CN104604691B - A kind of Millettia dielsiana tissue culture and rapid propagation method - Google Patents
A kind of Millettia dielsiana tissue culture and rapid propagation method Download PDFInfo
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- CN104604691B CN104604691B CN201510071050.3A CN201510071050A CN104604691B CN 104604691 B CN104604691 B CN 104604691B CN 201510071050 A CN201510071050 A CN 201510071050A CN 104604691 B CN104604691 B CN 104604691B
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Abstract
The invention discloses a kind of Millettia dielsiana tissue culture and rapid propagation method, the method is chosen by outer implant and is processed, initial culture, differentiation culture, enrichment culture, root culture define the Millettia dielsiana group culturation rapid propagating technology of complete set.It is short that the present invention has cultivation cycle, breeding coefficient is high, reproduction speed is fast, rooting efficiency is good, keep the features such as the excellent hereditary character of maternal plant, make Millettia dielsiana can carry out rapid propagation in factory, its growth coefficient is up to 15.0～18.0, with with seed germination, simple bud carries out tissue-culturing rapid propagation with the aseptic seedling that stem with bud is material acquisition and compares, its cultivation effect is good, propagation efficiency is high, decrease seed sprout growth in culture medium and become the stage of aseptic seedling, thus shortening cultivation cycle, medicinal for one, the good material viewing and admiring dual-purpose provides Fast-propagation production technology.
Description
Technical field
The invention belongs to Millettia dielsiana technical field of tissue culture, be specifically related to a kind of Millettia dielsiana tissue culture and rapid propagation method.
Background technology
Millettia dielsiana (MillettiadielsianaHarms) has another name called CAULIS SPATHOLOBI, big Fructus Crotonis, mountain HUDOU, and for the perennial half evergreen bejuco of Papilionaceae Millettia, great majority are all also in wild state.Its rattan, root are all pharmaceutically acceptable, can circulation of qi promoting, antipyretic, hemostasis, evil-wind expelling and dampness removing, dredge the meridian passage, seed also can as oil plant material, floral leaf is also beautiful, pattern is many, bright-colored, and the florescence is long, gardens should can be used for frame, artificial hillock, metope, Po Kan, the greening of quilt, roof, potted landscape etc., be a kind of good medicinal, view and admire dual-purpose garden plants.In recent years, owing to Millettia dielsiana is that a kind of important medicinal plants is (from the seventies, the Millettia dielsiana production for Chinese patent medicine is extensively excavated) in area, Guangdong and Guangxi Provinces, it is again that a class can as excellent ornamental plants in garden, so, quite being liked by numerous citizen at present, on market, the demand for Millettia dielsiana constantly increases, and makes Millettia dielsiana suffer the phenomenon of xcessive digging increasingly severe.How making various merits genetic stability in offspring individuals of the excellent maternal plant of Millettia dielsiana, the advantages such as breeding coefficient is high, reproduction speed is fast, rooting efficiency is good, numerous soon by plant tissue culture is most effective approach.The planting seed of Millettia dielsiana, rattan cuttage and press strip method are bred, and its breeding coefficient is all not high, and therefore, the tissue-culturing rapid propagation research work strengthening Millettia dielsiana has great importance.
About Millettia dielsiana tissue-culturing rapid propagation report 1 section (1, Huang Yanning, Peng Jinhui, Zhou Hongcan, Deng. the tissue culture and rapid proliferation [J] of Millettia dielsiana. Plant Physiology Communications, 2007.8,43 (4): 751.) and Master's thesis 1 section (2, Huang Yanning. the reproduction technique of Millettia dielsiana and Landscape Application research).Time in document 1 with ripe haricot for group training material, the limited time system of drawing materials, draw materials when being only annual pods mature in October, and the germination rate that seed is in culture medium is not high, each seed can only send a strain aseptic seedling, carrying out cutting again through aseptic seedling and carry out enrichment culture, breeding coefficient is not high, is 5.0.Time in document 2 with simple bud and stem with bud for group training material, when drawing materials, consumptive material is relatively more, it is necessary to pluck substantial amounts of edible tender branch, and breeding coefficient is not high, is 5.56.The present invention is with blade for group training material, clump bud inducement proliferation and differentiation is carried out by the training of wound healing group, can breed with the speed of tens times at short notice, really can realize the saying of " to my a piece of greenery; also your a piece of forest ", the propagation method being material with seed and stem with bud, its growth coefficient and speed are beyond one's reach at short notice.Meanwhile, utilizing the plumelet that the inventive method obtains, rooting rate is high, and transplanting survival rate is high, has been truly realized the development of industrialization.
At present, relevant Millettia dielsiana vane group culturation rapid propagating technology is not yet reported, and the method has convenience of drawing materials, consumptive material is few, cultivation cycle is short, breeding coefficient is high, reproduction speed is fast, rooting efficiency is good, keep the features such as the excellent hereditary character of maternal plant, makes Millettia dielsiana can carry out rapid propagation in factory.
Summary of the invention
It is an object of the invention to there are provided a kind of Millettia dielsiana tissue culture and rapid propagation method, tissue culture and rapid propagation method provided by the invention, have breeding coefficient height, reproduction speed is fast, rooting efficiency good, keep the features such as the excellent hereditary character of maternal plant, Millettia dielsiana high quality seedling can be obtained in the short term, and rapid propagation in factory can be carried out.
For reaching above-mentioned purpose, the present invention takes techniques below measure:
The technical solution adopted in the present invention is to be chosen and processed, just define for callus culture, differentiation culture, enrichment culture, root culture the Millettia dielsiana group culturation rapid propagating technology of complete set by outer implant.
A kind of Millettia dielsiana tissue culture and rapid propagation method, its step is as follows:
(1) outer implant is chosen and is processed:
Outer implant is chosen: growth selection is healthy and strong, be propagating materials without the Millettia dielsiana plant of pest and disease damage, and it is outer implant that mid or late March adopts the young sprout young leaflet tablet that one, biennial branch take out then, after sterilizing, is cut into the blockage of 1cm × 1cm.
It is robust growth that described outer implant material selects, young leaves without pest and disease damage, the just expansion on lignified edible tender branch that has no mechanical damage, do not have.
Outer implant processes: rinse well in placement bottle by the young leaflet tablet fetched with a small amount of detergent, bottleneck monolayer medical gauze is sealed, and is placed in tap water and rinses 60min, then pours out the water in bottle, material is placed in superclean bench, first with 75% ethanol postincubation 30s, carry out surface sterilization, then with aseptic water washing 5～6 times, then 5min is processed with 0.1%HgCl, use aseptic water washing 5～6 times again, then blade surrounding is excised, be then cut into the blockage of 1cm × 1cm.
(2) just for callus culture: the blade inoculation that bacterium of having gone out cuts block carries out initial culture in the culture bottle equipped with initial culture base, during inoculation, leaf back is downward, is close in culture medium.Material is cultivated and is all placed on the illumination cultivation indoor that temperature is 25 DEG C ± 2 DEG C, and light application time is 14h, and intensity of illumination is 1500～2000Lx, cultivates 25～35d and is proceeded in proliferation and differentiation culture medium by the callus cultivated.
The formula of described initial culture base is: MS+6-BA0.5mg/L+NAA1.5mg/L+IAA0.1mg/L+ agar 8g/L+ sucrose 30g/L, pH value is 5.5～6.0.
(3) differentiation culture: the fritter that the callus of initial culture is cut into 0.5cm × 0.5cm is inoculated into equipped with carrying out differentiation culture in the culture bottle of division culture medium, and pH value, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2).Calli Differentiation becomes the differentiation rate of bud clump to be 60%～70%.
The formula of described division culture medium is: MS+6-BA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L.
(4) enrichment culture: the bud clump of acquisition is cut into simple bud (bud length 0.5～1.0cm) and is inoculated in the culture bottle of proliferated culture medium and carries out enrichment culture, pH value, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2), form Multiple Buds.Treat that the high 0.5～1.0cm of bud can cut again, be placed in proliferated culture medium, under same culture conditions, proceed enrichment culture, expanding propagation plant.
The formula of described proliferated culture medium is: MS+6-BA2.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L.
(5) root culture: by the not final singling length on bud clump to 3～5cm, when having 2～4 complete blades, cuts into individual plant not final singling and is inoculated into and carries out root culture.Cultivation temperature, light application time, incubation time are all just identical for callus culture with (2), intensity of illumination is 800～1000Lux, rooting rate up to more than 90%, until not final singling root in root media have 2～3, root length be 2～5cm time prepare bottle outlet.
The formula of described root media is: MS+IBA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L, pH value is 5.5～6.0.
Operation after bottle outlet conventionally scheme carries out, be referred to Master's thesis (Huang Yanning. the reproduction technique of Millettia dielsiana and Landscape Application research)
Compared with prior art, it is an advantage of the current invention that:
nullMake public for the first time a kind of Millettia dielsiana tissue culture and rapid propagation method，The method is undertaken just for callus culture by blade、Differentiation culture、Enrichment culture、Root culture obtains breeding coefficient height、Reproduction speed is fast、Rooting efficiency is good、Keep the Millettia dielsiana high quality seedling of the features such as the excellent hereditary character of maternal plant，Realize rapid propagation in factory，Compared with carrying out tissue-culturing rapid propagation with the aseptic seedling that seed is material sprouting acquisition，Its growth coefficient is high，Up to 15.0～18.0，Reproduction speed is fast，Decrease seed sprout growth in culture medium and become the stage of aseptic seedling，Thus shortening cultivation cycle，With the outer implant of the blade blockage of a 1cm × 1cm at initial culture 30d，Differentiation、The each 30d of enrichment culture，Each breeding rate is 16 times of calculating，Can emerge according to practical condition for one end of the year and be about 42.95 hundred million strains，Medicinal for one、The good afforestation material viewing and admiring dual-purpose provides Fast-propagation production technology.
| Natural law (d) | 90 | 120 | 150 | 180 | 210 | 240 | 270 | 300 | 330 |
| Seedling number () | 1 | 16 | 256 | 4096 | 65536 | 1048576 | 16777216 | 268435456 | 4294967296 |
Accompanying drawing explanation
Fig. 1 is the blade initial culture effect schematic diagram of the Millettia dielsiana tissue-culturing rapid propagation of embodiment 1.
Fig. 2 is the blade cultivation effect schematic diagram of the Millettia dielsiana tissue-culturing rapid propagation of embodiment 1.
Fig. 3 is the rooting efficiency schematic diagram of the Millettia dielsiana blade tissue-culturing rapid propagation of embodiment 1.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1:
A kind of Millettia dielsiana tissue culture and rapid propagation method, comprises the following steps
(1) outer implant is chosen and is processed:
Outer implant is chosen: growth selection is healthy and strong, be propagating materials without the Millettia dielsiana plant of pest and disease damage, and it is outer implant that mid or late March adopts the young sprout young leaflet tablet that one, biennial branch take out then.
It is robust growth that described outer implant material selects, young leaves without pest and disease damage, the just expansion on lignified edible tender branch that has no mechanical damage, do not have.
Outer implant processes: rinse well in placement bottle by the young leaflet tablet fetched with a small amount of detergent, bottleneck monolayer medical gauze is sealed, and is placed in tap water and rinses 60min, then pours out the water in bottle, material is placed in superclean bench, first with 75% ethanol postincubation 30s, carry out surface sterilization, then with aseptic water washing 5～6 times, then 5min is processed with 0.1%HgCl, use aseptic water washing 5～6 times again, then blade surrounding is excised, be then cut into the blockage of 1cm × 1cm.
(2) just for callus culture: the blade inoculation that bacterium of having gone out cuts block carries out initial culture to initial culture base MS+6-BA0.5mg/L+NAA1.5mg/L+IAA0.1mg/L+ agar 8g/L+ sucrose 30g/L, pH value is 5.8, during inoculation, leaf back is downward, is close in culture medium.Culture medium is divided in the culture bottle of 350ml, every bottle of subpackage 40ml, 2 outer implant of every bottle graft kind, tightens sealing with plastic bottle closure.Material is cultivated and is all placed on the illumination cultivation indoor that temperature is 25 DEG C ± 2 DEG C, and light application time is 14h, and intensity of illumination is 1800Lx, cultivates 30d, is proceeded in proliferation and differentiation culture medium by the callus cultivated.
The culture bottle of described 350ml is the Clear glass bottles and jars that the group training without air-vent plastic bottle closure, superhigh temperature resistant is special.
(3) differentiation culture: the callus of initial culture is cut into the fritter of 0.5cm × 0.5cm and is inoculated into division culture medium MS+6-BA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L and carries out proliferation and differentiation cultivation, 5 calluss of every bottle graft kind.PH value, culture bottle, subpackage volume, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2).The bud ratio of callus is up to 65%.
(4) enrichment culture: the bud clump of acquisition is cut into simple bud (the long 0.8cm of bud) and is inoculated into proliferated culture medium MS+6-BA2.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L and carries out enrichment culture, 3 simple buds of every bottle graft kind.PH value, culture bottle, subpackage volume, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2).Shoot propagation coefficient is 18.0, it is possible to obtain substantial amounts of plant at short notice.Treat that bud height 0.8cm can cut again, be placed in proliferated culture medium, under same culture conditions, proceed enrichment culture, expanding propagation plant.
(5) root culture: by the not final singling length on bud clump to 3～5cm, when having 2～4 complete blades, cuts into individual plant not final singling and is inoculated into MS+IBA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L sucrose and carries out root culture.PH value, culture bottle, subpackage volume, every bottle graft kind number, cultivation temperature, light application time, incubation time are all just identical for callus culture with (2), intensity of illumination is 1000Lx, rooting rate is up to 92%, until not final singling root in root media have 2～3, root length get final product bottle outlet when being 2～5cm, after bottle outlet routinely operation (with reference to Master's thesis (Huang Yanning. the reproduction technique of Millettia dielsiana and Landscape Application research) in technical scheme), after 60 days, survival rate is 90.62%.
Above-described embodiment does not limit the present invention in any form, and the present invention is also not limited to the example above.The technical scheme that the mode with equivalent replacement or equivalent transformation of the art obtains, all falls within protection scope of the present invention.
Claims (2)
1. a Millettia dielsiana tissue culture and rapid propagation method, its step is as follows:
(1) outer implant is chosen and is processed:
Outer implant is chosen: growth selection is healthy and strong, be propagating materials without the Millettia dielsiana plant of pest and disease damage, and it is outer implant that mid or late March adopts the young sprout young leaflet tablet that one, biennial branch take out then, after sterilizing, is cut into the blockage of 1cm × 1cm；
(2) just for callus culture: the blade inoculation that bacterium of having gone out cuts block carries out initial culture in the culture bottle equipped with initial culture base, during inoculation, leaf back is downward, is close in culture medium；Material is cultivated and is all placed on the illumination cultivation indoor that temperature is 25 DEG C ± 2 DEG C, and light application time is 14h, and intensity of illumination is 1500～2000Lx, cultivates 25～35d and is proceeded in division culture medium by the fritter that the callus cultivated is cut into 0.5cm × 0.5cm；
The formula of described initial culture base is: MS+6-BA0.5mg/L+NAA1.5mg/L+IAA0.1mg/L+ agar 8g/L+ sucrose 30g/L, pH value is 5.5～6.0；
(3) differentiation culture: the fritter that the callus of initial culture is cut into 0.5cm × 0.5cm is inoculated into equipped with carrying out differentiation culture in the culture bottle of division culture medium, and pH value, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2)；
The formula of described division culture medium is: MS+6-BA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L；
(4) enrichment culture: the bud clump of acquisition is cut into simple bud, bud length 0.5～1.0cm, it is inoculated in the culture bottle of proliferated culture medium and carries out enrichment culture, pH value, cultivation temperature, light application time, intensity of illumination, incubation time are all just identical for callus culture with (2), form Multiple Buds；Treat that the high 0.5～1.0cm of bud can cut again, be placed in proliferated culture medium, under same culture conditions, proceed enrichment culture, expanding propagation plant；
The formula of described proliferated culture medium is: MS+6-BA2.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L；
(5) root culture: by the not final singling length on bud clump to 3～5cm, when having 2～4 complete blades, cuts into individual plant not final singling and is inoculated into and carries out root culture；Cultivation temperature, light application time, incubation time are all just identical for callus culture with (2), intensity of illumination is 800～1000Lux, rooting rate up to more than 90%, until not final singling root in root media have 2～3, root length be 2～5cm time prepare bottle outlet；
The formula of described root media is: MS+IBA3.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L, pH value is 5.5～6.0.
2. method according to claim 1, it is characterised in that:
It is robust growth that described outer implant material selects, young leaves without pest and disease damage, the just expansion on lignified edible tender branch that has no mechanical damage, do not have；
Outer implant processes: rinse well in placement bottle by the young leaflet tablet fetched with a small amount of detergent, bottleneck monolayer medical gauze is sealed, it is placed in tap water and rinses 60min, then the water in bottle is poured out, material is placed in superclean bench, first with 75% ethanol postincubation 30s, carry out surface sterilization, use aseptic water washing 5～6 times again, then use 0.1%HgCl2Process 5min, then with aseptic water washing 5～6 times, then by the excision of blade surrounding, it is then cut into the blockage of 1cm × 1cm.
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| CN106962188A (en) * | 2017-03-01 | 2017-07-21 | 防城港市防城区蚕种场 | Beautiful millettia root seed directly induces the method from bud and fast breeding seedling |
| CN110463612B (en) * | 2019-09-27 | 2023-05-02 | 玉林师范学院 | Primary callus and bud induction method of Yulang umbrella |
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| CN103039361B (en) * | 2013-01-11 | 2013-12-18 | 广西壮族自治区药用植物园 | Method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings |
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