CN104885942A - Chemical disinfection tissue culture method for rhododendron simsii planch - Google Patents
Chemical disinfection tissue culture method for rhododendron simsii planch Download PDFInfo
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Abstract
The invention relates to a chemical disinfection tissue culture method for rhododendron simsii planch. The chemical disinfection tissue culture method for rhododendron simsii planch comprises the steps of culture vessel disinfection, culture medium preparation, induction culture, enrichment culture, rooting culture and bottle seedling transplanting. According to the chemical disinfection tissue culture method for rhododendron simsii planch, the purpose of disinfection or bacteriostasis can be achieved by a chemical reagent; in the culture medium preparation process, a culture vessel and a culture medium do not need to be disinfected at high temperature and high pressure, the workload and the energy consumption can be reduced, and the rhododendron simsii planch tissue culture link can be simplified; the operation is simple, and the preparation is implemented according to different culture medium formulae; the practicability is high, the popularization is good, the tissue culture cost of rhododendron simsii planch can be effectively lowered, and the cost can be generally reduced by no less than 10%.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, be specifically related to a kind of azalea chemical disinfection tissue culture method, belong to biological technical field.
Background technology
Rhododendron (RhododendronLinn.) plant is world-renowned ornamental plants, being one of large famous flower of China ten, is also world-renowned ornamental flower. its gorgeous colorful pattern and proper potted flower moulding are always in occupation of one of fast-selling kind of China flowers in the Spring Festival.Azalea adopts seed, cuttage, press strip and grafting method breeding usually, but limits by factors such as kind or kind, season, maternal plant materials, is difficult to carry out a large amount of commercially producing.The culture in vitro system of ghent azalea (Rhododendron xx) is successfully established first from Anderon in 1975; tissue cultures has become the important means of azalea breeding, is widely used in the commercially producing of plasm resource protection, genetic breeding and clone nursery stock.For specific azalea kind, kind, the successful key of its in-vitro propagate is the selection of minimal medium, to the optimization of added hormone kind and proportioning in the induction and Multiplying culture of explant, and the regulation and control to envirment factor in rooting culture.But, due to azalea kind and interracial difference, the foundation of the rooting culture system of subculture Multiplying culture, root induction, plantlet in vitro in tissue culture procedures, be still urgent problem in tissue cultures, particularly Multiple Buds and the low problem of Differentiation ration of adventitious buds, be the bottleneck of tissue-culturing rapid propagation.
Cuckoo tissue cultures, can expand its reproduction coefficient on the one hand, thus realize biological control; On the other hand, on the basis of preserving its original merit, the Germ-plasma resources protection of cuckoo and transgenic research etc. can be realized.The cost of cuckoo tissue cultures mainly comprises medium, facilities and equipment, power supply cost, consumptive material and cost of labor needed for plantlet in vitro production.Ability normal growth in aseptic culture medium after conventional cuckoo method for tissue culture requires explant to be seeded in autoclave sterilization, power consumption is large, and manual operation cost is high.If transform medium by the method for chemical disinfection, making medium not needing to carry out autoclave sterilization, just can obtain the normal growth of cuckoo.Like this, can reduce the requirement of cuckoo tissue cultures to facilities and equipment on the one hand, especially do not need the pressure cooker equipment of the required configuration of autoclave sterilization, reduce cost of investment, power consumption also will significantly reduce; On the other hand, adopt this medium to carry out cuckoo tissue cultures, group training link greatly simplifies, and manually-operated speed significantly improves, thus reduces cost of labor.In addition, because improved medium self has sterilization, antibacterial or bacteriostasis, effectively can control the pollution problem in tissue culture procedures, can not produce cuckoo growth again and poison or suppress, namely do not affect the normal growth of cuckoo, radically simplify cuckoo tissue cultures.
Summary of the invention
The object of this invention is to provide a kind of azalea chemical disinfection tissue culture method.
The object of the invention is to be achieved through the following technical solutions.
A kind of azalea chemical disinfection tissue culture method of the present invention, comprises culture vessel sterilization, medium preparation, Fiber differentiation, Multiplying culture, culture of rootage and bottle transplantation of seedlings, it is characterized in that:
1. culture vessel sterilization: the stainless steel disc of blake bottle bottle cap and inoculation is soaked and is no less than 10h in the aqueous solution of 100mg/L clorox+2mg/L penicillin+100mg/L calcium propionate, saves backup;
2. medium preparation: inducing culture is ER+6 ~ 15mg/L ZT+5 ~ 15mg/L IAA+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Proliferated culture medium is: ER+1 ~ 3mg/L ZT+0.5 ~ 1.0mg/L IAA+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Root media is 1/2ER+0.5 ~ 1.5mg/LNAA+0.1 ~ 0.5IBA mg/L+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Described ER medium is nineteen sixty-five, ER medium disclosed in Eriksson; Described ZT is zeatin; Described NAA is 〆-methyl α-naphthyl acetate; Described IAA is heteroauxin; Described IBA is indolebutyric acid; During preparation medium, first claim sucrose and agar powder in each culture medium prescription, add the running water of medium cumulative volume 1/2, be heated to agar powder dissolve completely, after assorting other raw materials by each culture medium prescription again, constant volume, then use the HCl adjust pH to 5.8 of NaOH or 1mol/L of 1mol/L, be dispensed in the culture vessel of sterilizing, sealing, for subsequent use after cooled and solidified;
3. Fiber differentiation: get healthy and strong cuckoo terminal bud or bud, first tap water surface dirt, peels off outer bract, is soak 2 ~ 3s in 75% alcohol by volume ratio, then is 0.1%HgCl by weight ratio
2sterilization 6 ~ 10min, aseptic water washing 3 ~ 5 times; On superclean bench, peel off the bract of 2 ~ 3 layers, excision base portion brownization part, is inoculated on inducing culture, forms some budlets after 40d; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
4. Multiplying culture: proceed in proliferated culture medium by the budlet of Fiber differentiation, progressively induces Multiple Buds after 30d and develops into and do not have a crowd shoots of root; Crowd shoots is cut into the stem section of band bud, is transferred in new proliferated culture medium, every 30d switching once; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
5. culture of rootage: when the crowd shoots of Multiplying culture grows to 4 ~ 6cm, crowd shoots is cut into individual plant, is inoculated on root media, forms whole plant after 25d; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
6. bottle transplantation of seedlings: whole plant culture of rootage obtained takes out, and cleans the medium of root, after soaking 30min with the carbendazim of 800 times, transplant in cultivation matrix, described cultivation matrix is made up of peat soil and perlite, and its weight ratio is 2: 1; Booth upper strata 75% shading net hides, and the environmental temperature of transplanting is 22 ~ 28 DEG C.
Effect of the present invention:
Due to chemical disinfection tissue culture method of the present invention, be utilize chemical reagent to add in various medium, reach sterilizing or antibacterial effect, so the medium of preparation is without the need to autoclave sterilization.Therefore, in azalea Fiber differentiation, Multiplying culture, the impact in each stage of culture of rootage, except will considering the evaluation index commonly used, the problem of pollution rate also to be considered.
1. Fiber differentiation: confirm by experiment, the chemical disinfection tissue culture method of a kind of azalea of the present invention is compared with the azalea tissue culture method of routine, result is as shown in table 1, and survival rate, pollution rate and the lethality of cultivating at the explant in Fiber differentiation stage all do not have too big-difference.
Table 1 chemical disinfection tissue culture method of the present invention is on the impact of azalea Fiber differentiation
| Tissue culture mode | Average survival (%) | Average pollution rate (%) | Average mortality (%) |
| Conventional tissue culture method | 45 | 36 | 19 |
| Chemical disinfection tissue culture method | 50 | 33 | 17 |
2. Multiplying culture: chemical disinfection tissue culture method of the present invention and conventional organize comparing of the proliferation times of training azalea and pollution rate, result is as shown in table 2, and proliferation times and pollution rate two indexs all do not have too big-difference.See at the upgrowth situation of bottle seedling, the medium of chemical disinfection tissue culture method of the present invention due to without autoclave sterilization, hormone and nutritive element loss less, its azalea plantlet in vitro is greener, more strong.
Table 2 chemical disinfection tissue culture method of the present invention is on the impact of azalea proliferation times and pollution rate
| Tissue culture mode | Average proliferation multiple | Average pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 5.6 | 0.5 | Seedling is light green, weak |
| Chemical disinfection tissue culture method | 6.0 | 0 | Miao Lv, strong |
3. chemical disinfection tissue culture method of the present invention is on the impact of azalea rooting rate and pollution rate: in rooting process, chemical disinfection tissue culture method of the present invention is compared with the azalea tissue culture method of routine, result is as shown in table 3, and rooting rate and pollution rate all do not have too big-difference; From the upgrowth situation of bottle seedling, use method of the present invention, azalea plantlet in vitro stem is thicker, and blade is larger.
The azalea rooting situation of table 3 chemical disinfection tissue culture method of the present invention
4. cost analysis: chemical disinfection tissue culture method of the present invention organizes the cost analysis of training in table 4 with conventional.
Of the present invention group of training eliminates autoclaving link, simplifies group training program, improves operating efficiency.Calculate from the expense that can directly calculate, can save every year about 50,000 yuan (calculating to prepare 50L medium every day).In addition, some are also had to be not easy the project calculated, as pressure cooker maintenance, safety, save space etc.:
The easy tissue culture method of table 4 azalea of the present invention is cultivated and is organized with conventional the cost analysis table trained
Tool of the present invention has the following advantages and beneficial effect:
1. in azalea chemical disinfection tissue culture method of the present invention, culture vessel and medium, all without the need to autoclave sterilization, decrease workload and energy resource consumption, simplify azalea group training link, reduce azalea group training cost.
2. azalea chemical disinfection tissue culture method of the present invention, simple to operate, as long as by after the preparation of different culture medium prescriptions, practical, generalization is good.
3. azalea chemical disinfection tissue culture method of the present invention compares with conventional azalea tissue culture method, can reduce costs more than 10%.
Embodiment
In order to illustrate the present invention instead of restriction the present invention further, be illustrated below in conjunction with embodiment.
Embodiment one: a kind of azalea chemical disinfection tissue culture method
A kind of azalea chemical disinfection tissue culture method, comprises the following steps:
1. culture vessel sterilization: the stainless steel disc of blake bottle bottle cap and inoculation is soaked 10h in the aqueous solution of 100mg/L clorox+2mg/L penicillin+100mg/L calcium propionate, saves backup.
2. medium preparation: inducing culture is ER+10mg/L ZT+10mg/L IAA+80mg/L clorox+3mg/L penicillin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Proliferated culture medium is: ER+1mg/L ZT+1.0mg/L IAA+80mg/L clorox+2mg/L penicillin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Root media is 1/2ER+1.0mg/L NAA+0.5IBA mg/L+80mg/L clorox+2mg/L penicillin+80mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; During preparation medium, first claim sucrose and agar powder in each culture medium prescription, add the running water of medium cumulative volume 1/2, be heated to agar powder dissolve completely, after assorting other raw materials by each culture medium prescription again, constant volume, then use the HCl adjust pH to 5.8 of NaOH or 1mol/L of 1mol/L, be dispensed in the culture vessel of sterilizing, sealing, for subsequent use after cooled and solidified.
3. Fiber differentiation: get healthy and strong cuckoo bud or terminal bud, first tap water surface dirt, peels off outer bract, is soak 2 ~ 3s in 75% alcohol by volume ratio, then is 0.1%HgCl by weight ratio
2sterilization 8min, aseptic water washing 3 times.On superclean bench, peel off the bract of 2 ~ 3 layers, excision base portion brownization part, is inoculated on inducing culture, can forms a large amount of budlet after 40d.Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx.
4. Multiplying culture: proceed in proliferated culture medium by the budlet of Fiber differentiation, progressively induces Multiple Buds after 30d and develops into and do not have a crowd shoots of root.Crowd shoots is cut into the stem section of band bud, is transferred in new proliferated culture medium, every 30d switching once, can reach the effect of a large amount of propagation.Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx.
5. culture of rootage: when the crowd shoots of Multiplying culture grows to 4 ~ 6cm, crowd shoots is cut into individual plant, is inoculated on root media, forms whole plant after 25d; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx.
6. bottle transplantation of seedlings: whole plant culture of rootage obtained takes out, clean the medium of root, transplanting to peat soil and perlite ratio after soaking 30min with the carbendazim of 800 times is in the matrix of 2: 1, and booth upper strata 75% shading net hides, and the environmental temperature of transplanting is 22 ~ 28 DEG C.
Claims (4)
1. an azalea chemical disinfection tissue culture method, comprises culture vessel sterilization, medium preparation, Fiber differentiation, Multiplying culture, culture of rootage and bottle transplantation of seedlings, it is characterized in that:
(1) culture vessel sterilization: the stainless steel disc of blake bottle bottle cap and inoculation is soaked and is no less than 10h in the aqueous solution of 100mg/L clorox+2mg/L penicillin+100mg/L calcium propionate, saves backup;
(2) medium preparation: inducing culture is ER+6 ~ 15mg/L ZT+5 ~ 15mg/L IAA+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Proliferated culture medium is: ER+1 ~ 3mg/L ZT+0.5 ~ 1.0mg/L IAA+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Root media is 1/2ER+0.5 ~ 1.5mg/LNAA+0.1 ~ 0.5IBA mg/L+40 ~ 100mg/L clorox+2 ~ 5mg/L penicillin+50 ~ 100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8; Described ER medium is nineteen sixty-five, ER medium disclosed in Eriksson; Described ZT is zeatin; Described NAA is 〆-methyl α-naphthyl acetate; Described IAA is heteroauxin; Described IBA is indolebutyric acid; During preparation medium, first claim sucrose and agar powder in each culture medium prescription, add the running water of medium cumulative volume 1/2, be heated to agar powder dissolve completely, after assorting other raw materials by each culture medium prescription again, constant volume, then use the HCl adjust pH to 5.8 of NaOH or 1mol/L of 1mol/L, be dispensed in the culture vessel of sterilizing, sealing, for subsequent use after cooled and solidified;
(3) Fiber differentiation: get healthy and strong cuckoo terminal bud or bud, first tap water surface dirt, peels off outer bract, is soak 2 ~ 3s in 75% alcohol by volume ratio, then is 0.1%HgCl by weight ratio
2sterilization 6 ~ 10min, aseptic water washing 3 ~ 5 times; On superclean bench, peel off the bract of 2 ~ 3 layers, excision base portion brownization part, is inoculated on inducing culture, forms some budlets after 40d; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
(4) Multiplying culture: proceed in proliferated culture medium by the budlet of Fiber differentiation, progressively induces Multiple Buds after 30d and develops into and do not have a crowd shoots of root; Crowd shoots is cut into the stem section of band bud, is transferred in new proliferated culture medium, every 30d switching once; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
(5) culture of rootage: when the crowd shoots of Multiplying culture grows to 4 ~ 6cm, crowd shoots is cut into individual plant, is inoculated on root media, forms whole plant after 25d; Culturing room's temperature is 25 ~ 28 DEG C, illumination 12h, and intensity of illumination is 1200 ~ 1500lx;
(6) bottle transplantation of seedlings: whole plant culture of rootage obtained takes out, and cleans the medium of root, after soaking 30min with the carbendazim of 800 times, transplant in cultivation matrix, described cultivation matrix is made up of peat soil and perlite, and its weight ratio is 2: 1; Booth upper strata 75% shading net hides, and the environmental temperature of transplanting is 22 ~ 28 DEG C.
2. a kind of azalea chemical disinfection tissue culture method according to claim 1, it is characterized in that described inducing culture is ER+10mg/L ZT+10mg/L IAA+80mg/L clorox+3mg/L penicillin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8.
3. a kind of azalea chemical disinfection tissue culture method according to claim 1, it is characterized in that described proliferated culture medium is: ER+1mg/L ZT+1.0mg/L IAA+80mg/L clorox+2mg/L penicillin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8.
4. a kind of azalea chemical disinfection tissue culture method according to claim 1, it is characterized in that described root media is 1/2ER+1.0mg/L NAA+0.5IBA mg/L+80mg/L clorox+2mg/L penicillin+80mg/L calcium propionate+20g/L sucrose+3.0g/L agar powder, pH5.8.
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Cited By (3)
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| CN105248281A (en) * | 2015-11-03 | 2016-01-20 | 钟煜 | Preparation method of raspberry induction culture medium |
| CN106613995A (en) * | 2017-01-18 | 2017-05-10 | 福建农林大学 | Tissue culture method for culturing anoectochilus roxburghii by bacteriostatic culture medium |
| CN114847162A (en) * | 2022-05-19 | 2022-08-05 | 福建农林大学 | Azalea in vitro culture and micro-bud grafting method |
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