CN103535285B - The Fast-propagation of honeysuckle seedling and in-vitro conservation method - Google Patents
The Fast-propagation of honeysuckle seedling and in-vitro conservation method Download PDFInfo
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Abstract
Fast-propagation and the in-vitro conservation method of honeysuckle seedling are provided, comprise aseptic clonal foundation, Fast-propagation, culture of rootage, heeling in and transplanting of test-tube plantlet, Plantlet in vitro step.Honeysuckle has higher health care, views and admires, medicinal and ecological functions, is one of distinctive rare traditional Chinese medicine of China, and number has the good reputation of " penicillin " among Chinese medicine.The invention provides in-problem solution and best culture technique in the training of honeysuckle group.Method growth rate of the present invention reaches 1:4-6, transplanting survival rate reaches more than 90%, with paclobutrazol (PP333) and indolebutyric acid (IBA) with the use of the rooting efficiency obtaining 99%, the test-tube plantlet genetic stability that the blastogenesis obtained becomes, the aseptic clone of honeysuckle set up by the method is in incubation almost pollution-free generation, and explant very fast restoration ecosystem on medium, for large-scale industrialized production honeysuckle high quality seedling provides technical support.
Description
Affiliated field:
The invention belongs to biological technical field, particularly, relate to a kind of Fast-propagation and in-vitro conservation method of honeysuckle seedling.
Background technology:
Honeysuckle be there is higher health care, view and admire, species that medicinal and ecological functions are integrated, be one of distinctive rare traditional Chinese medicine of China, number has the good reputation of " penicillin " among Chinese medicine.Particularly role in the control of resist SARS in 2003, is subject to people and pays close attention to, become the focus development object in Caprifoliaceae resource plant.Modern pharmacological research shows: honeysuckle has broad-spectrum antiseptic, antiviral, antitumor, strengthens immune and antipyretic, the multiple biologically active such as anti-inflammatory, cholagogic protect the liver, reducing blood lipid, antifertility, hemostasis antiulcer.On market, both production and marketing thrive, dried flower flower bud price goes up year by year.But the seedling of seedling particularly high yield and high quality kind is in great shortage.The eighties in 20th century, people once started honeysuckle group training research, but until relevant research in recent years just increases gradually.But the Problems existing of honeysuckle group training at present limits the Fashion and Evolution of honeysuckle industry.Subject matter is: 1. explant sterilization difficulty; 2. subculture period short (1-2 month) and Plantlet in vitro nobody do; 3. test tube transplanting survival rate is low, and Test tube seedlings cost is high; 4. the real large-scale application of the test-tube plantlet of tissue cultures is little in what produce, the report of rarely seen Li Hong etc. (2007) honeysuckle group training factorial praluction and cultivation management technology, but the integral level of factory culturing seedling is not high, crucial production link technology is weak, cause reproduction coefficient low, greatly limit applying of famous and precious kind.So far, in prior art there are no the Fast-propagation of honeysuckle seedling and the report of in-vitro conservation method.
Summary of the invention:
For prior art above shortcomings part, the present invention aims to provide a kind of Fast-propagation and in-vitro conservation method of honeysuckle seedling.In-problem solution, technology and optimal culture condition in providing honeysuckle group to train, the integral level overcoming the factory culturing seedling that prior art exists is not high, the shortcoming that reproduction coefficient is low, for large-scale industrialized production honeysuckle high quality seedling provides technical support.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The Fast-propagation of honeysuckle seedling and in-vitro conservation method, comprise aseptic clonal foundation, Fast-propagation, culture of rootage, heeling in and transplanting of test-tube plantlet, Plantlet in vitro step, described aseptic clonal foundation is extracting honeysuckle budling or young stem, first use 70% ethanol disinfection 20s, to go out 6min with the 0.1% mercuric chloride aqueous solution again, clean with aseptic water washing, Fast-propagation is carried out in access medium, after young stem sterilization, remove blade, segment, be placed on medium 1. on cultivate 25d, take off bud that stem section sends medium 2. on cultivate 60d, (subculture cycle is 30d subculture 1 time) 3. goes up the cultivation through 30d at medium again, described culture of rootage be medium 3. on cultivate, after 3-4 clump bud/bottle graft kind 30d, choose budling height at more than 3cm, intercepts be placed on medium 4. upper cultivation 14d-17d start to take root, described test-tube plantlet to heel in transplanting be before transplanting transition, choose culture of rootage 20d test tube bottle seedling, first be placed in Simple pergola, move into after 10d and plant in seedling matrix, cover shifting out moisturizing above seedling with Polypropylence Sheet just transplanting seedlings in 1-2 week, grow after Xin Gen or young leaves until seedling and remove Polypropylence Sheet, and low level management moves into land for growing field crops after cultivating 30d, the culture type of described Plantlet in vitro vitro rooting in test tube seedling carries out Plantlet in vitro.
The Fast-propagation of described honeysuckle seedling and in-vitro conservation method, described medium adopts: minimal medium is that 1/2MS(MS macroelement reduces by half, all the other compositions and MS with), the medium that induction stem segment length goes out axillalry bud is 1. 1/2MS+BA0.5mg/L+NAA0.2mg/L; The medium of inducing young stem section clump Shoot propagation is 2. 1/2MS+BA0.5mg/L+IBA0.01mg/L and 3. 1/4MS+BA0.3mg/L; The medium of root induction is 4. 1/2MS+IBA3mg/L+PP3330.5mg/L; Medium 1. 2. 3. sugared concentration be 3%, medium 4. sugared concentration is 2%; Condition of culture is: all medium PH5.8, cultivation temperature 26 ± 2 DEG C, light application time 12h.d
-1, intensity of illumination 20-40 μm of ol.m
-2.S
-1.
The Fast-propagation of described honeysuckle seedling and in-vitro conservation method, described cultivation seedling matrix selects new loess and leaf mould half and half, mix, thimerosal is made again with 20 parts, 1 part of formaldehyde+water, water even by the cultivation seedling matrix mixed, take the photograph agglomerating to plant seedling matrix hand, hand scatters, and rolling into a ball also scatters is as the criterion, open after sealing 24 hours with Polypropylence Sheet, cultivation seedling matrix is loaded in bag of transplanting seedlings stand-by.
The Fast-propagation of described honeysuckle seedling and in-vitro conservation method, described transplanting is by test-tube plantlet in taking-up blake bottle, washes out the sticky agar of test-tube plantlet root, do not injure blade and the tip of a root with flowing water, moving into fills in the plastic sack of matrix, waters sufficient normal root water and is placed on the ground not having direct sunlight.
The Fast-propagation of described honeysuckle seedling and in-vitro conservation method, described test-tube plantlet to heel in transplanting be before transplanting transition, choose culture of rootage 20d, height 4.5-5.5cm, the test-tube plantlet of below root length 0.5cm, first be placed in topped asbestos shingle on canopy, all around 70% shading screen, canopy and divide in the Simple pergola of two, place blake bottle to add intense light irradiation, move into after 10d and plant in seedling matrix.
The Fast-propagation of described honeysuckle seedling and in-vitro conservation method, described Plantlet in vitro is with vitro rooting in test tube seedling light culture at 4 DEG C, and in incubation, under making the former condition of culture of honeysuckle culture, every 20d-30d subculture once, can extend subculture cycle to 1 year.
Compared with prior art, method of the present invention not only reduces workload, also avoid the possibility that continuous subculture causes hereditary change, also adds capacity in vitro Germplasm Bank, is also that preservation and the sustainable use of the excellent genes of honeysuckle provides guarantee.Growth rate reaches 1:4-6, transplanting survival rate reaches more than 90%, with paclobutrazol (PP333) and indolebutyric acid (IBA) with the use of the rooting efficiency obtaining 99%, the test-tube plantlet genetic stability that the blastogenesis obtained becomes, the aseptic clone of honeysuckle set up by the method is in incubation almost pollution-free generation, and explant can very fast restoration ecosystem on medium.
Embodiment:
Further illustrate essentiality content of the present invention by the specific embodiment of the invention below, but do not limit the present invention with this.
Embodiment 1:
One, aseptic clonal foundation
Material formal name used at school: honeysuckle Lonicera japonica Thnnb.
The sterilizing of explant material is the important step in group training, and because of stem of Flos Lonicerae hollow, pubescence and thin glandular hairs are dredged in outside, more difficult in the initial sterilizing of group training.Both required the microorganism thoroughly killing external-talent surface, again the least possible injury external-talent tissue and superficial cell, by reaching ideal effect to the research of sterilisation program, specific practice is as follows:
1, the honeysuckle Lonicera japonica Thnnb. of fine quality that field or land for growing field crops filter out high yield lives, and (plant type is compact for plant, each axil opens the plant that number of flowers reaches about 30), move in basin and be placed on indoor maintenance, first to bind the growing of (removing top stem apex) favourable axillalry bud, 10d waters 1 time, water is that 1/4MS(MS macroelement is reduced to 1/4, all the other compositions are identical but remove organic principle)+BA0.25mg/L composition, about 10d axillalry bud reveals, and be grown to new young stem, when young stem is about 4cm, take off stand-by.
2, (plant type is compact in the excellent strain of honeysuckle Lonicera japonica Thnnb. taked in field, each axil opens the plant that number of flowers reaches about 30) give birth to stem section then, being about 30cm inserts in the brown bottle of dress water, cultivate 1-2d and change water 1 time, water is running water, about 10d axillary bud growth is new young stem, when young stem is with 2-3 joint, cuts stand-by.
The budling (or young stem) that above-mentioned two kinds of modes are taken off first uses 70% ethanol disinfection 20s, discharges air between external-talent Mao Yumao, is convenient to disinfectant liquid and can enters and reach material sterilizing object thoroughly.To go out 6min with the 0.1% mercuric chloride aqueous solution again, clean with aseptic water washing, namely in accessible preprepared medium.The aseptic clone of honeysuckle set up by the method is in incubation almost pollution-free generation, and explant very fast restoration ecosystem on medium.
" blunt emerald green flower bud " breeding that one of them material source specifically selected of the present invention is cultivated in Hunan, after introducing a fine variety to Chongqing, screen again " the emerald green flower bud No. 1 of changing " cultivated, from in emerald green flower bud No. 1 plantation field, Chongqing, with the naked eye select plant type compact, each axil is opened the plant that number of flowers reaches about 30 and is made material.
Two, condition of culture: minimal medium is that 1/2MS(MS macroelement reduces by half, all the other compositions and MS are together), the medium that induction stem segment length goes out axillalry bud is: 1. 1/2MS+BA0.5mg/L (unit is as follows)+NAA0.2; The medium of young stem section clump Shoot propagation is induced to be: 2. 1/2MS+BA0.5+IBA0.01 and 3. 1/4MS+BA0.3; The medium of root induction is: 4. 1/2MS+IBA3+PP3330.5; Medium 1. 2. 3. sugared concentration be 3%, medium 4. sugared concentration is 2%.Condition of culture is: medium PH is 5.8, cultivation temperature 26 ± 2 DEG C, light application time 12h.d
-1, intensity of illumination 20-40 μm of ol.m
-2.S
-1.
Three, Fast-propagation
Children's stem, after sterilization, removes blade, is trimmed to the segment being about 1cm band 1 joint, is placed on medium and 1. above cultivates, and about after 25d cultivates, each stem director Duan Jie goes out axillalry bud 1-2, and inductivity is 100%, and the mean of the bud of every block of material is 1.7.Take off bud that stem section sends medium 2. on to cultivate 60d(subculture cycle be 30d), just built up honeysuckle Fast-propagation clone, multiplication rate can reach 1:3, more 2. goes up the cultivation through 30d at medium, and growth rate can reach 1:4-6.Reach the requirement of honeysuckle factorial praluction.Clump shoot regeneration mode has two kinds: one is that stem section axil grows, and takes the mode of aseptic cuttage, can breed down continuously after cutting; Another kind of mode is form Multiple Buds in stem segment base portion incision, produce position still at axil place, because it is sufficient to contact medium nutrition supply herein, bud occurs and grows very fast, so be the form appearance of Multiple Buds, the material that this medium is cultivated, produces without callus, illustrates that the test-tube plantlet inheritance that the blastogenesis that this Clonal regeneration mode obtains becomes is stable.
After the propagation clump bud bottle number of honeysuckle reaches requirement, (this seedling number will need producing seedling number and monthly planned production per year according to market decides.), control to deposit the female bottle number of frame very crucial, because deposit frame propagation bottle number too much in certain subculture cycle internal cause manpower or equipment deficiency, cause propagation not complete on time, overstock bottle seedling quality and decline, abandon and can cause waste.If it is few to deposit frame propagation bottle quantity, cause that production task is complete not to be become, loss economically and prestige can be brought to reduce.Therefore strictly to control to deposit frame and breed total bottle number.Will consider that propagation seedling growing way can occur in seedling incubation in addition bad, budling is withered, the phenomenon such as yellowing leaf and pollution, and bottle number bred by the frame of depositing of the seedling number increase by 10% or 20% when formulating the production schedule than actual demand.
Four, culture of rootage
Medium 3. on cultivate, after 3-4 clump/bottle bud grafting kind 30d, every bottle highly can be reached more than 3-4cm budling about 5, simultaneously also has propagation, the moon growth rate be 1:4.Choose budling height at more than 3cm, intercepts and be placed on that medium is 4. upper cultivates, 14d-17d just starts to take root, and along with incubation time extends, reach 99% rooting rate, every strain 2-5 bar root, root is about 0.2cm slightly, plant strain growth stalwartness.
In currently available technology report Rooting, the not high time-consuming length of rooting rate on the one hand, employ NAA in the medium on the other hand, while induction root occurs, produce a large amount of callus, the root that this test-tube plantlet produces is not the root of the endogenous origin produced by pericycle, does not exist and links closely, can not survive after transplanting between adventive root and plant.The invention with paclobutrazol (PP333) and indolebutyric acid (IBA) with the use of obtaining desirable rooting efficiency.
Five, the heeling in and transplanting of test-tube plantlet
Through the honeysuckle test-tube plantlet of culture of rootage, residing environment is superior, and as nutrition supply is sufficient, temperature humidity is suitable, and shifting out blake bottle will row autophyting growth, adds the change of temperature humidity in environment, is difficult to adapt to, causes test-tube seedling transplanting survival rate not high.For addressing this problem, the present invention, before transplanting transition, chooses culture of rootage 20d test tube bottle seedling (test-tube plantlet height about 5cm, below root length 0.5cm), puts on balcony built Simple pergola.Canopy is high 1 meter, and length is determined according to need, upper topped asbestos shingle, 70% shading screen all around of canopy, divides two in canopy, places blake bottle to add intense light irradiation, and about 10d just can move into and plant in seedling matrix.Plant seedling matrix and select new loess and leaf mould half and half, mix, thimerosal is made again with 20 parts, 1 part of formaldehyde+water, the cultivation seedling matrix mixed is watered even, take the photograph agglomerating hand group of scattering and also scatter to plant seedling matrix hand and be as the criterion, open after sealing 24 hours with Polypropylence Sheet, cultivation seedling matrix is loaded in bag of transplanting seedlings stand-by.Open bottle cap carefully take out test-tube plantlet in blake bottle by adding the bottle seedling after intense light irradiation.Do not injure blade and the tip of a root, with flowing water wash out test-tube plantlet root the agar that glues, move into and fill in the plastic sack of matrix, water sufficient normal root water and be placed on the ground not having direct sunlight.Cover shifting out above seedling at available Polypropylence Sheet in 1-2 week of just transplanting seedlings, moisturizing, progressively opens later, grows remove Polypropylence Sheet after Xin Gen or young leaves and low level management is cultivated about 30d and moved into land for growing field crops, survival rate more than 90% until seedling.
Six, Plantlet in vitro
First culture type in Plantlet in vitro is selected, the present invention at room temperature, the comparison of subculture cycle has been carried out (with culture yellowing leaf to honeysuckle Multiple Buds and test-tube plantlet, the withered of budling is standard more than 20%, whether determine subculture, result shows: Multiple Buds subculture cycle is 30d, and vitro rooting in test tube seedling subculture cycle is 4 months.The present invention carries out Plantlet in vitro with the culture type of vitro rooting in test tube seedling.Mainly take restrict growth conservation (Slow growth conservation) a middle or short term Plantlet in vitro mode, testing growth inhibitor CCC1mg/L (under unit with), 5,10; PP3330.5,1,5 and ABA0.5 after, do not obtain desirable result (test-tube plantlet growth is repressed has occurred callus simultaneously, yellowing leaf and the phenomenon such as budling is withered).The present invention adopts again the method (light culture 4 DEG C) reducing cultivation temperature to carry out Plantlet in vitro.Subculture cycle to 1 year can be extended.In incubation, under former condition of culture, honeysuckle culture 20d-30d must subculture.
To sum up, method of the present invention not only reduces workload, also avoid the possibility that continuous subculture causes hereditary change, also adds capacity in vitro Germplasm Bank, is also that preservation and the sustainable use of the excellent genes of honeysuckle provides guarantee.Growth rate reaches 1:4-6, transplanting survival rate reaches more than 90%, with paclobutrazol (PP333) and indolebutyric acid (IBA) with the use of the rooting efficiency obtaining 99%, the test-tube plantlet genetic stability that the blastogenesis obtained becomes, the aseptic clone of honeysuckle set up by the method is in incubation almost pollution-free generation, and explant material very fast restoration ecosystem on medium.
Claims (1)
1. the Fast-propagation of honeysuckle seedling and in-vitro conservation method, comprise aseptic clonal foundation, Fast-propagation, culture of rootage, heeling in and transplanting of test-tube plantlet, Plantlet in vitro step, described aseptic clonal foundation is extracting honeysuckle budling or young stem, first use 70% ethanol disinfection 20s, use 0.1% mercuric chloride aqueous solution sterilizing 6min again, clean with aseptic water washing, Fast-propagation is carried out in access medium, after young stem sterilization, remove blade, segment, be placed on medium 1. on cultivate 25d, take off bud that stem section sends medium 2. on cultivate 60d, subculture cycle is 30d subculture 1 time, 3. the cultivation through 30d is gone up again at medium, described culture of rootage be medium 3. on cultivate, after 3-4 clump bud/bottle graft kind 30d, choose budling height at more than 3cm, intercepts be placed on medium 4. upper cultivation 14d-17d start to take root, described test-tube plantlet to heel in transplanting be before transplanting transition, choose the test tube bottle seedling of culture of rootage 20d, first be placed in Simple pergola, move into after 10d and plant in seedling matrix, just transplanting seedlings in 1-2 week, covering shifting out moisturizing above seedling with Polypropylence Sheet, growing after Xin Gen or young leaves until seedling and removing Polypropylence Sheet, move into land for growing field crops after cultivating 30d, described Plantlet in vitro carries out Plantlet in vitro by the culture type of vitro rooting in test tube seedling,
Described medium adopts: minimal medium is 1/2MS (MS macroelement reduces by half, all the other compositions and MS with), and the medium that induction stem segment length goes out axillalry bud is 1. 1/2MS+BA0.5mg/L+NAA0.2mg/L; The medium of inducing young stem section clump Shoot propagation is 2. 1/2MS+BA0.5mg/L+IBA0.01mg/L and 3. 1/4MS+BA0.3mg/L; The medium of root induction is 4. 1/2MS+IBA3mg/L+PP3330.5mg/L; Medium 1. 2. 3. sugared concentration be 3%, medium 4. sugared concentration is 2%; Condition of culture is: all medium PH 5.8, cultivation temperature 26 ± 2 DEG C, light application time 12h.d
-1, intensity of illumination 20-40 μm of ol.m
-2.S
-1;
Described cultivation seedling matrix selects new loess and leaf mould half and half, mix, thimerosal is made again with 20 parts, 1 part of formaldehyde+water, the cultivation seedling matrix mixed is watered even, take the photograph agglomerating to plant seedling matrix hand, hand scatters, and rolling into a ball also scatters is as the criterion, and opens, load in bag of transplanting seedlings stand-by by cultivation seedling matrix with Polypropylence Sheet after sealing 24 hours;
Described transplanting is the test-tube plantlet will taken out in blake bottle, washes out the sticky agar of test-tube plantlet root, do not injure blade and the tip of a root with flowing water, move into fill matrix transplant seedlings in bag, water normal root water and be placed on the ground not having direct sunlight;
Described test-tube plantlet to heel in transplanting be before transplanting transition, choose culture of rootage 20d, height 4.5-5.5cm, the test-tube plantlet of below root length 0.5cm, first be placed in topped asbestos shingle on canopy, all around 70% shading screen, canopy and divide in the Simple pergola of two, place blake bottle to add intense light irradiation, move into after 10d and plant in seedling matrix;
Described Plantlet in vitro is with vitro rooting in test tube seedling light culture at 4 DEG C, and in incubation, under making the former condition of culture of honeysuckle culture, every 20d-30d subculture once, can extend subculture cycle to 1 year; Described honeysuckle culture refers to vitro rooting in test tube seedling; Described former condition of culture refers to culture of rootage condition.
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| CN105340750A (en) * | 2015-11-27 | 2016-02-24 | 山东中医药大学 | Honeysuckle tissue-culture-seedling culture medium and honeysuckle tissue-culture rapid propagation method |
| CN105393917B (en) * | 2015-11-30 | 2017-11-24 | 北京林业大学 | Clove leaf honeysuckle rapid propagation method |
| CN107410033B (en) * | 2017-09-29 | 2019-05-24 | 中国科学院昆明植物研究所 | The rapid propagation method of national spice berry snippings |
| CN115039695A (en) * | 2022-06-18 | 2022-09-13 | 天津博奥聚能生物科技有限公司 | Mitosis inhibitor, using method and method for preparing tetraploid honeysuckle |
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| JP2002173416A (en) * | 2000-12-06 | 2002-06-21 | Kao Corp | Hair cosmetic |
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| CN102972294A (en) * | 2012-12-04 | 2013-03-20 | 重庆市秀山红星中药材开发有限公司 | Rapid honeysuckle tissue culture propagation method |
| CN103125379A (en) * | 2011-11-25 | 2013-06-05 | 刘金峰 | Honeysuckle tissue culturing and honeysuckle tissue culture medium preparation method |
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| CN102657082A (en) * | 2012-04-28 | 2012-09-12 | 湖南农业大学 | In-vitro culture and planting regeneration and propagation method of Xianglei honeysuckle leaves and culture medium |
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