CN1600080A - Method for breeding mimosa - Google Patents
Method for breeding mimosa Download PDFInfo
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- CN1600080A CN1600080A CN 200410051960 CN200410051960A CN1600080A CN 1600080 A CN1600080 A CN 1600080A CN 200410051960 CN200410051960 CN 200410051960 CN 200410051960 A CN200410051960 A CN 200410051960A CN 1600080 A CN1600080 A CN 1600080A
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Abstract
A method for reproducing mimose includes disinfecting explant, geminating to obtain aseptic seedlings, inducing calli by use of the culture medium MS+2mg/L 2,4-D+0.2mg/L6-BA, inducing the generation of adventitious bud by use of the culture medium MS or B5+1.0-2.0 mg/L 6-BA+0-0.5 mg/L TD2, and rooting by use of the culture medium B5+2mg/L IAA or 1/2 MS+0.5-2 mg/L IBA or NAA.
Description
(1) technical field
The present invention relates to a kind of method for plant tissue culture, specifically, be meant a kind of method that adopts tissue culture technique that the sensitive plant cultured in vitro is bred fast.
(2) background technology
Plant Tissue Breeding is meant any organ, tissue or the cell with plant, carries out the process that aseptic culture is grown under the manual control condition.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 the plant of maternal biological nature of a large amount of maintenances and genetic character at short notice.It is reported that China plant of breeding fast has nearly thousand kinds.Normally used minimal medium has several, as MS, B5, White, N6 etc.In tissue culture, plant explants will be under the effect of plant growth substance, through inducing dedifferentiation, recover fissional ability, influencing processes such as breaking up, promote organ formation again.The size of plant explants reproduction coefficient directly affects the quality and quantity of producing the offspring plant.
Sensitive plant (Mimosa pudica L.), another name is known shy grass, Biophytum nanum H. Chuang sp. Nov. Ined.-B. Sensitivum auct. Non (L.) DC, is pulse family Mimosa perennial herb or fruticuli, often does and gives birth to cultivation in 1 year.Originate in the american torrid zone area, introduce China in one's early years, hot-zones such as Xishuangbanna, Yunnan Province, Dehong all have ease to give birth to.The pot plant cultivation is done in various places, often does experiment material in the plant teaching.The sensitive plant complete stool is slightly poisonous, hyoscine, have the calmness of calming the nerves, the hemostasis convergence effect, bright leaf is mashed external application can control belt-shaped blister.Contain a kind of plant amino acid-mimosine (mimosine) in the sensitive plant, be named as a kind of invertibity cell cycle mortifier recently, can put direct competitive at dna replication dna with deoxythymidine triphosphate.
The method for tissue culture of relevant sensitive plant vegetative propagation seedling at home as yet the someone report that an example report (Gharyal P K ﹠amp is only arranged in the world; Maheshwari S C, Plantlet formation intissue cultures of the sensitive plant Mimosa pudica L.International Journal ofPlant Physiology, 1982,105 (2): 179-182).Its method mainly comprises the steps: the sterilization of (1) explant: with the seed is explant, sterilizes earlier earlier before the breeding; (2) sprout aseptic seedling: on B5 medium, sprout aseptic seedling; (3) evoked callus: with aseptic shoot root, cotyledon, hypocotyl, blade, stem apex, be placed on additional have heteroauxin (being called for short IAA), methyl (being called for short NAA), 2 respectively, the 4-dichlorphenoxyacetic acid (is called for short 2,4-D), evoked callus in the B5 solid culture medium of 6-benzylaminopurine (being called for short 6-BA); (4) evoking adventive bud takes place: callus is transferred to differential medium B5+0.5mg/L 2, and evoking adventive bud takes place among the 4-D+1mg/L 6-BA, and its condition of culture is: 25 ± 2 ℃ of temperature; Intensity of illumination 750Lux; (5) cultivation is taken root: the indefinite bud that will highly be 3-7 centimetre cuts into individual plant, cultivates the same step of condition of culture (4) on additional 2mg/L IAA solid culture medium.But through test, this propagation method mainly exists uses hormone kind various, inconvenient operation, deficiency such as differentiation rate is low, rooting rate is low.
(3) Fa Ming content
The objective of the invention is to the deficiency at the existing method for tissue culture of sensitive plant, a kind of new method of quick breeding is provided, it can obviously improve the vegetative propagation coefficient of sensitive plant, and easy and simple to handle.
Known the kind of medium has certain influence to the differentiation of plant in regulating controlling plant material cultured in vitro process, and plant growth substance also plays an important role, and wherein influence is the most significant is auxins and cytokinin-like substance.In order to realize purpose of the present invention, the present invention comprises in above-mentioned existing method on the basis of five steps and carries out following improvement: in step (3), reduce hormone kind, the concentration of the growth hormone and the basic element of cell division in the raising medium is to improve the evoked callus generation rate; Reduce the kind of the used hormone of medium in the step (4), only use cytokinin, to improve the ability that evoking adventive bud takes place; Medium to step (5) improves.Concretely, concrete operations of the present invention are as follows:
(1) explant sterilization: place 65-75% ethanol to soak 20-60s (representing " second ", down together) in the sensitive plant seed, the 4-15min that sterilizes in 0.1% mercuric chloride (expression " minute ", down together), use aseptic water washing 3-6 time again;
(2) sprout aseptic seedling: the seed after the sterilization is inoculated on the 1/2MS medium and cultivated 15-30 days, sprouts aseptic seedling, and condition of culture is 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1(expression " hour/day ", down together);
(3) evoked callus: place prescription to be MS+2mg/L 2 aseptic seedling, evoked callus in the medium of 4-D+0.2mg/L6-BA, incubation time are 10-30 days, the same step of condition of culture (2);
(4) evoking adventive bud takes place: place prescription to obtain indefinite bud (highly being generally 3-7 centimetre), the same step of condition of culture (2) in 30-60 days for cultivating on the medium of MS or B5+1.0-2.0mg/L6-BA+0-0.5mg/L TDZ callus;
(5) cultivate and to take root: the resulting indefinite bud of step (4) is cut into individual plant, on the medium of B5+2mg/LIAA or 1/2MS+0.5-2mg/L IBA (indolebutyric acid) or NAA (methyl) medium, cultivated the same step of condition of culture (2) 10-30 days.
In the said method, in step (4) bud atomization, the concentration of hormone 6-BA is 2.0mg/L preferably.Medium preferably adopts 1/2MS+0.5-2mg/L IBA or NAA prescription in the step (5), it can be avoided, and hormone decomposes and unhandy shortcoming in the B5+2mg/L IAA prescription, that is: heteroauxin is having decomposition easily under the condition of light, and to adopt the method for filtration sterilization in operation, and making more convenient operation after adopting methyl or indolebutyric acid, rooting rate improves; The concentration of NAA 1.0mg/L preferably wherein.
In the method, MS and B5 medium are the most frequently used medium, and generally the book from relevant Plant Tissue Breeding all can find its prescription.
The present invention has following advantage and effect:
1. the experiment proved that compare with original method, the inductivity of the inventive method step (3) callus is 100%; In the step (4), adopt the method that improves cytokinin concentration, the bud number average of the induction frequency of indefinite bud and every explant generation is higher than former method; In step (5), adopt methyl, indolebutyric acid to replace original heteroauxin, the adventive root time of origin shortens, and rooting rate increases, and grows fine, and has guaranteed test-tube plantlet adventive root quality.
2. the inventive method is compared with existing conventional method, need not to increase task equipment, and is easy and simple to handle.
(4) concrete embodiment
Embodiment 1:
(1) sterilization of explant: get shy grass seed and place 65% ethanol to soak 20s, the 15min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 3 times again;
(2) sprouting of aseptic seedling: the seed that will sterilize is inoculated on the 1/2MS medium and cultivated 15 days, obtains aseptic seedling, and condition of culture is 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1(expression " hour/day ", down together);
(3) inducing of callus: get the aseptic seedling hypocotyl segment, being transferred to add has 2mg/L2, cultivates 30 days evoked callus, callus induction rate 100% on the MS medium of 4-D and 0.2mg/L 6-BA;
(4) differentiation of bud: being inoculated into after one month to add has on the MS medium of 1mg/L 6-BA and 0.5mg/L TDZ, and when cultivating two months, differentiation frequency is 40%, and the seedling number is 5-10 on the average every callus;
(5) formation of root: formation seedling, the switching go into to add on the B5 medium of 2.0mg/L IAA, in the time of 30 days, rooting rate is 50%.
And the control group that adopts original method to cultivate, under the condition of adding multiple hormone, the explant callus induction rate reaches 100%, but 80% callus generation root is arranged this moment, is unfavorable for further cultivation.The incidence of its indefinite bud is up on other explants: 31%, and only be that 8% differentiation is sprouted when being explant with the hypocotyl, growth is slow, obviously is worse than the inventive method.This programme adopts the hormone of less kind just can make callus induction rate reach 100%, does not have the formation of root this moment, and the indefinite bud occurrence frequency also is higher than contrast 9%.
Embodiment 2:
Other operation and control group are with embodiment 1, and different is: in the step (1), get shy grass seed and place 75% ethanol to soak 60s, the 4min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 6 times again; In the step (2), incubation time is 30 days; In the step (3), incubation time is 10 days; In the step (4), culture medium prescription is MS+2mg/L 6-BA, and incubation time is 30 days, and the differentiation adventitious buds rate reaches 60%; In the step (5), medium changes 1/2MS+1.0mg/L NAA into, and in the time of 20 days, rooting rate is 64.2%, and along with the prolongation of incubation time, rooting rate can be higher.
And the adventitious bud induction frequency of control group only is 8%, and the adventive root incidence is up to 50%, obviously is worse than the inventive method.
Embodiment 3:
Other operation and control group are with embodiment 1, and different is: in the step (1), get shy grass seed and place 70% ethanol to soak 30s, the 8min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 5 times again; In the step (4), the sensitive plant callus is transferred to add to be had on the B5 medium of 2mg/L 6-BA, and the differentiation adventitious buds rate reaches 70%; In the step (5), medium is 1/2MS+1mg/L NAA, and incubation time is 10 days, and rooting rate is 64.2%.
And control group step (4) adopts additional hormone 2, and the B5 medium of 4-D 0.5mg/L and 2mg/L 6-BA, adventitious bud induction frequency only are 8%, adopt the medium of B5+2mg/L IAA in the step (5), the adventive root incidence only is 50%, and growth is slow, obviously is worse than the inventive method.
Embodiment 4:
Other operation and control group are with embodiment 3, and different is: in the step (4), culture medium prescription is B5+1.0mg/L 6-BA+0.5mg/L TDZ; In the step (5), culture medium prescription is 1/2MS+0.5mg/LNAA, and rooting rate is 50%, remains basically stable with control group.
Embodiment 5:
Other operation and control group are with embodiment 3, and in the step (5), culture medium prescription is 1/2MS+2mg/L NAA, and rooting rate is 60%.And control group adventive root incidence only is 50%, and growth is slow, obviously is worse than the inventive method.
Embodiment 6:
Other operation and control group are with embodiment 3, and different is: in the step (5), medium is the additional 1/2MS that 2mg/L IBA is arranged, and rooting rate is 50%, and it is slower to grow.Control group adventive root incidence also is 50%, decomposes easily but IAA sees light, and needs filtration sterilization, complicated operation.
Embodiment 7:
Other operation and control group are with embodiment 3, and different is: in the step (5), medium has on the 1/2MS of 0.5mg/L IBA for additional, and rooting rate is 60%.And control group adventive root incidence only is 50%, obviously is worse than the inventive method.
Claims (4)
1, the propagation method of a kind of sensitive plant is characterized in that comprising the steps:
(1) explant sterilization: place 65-75% ethanol to soak 20-60s in the sensitive plant seed, the 4-15min that sterilizes in 0.1% mercuric chloride uses aseptic water washing 3-6 time again;
(2) sprout aseptic seedling: the seed after the sterilization is inoculated on the 1/2MS medium and cultivated 15-30 days, sprouts aseptic seedling, and condition of culture is 25 ± 2 ℃ of temperature, intensity of illumination 1500Lux, light application time 12hd
-1
(3) evoked callus: place prescription to be MS+2mg/L 2 aseptic seedling, evoked callus in the medium of 4-D+0.2mg/L6-BA, incubation time are 10-30 days, the same step of condition of culture (2);
(4) evoking adventive bud takes place: place prescription to obtain indefinite bud, the same step of condition of culture (2) in 30-60 days for cultivating on the medium of MS or B5+1.0-2.0mg/L6-BA+0-0.5mg/L TDZ callus;
(5) cultivate and to take root: the resulting indefinite bud of step (4) is cut into individual plant, on the medium of B5+2mg/LIAA or 1/2MS+0.5-2mg/L IBA or NAA medium, cultivated the same step of condition of culture (2) 10-30 days.
2, the method for claim 1 is characterized in that: in step (4) bud atomization, the concentration of hormone 6-BA is 2.0mg/L.
3, method as claimed in claim 1 or 2 is characterized in that: the prescription of medium is in the step (5): 1/2MS+0.5-2mg/LIBA or NAA.
4, want 5 described methods as right, it is characterized in that: in the step (5), the concentration of NAA is 1.0mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410051960 CN1288961C (en) | 2004-10-27 | 2004-10-27 | Method for breeding mimosa |
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| CN 200410051960 CN1288961C (en) | 2004-10-27 | 2004-10-27 | Method for breeding mimosa |
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| CN1600080A true CN1600080A (en) | 2005-03-30 |
| CN1288961C CN1288961C (en) | 2006-12-13 |
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| CN 200410051960 Expired - Fee Related CN1288961C (en) | 2004-10-27 | 2004-10-27 | Method for breeding mimosa |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112889828A (en) * | 2021-01-12 | 2021-06-04 | 碧沃丰生物科技(广东)股份有限公司 | Plant alkaloid algaecide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101946711B (en) * | 2010-08-28 | 2012-07-04 | 中国农业科学院草原研究所 | High-efficiency tissue culture and regeneration method for Medicago sativa |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112889828A (en) * | 2021-01-12 | 2021-06-04 | 碧沃丰生物科技(广东)股份有限公司 | Plant alkaloid algaecide and application thereof |
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Granted publication date: 20061213 Termination date: 20101027 |