CN102577976B - Simple tissue culture method for broussonetia papyrifera - Google Patents
Simple tissue culture method for broussonetia papyrifera Download PDFInfo
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Abstract
The invention relates to a simple tissue culture method for broussonetia papyrifera. The simple tissue culture method for the broussonetia papyrifera comprises the following steps: preparing a bacteriostatic agent, sterilizing a culture vessel, preparing a culture medium, performing induction culture, performing proliferation culture, performing rooting culture and transplanting a bottled plantlet. By the simple tissue culture method for the broussonetia papyrifera, the culture vessel and the culture medium are not required to be sterilized at high temperature and high pressure, so that the workload and the energy consumption are reduced, the tissue culture link of the broussonetia papyrifera is simplified, and tissue culture cost of the broussonetia papyrifera is lowered; the simple tissue culture method for the broussonetia papyrifera is easy to operate, high in practicality and good in popularization, and the culture medium is prepared according to different formulas and the bacteriostatic agent is added; and compared with the conventional tissue culture method for the broussonetia papyrifera, by the simple tissue culture method for the broussonetia papyrifera, the cost can be reduced by more 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 Simple tissue culture method for broussonetia papyrifera, belong to biological technical field.
Background technology
Paper mulberry (
Broussonetia papyriferaL) belong to Moraceae, be perennial broad-leaved arbor, have fast-growing, strong adaptability, distribution is wide, heat is high, rotation is short characteristics.Paper mulberry is short, instant effect of a kind of small investment, cycle, the long extraordinary nonwood forest trees of being benefited, and mainly is distributed in China the Yellow River, the Changjiang river and Pearl River Delta.The phloem fiber content of paper mulberry is high, and fiber is long and thin, best in quality, is the quality raw materials of manufacturing rice paper and coinage paper; Leaf is rich in protein, amino acid, vitamin, carbohydrate and trace element, is the fully natural green raw material of processing livestock feed.Accelerate paper mulberry and cultivate speed, enlarging Broussonetia papyrifera is the important foundation that guarantees rice paper manufacturing and feed processing industry sustainable development, also is the important channel that increases peasant income.
The paper mulberry tissue is cultivated, and can enlarge its reproduction coefficient on the one hand, thereby realizes biological control; On the other hand, can on the basis of preserving its original merit, realize producing in the Fast-propagation of paper mulberry seedling and strong sprout.The cost that the paper mulberry tissue is cultivated mainly comprises required medium, facilities and equipment, power supply cost, consumptive material and the cost of labor of group training seedling production.Conventional paper mulberry method for tissue culture requires seeded process, incubation all to carry out in aseptic culture medium, and aseptic sterilization power consumption is large, and the manual operation cost is high.If can be by the medium transformation, obtain both to have guaranteed that paper mulberry organizes normal growth, have again the medium of sterilization, antibiotic or bacteria resistance function, mushroom also just can not grow in medium.Because this improved medium does not need to carry out autoclave sterilization, can reduce on the one hand the cultivation of paper mulberry tissue to the requirement of facilities and equipment, especially do not need the pressure cooker equipment of the required configuration of autoclave sterilization, reduced cost of investment, power consumption is also with decrease; On the other hand, adopt this medium to carry out the paper mulberry tissue and cultivate, group training link is greatly simplified, and manually-operated speed significantly improves, thereby has reduced cost of labor.In addition, because improved medium self has sterilization, antibiotic or bacteriostasis, the pollution problem in the tissue culture procedures is effectively controlled, and the paper mulberry tissue is cultivated and be need not to carry out in strict gnotobasis, has fundamentally simplified the paper mulberry tissue and has cultivated.Therefore, the key of easy group of training of paper mulberry is the transformation of medium, specifically finds one or more can add in the medium, has both had broad spectrum activity sterilization, antibiotic or bacteriostasis, can not produce the paper mulberry growth again and poison or inhibition, namely not affect the material of paper mulberry growth.
Summary of the invention
The purpose of this invention is to provide a kind of Simple tissue culture method for broussonetia papyrifera.
The objective of the invention is to realize by the following method.
A kind of Simple tissue culture method for broussonetia papyrifera of the present invention comprises that cultivation, propagation cultivation, culture of rootage and bottle transplantation of seedlings are prepared, induced to preparation, culture vessel sterilization, the medium of bacteriostatic agent, it is characterized in that:
1. the preparation of bacteriostatic agent: the compound method that No. 1, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, and with the distilled water constant volume to 100 ml, be No. 1, bacteriostatic agent; The compound method that No. 2, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, add again liquor natrii hypochloritis 10～20 ml, and be settled to 100 ml with distilled water, be No. 2, bacteriostatic agent; The grand bactericide of described benefit training is comprised of A bottle medicament and B bottle medicament, is buied by market; Described liquor natrii hypochloritis is pure for analyzing, and the percentage by weight of active chlorine is not less than 0.5%;
2. immersion is no less than 10 h in aqueous solution culture vessel sterilization: be 0.3 %～0.5 %(V/V in the volume ratio of No. 1, bacteriostatic agent and water with blake bottle and bottle cap), saves backup;
3. medium preparation: inducing culture is MS+0.5～1.0 mg/L 6-BA+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Proliferated culture medium is MS+1.0 mg/L 6-BA+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Root media is 1/2MS+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Described MS medium is 1962, the disclosed MS medium of Murashige and Skoog; Described 6-BA refers to the amino fast cry of certain animals of 6-benzyl; Described NAA ， is Zhied 〆-methyl α-naphthyl acetate; During the preparation medium, first not with No. 2, bacteriostatic agent, after assorting other raw materials by each culture medium prescription, with the distilled water constant volume, be heated to agar powder and dissolve fully, then press each culture medium prescription and add bacteriostatic agent No. 2, with the NaOH of 1 mol/L or the HCl adjust pH to 5.8 of 1 mol/L, divide and install in the culture vessel, sealing, cooled and solidified, for subsequent use;
4. induce cultivation: after the paper mulberry branch that gathers was disinfected in alcohol, with the mercuric chloride solution of 0.1 %, 6 min that sterilize, taking-up was inoculated on the inducing culture with behind the aseptic water washing; Culturing room's temperature is 28 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
5. propagation is cultivated: the bud seedling that induces is cut into stems with bud is inoculated on the proliferated culture medium, 30 d transfer once; Culturing room's temperature is 26 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
6. culture of rootage: when seedling length to 2～4 cm, the seedling of will growing thickly is cut into individual plant, is inoculated on the root media, and 25 d transplant;
Culturing room's temperature is 25 ℃, illumination 12 h, and intensity of illumination is 1500 lx;
7. bottle transplantation of seedlings: behind culture of rootage 25 d, the seedling of will taking root takes out, and cleans the medium of root, transplants to booth after soaking 30 min with 800 times carbendazim, and the booth upper strata hides with shading net, and the environmental temperature of transplanting is 25 ℃.
Effect of the present invention:
1. Simple tissue culture method for broussonetia papyrifera of the present invention is bred the impact of multiple and pollution rate on paper mulberry: because Simple tissue culture method for broussonetia papyrifera of the present invention, the medium of preparation is to need not autoclave sterilization, so in breeding, except considering the rate of increase, also will consider the problem of pollution rate.Confirm by experiment, Simple tissue culture method for broussonetia papyrifera of the present invention is compared with the paper mulberry tissue culture method of routine, and the rate of increase and pollution rate all do not have too big-difference.From the upgrowth situation of bottle seedling, the medium of Simple tissue culture method for broussonetia papyrifera of the present invention is because without autoclave sterilization, hormone and nutritive element loss are less, and its paper mulberry group training seedling is greener, more strong.Simple tissue culture method for broussonetia papyrifera of the present invention is as shown in table 1 on the impact of paper mulberry propagation multiple and pollution rate.
Table 1 Simple tissue culture method for broussonetia papyrifera of the present invention is on the impact of paper mulberry propagation multiple and pollution rate
| Tissue culture mode | The propagation multiple | Pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 2.9 | 0 | Seedling is light green, a little less than |
| Easy tissue culture method | 3.0 | 0 | Seedling is green, strong |
2. Simple tissue culture method for broussonetia papyrifera of the present invention is on the impact of paper mulberry rooting rate and pollution rate: in rooting process, Simple tissue culture method for broussonetia papyrifera of the present invention is compared with the paper mulberry tissue culture method of routine, the result is as shown in table 2, 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, paper mulberry group training seedling stem is thicker, and blade is larger.
The paper mulberry rooting situation that table 2 Simple tissue culture method for broussonetia papyrifera of the present invention is cultivated
| Tissue culture mode | Rooting rate (%) | Pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 100 | 0 | Stem is thin, and blade is little |
| Easy tissue culture method | 100 | 2.0 | Stem is thick, and blade is large |
3. different bacteriostatic agents and NAA concentration are as shown in table 3 on the impact of rooting rate and pollution rate in the Simple tissue culture method for broussonetia papyrifera of the present invention, in the Simple tissue culture method for broussonetia papyrifera of the present invention, add 4 ml bacteriostatic agents in every liter of medium than 3 ml and 5 ml bacteriostatic agents, pollution rate slightly reduces sometimes, but difference is not obvious; Rooting rate there is no difference.From different NAA concentration, for well, plant strain growth is better with 0.1 mg/L, and the leaf look green, and root is sturdy.Therefore, that rooting rate and pollution rate are affected difference is not obvious for variable concentrations NAA and bacteriostatic agent.
Different bacteriostatic agents and NAA concentration are on the impact of rooting rate and pollution rate in table 3 Simple tissue culture method for broussonetia papyrifera of the present invention
Annotate: other composition of medium is: 1/2MS+20 g/L sucrose+3.5 g/L agar powders, pH5.8;
4. No. 2 impacts on paper mulberry propagation bud seedling pollution of No. 1, bacteriostatic agent and bacteriostatic agent in the medium of the present invention: we have designed the comparative trial of No. 2, No. 1, bacteriostatic agent and bacteriostatic agent, and the result is as shown in table 4.Result of the test shows, for well, paper mulberry propagation bud seedling pollution rate is 0 with No. 2, bacteriostatic agent, and uses in the situation of No. 1, bacteriostatic agent, and pollution rate is 37.5 %.
No. 2 impacts on paper mulberry propagation bud seedling pollution of No. 1, bacteriostatic agent and bacteriostatic agent in table 4 medium of the present invention
| Bacteriostatic agent | Bud seedling number | Pollute number | Pollution rate (%) |
| No. 1, bacteriostatic agent | 80 | 30 | 37.5 |
| No. 2, bacteriostatic agent | 80 | 0 | 0.00 |
Remarks: the concentration that No. 2, No. 1, bacteriostatic agent and bacteriostatic agent is all 4 ml/L.
The present invention has following beneficial effect:
1. in the Simple tissue culture method for broussonetia papyrifera of the present invention, culture vessel and medium all need not autoclave sterilization, have reduced workload and energy resource consumption, have simplified paper mulberry group training link, have reduced paper mulberry group training cost.
2. Simple tissue culture method for broussonetia papyrifera of the present invention is simple to operate, as long as by after the different culture medium prescription preparations, add bacteriostatic agent and get final product, practical, generalization is good.
3. Simple tissue culture method for broussonetia papyrifera of the present invention contrasts with the paper mulberry tissue culture method of routine, can reduce more than cost 10 %.
Embodiment
In order further to illustrate the present invention rather than restriction the present invention, be illustrated below in conjunction with embodiment.
Embodiment one: a kind of Simple tissue culture method for broussonetia papyrifera
A kind of Simple tissue culture method for broussonetia papyrifera may further comprise the steps:
1. the preparation of bacteriostatic agent: the compound method that No. 1, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, and with the distilled water constant volume to 100 ml, be No. 1, bacteriostatic agent; The compound method that No. 2, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, add again liquor natrii hypochloritis 15 ml, and with the distilled water constant volume to 100 ml, be No. 2, bacteriostatic agent;
2. immersion 10 h in aqueous solution culture vessel sterilization: be 0.4 %(V/V in the volume ratio of No. 1, bacteriostatic agent and water with blake bottle and bottle cap) save backup;
3. medium preparation: inducing culture is MS+0.8 mg/L 6-BA+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Proliferated culture medium is MS+1.0 mg/L 6-BA+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Root media is 1/2MS+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; During the preparation medium, first not with No. 2, bacteriostatic agent, after assorting first other raw materials by each culture medium prescription, with the distilled water constant volume, be heated to agar powder and dissolve fully, then press each culture medium prescription and add bacteriostatic agent No. 2, with the NaOH of 1 mol/L or the HCl adjust pH to 5.8 of 1 mol/L, divide and install in the culture vessel, sealing, cooled and solidified, for subsequent use;
4. induce cultivation: after the paper mulberry branch that gathers was disinfected in alcohol, with the mercuric chloride solution of 0.1 %, 6 min that sterilize, taking-up was inoculated on the inducing culture with behind the aseptic water washing; Culturing room's temperature is 28 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
5. propagation is cultivated: the bud seedling that induces is cut into stems with bud is inoculated on the proliferated culture medium, 30 d transfer once; Culturing room's temperature is 26 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
6. culture of rootage: when seedling length to 2～4 cm, the seedling of will growing thickly is cut into individual plant, is inoculated on the root media, and 25 d transplant;
Culturing room's temperature is 25 ℃, illumination 12 h, and intensity of illumination is 1500 lx;
7. bottle transplantation of seedlings: behind culture of rootage 25 d, the seedling of will taking root takes out, and cleans the medium of root, transplants to booth after soaking 30 min with 800 times carbendazim, and the booth upper strata hides with shading net, and the environmental temperature of transplanting is 25 ℃.
The above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. a Simple tissue culture method for broussonetia papyrifera comprises that cultivation, propagation cultivation, culture of rootage and bottle transplantation of seedlings are prepared, induced to preparation, culture vessel sterilization, the medium of bacteriostatic agent, it is characterized in that:
(1) preparation of bacteriostatic agent: the compound method that No. 1, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, and with the distilled water constant volume to 100 ml, be No. 1, bacteriostatic agent; The compound method that No. 2, bacteriostatic agent: mix with B bottle medicament after in the grand bactericide A bottle medicament of benefit training, adding 20 ml distilled water dilutings, add again liquor natrii hypochloritis 10～20 ml, and be settled to 100 ml with distilled water, be No. 2, bacteriostatic agent; Described liquor natrii hypochloritis is pure for analyzing, and the percentage by weight of active chlorine is not less than 0.5%;
(2) culture vessel sterilization: be that immersion is no less than 10 h in the aqueous solution of 0.3 %～0.5 % with blake bottle and bottle cap in the volume ratio of No. 1, bacteriostatic agent and water, save backup;
(3) medium preparation: inducing culture is MS+0.5～1.0 mg/L 6-BA+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Proliferated culture medium is MS+1.0 mg/L 6-BA+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Root media is 1/2MS+0.1～0.5 mg/L NAA+3～5 ml/L bacteriostatic agents No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8; Described MS medium is 1962, the disclosed MS medium of Murashige and Skoog; Described 6-BA refers to the amino fast cry of certain animals of 6-benzyl; Described NAA ， is Zhied 〆-methyl α-naphthyl acetate; During the preparation medium, first not with No. 2, bacteriostatic agent, after assorting other raw materials by each culture medium prescription, with the distilled water constant volume, be heated to agar powder and dissolve fully, then press each culture medium prescription and add bacteriostatic agent No. 2, with the NaOH of 1 mol/L or the HCl adjust pH to 5.8 of 1 mol/L, divide and install in the culture vessel, sealing, cooled and solidified, for subsequent use;
(4) induce cultivation: after the paper mulberry branch that gathers was disinfected in alcohol, with the mercuric chloride solution of 0.1 %, 6 min that sterilize, taking-up was inoculated on the inducing culture with behind the aseptic water washing; Culturing room's temperature is 28 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
(5) propagation is cultivated: the bud seedling that induces is cut into stems with bud is inoculated on the proliferated culture medium, 30 d transfer once; Culturing room's temperature is 26 ℃, illumination 12 h, and intensity of illumination is 1400 lx;
(6) culture of rootage: when seedling length to 2～4 cm, the seedling of will growing thickly is cut into individual plant, is inoculated on the root media, and 25 d transplant; Culturing room's temperature is 25 ℃, illumination 12 h, and intensity of illumination is 1500 lx;
The bottle transplantation of seedlings: behind culture of rootage 25 d, the seedling of will taking root takes out, and cleans the medium of root, transplants to booth after soaking 30 min with 800 times carbendazim, and the booth upper strata hides with shading net, and the environmental temperature of transplanting is 25 ℃.
2. a kind of Simple tissue culture method for broussonetia papyrifera according to claim 1 is characterized in that inducing culture is MS+0.8 mg/L 6-BA+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8.
3. a kind of Simple tissue culture method for broussonetia papyrifera according to claim 1 is characterized in that proliferated culture medium is MS+1.0 mg/L 6-BA+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8.
4. a kind of Simple tissue culture method for broussonetia papyrifera according to claim 1 is characterized in that root media is 1/2MS+0.1 mg/L NAA+4 ml/L bacteriostatic agent No. 2+20 g/L sucrose+3.5 g/L agar powders, pH5.8.
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| CN104982332B (en) * | 2015-06-19 | 2017-02-01 | 大连中植环境生物科技有限公司 | Method for preparing paper mulberry somatic embryo and application thereof to paper mulberry expanding propagation |
| CN105165627B (en) * | 2015-10-22 | 2018-08-17 | 贵州大学 | A kind of rosewood tissue culture sterilization formula and tissue culture method |
| CN105532450A (en) * | 2015-12-05 | 2016-05-04 | 天水德农供销种业开发有限公司 | Hybrid paper mulberry industrial tissue culture and breeding method |
| CN107125132A (en) * | 2017-03-27 | 2017-09-05 | 河池乐康生态农业科技有限公司 | One kind hybridization paper mulberry quick breeding method for tissue culture |
| CN107129365A (en) * | 2017-05-18 | 2017-09-05 | 中科天华生物科技有限公司 | A kind of culture medium for paper mulberry nursery |
| CN108812260B (en) * | 2018-05-04 | 2020-11-17 | 杭州早稻田生物技术有限公司 | Tissue culture and rapid seedling method for paper mulberry root hairs |
| CN109168858A (en) * | 2018-08-23 | 2019-01-11 | 云南兴构农业产业发展有限公司 | Tame and docile seedling-growing method for paper mulberry tissue-cultured seedling |
| CN109220800A (en) * | 2018-10-18 | 2019-01-18 | 楚雄师范学院 | A method of effectively improving hybridization paper mulberry tissue-cultured seedling rooting rate |
| CN113598047B (en) * | 2021-08-04 | 2023-03-31 | 广西壮族自治区林业科学研究院 | High-protein hybrid paper mulberry efficient tissue culture seedling raising method |
| CN115589945A (en) * | 2022-10-10 | 2023-01-13 | 长江大学（Cn） | Method for openly culturing ginger test-tube plantlets |
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