.8, two medium replace subculture and carry out, and every 20d subculture is once bred frequency averaging and reached 15, and growth coefficient uses the test-tube plantlet enrichment culture to improve more than 3 times more separately;
(3) rooting of vitro seedling acclimatization and transplants:
(3.1) strengthening seedling and rooting is cultivated:
Above-mentioned clump bud length of growing healthy and strong is cut into individual plant to 4cm is transferred on the strengthening seedling and rooting medium, cultivate 30d, guarantee that cauline leaf grows normally, root growth is healthy and strong, and it is above and when 3-4 bar lateral root is arranged that root grows to 1.5cm, gets final product acclimatization and transplants;
(3.2) acclimatization and transplants:
At first the test-tube plantlet bottle cap is opened, adapted to 1d, move on to the shading screen plastic tunnel greenhouse experiment lower refining seedling 2-3d of obscurity 70-80% afterwards in culturing room; With tweezers seedling is pressed from both sides out; The medium that clear water flush away base portion is residual is transplanted to and is equipped with in the cave dish that mixes seedling medium, utilizes intermittent spraying device or artificial water spray to keep relative moisture more than 80%; Behind domestication refining seedling 5-10d, treating that the eruption of new root sprouting reduces to water number of times and be transplanted to flowerpot gradually carries out Routine Management.
The composition of cave dish or flowerpot mixing seedling medium is counted with volume ratio: vermiculite: turfy soil: garden mould=3:5:2.
Embodiment 3
The fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement may further comprise the steps:
(1) set up photinia glabra sterile test tube seedling regenerating system:
(1.1) selection and material processed: choose robust growth, individual plant that The Characters is good the fine morning in spring, spray the branch of choosing with 5% carbendazim, sprinkling once weekly; Sprayed one month continuously, the firm anosis worm spray of sprouting of clip comprises front end not lignification and semi-lignified part afterwards; Remove unnecessary cauline leaf, need be partly with liquid detergent solution soaking 5min, after cleaning with cotton balls is careful then; Again with flowing water flushing 1 hour; Use 2%NaClO solution soaking 20min at last, clean with aseptic water washing again, with subsequent use behind the sterilization filter paper suck dry moisture;
(1.2) selection sterilization and bud start the sprouting cultivation: the material of the described processing of step (1.1) is placed on the superclean bench alcohol disinfecting 15s with 75%, aseptic water washing 6 times; Again with 0.1% mercuric chloride sterilization 15min; Aseptic water washing 8 times, with sterilization filter paper suck dry moisture, then about clip 2cm with the stem section of an axillary-bud or top-bud; Vertically insert bud with ecological hypomere and start on the medium, bud starts medium and is improvement MS+0.5mgL
-1BA+1.0mgL
-1KT+0.1mgL
-1NAA+7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8,3 explants of every bottle graft kind are induced terminal bud and axillary bud sprouting; At 25 ± 2 ℃ of culturing room's temperature daytimes, 20 ± 2 ℃ of nights, intensity of illumination 1500-2000 lx; Under the condition of light application time 14 h/d, carry out the cultivation of 20d, stem segment with axillary buds begins to sprout; Stem apex obviously extends, and 50d left and right sides bud point grows up to the young sprout about 4cm, forms the sterile test tube seedling;
(1.3) the sterile test tube seedling proliferation is cultivated: the sterile test tube seedling that step (1.2) is induced removes radical leaves, is cut into the stem section or the stem apex that contain 1-2 axillalry bud and is seeded on the test-tube plantlet proliferated culture medium, and the test-tube plantlet proliferated culture medium is improvement MS+2.0mgL
-1BA+2.0mgL
-1KT+0.1～0.5mgL
-1NAA+7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.9, general basal part of stem has more callus to produce and goes out 4.5 budlets from the callus director, and subculture was 1 time in 28 days, and the concentration of the NAA growth hormone through above-mentioned test-tube plantlet proliferated culture medium is at 0.1～0.5mgL
-1Adjustment in the control range changes, and constantly cuts breeding through basal part of stem bud clump, can obtain a large amount of sterile test tube seedlings, sets up photinia glabra sterile test tube seedling regenerating system;
(2) set up the fast traditional font of the high frequency of excised leaf body embryo and adventitious bud inducing system:
Launching leaf with test-tube plantlet the 3rd to the 6th is explant, and the short petiole of blade band cuts blade paripheral zone zigzag leaf margin, and blade is along master pulse crosscut 3 cuttves, and blade back is close to the inducing culture placement down, is seeded in MS+2.0 mgL
-1NAA+0.5 mgL
-12,4-D+0.5mgL
-1BA+6.5gL
-1Agar+30gL
-1On the medium of sucrose, pH value 5.8 is secretly cultivated 15d, the formation of evoking adventive bud;
Above-mentioned explant is transferred to MS+2.0mgL
-1BA+1.0mgL
-1IBA+2.0mgL
-1KT+6.5gL
-1Agar+30gL
-1On the medium of sucrose, pH value 5.9 is cultivated 40d at culturing room light, middle subculture once, intensity of illumination 1000-1500lx.Through above-mentioned cultivation, can form many buds of growing thickly on the explant, the bud height can have from 2cm to 5cm;
Induce the bud seedling of formation to be cut into the stem section or the stem apex that contain 1-2 axillalry bud above-mentioned steps and be transferred to MS+1.0mgL
-1BA+1.0mgL
-1KT+0.1mgL
-1NAA+6.5gL
-1Agar+30gL
-1On the medium of sucrose, or MS+2.0mgL
-1BA+IBA 1.0mgL
-1+ 2.0mgL
-1KT+6.5gL
-1Agar+30gL
-1On the medium of sucrose, pH value 5.8, two medium replace subculture and carry out, and every 20d subculture is once bred frequency averaging and reached 10, and growth coefficient uses the test-tube plantlet enrichment culture to improve more than 2 times more separately;
(3) rooting of vitro seedling acclimatization and transplants:
(3.1) strengthening seedling and rooting is cultivated:
Above-mentioned clump bud length of growing healthy and strong is cut into individual plant to 3cm is transferred on the strengthening seedling and rooting medium, cultivate 35d, guarantee that cauline leaf grows normally, root growth is healthy and strong, and it is above and when 3-4 bar lateral root is arranged that root grows to 1.5cm, gets final product acclimatization and transplants;
(3.2) acclimatization and transplants:
At first the test-tube plantlet bottle cap is opened, adapted to 1d, move on to the shading screen plastic tunnel greenhouse experiment lower refining seedling 2-3d of obscurity 70-80% afterwards in culturing room; With tweezers seedling is pressed from both sides out; The medium that clear water flush away base portion is residual is transplanted to and is equipped with in the cave dish or flowerpot that mixes seedling medium, utilizes intermittent spraying device or artificial water spray to keep relative moisture more than 80%; Behind domestication refining seedling 5-10d, treat that the eruption of new root sprouting reduces gradually to water number of times and carry out Routine Management.
The composition of cave dish or flowerpot mixing seedling medium is counted with volume ratio: vermiculite: turfy soil: garden mould=3:5:2.
Among 3 wherein above-mentioned embodiment, improvement MS comprises grand nutrition element, micronutrient element and organic reagent, and wherein, the component of grand nutrition element is following with its corresponding concentration:
Potassium nitrate 1900 mg/L;
Ammonium sulfate 1650 mg/L;
Epsom salt 370mg/L;
Calcium chloride dihydrate 440 mg/L;
Anhydrous potassium dihydrogenphosphate 170 mg/L;
Disodium ethylene diamine tetraacetate 37.3 mg/L;
Ferrous sulfate heptahydrate 27.8 mg/L.
The component of micronutrient element is following with its corresponding concentration:
Four water manganese sulphates, 22.3 mg/L;
Zinc sulphate 8.6 mg/L;
Boric acid 6.2 mg/L;
KI 0.83 mg/L;
Sodium molybdate 0.25 mg/L;
Copper sulphate 0.025 mg/L;
Cobalt chloride 0.025 mg/L.
The component of organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 10.0 mg/L;
Nicotinic acid 1.0 mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100.0 mg/L.
The present invention utilizes the photinia glabra of the fast numerous seminal propagation difficulty of hot research plant somatocyte embryo generation technique in the plant cell and tissue culture in recent years to be only the fundamental way that addresses the above problem; It not only can solve the problem that the photinia glabra germplasm is degenerated; The purpose that reaches good seed detoxification, purification and rejuvenation and breed fast; And be the important channel of highly efficient regeneration seed source, still carry out the desirable acceptor material of genetic transformation or mutation breeding seed selection new varieties.
Below disclose the present invention with preferred embodiment, so it is not in order to restriction the present invention, and all employings are equal to replacement or the technical scheme that obtained of equivalent transformation mode, all drop within protection scope of the present invention.
Claims (2)
1. the fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement is characterized in that, may further comprise the steps:
(1) set up photinia glabra sterile test tube seedling regenerating system:
(1.1) selection and material processed: choose robust growth, individual plant that The Characters is good the fine morning in spring, spray the branch of choosing with 5% carbendazim, sprinkling once weekly; Sprayed one month continuously, the firm anosis worm spray of sprouting of clip comprises front end not lignification and semi-lignified part afterwards; Remove unnecessary cauline leaf, need be partly with liquid detergent solution soaking 5-10min, after cleaning with cotton balls is careful then; Again with flowing water flushing 1 hour; Use 2%NaClO solution soaking 15-20min at last, clean with aseptic water washing again, with subsequent use behind the sterilization filter paper suck dry moisture;
(1.2) selection sterilization and bud start the sprouting cultivation: the material that step (1.1) was handled places on the superclean bench, the alcohol disinfecting 15-20s with 75%, and aseptic water washing 5-6 time is again with 0.1% mercuric chloride sterilization 8-15min; Aseptic water washing 6-8 times, with sterilization filter paper suck dry moisture, clip 1-2cm is with the stem section of an axillary-bud or top-bud then; Vertically insert bud with ecological hypomere and start on the medium, 3 explants of every bottle graft kind are induced terminal bud and axillary bud sprouting; At 25 ± 2 ℃ of culturing room's temperature daytimes, 20 ± 2 ℃ of nights, intensity of illumination 1500-2000 lx; Under the condition of light application time 14 h/d, carry out the cultivation of 15-20d, stem segment with axillary buds begins to sprout; Stem apex obviously extends, and the bud point grows up to the young sprout of 2-4cm behind the 40-50d, forms the sterile test tube seedling;
(1.3) the sterile test tube seedling proliferation is cultivated: the sterile test tube seedling that step (1.2) is induced removes radical leaves; Being cut into the stem section or the stem apex that contain 1-2 axillalry bud is seeded on the test-tube plantlet proliferated culture medium; In 25-28d follow-up generation 1 time,, the NAA concentration through above-mentioned test-tube plantlet proliferated culture medium is at 0.1～0.5mgL
-1Scope in adjustment change, obtain a large amount of sterile test tube seedlings, set up photinia glabra sterile test tube seedling regenerating system;
(2) set up the fast traditional font of the high frequency of excised leaf body embryo and adventitious bud inducing system:
(2.1) formation of inductor embryo and indefinite bud: launching leaf with test-tube plantlet the 3rd to the 6th is explant; The short petiole of blade band; Blade paripheral zone zigzag leaf margin is cut; Do following processing afterwards: blade is cut into left and right sides two halves along master pulse, backsight blade size row dry the 1-2 cutter or with blade along master pulse crosscut 2-3 cutter; Be seeded in the first inductor embryo and/or indefinite bud and form medium; Perhaps, the second inductor embryo and/or indefinite bud form on the medium, and the medium that requires the explant blade back to be close to is separately down placed; In dark culturing 7-10d; The formation of inductor embryo, in dark culturing 15-20d, the formation of evoking adventive bud;
(2.2) growth of body embryo and indefinite bud and differentiation: with the first inductor embryo and/or indefinite bud form medium and, the second inductor embryo and/or indefinite bud form the explant of cultivating on the medium and are transferred on the first body embryo and/or indefinite bud growth and the differential medium; Put into climatic cabinate then; When temperature is 10 ± 2 ℃, secretly cultivates and change culturing room over to after the 3-5d and secretly cultivate 35-40d, or directly secretly cultivate 40-45d in culturing room; Middle subculture once; Cultivation through the above-mentioned first body embryo and/or indefinite bud growth and differential medium forms many buds of growing thickly on explant, the bud height is at 2cm～5cm;
(2.3) the fast numerous cultivation of adventitious buds proliferation: stem section or stem apex that above-mentioned steps (2.2) is induced the bud of growing thickly of formation to be cut into to contain 1-2 axillalry bud are transferred to the test-tube plantlet proliferated culture medium; Perhaps; Replace subculture on the second body embryo and/or indefinite bud growth and the differential medium; Breed fast numerous cultivation, every 18-22d subculture once, the propagation frequency is 10-15;
(3) rooting of vitro seedling acclimatization and transplants:
(3.1) strengthening seedling and rooting is cultivated:
Above-mentioned steps (2.3) the grow thickly bud of healthy and strong length behind the 3-4cm that grow is cut into individual plant and is transferred on the strengthening seedling and rooting medium, cultivate 25-35d, guarantee that cauline leaf grows normally, root growth is healthy and strong, and root grows to 1.5cm when above, carries out acclimatization and transplants;
(3.2) acclimatization and transplants:
At first the test-tube plantlet bottle cap is opened, after culturing room adapts to 1d, moved on to the shading screen plastic tunnel of obscurity 70-80%, at greenhouse experiment lower refining seedling 2-3d; With tweezers seedling is pressed from both sides out, the medium that clear water flush away base portion is residual is transplanted to and is equipped with in the cave dish that mixes seedling medium; Utilize intermittent spraying device or artificial water spray to keep relative moisture more than 80%; Behind domestication refining seedling 5-10d, after new root sprouting eruption, reducing gradually waters number of times and be transplanted to flowerpot carries out Routine Management;
Described bud starts medium: improvement MS+0.5～1.0 mgL
-1BA+0.5～1.0mgL
-1KT+0.1～0.2mgL
-1NAA+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described test-tube plantlet proliferated culture medium is: improvement MS+1.0～2.0mgL
-1BA+1.0～2.0mgL
-1KT+0.1～0.5mgL
-1NAA+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described first inductor embryo and/or indefinite bud form medium: improvement MS+0.5mgL
-12,4-D+0.5～1.0mgL
-1BA+0.5～2.0mgL
-1NAA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described second inductor embryo and/or indefinite bud form medium: improvement MS+0.1mgL
-12,4-D+0.5mgL
-1BA+10～30 mgL
-1NAA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described first body embryo and/or indefinite bud growth with differential medium are: improvement MS+2.0～4.0mg L
-1BA+3.0～3.5gL
-1Phytagel+20gL
-1Sucrose, and the pH value is 5.8～6.0;
Described second body embryo and/or indefinite bud growth with differential medium are: MS+2.0mgL
-1BA+1.0 mgL
-1IBA+2.0 mgL
-1KT+6.0～7.0gL
-1Agar+30gL
-1Sucrose, and the pH value is 5.8～6.0;
Described strengthening seedling and rooting medium is: 1/2 improvement MS+0.2～0.5mgL
-1IBA+0～0.05mgL
-1NAA+6.0～7.0gL
-1Agar+20gL
-1Sucrose, and the pH value is 5.8～6.0,
Described improvement MS comprises grand nutrition element, micronutrient element and organic reagent,
Wherein, the component of described grand nutrition element is following with its corresponding concentration:
Potassium nitrate 1900 mg/L;
Ammonium sulfate 1650 mg/L;
Epsom salt 370mg/L;
Calcium chloride dihydrate 440 mg/L;
Anhydrous potassium dihydrogenphosphate 170 mg/L;
Disodium ethylene diamine tetraacetate 37.3 mg/L;
Ferrous sulfate heptahydrate 27.8 mg/L,
The component of described micronutrient element is following with its corresponding concentration:
Four water manganese sulphates, 22.3 mg/L;
Zinc sulphate 8.6 mg/L;
Boric acid 6.2 mg/L;
KI 0.83 mg/L;
Sodium molybdate 0.25 mg/L;
Copper sulphate 0.025 mg/L;
Cobalt chloride 0.025 mg/L,
The component of described organic reagent is following with its corresponding concentration:
Thiamine hydrochloride 10.0 mg/L;
Nicotinic acid 1.0 mg/L;
Puridoxine hydrochloride 1.0mg/L;
Inositol 100.0 mg/L.
2. the fast numerous cultural method of photinia glabra excised leaf somatic embryo inducement according to claim 1 is characterized in that the composition of described mixing seedling medium is counted with volume ratio: vermiculite: turfy soil: garden mould=3:5:2.
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| CN102379241B (en) * | 2011-08-02 | 2013-03-27 | 上海交通大学 | Rapid propagation method for Photinia fraseri tissue culture |
| CN102422814A (en) * | 2011-10-11 | 2012-04-25 | 山西腾达种业有限公司 | Transplanting method for photinia serrulata breeding |
| CN102415339A (en) * | 2011-10-11 | 2012-04-18 | 山西腾达种业有限公司 | Rapid propagation method of photinia fraseri |
| CN102835314B (en) * | 2012-09-20 | 2014-10-08 | 重庆文理学院 | Photinia serrulata tissue culture seedling rooting culture medium and culture method for photinia serrulata tissue culture seedling rooting |
| CN102925481A (en) * | 2012-11-30 | 2013-02-13 | 山东省林业科学研究院 | Genetic transformation method for embryonic callus of photinia serrulata lamina through gene gun mediation |
| CN103636400B (en) * | 2013-12-24 | 2015-05-27 | 长春大学 | Autologous rapid propagation method for Changbai Mountain cacalia firma kom |
| CN103805631A (en) * | 2014-01-07 | 2014-05-21 | 泰安市泰山林业科学研究院 | Method for efficient gene transformation of photinia serrulata |
| CN105557111A (en) * | 2015-12-15 | 2016-05-11 | 山水园林股份有限公司 | Rapid propagation method and planting method of salix chaenomeloides |
| CN105409777A (en) * | 2015-12-16 | 2016-03-23 | 青岛百瑞吉生物工程有限公司 | Growth and differentiation culture medium for somatic embryo of pear tree |
| CN105660396B (en) * | 2016-01-12 | 2018-05-25 | 山东省林业科学研究院 | A kind of rapid propagation method of Chinese scholar tree excised leaf somatic embryo inducement |
| CN106857251B (en) * | 2017-01-22 | 2018-10-09 | 浙江农林大学 | A kind of Phoebe bournei somatic embryo and adventitious bud inducing method |
-
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Non-Patent Citations (5)
| Title |
|---|
| 何家涛.红叶石楠离体培养与高效再生体系建立.《湖北农业科学》.2010,第49卷(第7期), * |
| 朱玉球,等.光叶石楠组培快速繁殖.《浙江林学院学报》.2005,第22卷(第2期), * |
| 邓小梅，等.红叶石楠"红罗宾"的组培高效再生***的建立.《江西林业科技》.2004,(第4期), |
| 邓小梅,等.红叶石楠"红罗宾"的组培高效再生***的建立.《江西林业科技》.2004,(第4期), * |
| 黄新,等.石楠的组织培养研究.《石楠的组织培养研究》.2004,第32卷(第6期), * |
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