CN1669409A - Outside sprout-cultivating-bottle radication method for saussurea involucrate set - Google Patents
Outside sprout-cultivating-bottle radication method for saussurea involucrate set Download PDFInfo
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- CN1669409A CN1669409A CN 200510072449 CN200510072449A CN1669409A CN 1669409 A CN1669409 A CN 1669409A CN 200510072449 CN200510072449 CN 200510072449 CN 200510072449 A CN200510072449 A CN 200510072449A CN 1669409 A CN1669409 A CN 1669409A
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Abstract
The invention relates to a method of Xinjiang saussurea involucrate group culture sprout bottle ectotrophic root, it comprises the following steps: match the MS or B5 or White or Nitsh or N6 well in the common condition without sterilization, mix them with supporting agent vermiculite or cellulose or chip or coconut furfuryl, separately load into the incubator, then wash the unrooted saussurea involucrate group culture sprout from the gelose with water, move the sprout into the incubator, semi-open culture for 10 to 15 days, then the saussurea involucrate sprout can arise good roots. The invention combines the arising root and growing seed as the first step, the gained saussurea involucrate root is well growled without exercise and domestication, it shortens the time, saves a good deal of cost, and is good to spread in mass production.
Description
Technical field
The present invention relates to a kind of outside sprout-cultivating-bottle radication method for saussurea involucrate set
Background technology
Saussurea involucrata (Saussurea involucrata.et kir) is one of famous and precious Chinese herbal medicine, and Xinjiang is one of main place of production.Saussurea involucrata in the market mainly comes from wild, and this has just destroyed fragile ecology greatly; The growth of the suitable saussurea involucrata in many areas, Xinjiang, therefore in recent years the artificial cultivation of saussurea involucrata carry out larger, wherein the effect of tissue culture technique in the extensive artificial planting of saussurea involucrata more and more is much accounted of.
According to the tissue culture of relevant document, following report is arranged at present about Xinjiang Saussurea involucrate:
Watt Cuba promise baby waits with the Xinjiang tender Xinjiang Saussurea involucrate plant different parts of children and makes explant, is inoculated in MS cultivations that contains different hormonal readinesses, promptly forms callus through cultivation in 15 days; Callus is transferred to additional KT, on the MS medium of BA, 2-3 after week bud begin differentiation; (0.1～1.0mg/L)+active carbon (5g/L) is the root media root induction with 1/2MS+IAA; With the exercise of the seedling that forms in the enterprising line number of agarose medium sky, from test tube, be transplanted in the flowerpot again, keep air themperature with plastic film.And handled at low temperatures some days, the survival rate of Yi Zaiing is about 50% like this.
As explant, on medium MS+6-BA (2mg/L)+NAA (0.5mg/L), begin to sprout about 10 days callus occurs to prince's rosy clouds etc. with cotyledon; Callus forwarded on bud differential medium MS+6-BA (0.5mg/L)+NAA (0.5mg/L) grow the bud of growing thickly very soon, the bud of growing thickly of 3-4 centimeter length is cut into to change behind the simple bud plants on root media 1/2MS+IAA (0.5mg/L)+active carbon (0.1%), the most bud of growing thickly in the left and right sides can grow 2-5 bar root in 30 days, form whole plant, opened the bottleneck hardening during transplanting 3 days, plant after clean in flowerpot, keep certain humidity, survival rate can reach about 50%.
Song Huifeng etc. as explant, obtain test-tube plantlet with the Xinjiang Saussurea involucrate shoot tip meristem; On root media 1/2MS+IBA (0.5mg/L), there are 5～6 roots to form about 20 days, opened the bottle cap hardening during transplanting 3 days, be transplanted in the nutritious bag of forest soil after cleaning, cover with film, temperature remains on about 20 ℃, and survival rate can reach about 80%.
Piao Rizi etc. with the aseptic blade of Xinjiang Saussurea involucrate as explant, the test-tube plantlet that obtains root induction on the root media of 1/2MS+IAA (0.2mg/L)+active carbon (0.5%), when root is about 1 centimetre of left and right sides, to transition cultivating matrix (humus soil: river sand=2: 1) go up transplanting, keep humidity, survival rate is 72%.
From above traditional rooting method for tissue culture as can be seen, conventional method is utilized the light Mixed culture usually, with gelatinizing agent such as agarose, acetyl gellan gum (Gelrite) as support substance, add sugar as sucrose, glucose etc. as carbon source, plant is placed cultivation under the light.There is following problem in this method:
1, because the existence of polysaccharide causes the pollution of medium, container and internal environment, need sterilization through autoclaving and other methods, the transplanting of plant tissue or successive transfer culture process be cleaning platform and suchlike sterile working very carefully, could avoid as much as possible then polluting, such process expends a large amount of labours, and need the long time, so this method has more problem.
2, under most situation, moisture is more in gel, and the root of inducing is transplanted to the easy dehydration of seedling in the soil, poor growth, root growth postpones, and because medium (gel) is the non-pneumatic phase, the root that obtains is a water root, generally can not transplant in soil, need through hardening, transplanting survival rate is lower;
3, because the polysaccharide in the required carbon source medium of great majority growth, and it is lower to photosynthetic dependence, so the growth of leaf is less, need the adaptation of long period after being transplanted in the soil, the regeneration plant of gained needs for a long time, the domestication step of labor intensive is absolutely necessary, can the light autophyting growth until plant; The plant that can not adapt to such domestication in addition is very easily dead.
In order to improve these problems, the someone has proposed certain methods, for example reports (Japanese publication (kokai) 4-311326 with the method for bactericide as the existing people of the method that overcomes problem 1,9-235208,7-255305,7-255306,3-297331,10-313717).
There is report to disclose the method for problem 2 in addition, it is reported that Chinese publication (CN1284261A) discloses materials such as utilizing vermiculite, cellulose as cultivating support substance, add the nutrient solution that contains a certain amount of bactericide,
But in the said method, owing to be to have used bactericide, plant is had the obvious impairment effect in incubation, delayed the growth of plant, this problem has also limited the application of these methods greatly.And the present invention is to the committed step in the Xinjiang Saussurea involucrate tissue culture---take root and done careful research, and obtained a kind of new method.
Summary of the invention
The outside sprout-cultivating-bottle method that the purpose of this invention is to provide a kind of easy and simple to handle, with low cost, extraordinary saussurea involucrate tissue culture sprout of effect.This method need not traditional sterile working step, makes the saussurea involucrate set seedling rooting become simple, and practicality is very strong.In addition, this method will be taken root and hardening is combined into a step, and the snow lotus root upgrowth situation that obtains is good, need not to take exercise and domestication, shortened the time, save a large amount of costs, pollute with temperature control, take root on the medium of unrooted saussurea involucrata seedling in open environment that the group training is obtained.
Outside sprout-cultivating-bottle radication method for saussurea involucrate set of the present invention follows these steps to carry out:
A, under common environment, MS or B5 or White or Nitsh or N6 are added the plant hormone medium and prepare, need not sterilization, after Supporting Media vermiculite or cellulose or wood chip or coconut palm chaff mix in 5: 4 ratios, be sub-packed in the container of cultivation, cultivation temperature 10-25 ℃, the growth of control bacterium makes that the tissue cultivating seedling on open sugar-free culture-medium is taken root, and carries out hardening simultaneously;
B, will wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is in the container that contains the Supporting Media medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
C, culture environment condition are: illumination is 1000-5000lex; CO
2Concentration maintains in the normal atmosphere or is higher, as 300-2000 μ mol/mol; Temperature is 20-24 ℃; Humidity is 40-80%, and incubation time is 10-25 days, and the saussurea involucrata seedling can be taken root.
The characteristics of outside sprout-cultivating-bottle radication method for saussurea involucrate set of the present invention are: replaced agar with vermiculite, because vermiculite can reuse, therefore cost has greatly been descended; Need not to carry out environment and medium, container, plant etc. are sterilized and sterile working, save the labour in a large number, increase work efficiency; And the growing way of seedling obviously is better than taking root on traditional closed agar medium, and root growth is very fast, the well developed root system of formation, and seedling can enter in the soil without domestication, the survival rate height; Can avoid simultaneously the phenomenon of the growth of the above-mentioned use bactericide control seedling of mentioning again.
Outside sprout-cultivating-bottle radication method for saussurea involucrate set of the present invention, this method is utilized the growth of suitable cultivation temperature (10-25 ℃) control bacterium, make the tissue cultivating seedling on open culturing base (sugar-free) take root, and carry out hardening simultaneously, the regeneration plant of gained, well developed root system, amount of growth is bigger, owing to take root under open environment, does not provide sugar as carbon source, plant is an autotrophy, and survival rate greatly improves in the time of therefore in being transplanted to soil.
Container: with various open containers can: such as the bigger box of various vials, plastic bottle, volume and basin (material is not limit, but will keep the top can printing opacity) or the like;
Medium holder: be not with traditional gelatinizers such as agar, use the material of Supporting Medias such as vermiculite, cellulose, wood chip, coconut palm chaff instead; Wherein the performance of vermiculite is more suitable for the present invention and uses, because vermiculite is lighter, suction, water retention capacity are strong, and the space is big, and be beneficial to plant and produce aerial root, rather than water root, this transplant survival to seedling is most important.
Medium: according to concrete experimental selection, all liquid nutrient mediums (removing sugared composition) can use, as MS, B5, White, Nitsh, N6 (above-mentioned medium is all removed sugared composition) or the like.
Condition of culture: illumination: be controlled at 1000-5000lex, can suitably regulate according to the culture situation.
CO
2Concentration: maintain CO in the normal atmosphere
2Concentration gets final product, or CO
2Concentration is higher, and as 300-2000 μ mol/mol, the culture upgrowth situation can be better.
Temperature: because environmental temperature when higher, has been accelerated the pollution of medium, be unfavorable for operation, the temperature of culture environment is controlled between 10-25 ℃; In this temperature range, plant grows fine, and during plant growing (10-25 days), microbial reproduction speed is slower, and medium seldom has the situation of pollution to take place.
Humidity: be controlled at 40-80%, avoid the humidity of culture environment excessive or too small.
In the present invention, the aseptic saussurea involucrata seedling (unrooted) that will obtain by the normal structure cultural method selects wherein relatively more healthy and stronger no offspring (more than the height of seedling degree 4cm, having more than the 4-5 sheet leaf) to take root.The saussurea involucrata seedling is taken out (in conventional environment from agar medium, need not aseptic condition), wash with running water, attention cleans up the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling, then transplantation of seedlings is supported in the container of composition to containing above-mentioned medium, opening is cultivated, in above-mentioned culture environment, cultivate, general about 10-25 days, just can bear desirable root (more than the general long 3cm of root, radical reaches more than 10), and vegetation growth state is good, and transplanting survival rate is better than traditional agar rooting method.
Effect: because the present invention need not traditional sterile working, make the saussurea involucrate set seedling rooting become simple, practicality is very strong, is beneficial to large-scale promotion application.
Embodiment
Respectively choose 100 strain saussurea involucrate tissue culture sprouts (unrooted), carry out the comparison of rooting method in outside sprout-cultivating-bottle method of the present invention and the traditional bottle.
Embodiment 1 (experimental implementation of the present invention)
Choose 100 strain saussurea involucrate tissue culture sprouts (unrooted), under common environment (need not aseptic), MS medium (sugar-free) is prepared, need not sterilization, mix in 5: 4 ratios with the Supporting Media vermiculite, make the medium that mixes and vermiculite can not be too dry or moistening after, be sub-packed in the container of cultivation, 10 ℃ of cultivation temperature, the growth of control bacterium makes that the tissue cultivating seedling on open sugar-free culture-medium is taken root, and carries out hardening simultaneously;
To wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is supported in the container of composition to containing Supporting Media vermiculite medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
The culture environment condition is: illumination 1000lex; CO
2Concentration maintains 300 μ mol/mol; Temperature is 20 ℃; Humidity is 40%, and incubation time is 10 days, and the saussurea involucrata seedling can be taken root.
Embodiment 2 (experimental implementation of the present invention)
Choose 100 strain saussurea involucrate tissue culture sprouts (unrooted), under common environment (need not aseptic), B5 added plant hormone (sugar-free) medium, need not sterilization, after the Supporting Media cellulose mixes in 5: 4 ratios, be sub-packed in the container of cultivation 15 ℃ of cultivation temperature, the growth of control bacterium, make the tissue cultivating seedling on open sugar-free culture-medium take root, and carry out hardening simultaneously;
To wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is in the container that contains Supporting Media cellulose medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
The culture environment condition is: illumination is 2000lex; CO
2Concentration is kept 1000mol/mol; Temperature is 22 ℃; Humidity is 60%, and incubation time is 15 days, and the saussurea involucrata seedling can be taken root.
Embodiment 3 (test operation of the present invention)
Under common environment, (need not aseptic) White is added plant hormone (sugar-free) medium, need not sterilization, after the Supporting Media wood chip mixes in 5: 4 ratios, be sub-packed in the container of cultivation, 20 ℃ of cultivation temperature, the growth of control bacterium makes that the tissue cultivating seedling on open culturing base sugar-free is taken root, and carries out hardening simultaneously;
To wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is in the container that contains the Supporting Media sawdust medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
The culture environment condition is: illumination is 4000lex; CO
2Concentration maintains 1500mol/mol; Temperature is 24 ℃; Humidity is 70%, and incubation time is 20 days, and the saussurea involucrata seedling can be taken root.
Embodiment 4 (test operation of the present invention)
Choose 100 strain saussurea involucrate tissue culture sprouts (unrooted), under common environment, (need not aseptic) Nitsh is added plant hormone (sugar-free) medium, need not sterilization, after Supporting Media coconut palm chaff mixes in 5: 4 ratios, be sub-packed in the container of cultivation 25 ℃ of cultivation temperature, the growth of control bacterium, make the tissue cultivating seedling on open sugar-free culture-medium take root, and carry out hardening simultaneously;
To wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is in the container that contains Supporting Media coconut palm chaff medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
The culture environment condition is: illumination is 5000lex; CO
2Concentration maintains 2000mol/mol; Temperature is 24 ℃; Humidity is 80%, and incubation time is 25 days, and the saussurea involucrata seedling can be taken root.
Embodiment 5 (operated in accordance with conventional methods)
With taking root of 100 saussurea involucrata seedlings, the go forward side by side horizontal high voltage sterilization of sterile environment, preparation medium is operated on superclean bench, and whole operation needs 3 hours, and culture environment is illumination 3000lex, and temperature is controlled between 20 ℃; Seedling was growth on the agar medium about 30 days, and seedling can bear root; This transplantation of seedlings is performed physical exercise on vermiculite and forest soil mixed-matrix, can in soil, transplant in about about 10 days.
Embodiment 6 (operated in accordance with conventional methods)
With taking root of 100 saussurea involucrata seedlings, the go forward side by side horizontal high voltage sterilization of sterile environment, preparation medium is operated on superclean bench, and whole operation needs 4 hours.Culture environment is illumination 3000lex, and temperature is controlled between 24 ℃; Seedling was growth on the agar medium about 40 days, and seedling can bear root; This transplantation of seedlings is performed physical exercise on vermiculite and forest soil mixed-matrix, can in soil, transplant in about about 10 days.
The present invention and conventional method relatively see Table
Find out that from the contrast experiment outside sprout-cultivating-bottle radication method for saussurea involucrate set of the present invention can be eliminated the sterilization process of labor intensive, need not in gnotobasis, to operate.In addition, the snow lotus root upgrowth situation is good, and the seedling that obtains need not be taken exercise and tame, and has shortened the time, saves a large amount of costs, and practicality is extremely strong.In the experimental implementation of invention, need not carry out disinfection to culture environment and the apparatus of manipulating, whole operation is simple, quick, only needs about 1 hour, cultivates in above-mentioned culture environment about 10～15 days, and the saussurea involucrata seedling can bear desirable root.
Claims (1)
1, a kind of outside sprout-cultivating-bottle radication method for saussurea involucrate set is characterized in that following these steps to carrying out:
A, under common environment, MS or B5 or White or Nitsh or N6 are added the plant hormone medium, need not sterilization, after Supporting Media vermiculite or cellulose or wood chip or coconut palm chaff mix in 5: 4 ratios, be sub-packed in the container of cultivation, cultivation temperature 10-25 ℃, the growth of control bacterium makes that the tissue cultivating seedling on open sugar-free culture-medium is taken root, and carries out hardening simultaneously;
B, will wash with running water from the unrooted saussurea involucrate tissue culture sprout that agar takes out then, the sugar culture-medium composition that contains that adheres on the saussurea involucrata seedling is cleaned up, transplantation of seedlings is in the container that contains the Supporting Media medium, semi-open, the film that seals above the blake bottle is covered half cultivation;
C, culture environment condition are: illumination is 1000～5000lex; CO
2Concentration maintains in the normal atmosphere or is higher, as 300-2000 μ mol/mol; Temperature is 20-24 ℃; Humidity is 40-80%, and incubation time is 10-25 days, and the saussurea involucrata seedling can be taken root.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139571B (en) * | 2007-08-29 | 2011-09-14 | 清华大学 | Method for producing saussurea involucrate secondary metabolites |
| CN102630443A (en) * | 2012-03-02 | 2012-08-15 | 中国热带农业科学院橡胶研究所 | Method for inducing new root of rubber tree |
| CN106359049A (en) * | 2016-08-29 | 2017-02-01 | 江西九草铁皮石斛科技协同创新有限公司 | Aerial fog type seedling hardening method of dendrobium officinale tissue culture seedlings |
| CN107439181A (en) * | 2017-08-03 | 2017-12-08 | 阜康国有林管理局 | Xinjiang Saussurea involucrate Proterozoic structure of community rationally and fast breeding method |
-
2005
- 2005-05-10 CN CN 200510072449 patent/CN1669409A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139571B (en) * | 2007-08-29 | 2011-09-14 | 清华大学 | Method for producing saussurea involucrate secondary metabolites |
| CN102630443A (en) * | 2012-03-02 | 2012-08-15 | 中国热带农业科学院橡胶研究所 | Method for inducing new root of rubber tree |
| CN106359049A (en) * | 2016-08-29 | 2017-02-01 | 江西九草铁皮石斛科技协同创新有限公司 | Aerial fog type seedling hardening method of dendrobium officinale tissue culture seedlings |
| CN107439181A (en) * | 2017-08-03 | 2017-12-08 | 阜康国有林管理局 | Xinjiang Saussurea involucrate Proterozoic structure of community rationally and fast breeding method |
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