CN103444551B - To induce the tissue cultures test-tube plantlet preparation method that lamiophlomis rotata seed germination seedling is explant - Google Patents
To induce the tissue cultures test-tube plantlet preparation method that lamiophlomis rotata seed germination seedling is explant Download PDFInfo
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- CN103444551B CN103444551B CN201310436768.9A CN201310436768A CN103444551B CN 103444551 B CN103444551 B CN 103444551B CN 201310436768 A CN201310436768 A CN 201310436768A CN 103444551 B CN103444551 B CN 103444551B
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- lamiophlomis rotata
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Abstract
The invention discloses a kind of to induce the tissue cultures test-tube plantlet preparation method that lamiophlomis rotata seed germination seedling is explant, the method be first by lamiophlomis rotata seed through variable concentrations GA3 Fiber differentiation, spray through carbendazim again, through explant sterilization, be inoculated in MS+6-BA0.5mgL
-1+ NAA 1.0mgL
-1+ sucrose 30gL
-1+ agar 7.0gL
-1in carry out just for induction of callus, after cultivating 30d, namely explant surface grows the quality callus closely of white or yellow-white.Through callus after bud inducement is cultivated and root induction is cultivated, formative tissue culture test tube seedling.The method well solves, and during with lamiophlomis rotata strain for explant, pollution rate is large, not easily produces the problem of aseptic seedling; Establish simultaneously and be obstructed in low altitude area lamiophlomis rotata sexual reproduction, adopt tissue cultures to obtain the method for test tube seedling.
Description
Technical field
The present invention relates to a kind of method that lamiophlomis rotata tissue cultures obtains test-tube plantlet, particularly relate to a kind of tissue cultures test-tube plantlet preparation method being explant with lamiophlomis rotata Seed inducement seedling.
Background technology
Lamiophlomis rotata Lamiophlomis rotata (Benth.) Kudo is Labiatae, lamiophlomis rotata platymiscium, and this genus only has lamiophlomis rotata kind.Main product Tibet, Qinghai, Gansu, western Sichuan and northwestern Yunnan Province; Be born in the rubble beach of intensity weathering on plateau or high mountain or stone matter alpine meadow, flood land, height above sea level 2700-4500 rice.Have typical alpine plant feature, as plant is short, stem is short, well developed root system, and blade is comparatively large, pastes ground and launches.These features and its growing environment are severe cold region, dry wind is large, radiation is strong and day and night temperature is large etc. relevant.
Lamiophlomis rotata belongs to traditional Tibetan medicine, all herbal medicine among the people, controls traumatic injury, arthralgia and myalgia, the stagnation of the circulation of vital energy are sprained one's back, flows yellow water after edema, yellow water is amassed in joint, cancellous bone inflammation.In addition according to Qinghai, big white mouse femoral artery, arteria brachialis, arteria carotis crosscut are broken hemostasis trial, think that this kind has good haemostatic effect.Along with the memory space of wild lamiophlomis rotata progressively reduces, the research of lamiophlomis rotata is also more and more come into one's own.Also part is had to carry out to the research work of lamiophlomis rotata being introduced a fine variety to low altitude area.As Jin Lan etc. by by lamiophlomis rotata rhizome bud from height above sea level 4300m be wildly transplanted to the Experimental Base of height above sea level 2366m and 3100m after, to the Pollen Activity of transplanting ground lamiophlomis rotata, pollen germination and the rate that naturally terminates are added up, found that lamiophlomis rotata be transplanted to comparatively after low altitude area Pollen Activity low, without pollen germination on column cap, Natural seed setting rate is 0.Therefore, in low altitude area, the seedling of lamiophlomis rotata is obtained, more feasible in Plant Tissue Breeding mode.Then, lamiophlomis rotata strain stem is short, about 1cm, and blade is large, and tool wrinkles, and gather fine hair.When these features make employing lamiophlomis rotata strain be explant, it is very large that sterilizing obtains aseptic seedling difficulty.
Summary of the invention
The object of the present invention is to provide a kind of to induce the lamiophlomis rotata tissue cultures test-tube plantlet preparation method that lamiophlomis rotata seed germination seedling is explant.
For achieving the above object, the solution that the present invention adopts comprises the following steps:
(1) choose results then healthy full, the great-hearted lamiophlomis rotata seed of tool, is seeded in and is soaked with GA
350mgL
-1filter paper on carry out vernalization, grow to after about 1cm until seedling, the carbendazim spraying 800 times of liquid carries out the surface sterilization sterilization pretreatment of plant, then collects seedling.
(2) first the lamiophlomis rotata seedling as explant is invaded bubble 30min with 800 times of carbendazim solutions, use tap water about 1h again, put into the alcohol disinfecting 10s that superclean bench concentration is 75%, aseptic water washing 3 times, then be the mercuric chloride sterilization 5min of 0.1% by concentration, finally wash 4 ~ 6 times with sterile water concussion, the moisture blotting seedling surface with aseptic filter paper is for subsequent use;
(3) aseptically on lamiophlomis rotata seedling, mark wound with scalpel, be inoculated in callus inducing medium: MS+6-BA 0.5mg.L-1+NAA 1.0mgL
-1+ sucrose 30gL
-1+ agar 4.8gL
-1in carry out callus induction, the pH value adopted is 6.0, and cultivation temperature 20 DEG C, lighting delay number 10h/d, intensity of illumination are 1000 ~ 1500Lx, incubation time 30d;
(4) after 30d, get colors yellowish green, have the callus of strumae fine texture as explant, access inducing clumping bud medium: MS+6-BA 1.0 ~ 3.0mg+NAA 0.1 ~ 3mgL
-1+ sucrose 30gL
-1+ agar 4.8gL
-1in carry out the induction of Multiple Buds, the Medium's PH Value adopted is 6.0, and cultivation temperature is 20 DEG C, lighting delay number 12h/d, intensity of illumination are 1200 ~ 1800Lx, and after cultivating 35d, callus induction differentiates a large amount of Multiple Buds.
(5) Fiber differentiation of root, when Multiple Buds grows to 2cm, is divided into simple bud by Multiple Buds, is transferred to root media: 1/2MS+IBA 0.5mgL
-1+ NAA 0.1mgL
-1+ sucrose 15gL
-1+ agar 4.8gL
-1, after middle root induction 20d, when adventive root grows to 2-3cm, namely can be moved in the green house of high altitude localities and carry out acclimatization and transplants.
Adopt such scheme, the present invention for explant, can effectively reduce the postvaccinal pollution rate of explant with seed germination seedling, obtains lamiophlomis rotata fast organize plumule test-tube plantlet in low altitude area.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
(1) choose full, vital lamiophlomis rotata seed, after clean water, dry in the shade.Put into and soak into 50gL
-1on the filter paper of GA3,20 DEG C of cultivations, induction is sprouted until radicle and plumule breakthrough seed coat grow, average germination rate 31.4%.4d after growing, start often daily 800 times of carbendazim and spray application to plate, after spraying 4d continuously, collect seedling, clean with tap water, then dry in the shade, be the alcohol disinfecting 15s of 75% again in super-clean bench concentration, then be the mercuric chloride sterilization 5min of 0.1% by concentration, finally wash 5 times with sterile water concussion, wash the rear moisture repeatedly blotting seedling surface with sterilized filter paper;
(2) by the seedling aseptic operation cutter after disinfecting in cotyledon and stem junction, mark wound, then the surface of a wound is downward, is inoculated in MS+6-BA 0.5mgL
-1+ NAA 1.0mgL
-1+ sucrose 30gL
-1+ agar 7.0gL
-1in carry out Initial culture, the pH 7.0 adopted, cultivation temperature 20 DEG C, illumination every day 8h, intensity of illumination are 1200Lx.Incubation time 30d, explant generally expands, and grow white and flaxen callus, adding up its callus induction rate is 58.2%.
(3) callus cutting is 1cm by the relative dense callus of turning out after choosing Initial culture
3size is block, then is accessed bud inducement medium MS+6-BA 2.0mgL
-1+ NAA 0.5mgL
-1+ sucrose 30gL
-1+ agar 7.0gL
-1in cultivate, the medium pH 7.0 adopted, cultivation temperature is 20 DEG C, illumination every day 10h, intensity of illumination are 1500Lx, cultivates after 35d, and callus induction differentiation is sprouted, bud ratio 48.6%.
(4) culture of rootage, by inducing the plant corpus sprouted, is transferred to root media, 1/2 MS+6-BA 1.0mgL
-1+ NAA 0.5mgL
-1+ sucrose 20gL
-1+ agar 7.0gL
-1, start to grow adventive root after Fiber differentiation 20d, test-tube plantlet planting percent 36.7%.
Embodiment 2
Seed inducement seedling in embodiment 1 step (1) changed into, seed is after 50mg/L GA3 soaks 1 day, and receive on the filter paper after clear water immersion, induction is sprouted, average germination rate 42.2%.Other step is with embodiment 1, and Fiber differentiation about 90d obtains tissue cultures test-tube plantlet, and planting percent is 37.1%.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (1)
1., to induce the tissue cultures test-tube plantlet preparation method that lamiophlomis rotata seed germination seedling is explant, it is characterized in that comprising the steps:
(1) choose results then healthy full, the great-hearted lamiophlomis rotata seed of tool, is seeded in and is soaked with GA
350mgL
-1filter paper on carry out vernalization, grow to after about 1cm until seedling, the carbendazim spraying 800 times of liquid carries out the surface sterilization sterilization pretreatment of plant, then collects seedling;
(2) first the lamiophlomis rotata seedling as explant is soaked 30min with 800 times of carbendazim solutions, use tap water 1h again, put into the alcohol disinfecting 10s that superclean bench concentration is 75%, aseptic water washing 3 times, then be the mercuric chloride sterilization 5min of 0.1% by concentration, finally wash 4 ~ 6 times with sterile water concussion, the moisture blotting seedling surface with aseptic filter paper is for subsequent use;
(3) aseptically on lamiophlomis rotata seedling, mark wound with scalpel, be inoculated in callus inducing medium: MS+6-BA 0.5mgL
-1+ NAA 1.0mgL
-1+ sucrose 30gL
-1+ agar 4.8gL
-1in carry out callus induction, the pH value adopted is 6.0, cultivation temperature 20 DEG C, lighting delay number 10h/d, intensity of illumination are 1000 ~ 1500Lx, incubation time 30d;
(4) after 30d, get colors yellowish green, have the callus of strumae fine texture as explant, access inducing clumping bud medium: MS+6-BA 1.0 ~ 3.0mgL
-1+ NAA 0.1 ~ 3mgL
-1+ sucrose 30gL
-1+ agar 4.8gL
-1in carry out the induction of Multiple Buds, the Medium's PH Value adopted is 6.0, cultivation temperature is 20 DEG C, lighting delay number 12h/d, intensity of illumination are 1200 ~ 1800Lx, and after cultivating 35d, callus induction differentiates a large amount of Multiple Buds;
(5) Fiber differentiation of root, when Multiple Buds grows to 2cm, is divided into simple bud by Multiple Buds, is transferred to root media: 1/2MS+IBA 0.5mgL
-1+ NAA 0.1mgL
-1+ sucrose 15gL
-1+ agar 4.8gL
-1after middle root induction 20d, when adventive root grows to 2-3cm, move in the green house of high altitude localities and carry out acclimatization and transplants.
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| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
| CN106612705A (en) * | 2016-08-31 | 2017-05-10 | 中国农业科学院兰州畜牧与兽药研究所 | Technology for improving wild lamiophlomis rotata seed germination |
| CN110432150B (en) * | 2019-09-06 | 2020-11-10 | 甘肃省农业科学院生物技术研究所 | Method for obtaining and rapidly proliferating aseptic seedlings of lamiophlomis rotata |
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| CN102657086A (en) * | 2012-05-10 | 2012-09-12 | 中国科学院西北高原生物研究所 | Method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata |
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