CN1157107C - Fast propagating method of Dendrobium nobile Lindl - Google Patents
Fast propagating method of Dendrobium nobile Lindl Download PDFInfo
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Abstract
The present invention discloses a rapid propagation method of dendrobium stems. A step of cespitose mud propagation culture is added to a step of cespitose mud induction of the existing five-step method, in the step of cespitose mud propagation culture, a culture medium which is prepared from 1/2 MS, 0.1 to 0.2 mg/L of NAA, 100 ml/l of 50% banana juice, 25 to 30 g/l of sucrose and 0.5 g/l of active carbon is adopted, the culture condition is from 25 to 28 DEG C, the light intensity is from 2000 to 3000Lx, the culture lasts for 25 to 30 days, and the light irradiation lasts for 10 to 12 hours/ day. The method of the present invention has the advantages of rapid propagation, low cost, regular seedling growth, simple and convenient seedling adaptation, good genetic stability, easy acquisition of explants, etc.
Description
(1) technical field
The present invention relates to a kind of method for quickly breeding of medicinal dendrobium, specifically, be meant a kind of method that adopts tissue culture technique that HERBA DENDROBII is bred fast.
(2) background technology
Plant Tissue Breeding is meant various organs, tissue or the cell with plant, carries out aseptic culture under the manual control condition, by the process of growing, and the whole plant that generation can be used to breed, and quantitatively significantly increased.This technology is drawn materials few, and it is low to cultivate the vegetable material cost, and growth cycle is short, convenient management.Utilize this quick propagating technology of Plant Tissue Breeding, can multiply a large amount of plant at short notice, and can keep maternal genetic character preferably.The plant that breeds fast by tissue culture technique in China has nearly thousand kinds.
The stem of noble dendrobium is a kind of traditional rare Chinese medicine of China, because growth conditions requires harshness, it is very slow to grow under the natural conditions, and a large amount of excavating at present makes some important medicinal dendrobium kinds endangered.According to investigations, kind surplus the medicinal dendrobium that circulates on the existing market has 30, and the most precious with wherein Dendrobidium huoshanness, dendrobium candidum, HERBA DENDROBII.The HERBA DENDROBII medical value is very high, and the present market price is 7-10 unit/gram.Simultaneously, the HERBA DENDROBII pattern is gorgeous, and the florescence is long, is suitable for doing potted flower culture, therefore, has value of exploiting and utilizing.
To the exploitation of the stem of noble dendrobium, breeding is the first step fast.At present more [as Ceng Songjun, formula monarch, Tissue Culture and Rapid Propagation of Dendrobium, traditional Chinese medicine, 1996,19 (10): 490-491 relevant for Dendrobidium huoshanness, the fast numerous technology report of candidum tissue culturing; Xu Yunchang, Yu Liwen etc., the cultivation of Dendrobidium huoshanness seed test-tube plantlet, Anhui Agriculture College's journal, 1985 (1): 95-97; Zhao Tianbang, Chen Zhixiu etc., stem of noble dendrobium tissue culture and culture technique research, Agricultural University Of He'nan's journal, 1994,28 (2): 128-133 etc.], we can say quite ripe from the selection of explant, the aspects such as screening of medium.Generally be to be material with ripe or immature seed, hormone being arranged or not having on the medium of hormone and sprout, form protocorm, protocorm continues amplification or Cheng Miao.Specifically comprise five steps:
(1) selection of explant and sterilization: the tender stem apex that has of selecting fruit or germinating then usually, fruit sterilization is generally with 70% ethanol sterilization after 1-2 minute, again with 3% disappear clean clever sterilization 20-25 minute, and aseptic water washing 3-5 time, cut fruit, seed is sprinkling upon media surface gets final product.Stem apex is then earlier clean with aseptic water washing, and with 10 seconds of 70% ethanol disinfection, with the 3% clean clever sterilization 20 minutes that disappears, aseptic water washing 4-5 time is seeded in stem apex on the medium again.
(2) sprouting of axillalry bud: adopt N
6Medium adds the 6-BA of 1mg/L and the NAA of 0.5mg/L, at 25 ± 1 ℃, illumination 1500-2000Lx, light application time is to cultivate under 8-10 hour/day the condition, until the seedling that forms the band leaf.
(3) inducing clumping bud: seedling is seeded in the 1/2N that contains 2mg/L NAA
6Cultivate on the medium, until the formation bud of growing thickly, the same step of condition of culture (2).
(4) become seedling to cultivate: usually the indefinite bud or the bud of growing thickly to be inoculated into MS or N
6On the medium of+10% bananas juice, took root in 29-31 days, form whole plant, the same step of condition of culture (2).
(5) practice seedling and transplanting: elder generation after indoor exercise 20-25 days, cleans the medium on the root with tissue cultivating seedling, after 0.1% carbendazim sterilization, cultivate in the liver moss that lives, water sufficient water after planting, often manually spray later on, keep environment moistening, spilt one time of nutrition liquid every 10-15 days.Survival rate can reach 95-97%.
Said method and following about description of the invention in, MS and N
6Medium is this area medium commonly used, all can find its prescription from the book of relevant Plant Tissue Breeding.BA is a 6-benzyladenine, and NAA is a methyl, and Lx is the unit of light intensity---lux.
But said method is material with the seed, because the Dendrobium plant under nature, is main modes of reproduction to nourish and generate mainly, many though bloom, but the pollination ripening rate is very low, makes the acquisition of seed have certain difficulty, and inoculation time is subjected to strict restriction; Selecting young tender stem apex is material, and not only inoculation time is restricted, and material quantity is limited, also can influence the normal growth of cultivation plant significantly; Be unfavorable for the renewal of material.In addition, seedling is technical practicing, and most methods are had relatively high expectations, as require to be planted on the liver moss alive, could obtain higher survival rate etc., so be difficult to large-scale production.
At present, do not appear in the newspapers so far about rapid propagation in vitro research of HERBA DENDROBII, because HERBA DENDROBII is the same with most orchids, seed is little, and germination rate is extremely low under the natural conditions, relies on traditional modes of reproduction, can't solve the quick breeding problem of HERBA DENDROBII.
(3) Fa Ming content
The objective of the invention is to deficiency at existing stem of noble dendrobium method for tissue culture, make improvements, a kind of method that can be fit to HERBA DENDROBII, adopt tissue culture technique to breed fast is provided, this method can be with the sapling multiplication batch production of HERBA DENDROBII, obtain to satisfy the needed seedling of large-scale production at short notice, have also that cost is low, seedling early growth is neat, practice simple and easy to do, the hereditary characteristics such as good stability of seedling-growing method, all can obtain required explant throughout the year simultaneously.
Method of the present invention is to increase " adventitious buds proliferation cultivation " step on the basis of existing stem of noble dendrobium tissue culture technique, and each step is improved, to improve the efficient of breeding.Concretely, method for quickly breeding of the present invention comprises the steps:
(1) selection of explant and sterilization: selecting biennial stem section for use is explant, at first the carbendazim solution with 300-500mg/L soaked 2-4 hour, use 70% ethanol sterilization 1-2 minute then, use 0.1% mercury chloride sterilization 15-20 minute again, use 12% clorox sterilization 0.5-1 minute at last.
(2) sprouting of axillalry bud: the explant after sterilization treatment is incubated in the following medium: 1/2MS+0-1mg/L BA+0-1mg/L NAA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH is 5.6-5.8; Condition of culture is 25 ± 1 ℃ of temperature, illumination 1500-2000Lx; Light application time is 8-10 hour/day, and incubation time is 28-32 days.
(3) inducing clumping bud: will be incubated in the following medium through the material of axillary bud sprouting: 1/2MS+0.1-0.5mg/L BA+50% bananas juice 100mL/L+ sucrose 25-30g/L+ active carbon 0.5g/L, pH is 5.6-5.8; Condition of culture is temperature 25-28 ℃, light intensity 1500-2000Lx; Light application time is 10-12 hour/day, and incubation time is 28-32 days.
(4) adventitious buds proliferation is cultivated: will cultivate in following medium through the material of inducing clumping bud: 1/2 MS+NAA 0.1-0.2mg/L+50% bananas juice 100mL/L+ sucrose 25-30g/L+ active carbon 0.5g/L, and pH is 5.6-5.8; Condition of culture is temperature 25-28 ℃, light intensity 2000-3000Lx; Incubation time is 25-30 days, and light application time is 10-12 hour/day.
(5) become seedling to cultivate: the culture medium prescription that is adopted is: MS or N
6+ 10% bananas juice+0.1-0.2mg/L NAA+1-2mg/LBA; Condition of culture is 25 ± 1 ℃ of temperature, illumination 1500-2000Lx; Light application time is 8-10 hour/day, and incubation time is 60-90 days.
(6) practice seedling and transplanting: no longer carry out indoor experienced seedling, but will clean medium the bottle seedling dried in the air in the cool 20-30 hour, slowly reduce its water content; Changing the liver moss that lives is the commodity fountain moss, does not water in 7-10 days in the cultivation back, with the rotten problem after the minimizing cultivation; After one month, begin fertilising, fertilising 2 times weekly is with composite fertilizer's foliage-spray of 0.3-0.5%.Like this, not only can shorten about the cycle two weeks, can also remove dependence from, make large-scale industrialized production become possibility the liver moss that lives.
In foregoing invention, in the step (2), hormone concentration preferably adopts 0.1mg/L NAA+1mg/L BA; In the step (3), the concentration of BA is 0.5mg/L preferably; In the step (4), the concentration of NAA is 0.2mg/L preferably; In step (6), the time of drying in the air is preferably 24 hours, the preferably 10 days time of not watering after the cultivation.
In the method, be material owing to adopt trophosome, rather than utilize seed, therefore guaranteed hereditary stability as material.Utilizing the sharpest edges of biennial stem section as explant, is whenever to draw materials in can be at all seasons, and the quantity of material abundance.Increase adventitious buds proliferation, can improve fast numerous efficient greatly, and practiced the improvement of seedling-growing method, not only guaranteed higher survival rate, also made this technology satisfy the requirement of large-scale promotion and batch production production.
The inventive method has following advantage or effect:
1, it is slow that this method has overcome traditional propagation method reproduction speed, is difficult to satisfy the deficiency of large-scale production demand.The sapling multiplication batch production of HERBA DENDROBII can be able to be bred at short notice and satisfies the needed seedling of large-scale production;
2, this method has characteristics such as cost is low, seedling early growth is neat;
3, owing to adopt ripe stem section, guaranteed hereditary stability,, upgraded at any time in can be at all seasons simultaneously because its quantity is many as explant.
(4) concrete embodiment
Below in conjunction with specific embodiment the present invention is further detailed:
Embodiment 1:
(1) selection of explant and sterilization: the biennial stem Duan Xianyong hairbrush that will adopt back dips in liquid detergent the dirt on surface is cleaned, peel off residual leaf sheath, be cut into scalpel and have 1-4 axillalry bud, long 3-5 centimetre section, carbendazim solution with 300mg/L soaked 2 hours, often shake therebetween, then discard carbendazim solution, material is rinsed well with flowing water, added 70% ethanol 1 minute, inclining behind the ethanol with 0.1% mercury chloride sterilization 20 minutes, and aseptic water washing 3-5 time takes out material, be cut into scalpel and have an axillalry bud, length is the segment about 1 centimetre, uses 12% clorox sterilization 1 minute again, inoculation.Its pollution rate is 16.7%, and the pollution rate as a result of conventional method is up to 95.2%, and to compare gap very big with method of the present invention.
(2) sprouting of axillalry bud: induction time is 28 days, and condition of culture is 25 ℃, light intensity 2000Lx, light application time 10 hours/day.Culture medium prescription is: 1/2MS+0.1mg/LNAA+1mg/LBA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH5.6.The result is in 88 explant materials, and the axillary bud sprouting rate is 25%, significantly better than 9.7% of control group.
(3) grow thickly the inducing of bud: induction time is 28 days, and condition of culture is 28 ℃, light intensity 1500Lx, light application time 12 hours/day.Culture medium prescription is: 1/2MS+0.5mg/LBA+50% bananas juice 100mL/L+ sucrose 30g/L+ active carbon 0.5g/L, pH5.8.In 50 materials of inoculation, the inducing clumping bud rate is 18%, and contrast is 4%, and method of the present invention is significantly better than control group.
(4) the grow thickly propagation of bud: adopt medium to be: 1/2MS+0.2mg/LNAA+50% bananas juice 100mL/L+ sucrose 30g/L+ active carbon 0.5g/L, pH5.8.Condition of culture is 28 ℃, light intensity 3000Lx, light application time 10 hours/day.Can make the quantity of the bud of growing thickly increase by 17.2 times in 30 days, reproductive efficiency obviously improves.
(5) become seedling to cultivate: medium adopts MS+0.1mg/LNAA+1mg/L BA, and condition of culture is 25 ℃, illumination 2000Lx, and light application time is 8 hours/day, and incubation time is 60-90 days, and average height of seedling is 4.6cm, obviously is better than the 2.5cm that contrasts.
(6) practice seedling and transplanting: clean bottle seedling, dried in the cool 24 hours, plant in wetting fountain moss, begin to water after 10 days, after one month, begin composite fertilizer's foliage-spray, 2 times weekly with 0.3%.Survival rate is 96.3%, and 95-97% is close with contrast, but this method has solved the restriction that must plant in the liver moss that lives, for condition has been created in large-scale industrialized production.
Comprehensive above each step, method of the present invention, not only in the convenience of drawing materials, the germination rate of axillalry bud, the grow thickly inductivity of bud, aspects such as propagation multiple and bottle seedling quality obviously are better than contrast, and are large-scale industrialized production, the feasible experienced seedling and the method for transplanting are provided, have had remarkable advantages compared with the control.
Embodiment 2:
Other is with embodiment 1, and the method for disinfection of step (1) is: 300mg/L carbendazim 4 hours+70% ethanol 1 minute+0.1% mercury chloride 15 minutes+12% clorox 0.5 minute.The result is in 859 explant materials of inoculation, and pollution rate is 47.7%, significantly better than control group.
Embodiment 3:
Other is with embodiment 1, and the method for disinfection of step (1) is: 500mg/L carbendazim 2 hours+70% ethanol 1 minute+0.1% mercury chloride 15 minutes+12% clorox 1 minute.The result is in 633 explant materials of inoculation, and pollution rate is 45.2%, significantly better than control group.
Embodiment 4:
Other is with embodiment 1, and in the step (2), induction time is 32 days, and condition of culture is 25 ℃, light intensity 1500Lx, light application time 8 hours/day.Culture medium prescription is: 1/2MS+1mg/LBA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH5.8.The result is in 88 explant materials, and the axillary bud sprouting rate is 25%, significantly better than 9.7% of control group.
Embodiment 5:
Other is with embodiment 1, and the used hormone of medium of step (2) only adopts 1mg/L NAA.In the step (3), induction time is 32 days, and condition of culture is 25 ℃, light intensity 2000Lx, light application time 10 hours/day.Culture medium prescription is: 1/2MS+0.3mg/L BA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH5.6.The result is in 35 inoculation materials, and the inducing clumping bud rate is 5.7%, is better than 4% of control group.
Embodiment 6:
Other is with embodiment 1, and in the step (3), induction time is 30 days, and condition of culture is 28 ℃, light intensity 1500Lx, light application time 12 hours/day.Culture medium prescription is: 1/2MS+0.1mg/L BA+50% bananas juice 100mL/L+ sucrose 30g/L+ active carbon 0.5g/L, pH5.8.The result is in 50 materials of inoculation, and the inducing clumping bud rate is 18%, significantly better than control group.
Embodiment 7:
Other is with embodiment 1, and in the step (4), incubation time is 25 days.Condition of culture is 25 ℃, light intensity 2000Lx, light application time 12 hours/day.Medium is: 1/2MS+0.1mg/L NAA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH5.6.The result is in 30 bottles of materials of inoculation, and the propagation multiple is 2.3, significantly better than 1.2 of control group.
Embodiment 8:
Other is with embodiment 1, and in the step (4), incubation time is 25 days.Condition of culture is 28 ℃, light intensity 3000Lx, light application time 10 hours/day.Medium is: 1/2MS+0.2mg/L NAA+50% bananas juice 100mL/L+ sucrose 30g/L+ active carbon 0.5g/L, pH5.8.In 30 bottles of materials of result's inoculation, the propagation multiple is 17.2, significantly better than control group.
Embodiment 9:
Other is with embodiment 1, and the medium of step (5) adopts N
6+ 10% bananas juice+0.2mg/LNAA+2mg/LBA, condition of culture are 25 ℃, illumination 1500Lx, and light application time is 10 hours/day, and incubation time is 60-90 days, and the height of seedling of bottle seedling is 4.6cm as a result, significantly better than the 2.5cm of control group.
Embodiment 10:
Other is with embodiment 1, in the step (6), to reach the bottle seedling of bottle outlet standard, pull out blake bottle gently, carefully clean accompanying medium, in the cool after cool 20 hours, it is cultivated on culture matrix, and culture matrix is selected commercial fountain moss for use, the not trickle of planting within back 7 days, after one month, begin fertilising.Fertilising 2 times weekly, the composite fertilizer's foliage-spray with 0.3%.Survival rate is 83.5% as a result.
Embodiment 11:
Other is with embodiment 10, bottle seedling cool 30 hours in the cool, and the not trickle of planting within behind the fountain moss 10 days, composite fertilizer's concentration is 0.5%.Survival rate is 96.3% as a result, and is close with control group.
According to statistics to embodiment 1-11, this method is finished to practicing seedling from the explant sterilization, concrete ratio (mean value) is as follows: as inoculation explant quantity is 100, not microbiological contamination quantity is 83.3 after the sterilization, through cultivation 20.825 axillary bud sproutings are arranged, inducing the bud quantity of growing thickly of generation is 3.748, and propagation one is on behalf of 64.47, two on behalf of 1108.96, and three on behalf of 19074.
Claims (2)
1, a kind of method for quickly breeding of HERBA DENDROBII is characterized in that comprising the steps:
(1) selection of explant and sterilization: selecting biennial stem section for use is explant, at first the carbendazim solution with 300-500mg/L soaked 2-4 hour, use 70% ethanol sterilization 1-2 minute then, use 0.1% mercury chloride sterilization 15-20 minute again, use 12% clorox sterilization 0.5-1 minute at last;
(2) sprouting of axillalry bud: the explant after sterilization treatment is incubated in the following medium: 1/2MS+0-1mg/L BA+0-1mg/L NAA+50% bananas juice 100mL/L+ sucrose 25g/L+ active carbon 0.5g/L, pH is 5.6-5.8; Condition of culture is 25 ± 1 ℃ of temperature, illumination 1500-2000Lx; Light application time is 8-10 hour/day, and incubation time is 28-32 days;
(3) inducing clumping bud: will be incubated in the following medium through the material of axillary bud sprouting: 1/2MS+0.1-0.5mg/L BA+50% bananas juice 100mL/L+ sucrose 25-30g/L+ active carbon 0.5g/L, pH is 5.6-5.8; Condition of culture is temperature 25-28 ℃, light intensity 1500-2000Lx; Light application time is 10-12 hour/day, and incubation time is 28-32 days;
(4) adventitious buds proliferation is cultivated: will cultivate in following medium through the material of inducing clumping bud: 1/2 MS+NAA 0.1-0.2mg/L+50% bananas juice 100mL/L+ sucrose 25-30g/L+ active carbon 0.5g/L, and pH is 5.6-5.8; Condition of culture is temperature 25-28 ℃, light intensity 2000-3000Lx; Incubation time is 25-30 days, and light application time is 10-12 hour/day;
(5) become seedling to cultivate: the culture medium prescription that is adopted is: MS or N
6+ 10% bananas juice+0.1-0.2mg/L NAA+1-2mg/LBA; Condition of culture is 25 ± 1 ℃ of temperature, illumination 1500-2000Lx; Light application time is 8-10 hour/day, and incubation time is 60-90 days;
(6) practice seedling and transplanting: the bottle seedling that will clean medium dried in the air 20-30 hour in the cool, slowly reduced its water content, it was cultivated in the commodity fountain moss then, do not water in 7-10 days in the cultivation back, after one month, beginning is applied fertilizer weekly 2 times, with composite fertilizer's foliage-spray of 0.3-0.5%.
2, propagation method as claimed in claim 1 is characterized in that: in step (2), hormone concentration adopts 0.1mg/L NAA+1mg/L BA; In the step (3), the concentration of BA is 0.5mg/L; In step (4), the concentration of NAA is 0.2mg/L; In step (6), the time of drying in the air is 24 hours, and the time of not watering after the cultivation is 10 days.
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| CNB021347549A CN1157107C (en) | 2002-09-17 | 2002-09-17 | Fast propagating method of Dendrobium nobile Lindl |
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| CNB021347549A CN1157107C (en) | 2002-09-17 | 2002-09-17 | Fast propagating method of Dendrobium nobile Lindl |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328951C (en) * | 2005-01-27 | 2007-08-01 | 合肥工业大学 | Dendrobe protocorm hormoneless cultivation method |
| CN101213941B (en) * | 2008-01-18 | 2011-01-12 | 中国科学院昆明植物研究所 | Fast replication and in-vitro conservation method for dendrobium |
| CN101889547B (en) * | 2009-05-22 | 2012-09-26 | 云南省德宏热带农业科学研究所 | Aseptic and rapid propagation method of dendrobium devonianum seeds |
| CN101695272B (en) * | 2009-10-26 | 2011-03-30 | 重庆市中药研究院 | Natural reproduction method for dendrobium |
| CN103392593B (en) * | 2013-06-20 | 2015-08-19 | 武汉久源生物医药科技有限公司 | A kind of quickly tissue culture propagation method of Dendrobidium huoshanness seedling |
| CN105532473B (en) * | 2016-01-28 | 2018-01-02 | 遵义医学院 | A kind of tissue culture method of HERBA DENDROBII seedling |
| CN108432599A (en) * | 2018-05-17 | 2018-08-24 | 河南福瑞滋生物科技有限公司 | The cultivation matrix and preparation method and tissue culture method of a kind of HERBA DENDROBII |
| CN110972944B (en) * | 2019-12-18 | 2022-12-06 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
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