CN101790960A - Tissue culture method of fructus evodiae - Google Patents
Tissue culture method of fructus evodiae Download PDFInfo
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Abstract
The invention discloses a tissue culture method of fructus evodiae, comprising the following steps of: 1) washing the bud of the fructus evodiae by water flow; 2) carrying out conventional sterilization, and then cutting the bud of the fructus evodiae into small sections of bud I with the length of 0.5-1.0 cm, and inoculating the bud I onto an adventitious bud induction culture medium for cultivating; 3) when the small sections of bud I grow up to be adventitious bud with the length being the same as that of a plantlet I, cutting the plantlet I into stem sections I, and then inoculating the stem sections I onto an enrichment medium for cultivating; 4) when the stem sections I grow up to be adventitious bud with the length being the same as 2.0-5.0 cm of a plantlet II, cutting the plantlet II into stem sections II, and then inoculating the stem sections II onto the enrichment medium for cultivating; 5) when the stem sections II grow up to be adventitious bud with the length being the same as 3.0-5.0 cm of a plantlet III, cutting the plantlet III, and inoculating the plantlet III into a rooting medium; and 6) when the plantlet III outgrows at least three roots which are longer than 2 cm, obtaining seedling which can be taken out of a bottle and planted. The tissue culture method can be used for obtaining a great deal of the bud of the fructus evodiae.
Description
Technical field
The present invention relates to a kind of method of evodia rutaecarpa tissue culture.
Background technology
Evodia rutaecarpa is one of them kind of Rutaceae Evodia, and fruit medicine is hot in nature, flavor is hot, bitter, has eliminating cold to stop pain, stopping nausea and vomiting by lowering the adverse flow of QI, effects such as supporing yang antidiarrheal are used for the treatment of headache, colic, beriberi, dysmenorrhoea, abdominal distention, vomiting acid regurgitation, aphtha etc., and medical value is very high; Be born under mountain region, warm area, roadside or the sparse woods, it is distributed in ground such as Guangdong, Guangxi, Guizhou, Yunnan, Sichuan, Shaanxi, Hunan, Hubei, Fujian, Zhejiang, Jiangxi.
The evodia rutaecarpa flower unisexuality, dioecism, seedling stage male and female plant be difficult for to differentiate and not homophyletic be that fruit active constituent content difference is big, cause that producing raises variety and mix serious, the unit are fruit yield is low; And the traditional seed or the seedling raising manners of transplanting a cutting, reproduction coefficient is low, only is 40-55% (and the staminiferous plant rate is up to 50%) and about 10% respectively; Badly influence the large-scale application of improved seeds and the uniformity and the stability of medical material quanlity.
Summary of the invention
The technical problem to be solved in the present invention provides the method for tissue culture of a kind of evodia rutaecarpa, adopts this method can obtain the seedling of a large amount of evodia rutaecarpas.
In order to solve the problems of the technologies described above, the invention provides a kind of method of evodia rutaecarpa tissue culture, may further comprise the steps successively:
1), the evodia rutaecarpa tender shoots of getting no scab, robust growth carries out the flowing water flushing;
2), the tender shoots after the flushing of above-mentioned flowing water is sterilized through routine, be cut into the long segment tender shoots I of 0.5～1.0cm then, above-mentioned segment tender shoots I be inoculated on the evoking adventive bud medium cultivate; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
3), when treating that the indefinite bud that grows on the segment tender shoots I grows to the high seedling I of 2.0～3.0cm, extract seedling I; This seedling I is cut into the long stem section I of 0.5～1.2cm, above-mentioned stem section I is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
4), II when treating that the indefinite bud that grows on the stem section I grows to the high seedling of 2.0～5.0cm, extract seedling II; This seedling II is cut into the long stem section II of 0.8～1.2cm, above-mentioned stem section II is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
5), when treating that the indefinite bud that grows on the stem section II grows to the high seedling III of 3.0～5.0cm, extract seedling III; III is inoculated in the root media with this seedling;
6), when treating that above-mentioned seedling III grows at least 3 roots greater than 2cm, but the seedling of bottle outlet plantation.
Improvement as the tissue culture method of evodia rutaecarpa of the present invention: the evoking adventive bud medium step 2) is: MS minimal medium+BA 0.5～1.0mg/l+IBA 0.1～0.5mg/l+ sugar 20～30g/l+ agar 5～9g/l, pH is 5.5～6.0.
The preparation method of evoking adventive bud medium is specific as follows: with the MS minimal medium as the basis, add 6-benzyladenine (BA), second diindyl butyric acid (IBA), sugar and agar respectively, evenly mix, utilizing the KOH of 1mol/L or the HCl adjusting pH of 1mol/L is 5.5～6.0; Add 0.5～1.0mg BA, 0.1～0.5mg IBA, 20～30g sugar and 5～9g agar in the MS minimal medium of every 1L.
Further improvement as the tissue culture method of evodia rutaecarpa of the present invention: the proliferated culture medium in step 3) and the step 4) is: MS minimal medium+BA 0.3～1.0mg/l+IBA 0.1～0.3mg/l+ sugar 20～30g/l+ agar 5～9g/l, pH is 5.5～6.0.
The preparation method of proliferated culture medium is specific as follows: with the MS minimal medium as the basis, add 6-benzyladenine (BA) and second diindyl butyric acid (IBA), sugar and agar respectively, evenly mix, utilizing the KOH of 1mol/L or the HCl adjusting pH of 1mol/L is 5.5～6.0; The MS minimal medium of every 1L adds 0.3～1.0mg BA, 0.1～0.3mg IBA, 20～30g sugar and 5～9g agar.
Further improvement as the tissue culture method of evodia rutaecarpa of the present invention: the root media in the step 5) is: 1/2MS minimal medium+NAA 0.2～0.5mg/l+ sugar 30g/l+ agar 5～9g/l, pH is 5.5～6.0.
The preparation method of root media is specific as follows: with 1/2MS minimal medium (content of all substances is half of MS minimal medium in the ie in solution) as the basis, add methyl (NAA), sugar, agar respectively, evenly mix, utilizing the KOH of 1mol/L or the HCl adjusting pH of 1mol/L is 5.5～6.0; The 1/2MS minimal medium of every 1L adds 0.2～0.5mgNAA, 30g sugar and 5～9g agar.
Further improvement as the tissue culture method of evodia rutaecarpa of the present invention: step 1) is: the evodia rutaecarpa tender shoots is put into beaker, running water flushing 1～2 hour.
The source of the evodia rutaecarpa tender shoots that the present invention is used is available from Zhejiang starlight agricultural development Co., Ltd.
The tissue culture method of evodia rutaecarpa of the present invention (method of cultured in vitro) belongs to a kind of evodia rutaecarpa method for tissue culture of breeding fast of inducing.According to the totipotent principle of cell in the Plant Tissue Breeding, can produce the high quality seedling that a large amount of genetic backgrounds are identical, growing way is consistent at short notice, and rely on that the laboratory can realize the anniversary high quality seedling is provided for a long time.In the method for the invention, the evodia rutaecarpa tender shoots of stalwartness is sterilized earlier, induce by suitable inducing culture again to obtain a certain amount of aseptic seedling.Adopt method of the present invention, but the general seedling that only needs can obtain in 60～80 days the bottle outlet plantation; Therefore the reproduction coefficient in the evodia rutaecarpa test-tube plantlet anniversary is 5 in theory
12Doubly.Evodia rutaecarpa tissue culture method of the present invention is a kind of factor affecting such as season that is not subjected to, and the method for high-quality evodia rutaecarpa seedling is provided efficiently, fast, can quicken improved variety popularization speed, improves field kind plantation output.
The seedling of gained of the present invention can adopt following method to plant: shift out blake bottle having at least 3 seedlings of being longer than the 2cm root, clean its root medium, be transplanted in the matrix (is that 3: 1: 1 peat, perlite and vermiculite formed by volume ratio), cultivate in the climatic cabinate chamber, condition of culture: 20～25 ℃ of temperature, humidity 85%～90%, illumination in 14～16 hours, intensity of illumination 15～25 μ mol m
-2.s
-1, 10～8 hours dark cultivations, above-mentioned illumination and dark the cultivation are hocketed; After 3～5 weeks, illumination in 14～16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, 10～8 hours dark cultivations, above-mentioned illumination and dark the cultivation are hocketed.
After 2 months, survival rate reaches more than 90%.
To 10 strains available from Zhejiang starlight agricultural development Co., Ltd is wzy1～wzy10, at first obtains seedling according to method of the present invention, and then seedling is planted according to the method described above, and survival rate all can reach more than 90%.
Embodiment
The tissue culture method of embodiment 1, a kind of evodia rutaecarpa, carry out following steps successively:
1), the evodia rutaecarpa tender shoots of getting no scab, robust growth is put into beaker, running water flushing 1～2 hour;
2) after the routine sterilization, (be 0.1%w/v HgCl, with above-mentioned tender shoots
2Handle after 6～10 minutes, aseptic water washing 5～8 times blots with aseptic filter paper then), be cut into the segment tender shoots I about 1cm, be inoculated on the evoking adventive bud medium and cultivate; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
The evoking adventive bud medium is: MS minimal medium+BA 0.8mg/l+IBA 0.3mg/l+ sugar 25g/l+ agar 7g/l, pH is 5.7.
3), when treating that the indefinite bud that grows on the segment tender shoots I grows to the high seedling I of 2.0～3.0cm, extract seedling I; This seedling I is cut into stem section I about 1cm, above-mentioned stem section I is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is: MS minimal medium+BA 0.7mg/1+IBA 0.2mg/l+ sugar 25g/l+ agar 7g/l, pH is 5.5～6.0.
4), II when treating that the indefinite bud that grows on the stem section I grows to the high seedling of 2.0～5.0cm, extract seedling II; This seedling II is cut into the long stem section II of 0.8～1.2cm, above-mentioned stem section II is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is with the proliferated culture medium of step 3).
5), when treating that the indefinite bud that grows on the stem section II grows to the high seedling III of 3.0～5.0cm, extract seedling III; III is inoculated in the root media with this seedling; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Root media is: 1/2MS minimal medium+NAA 0.4mg/l+ sugar 30g/l+ agar 7g/l, pH is 5.5～6.0.
6), when treating that above-mentioned seedling III grows at least 3 roots greater than 2cm, but the seedling of bottle outlet plantation.
According to said method, only needed 60～80 days can obtain test-tube plantlet (seedling); Therefore adopt method of the present invention can obtain a large amount of test-tube plantlets for a long time.
The tissue culture method of embodiment 2, a kind of evodia rutaecarpa, carry out following steps successively:
1), the evodia rutaecarpa tender shoots of getting no scab, robust growth is put into beaker, running water flushing 1～2 hour;
2) after the routine sterilization, (be 0.1%w/v HgCl, with above-mentioned tender shoots
2Handle after 6～10 minutes, aseptic water washing 5～8 times blots with aseptic filter paper then), be cut into the segment tender shoots I about 1cm, be inoculated on the evoking adventive bud medium and cultivate; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
The evoking adventive bud medium is: MS minimal medium+BA 0.5mg/l+IBA 0.5mg/l+ sugar 20g/l+ agar 5g/l, pH is 5.7.
3), when treating that the indefinite bud that grows on the segment tender shoots I grows to the high seedling I of 2.0～3.0cm, extract seedling I; This seedling I is cut into stem section I about 1cm, above-mentioned stem section I is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is: MS minimal medium+BA 1.0mg/l+IBA 0.1mg/l+ sugar 20g/l+ agar 5g/l, pH is 5.7.
4), II when treating that the indefinite bud that grows on the stem section I grows to the high seedling of 2.0～5.0cm, extract seedling II; This seedling II is cut into the long stem section II of 0.8～1.2cm, above-mentioned stem section II is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is with the proliferated culture medium of step 3).
5), when treating that the indefinite bud that grows on the stem section II grows to the high seedling III of 3.0～5.0cm, extract seedling III; III is inoculated in the root media with this seedling; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Root media is: 1/2MS minimal medium+NAA 0.2mg/l+ sugar 30g/l+ agar 9g/l, pH is 5.7.
6), when treating that above-mentioned seedling III grows at least 3 roots greater than 2cm, but the seedling of bottle outlet plantation.
According to said method, only needed 60～80 days can obtain test-tube plantlet; Therefore adopt method of the present invention can obtain a large amount of test-tube plantlets for a long time.
The method of embodiment 3, a kind of evodia rutaecarpa cultured in vitro, carry out following steps successively:
1), the evodia rutaecarpa tender shoots of getting no scab, robust growth is put into beaker, running water flushing 1～2 hour;
2) after the routine sterilization, (be 0.1%w/v HgCl, with above-mentioned tender shoots
2Handle after 6～10 minutes, aseptic water washing 5～8 times blots with aseptic filter paper then), be cut into the segment tender shoots I about 1cm, be inoculated on the evoking adventive bud medium and cultivate; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
The evoking adventive bud medium is: MS minimal medium+BA 1.0mg/l+IBA 0.1mg/l+ sugar 30g/l+ agar 9g/l, pH is 5.5～6.0.
3), when treating that the indefinite bud that grows on the segment tender shoots I grows to the high seedling I of 2.0～3.0cm, extract seedling I; This seedling I is cut into stem section I about 1cm, above-mentioned stem section I is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is: MS minimal medium+BA 0.3mg/l+IBA0.3mg/l+ sugar 30g/l+ agar 9g/l, pH is 5.5～6.0.
4), II when treating that the indefinite bud that grows on the stem section I grows to the high seedling of 2.0～5.0cm, extract seedling II; This seedling II is cut into the long stem section II of 0.8～1.2cm, above-mentioned stem section II is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Proliferated culture medium is with the proliferated culture medium of step 3).
5), when treating that the indefinite bud that grows on the stem section II grows to the high seedling III of 3.0～5.0cm, extract seedling III; III is inoculated in the root media with this seedling; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
Root media is: 1/2MS minimal medium+NAA0.5mg/l+ sugar 30g/l+ agar 5g/l, pH is 5.5～6.0.
6), when treating that above-mentioned seedling III grows at least 3 roots greater than 2cm, but the seedling of bottle outlet plantation.
According to said method, only needed 60～80 days can obtain test-tube plantlet; Therefore adopt method of the present invention can obtain a large amount of test-tube plantlets for a long time.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (5)
1. the tissue culture method of an evodia rutaecarpa is characterized in that may further comprise the steps successively:
1), the evodia rutaecarpa tender shoots of getting no scab, robust growth carries out the flowing water flushing;
2), the evodia rutaecarpa tender shoots after the flushing of above-mentioned flowing water is sterilized through routine, be cut into the long segment tender shoots I of 0.5～1.0cm then, above-mentioned segment tender shoots I be inoculated on the evoking adventive bud medium cultivate; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
3), when treating that the indefinite bud that grows on the segment tender shoots I grows to the high seedling I of 2.0～3.0cm, extract seedling I; This seedling I is cut into the long stem section I of 0.5～1.2cm, above-mentioned stem section I is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
4), when treating that the indefinite bud that grows on the stem section I grows to the high seedling II of 2.0～5.0cm, extract seedling II; This II of growing sturdily for a short time is cut into the long stem section II of 0.8～1.2cm, above-mentioned stem section II is inoculated on the proliferated culture medium cultivates; Condition of culture is: illumination in 16 hours, intensity of illumination 30～40 μ mol m
-2.s
-1, temperature is 26～28 ℃; 8 hours dark cultivations, temperature is 20～22 ℃; Above-mentioned illumination and dark the cultivation are hocketed;
5), when treating that the indefinite bud that grows on the stem section II grows to the high seedling III of 3.0～5.0cm, extract seedling III; This little bent III is inoculated in the root media;
6), when treating that above-mentioned seedling III grows at least 3 roots greater than 2cm, but the seedling of bottle outlet plantation.
2. the tissue culture method of evodia rutaecarpa according to claim 1, it is characterized in that: step 2) in the evoking adventive bud medium be: MS minimal medium+BA 0.5～1.0mg/l+IBA 0.1～0.5mg/l+ sugar 20～30g/l+ agar 5～9g/l, pH is 5.5～6.0.
3. the tissue culture method of evodia rutaecarpa according to claim 2, it is characterized in that: the proliferated culture medium in step 3) and the step 4) is: MS minimal medium+BA 0.3～1.0mg/l+IBA 0.1～0.3mg/l+ sugar 20～30g/l+ agar 5～9g/l, pH is 5.5～6.0.
4. the tissue culture method of evodia rutaecarpa according to claim 3, it is characterized in that: the root media in the step 5) is: 1/2MS minimal medium+NAA 0.2～0.5mg/l+ sugar 30g/l+ agar 5～9g/l, pH is 5.5～6.0.
5. the tissue culture method of evodia rutaecarpa according to claim 4, it is characterized in that: described step 1) is: the evodia rutaecarpa tender shoots is put into beaker, running water flushing 1～2 hour.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599061A (en) * | 2012-03-29 | 2012-07-25 | 常熟市海虞茶叶有限公司 | Method for rapid propagating tissue of cornus alba |
| CN106912378A (en) * | 2017-03-13 | 2017-07-04 | 玉林师范学院 | A kind of rapid propagation method of south of the Five Ridges medicinal and edible plant evodia lepta |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491216B (en) * | 2009-03-09 | 2011-11-09 | 浙江省农业科学院 | Evodia fruit tissue-culture quick propagation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599061A (en) * | 2012-03-29 | 2012-07-25 | 常熟市海虞茶叶有限公司 | Method for rapid propagating tissue of cornus alba |
| CN106912378A (en) * | 2017-03-13 | 2017-07-04 | 玉林师范学院 | A kind of rapid propagation method of south of the Five Ridges medicinal and edible plant evodia lepta |
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