CN100333638C - Method for inducing, breeding and transplanting triperygium wilfordii cluster buds - Google Patents
Method for inducing, breeding and transplanting triperygium wilfordii cluster buds Download PDFInfo
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- CN100333638C CN100333638C CNB2005100280395A CN200510028039A CN100333638C CN 100333638 C CN100333638 C CN 100333638C CN B2005100280395 A CNB2005100280395 A CN B2005100280395A CN 200510028039 A CN200510028039 A CN 200510028039A CN 100333638 C CN100333638 C CN 100333638C
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Abstract
The present invention relates to a method for tripterygium wilfordii asexual propagation. The method comprises the steps of induction, proliferation, strong seedlings, rooting, transplantation, etc. for cluster buds. Adopting the method of the present invention can protect wild tripterygium wilfordii resources, which provides a new way capable of supplying tripterygium wilfordii raw materials continuously.
Description
Technical field
The invention belongs to biological technical field.Specifically, the present invention relates to the vegetative method of a kind of thunder godvine, the inducing of the bud that is specifically related to grow thickly, propagation, strong sprout, take root and transplanting and other steps.
Background technology
Thunder godvine (Tripterygum wilfordii Hook f.) is the Celastraceae tripterygium plant.Thunder godvine is just progressively gone to the world with its strong drug action, widely applicable characteristics as the peculiar medicinal material of a kind of China, is expected to become the good medicine of capturing all kinds of chronic diseases.
Thunder godvine is perennial woody climber, poor growth, and unrestricted a large amount of diggings have made wild resource be subjected to heavy damage.In recent years, domestic units concerned adopt seed and division propagation mode to carry out artificial planting, but its reproduction coefficient is low, is difficult to form at short notice large-scale production.In addition, since the seventies in 20th century, Japan, Canada and the scientific research personnel of medicine institute of the Chinese Academy of Sciences have carried out the research of thunder godvine tissue and cell culture batch production production triptolide, from cultured products, isolate the purpose compound, but because of its complex process, the cost height is not seen the report that is applied to industrialization so far.The applicant has invented " preparation technology of tripterygium wilfordii regenerated plant ", and this invention mainly provides a kind of technology that thunder godvine genetic improvement, the technology that improves the purpose compounds content and regeneration plant thereof are induced and bred that is used for.
Plant rapid propagation in vitro technology can make the approach of purpose plant by the blastogenesis bud, produces a large amount of tissue cultured test-tube seedlings in a short time.Test-tube plantlet can directly be used as medicinal raw material; Also can be used as seedling, for large-scale planting.Each plant, particularly wild plant all have its special requirement to cultured in vitro, have only suitable cultural method just can induce to grow thickly in a large number bud to form, could the numerous coefficient of existing higher expansion, can guarantee the quality of fast propagating seedling again.And suitable transplanting method could obtain higher test-tube seedling transplanting survival rate.Up to now, do not see thunder godvine cultured in vitro inducing clumping bud, expand research and patent report numerous and that transplant.
Summary of the invention
Therefore, the present invention relates to the vegetative method of a kind of thunder godvine, described method comprises:
(1) get thunder godvine branch or seed seedling as initial donor material, sterilization, aseptic water washing, behind the suck dry moisture, getting tender tissue is explant, is inoculated in the inducing clumping bud medium, the inducing of the bud of growing thickly; Wherein, this inducing clumping bud medium is added with the basic element of cell division, sucrose 20-40g/L, the agar 5-8g/L of 0.01-1.0mg/L, pH 5-6 based on the MS medium;
(2) get the segment of the shoot band top/axillalry bud that step (1) obtains, change the expansion breeding culture medium over to, the propagation of the bud of growing thickly; Wherein, this expansion breeding culture medium is added with the 0.01-1.0mg/L basic element of cell division, sucrose 20-40g/L, agar 5-8g/L, pH 5-6 based on the MS medium.
In one embodiment, described method also comprises step (3):
(3) get the segment of the triperygium wilfordii cluster seedling band top/axillalry bud of step (2) gained, change the strong seedling culture base over to, cultivate; Wherein, this strong seedling culture base is added with caseinhydrolysate 200-1000mg/L, sucrose 20-40g/L, agar 5-8g/L based on the MS medium, and pH regulator is to 5-6.
In one embodiment, also comprise after the described step (3):
(4) get the segment of the triperygium wilfordii cluster seedling band bud that step (3) obtains, change root media over to, root media is the MS medium that macroelement reduces by half, and adds sucrose 20-40g/L, agar 5-8g/L, and pH regulator induces it to take root to 5-6;
(5) the room temperature hardening is 2～3 days, and the seedling of taking root of getting step (4) gained moves in the soil transplants; Wherein, the humidity of transplanting subenvironment is 60%-90%, and temperature remains on more than 5 ℃.
In one embodiment, the basic element of cell division of described inducing clumping bud medium, the use of expansion breeding culture medium is selected from one or more in 6-benzyl aminopurine, 6-chaff aminopurine, zeatin, isopentennyladenine, the phenyl thiadiazolyl group urea.In one embodiment, also can contain in the above-mentioned basic element of cell division one or more in the described strong seedling culture base.In another embodiment, above-mentioned root media also can contain one or both in growth hormone such as methyl and the indole-3-butyric acid.
In a preferred embodiment, the basic element of cell division that uses in the above-mentioned steps (1) is 6-benzyl aminopurine and 6-chaff aminopurine, and its consumption is respectively 0.2-0.3mg/L.
In a preferred embodiment, the basic element of cell division that uses in the above-mentioned steps (2) is 6-benzyl aminopurine and 6-chaff aminopurine, and its consumption is respectively 0.1-0.15mg/L.
In one embodiment, the pH of soil is 5.0-7.0 in the above-mentioned steps (5).
In one embodiment, the soil in the above-mentioned steps (5) is humus soil, as Black Hills mud, peat etc.
In one embodiment, the cultivation temperature of above-mentioned steps (1) and (2) is 18-30 ℃, and light application time is 6-12 hour.
In one embodiment, the humidity described in the above-mentioned steps (5) is 70-80%.
The present invention can protect wild thunder godvine resource, and the new way of a sustainable supply thunder godvine raw material is provided.Traditional thunder godvine propagation method adopts cottage propagation usually, promptly utilizes kind of a stem, kind root to carry out vegetative propagation.Theoretically, vegetative propagation can keep the kind of this plant.The present invention also is a kind of asexual reproduction method, but can obtain more seedling and plant in the shorter time, and can carry out standardization, large-scale production under controlled condition.
Description of drawings
Fig. 1 shows the thunder godvine tissue cultivating seedling of taking root that adopts the inventive method to obtain.
Fig. 2 shows the thunder godvine plant of the transplant survival that adopts the inventive method acquisition.
Embodiment
In the present invention, the step of inducing of triperygium wilfordii cluster buds comprises: get thunder godvine branch or seed seedling as initial donor material, and sterilization, aseptic water washing behind the suck dry moisture, is chosen explant, is inoculated in the inducing clumping bud medium, the inducing of the bud of growing thickly.
Can use the surface sterilizing agent commonly used of various this areas to carry out disinfection, as 75% alcohol, 4% Anaprox, 2% clorox, 0.1% mercury chloride etc.As long as can reach the purpose of obtaining aseptic explant.
The stem section of desirable tender shoots or band axillalry bud also can use other position as young tender stem section, blade as explant.
The prescription of inducing clumping bud medium that is used for this step is usually based on the MS medium, and other adds the 0.01-1mg/L basic element of cell division, sucrose 20-40g/L, agar 5-8g/L, and pH regulator is to 5-6.In one embodiment, the consumption of sucrose is 20-40g/L, can also be 25-35g/L, for example is 30g/L.In one embodiment, the consumption of agar can be 5-8g/L, for example is 6g/L.Can be as 5.0-6.0 with pH regulator, perhaps be for example 5.4-5.6.
In the optional 6-benzyl aminopurine freely of the basic element of cell division (hereinafter to be referred as 6-BA), 6-chaff aminopurine (hereinafter to be referred as KT), zeatin (hereinafter to be referred as ZT), isopentennyladenine (hereinafter to be referred as 2ip), the phenyl thiadiazolyl group urea (hereinafter to be referred as TDZ) one or more.These basic elements of cell division are commercially available, as buying from companies such as Sigma, Fluka.The consumption of the basic element of cell division is generally 0.01-1.0mg/L in the inducing clumping bud medium, as 0.05-1.0mg/L, 0.1-0.8mg/L, 0.1-0.5mg/L etc.Its consumption of the concrete basic element of cell division is not necessarily identical, and for example, in one embodiment, the employed basic element of cell division is 6-BA, and its consumption is 0.2-0.4mg/L, can also be 0.2-0.3mg/L.In one embodiment, the basic element of cell division is KT, and its consumption is 0.2-0.4mg/L, can also be 0.2-0.3mg/L.In another embodiment, the employed basic element of cell division is 6-BA and KT, and its consumption is respectively 0.2-0.3mg/L.
Cultivation temperature can be 18～30 ℃.In one embodiment, cultivation temperature can be 20-25 ℃, for example is 22-25 ℃.Give illumination in 6～12 hours every day, for example 10-12 hour illumination.After so cultivating 20～50 days (for example 30-40 days), 95% explant can induce the bud of growing thickly.
Afterwards, get the segment of each shoot band top/axillalry bud, breed.This step uses the expansion breeding culture medium, and this medium is based on the MS medium, and other adds the 0.01-0.5mg/L basic element of cell division, sucrose 20-40g/L, agar 5-8g/L, and pH regulator is to 5-6.In one embodiment, the consumption of sucrose is 20-40g/L, can also be 25-35g/L, for example is 30g/L.In one embodiment, the consumption of agar can be 5-8g/L, for example is 6g/L.Can be as 5.0-6.0 with pH regulator, perhaps be for example 5.4-5.6.
Among the optional 6-BA freely of the basic element of cell division, KT, ZT, 2ip, the TDZ one or more, its consumption is generally 0.01-0.5mg/L, as 0.05-0.5mg/L, 0.02-0.3mg/L, 0.1-0.2mg/L etc.In one embodiment, the employed basic element of cell division is 6-BA, and consumption is 0.05-0.2mg/L, can also be 0.08-0.15mg/L.In one embodiment, the employed basic element of cell division is KT, and consumption is 0.05-0.2mg/L, can also be 0.08-0.15mg/L.In another embodiment, the employed basic element of cell division is 6-BA and KT, and consumption is respectively 0.1-0.15mg/L.
The cultivation temperature of this step can be 18～30 ℃.In one embodiment, cultivation temperature can be 20-25 ℃, for example is 22-25 ℃.Give illumination in 6～12 hours every day, for example 10-12 hour illumination.After so cultivating 20～50 days (for example 30-40 days), can obtain to reach growth coefficient 6.0 or more (growth coefficient=through the band bud segment number of the one-step growth cultivation bud number/cultivation of growing thickly that obtains).
Then, the segment of the triperygium wilfordii cluster seedling band top/axillalry bud of the propagation of learning from else's experience changes in the strong seedling culture base, carries out strong sprout.The strong seedling culture base is based on the MS medium, and other adds caseinhydrolysate 200-1000mg/L, sucrose 20-40g/L, agar 5-8g/L, and pH regulator is to 5-6.In one embodiment, the consumption of caseinhydrolysate is 400-600mg/L, for example 500mg/L.In one embodiment, the consumption of sucrose is 20-40g/L, can also be 25-35g/L, for example is 30g/L.In one embodiment, the consumption of agar can be 5-8g/L, for example is 6g/L.Can be as 5.0-6.0 with pH regulator, perhaps be for example 5.4-5.6.
Can add one or more the basic element of cell division that is selected among 6-BA, KT, ZT, 2ip, the TDZ in the strong seedling culture base, its consumption is generally 0.01-0.5mg/L, as 0.01-0.3mg/L, 0.05-0.5mg/L etc.In one embodiment, the employed basic element of cell division is 6-BA, and consumption is 0.05-0.2mg/L, can also be 0.1-0.15mg/L.In one embodiment, the employed basic element of cell division is KT, and consumption is 0.05-0.2mg/L, can also be 0.1-0.15mg/L.In another embodiment, the employed basic element of cell division is 6-BA and KT, and consumption is respectively 0.1-0.15mg/L.
The cultivation temperature of this step can be 18～30 ℃.In one embodiment, cultivation temperature can be 20-25 ℃, for example is 22-25 ℃.Give illumination in 6～12 hours every day, for example 10-12 hour illumination.After so cultivating 15～40 days (for example 20-30 days), can make the bud color and luster of growing thickly dark green, the stem stalk is sturdy, and it is vigorous to grow, and plays the effect in strong sprout.
Afterwards, the segment of the triperygium wilfordii cluster seedling band bud of desirable stalwartness changes root media over to, cultivates 15-40 days under cultivation temperature 18-30 ℃, the condition of illumination in 6～12 hours every day.
The MS medium that employed root media reduces by half with macroelement, other adds sucrose 20-40g/L, agar 5-8g/L, and PH is adjusted to 5-6.In one embodiment, the consumption of sucrose is 20-40g/L, can also be 25-35g/L, for example is 30g/L.In one embodiment, the consumption of agar can be 5-8g/L, for example is 6g/L.Can be as 5.0-6.0 with pH regulator, perhaps be for example 5.4-5.6.In addition, in this medium, also can add in growth hormone methyl (NAA) and the indole-3-butyric acid (IBA) one or both.The consumption of methyl is 0.01-0.5mg/L, is generally 0.1-0.5mg/L, and the consumption of indole-3-butyric acid is 0.1mg/L-1.0mg/L, is generally 0.5-1.0mg/L.
Cultivation temperature can be 20-25 ℃, for example is 22-25 ℃.Light application time can be as 8-12 or 10-12 hour.After so cultivating 15～40 days (for example 20-30 days), can induce thunder godvine to take root, rooting rate nearly 100%.
Before transplanting the thunder godvine tissue cultivating seedling, open the culture dish bottle cap of the seedling of taking root, at room temperature hardening (making the tissue cultivating seedling that grows in the enclosed environment progressively adapt to the exercise process of open external environment) is 2～3 days, getting the seedling of taking root moves in the soil, the humidity of subenvironment remains on 60%-90%, soil conservation moisture state, preferably ponding not.Transplant 5～20 ℃ of temperature, be preferably 10-15 ℃, survival rate is more than 70%.After 40-60 days, transplanted seedling can be planted in the land for growing field crops.
The soil of transplanting usefulness can be slant acidity or neutrality, and for example, its pH can be 5-7, is generally 5.0-6.5, is more typically 5.0-5.5.Soil can be humus soil, as mountain mud, peat.The humidity of soil subenvironment can remain on as 70-80%.
The MS medium that the present invention uses is international medium, is commercially available, as buying from companies such as Sigma, Fluka.Also can prepare voluntarily according to the prescription of standard.Macroelement in the MS medium, trace element, molysite, organic element are like hereinafter defining.The sucrose that is added in the MS medium also can use other disaccharides to replace, and perhaps also can use glucose to replace.
In a preferred embodiment of the invention, get wild thunder godvine spray or seed seedling and make initial donor material, get its tender shoots for growing body outward, through surface sterilization sterilization 10～20 minutes, behind the aseptic water washing several, under the aseptic condition, place in the inducing culture, cultivation through about 35～40 days, 22～25 ℃ of cultivation temperature obtain to grow thickly bud.After the bud of waiting the to grow thickly elongation, get the band bud segment of its each shoot, change the expansion breeding culture medium over to, through about 40～50 days, cultivate, 22～25 ℃ of cultivation temperature, obtain to expand the new branch of numerous bud clump and elongation, expand numerous growth coefficient each time and can reach 5～6 times, repeat to expand numerous process and can reach quick breeding purpose.Get and expand numerous seedling, change the strong seedling culture base over to, 22～25 ℃, illumination 10～12 hours every days was cultivated 20～30 days, changed healthy seedling over to root media, and 22～25 ℃, illumination 10～12 hours every days was cultivated 20～30 days.Open the culture dish bottle cap of the seedling of taking root, at room temperature hardening is 2～3 days, gets the seedling of taking root and moves in the soil, and the humidity of subenvironment remains on 70%～80%, and soil will keep moisture state, but can not ponding.Transplant temperature with 10～20 ℃ of the bests, after 40～60 days, transplanted seedling can move into the land for growing field crops.
Below in conjunction with specific embodiment, to further specify technical characterictic of the present invention.Should be understood that following embodiment only to be used to the present invention is described and be not used in the scope of the present invention that limits.
Among the following embodiment of the present invention the prescription of used MS medium be (unit: mg/L):
Macroelement:
NH
4NO
4 1650
KNO
3 1900
CaCl
2.2H
2O 440
MgSO
4.7H
2O 370
KH
2PO
4 170
Trace element:
KI 0.83
H
3BO
3 6.2
MnSO
4.4H
2O 22.3
ZnSO
4.7H
2O 8.6
Na
2MoO
4.2H
2O 0.25
CuSO
4.5H
2O 0.025
CoCl
2.6H
2O 0.025
Molysite:
Na
2EDTA 37.3
FeSO
4.7H
2O 27.8
Organic element:
Glycine 2.0
Thiamine?HCl 0.1
Phridoxine?HCl 0.5
Nicotinic?acid 0.5
Myo-inositol 100.0
Inducing of embodiment 1 triperygium wilfordii cluster buds
Get that thunder godvine sprouts new buds then or the seed seedling as initial donor material, with 4% Anaprox sterilization 10 minutes, aseptic water washing was repeatedly, behind the suck dry moisture, the stem section of getting tender shoots or band axillalry bud is an explant, is inoculated in the MS medium that contains 6-BA 0.2mg/L, KT 0.2mg/L, and other adds sucrose 30g/L, agar 6g/L, PH is adjusted to 5.6, grow thickly the inducing of bud, 22～25 ℃, illumination 10～12 hours every days, cultivate after 30～40 days, 95% the outer body of growing can induce the bud of growing thickly.
The propagation of embodiment 2 triperygium wilfordii cluster buds
After the bud elongation of growing thickly to be induced, get the segment of each shoot band top/axillalry bud, change the expansion breeding culture medium over to, its minimal medium is MS, and other adds basic element of cell division 6-BA0.1mg/L, KT0.1mg/L, sucrose 30g/L, agar 6g/L, PH is adjusted to 5.6, the grow thickly propagation of bud, 22～25 ℃ of cultivation temperature, illumination 10～12 hours every days, cultivate after 30～40 days, its growth coefficient can reach more than 6.0.
The strong sprout of embodiment 3 triperygium wilfordii cluster buds
In the MS medium that adds, add caseinhydrolysate 500mg/L, other adds basic element of cell division 6-BA0.1mg/L, KT0.1mg/L, sucrose 30g/L, agar 6g/L, PH is adjusted to 5.6,22～25 ℃, illumination 10～12 hours every days was cultivated 20～30 days, obtained the dark green bud of growing thickly of color and luster, the stem stalk is sturdy, and it is vigorous to grow.
The strong sprout of embodiment 4 triperygium wilfordii cluster buds
Add caseinhydrolysate 500mg/L in the MS medium that adds, other adds sucrose 30g/L, agar 6g/L, and PH is adjusted to 5.6,22～25 ℃, and illumination 10～12 hours every days was cultivated 20～30 days, obtained the dark green bud of growing thickly of color and luster, and the stem stalk is sturdy, and it is vigorous to grow.
Taking root of embodiment 5 triperygium wilfordii cluster buds
Get the segment of healthy and strong triperygium wilfordii cluster seedling band bud, change the MS medium that macroelement reduces by half over to, the consumption of methyl is 0.05mg/L, and the consumption of indole-3-butyric acid is 0.5mg/L.Other adds sucrose 30g/L, agar 6g/L, and PH is adjusted to 5.6,22～25 ℃, and illumination 10～12 hours every days was cultivated 20～30 days, can induce tissue cultivating seedling to take root, rooting rate nearly 100%.
Taking root of embodiment 6 triperygium wilfordii cluster buds
Get the segment of healthy and strong triperygium wilfordii cluster seedling band bud, change the MS medium that macroelement reduces by half over to, other adds sucrose 30g/L, agar 6g/L, PH is adjusted to 5.6,22～25 ℃, illumination 10～12 hours every days, cultivated 20～30 days, and can induce tissue cultivating seedling to take root, rooting rate nearly 100%.
The transplanting of embodiment 7 thunder godvine tissue cultivating seedling
Open the culture dish bottle cap of the seedling of taking root, at room temperature hardening is 2～3 days, and getting the seedling of taking root, to move into pH be that the humidity of subenvironment remains on 70%～80% in the mountain mud of 5.0-5.5, soil conservation moisture state, but ponding not.Transplant temperature with 10～20 ℃ of the bests, about 40～60 days after, transplanted seedling can be planted in the land for growing field crops.This transplants and obtains 80% survival rate.
Claims (9)
1. vegetative method of thunder godvine is characterized in that described method comprises:
(1) gets thunder godvine branch, seed seedling, stem section or blade as initial donor material, sterilization, aseptic water washing, behind the suck dry moisture, getting tender tissue is explant, is inoculated in the inducing clumping bud medium, grow thickly the inducing of bud obtains the shoot of band terminal bud or axillalry bud; Wherein, this inducing clumping bud medium is added with the basic element of cell division, sucrose 20-40g/L, the agar 5-8g/L of 0.05-1.0mg/L, pH 5-6 based on the MS medium;
(2) get the band terminal bud of step (1) acquisition or the shoot segment of axillalry bud, change the expansion breeding culture medium over to, the propagation of the bud of growing thickly obtains the triperygium wilfordii cluster seedling; Wherein, this expansion breeding culture medium is added with the 0.01-0.5mg/L basic element of cell division, sucrose 20-40g/L, agar 5-8g/L, pH 5-6 based on the MS medium;
(3) get the band terminal bud of triperygium wilfordii cluster seedling of step (2) gained or the segment of axillalry bud, change the strong seedling culture base over to, cultivate, obtain the triperygium wilfordii cluster seedling; Wherein, this strong seedling culture base is added with caseinhydrolysate 200-1000mg/L, sucrose 20-40g/L, agar 5-8g/L based on the MS medium, and pH regulator is to 5-6;
(4) get the segment of the band bud of the triperygium wilfordii cluster seedling that step (3) obtains, change root media over to, obtain to take root seedling; Wherein, root media is the MS medium that macroelement reduces by half, and adds sucrose 20-40g/L, agar 5-8g/L, and pH regulator induces it to take root to 5-6.
2. the method for claim 1 is characterized in that, step also comprises after (4):
(5) the room temperature hardening is 2～3 days, and the seedling of taking root of getting step (4) gained moves in the soil transplants; Wherein, the humidity of transplanting subenvironment is 60%-90%, and temperature remains on more than 5 ℃.
3. the method for claim 1, it is characterized in that the basic element of cell division that uses in described inducing clumping bud medium and the expansion breeding culture medium is selected from 6-benzyl aminopurine, 6-chaff aminopurine, zeatin, isopentennyladenine, phenyl thiadiazolyl group urea or their combination.
4. method as claimed in claim 3, it is characterized in that the basic element of cell division that uses in the step (1) is 6-benzyl aminopurine and 6-chaff aminopurine, wherein, the consumption of 6-benzyl aminopurine is 0.2-0.3mg/L, and the consumption of 6-chaff aminopurine is 0.2-0.3mg/L.
5. method as claimed in claim 3, it is characterized in that the basic element of cell division that uses in the step (2) is 6-benzyl aminopurine and 6-chaff aminopurine, wherein, the consumption of 6-benzyl aminopurine is 0.1-0.15mg/L, and the consumption of 6-chaff aminopurine is 0.1-0.15mg/L.
6. method as claimed in claim 2 is characterized in that, the pH of soil is 5.0-7.0 in the step (5).
7. method as claimed in claim 2 is characterized in that, the soil in the step (5) is humus soil.
8. the method for claim 1 is characterized in that, the cultivation temperature of step (1) and (2) is 18-30 ℃, and light application time is 6-12 hour.
9. method as claimed in claim 2 is characterized in that, described humidity is 70-80%.
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| CN1366810A (en) * | 2002-03-01 | 2002-09-04 | 上海延农生物工程有限公司 | Tripterygium wilfordii regenerated plant and its preparation process |
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| CN1366810A (en) * | 2002-03-01 | 2002-09-04 | 上海延农生物工程有限公司 | Tripterygium wilfordii regenerated plant and its preparation process |
Non-Patent Citations (1)
| Title |
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| 雷公藤扦插繁殖 林刚，严宜昌，中药材，第20卷第4期 1997 * |
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