CN1843097A - High effective propagation of blattbulume stem node and knottiness axillary bud tissue culture seedling - Google Patents
High effective propagation of blattbulume stem node and knottiness axillary bud tissue culture seedling Download PDFInfo
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- CN1843097A CN1843097A CN 200610041910 CN200610041910A CN1843097A CN 1843097 A CN1843097 A CN 1843097A CN 200610041910 CN200610041910 CN 200610041910 CN 200610041910 A CN200610041910 A CN 200610041910A CN 1843097 A CN1843097 A CN 1843097A
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Abstract
The invention discloses a method of fast tissue culturing phyllanthus urinaria. The technological character is in that it takes the stem and branch of phyllanthus urinaria as explant, comprising following steps: (1) inducing bud sprout: inoculating the segment of stem and branch of phyllanthus urinaria into bud sprout inducing culture medium MSP1 for germ-free bud sprout differentiation; (2) breeding bud sprout: culturing the segment of stem and branch of said bud sprout on bud sprout breeding culturing medium MSP2, germinating clump sprout which can be used for next breeding process; (3) root growing of tissue culturing sprout: inoculating the single tissue culturing sprout on root growing culturing medium MSP3. The invention provides a new source for explant, which greatly increases breeding radix number. It also shortens the axillary bud generating time through improving culturing medium content and hormone type and proportion content, and inhibits the browning reaction of explant and bud sprout, which greatly increases the breeding coefficient for each explant.
Description
Technical field
The present invention relates to a kind of Common Leafflower Herb tissue culture and rapid propagation method, belong to biological group and cultivate the seedling technical field.
Background technology
At present, hepatitis carrier's quantity is very surprising.A tree name World Health Organization investigation statistics, the whole world reach 4～500,000,000 people, have every year 1000000 people to die from hepatitis B virus infection approximately.China has 10% population to suffer from this disease approximately, highly effective medicine is not arranged so far as yet, and the course of treatment is longer.Euphorbiaceae (Euphorbiaceae) plant Common Leafflower Herb (Phyllanthus urinaria L) as one of Chinese medicine anti-hepatic-B virus medicine clinically all carried out furtheing investigate from testing in recent years [Zhong Ying, Zuo Chunxu, Li Fengqin, etc.The research of Common Leafflower Herb chemical analysis and anti-hepatitis B virus activity thereof.CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998,23 (6): 363.].Discover except that having antivirus action, also can improve effects such as immunity, protection hepatocellular injury, anti-hepatic fibrosis, anticancer change, be expected to become a new profit growth point of pharmacy industry, demand will constantly increase.
China's Common Leafflower Herb still is in wild state at present, and [the Zhao Yonghua fourth is won the development and use of the clear China of Yang Chun Leafflower resources of medicinal plant seldom to carry out artificial cultivation.Biology circular 2000,35 (12): 39-40.], and percentage of seedgermination is not high, plant strain growth slow [Unander DW, Bryan HH, Lance CJ ﹠amp; McMillan RT Factors affecting germination and standestablishment of Phyllanthus amarus (Euphorbiaceae) .Econ.Bot.1995,49:49-55.].For satisfying Phytochemistry and pharmacology further research and the growing need of pharmacy industry better, overcome the scarcity of resources of manually excessively gathering and causing, should adopt positive effective measures, enable the sustainable use development.
Adopt in vitro tissue to cultivate fast breeding technique, can obtain a large amount of high-quality test-tube plantlets in a short time, be not subjected to the time season limit; whole year production; save the soil, protection Common Leafflower Herb natural resources satisfies Phytochemistry and pharmacology further research and the growing need of pharmacy industry better.
The research of relevant Common Leafflower Herb tissue culture quick breeding is less relatively.People [Elizabete Catapan, M ' arcio Lu ' is, Busi da Silva, F ' abio Netto Moreno ﹠amp such as Elizabete Catapan; Ana Maria Vianal Micropropagation, callusand root culture of Phyllanthus urinaria (Euphorbiaceae) Plant Cell, Tissue and Organ Culture2002,70:301-309.] be explant with Common Leafflower Herb node (node), adopt the rapid propagation in vitro method, on the MS medium of additional 5.0 micromole's kinetin KT, obtained test-tube plantlet.Obviously,, can enlarge the scope of application and the kind of explant, improve the breeding radix greatly if can utilize stipes and minor matters to be explant.
Summary of the invention
The purpose of this invention is to provide a kind of Common Leafflower Herb tissue culture and rapid propagation method, stipes and/or minor matters with Common Leafflower Herb 40～70d plant in age are explant, through the bud seedling induce, propagation and tissue cultivating seedling is taken root, practice links such as seedling and transplanting, obtain a large amount of tissue cultivating seedling fast, to overcome the scarcity of resources of manually excessively gathering and causing.
The Common Leafflower Herb tissue culture and rapid propagation method that the present invention proposes is an explant with the stipes and the minor matters of Common Leafflower Herb, through the bud seedling induce, seedling and bud proliferation, tissue cultivating seedling are taken root, hardening and transplanting, concrete steps are as follows:
(1) the bud seedling is induced: the segment of Common Leafflower Herb stipes and minor matters is inoculated in the upward aseptic bud seedling of differentiation of bud seedling inducing culture MSP1;
(2) seedling and bud proliferation: aseptic bud seedling stipes and minor matters segment are incubated on the seedling and bud proliferation medium MSP2, sprout the seedling of growing thickly, and the seedling of growing thickly can be used for the breeding of next round;
(3) tissue cultivating seedling is taken root: individual plant group training bud seedling is inoculated on the root media MSP3 takes root.
Nutrient media components and content proportioning are as follows in the above-mentioned steps:
Bud seedling inducing culture MSP1:
MS medium mineral salt
The organic matter of B5 medium
Vc 50～150mg/L
CH 200～400mg/L
6-BA 0.5～3.0mg/L
IBA 0.2～0.8mg/L
AgNO
3 2～10mg/L
Sucrose 30～50g/L
Agar 6.5g/L
PH value 5.8～6.2
Seedling and bud proliferation medium MSP2:
MS medium mineral salt
The B5 medium organic matter
Vc 100mg/L
CH 300mg/L
6-BA 0.5～2.0mg/L
IBA 0.2～1.0mg/L
Sucrose 30g/L
Agar 6.5g/L
PH value 5.8～6.2
Root media MSP3:
MS or 1/2MS medium
IBA 0.2～0.5mg/L
Sucrose 20～30g/L
Agar 6.5g/L
PH value 5.8～6.2
It is that 70% ethanol soaks 30s, 0.1%HgCl with Common Leafflower Herb stipes and the flushing of minor matters flowing water that the bud seedling is induced
2Surface sterilization 7～10min, aseptic water washing cut into the long segment of 2～3cm for several times then in the sterile water that contains 50～150mg/L Vc, be inoculated on the bud seedling inducing culture MSP1, differentiate aseptic bud seedling behind the 10d successively.
Seedling and bud proliferation is that the aseptic bud seedling stipes and the minor matters of will grow on the bud seedling inducing culture are cut into the segment of being with 1-2 sheet leaf, is inoculated in seedling and bud proliferation medium MSP2 and goes up cultivation 20～30d.
Tissue culture plant inoculation is taken root on root media MSP3, selects the whole plant of robust growth, opens wide bottle cap hardening 5d, transplants in containing vermiculite: humus soil: in the basin alms bowl of rural area soil=2: 1: 1, overlay film is preserved moisture and is cultivated 5～7d, the natural lighting growth.
The bud seedling is induced, 25 ℃ of the temperature of propagation and tissue cultivating seedling culture of rootage, illumination every day 12h, intensity of illumination 2000Lux.
Advantage of the present invention and good effect: the present invention is an explant with stipes and minor matters, the scope of application and the kind of explant have not only been innovated, improved the breeding radix greatly, and by improved culture medium composition and hormone kind and proportioning content, shortened the axillalry bud time of origin, the browning phenomenon that has suppressed explant and bud seedling has improved bud seedling quality, thereby the reproduction coefficient of every strain explant is increased substantially.Can breed a large amount of tissue cultured test-tube seedlings quickly and efficiently with the method, every 25d is an one-period, and the reproduction coefficient of each sections is 9～15, and the reproduction coefficient of every Common Leafflower Herb can reach 130～200.This high efficiency quick breeding system is the raw material basis that the further development and use of Common Leafflower Herb provide continually, the four seasons can be used.
Description of drawings
Fig. 1 is the bud seedling of Common Leafflower Herb explant induction.
Fig. 2 is the tissue cultivating seedling regeneration of plantlet.
Embodiment
The Chinese of the abbreviation correspondence that the present invention uses is as follows:
Vc (vitamin C) CH (lactoalbumin hydrolysate) 6-BA (6-benzyl purine)
IBA (indolebutyric acid)
The MS medium mineral salt that the present invention uses are formed with content as follows: MgSO according to the preparation of handbook universal method
47H
2O:370mg/L, CaCl
22H
2O:440mg/L, KNO
3: 1900mg/L, NH
4NO
3: 1650mg/L, KH
2PO
4: 170mg/L, MnSO
44H
2O:22.3mg/L, KI:0.83mg/L, ZnSO
47H
2O:8.6mg/L, CoCl
26H
2O:0.025mg/L, CuSO
45H
2O:0.025mg/L, H
3BO
3: 6.2mg/L, Na
2MoO
42H
2O:0.25mg/L, FeSO
47H
2O:27.8mg/L, Na
2-EDTA:37.3mg/L.
B5 medium organic matter: i.e. inositol: 100mg/L, Cobastab
1: 10mg/L, Cobastab
6: 1mg/L, nicotinic acid: 1mg/L.
Further describe the present invention by the following examples.
Embodiment 1: 4～5 stipes of healthy and strong plant top inverse and the minor matters in 60d age of planting experimentally with Chinese medicine garden, botanical garden, Xi'an are explant, remove blade, the flowing water flushing, and 70% ethanol soaks and shakes 30s, 0.1%HgCl
2Surface sterilization 9min, aseptic water washing 6 times, in the sterile water that contains 50mg/L Vc, cut into the segment of 2 leaves of tool then, level or vertically be positioned over the organic matter+Vc100mg/L+CH300mg/L+6-BA 1.5mg/L+IBA0.2mg/L+AgNO of MS medium mineral salt+B5 medium
3On the medium of 10mg/L+40g/L sucrose, axillalry bud begins germination and growth behind the 5d, can grow up to high 6cm behind the 25d, contains 6～8 leaves, the aseptic seedling (shown in Figure 1) of 2～4 lateral buds of tool.
The stipes of sterile test tube seedling and minor matters are cut into segment with 2 leaves, be inoculated on the medium of MS medium mineral salt+B5 medium organic matter+Vc100mg/L+CH300mg/L+6-BA1.0mg/L+IBA0.5mg/L+30g/L sucrose, carry out the further enrichment culture of bud seedling, behind the 25d, the axillalry bud of each stem section can be sprouted again and grow up to the high 5～7cm of stem, is with 5～8 leaves, the seedling of growing thickly of 2～4 lateral buds.The seedling of these propagation can be used for the breeding of next round again.Can breed a large amount of tissue cultivating seedling quickly and efficiently with the method, every 25d is an one-period, and each segment can be induced and be produced 10～15 bud seedlings, and the strain plant can breed 138～160 sterile test tube bud seedlings under every leaf.
Rooting and transplant if desired cuts the above tissue cultivating seedling of high about 2cm, is inoculated on the root media of 1/2MS+IBA0.5+20g/L sucrose, and strengthening seedling and rooting under 25 ℃, 2000Lx light, root begins formation behind 5～7d, and rooting rate reaches 100% (shown in Figure 2).After cultivating 15d, select robust growth,, open wide bottle cap hardening 5d, transplant that (the nutrition local soil type of basin alms bowl becomes vermiculite: humus soil: rural area soil=2: 1 in the basin alms bowl with the plant of 3 above roots; 1) in, plastic covering film on it is preserved moisture behind the cultivation 7d, removes film, well-grown under the natural daylight.Transplanting survival rate is 90.43%.
Embodiment 2: the healthy and strong plant stipes and the minor matters in 40d age of planting experimentally with Chinese medicine garden, botanical garden, Xi'an are explant, remove blade, the flowing water flushing, and 70% ethanol soaks and shakes 30s, 0.1%HgCl
2Surface sterilization 8min, aseptic water washing 6 times, in the sterile water that contains 150mg/L Vc, cut into the segment of 2 leaves of tool then, level or vertically be positioned over the organic matter+Vc100mg/L+CH300mg/L+6-BA1.0mg/L+IBA0.2mg/L+AgNO of MS medium mineral salt+B5 medium
3On the medium of 10mg/L+40g/L sucrose, axillalry bud begins germination and growth behind the 5d, can grow up to high 4～6cm about 30d, contains 5～7 leaves, the aseptic seedling of 2 lateral buds of tool.
With the stipes of sterile test tube seedling and minor matters be cut into 2 leaves and section, be inoculated on the medium of organic matter+Vc100mg/L+CH300mg/L+6-BA1.5mg/L+IBA0.5mg/L+30g/L sucrose of MS medium mineral salt+B5 medium, carry out the further enrichment culture of bud seedling, behind the 25d, the axillalry bud of each stem section can be sprouted again and grow up to about the high 5cm of stem, is with 5～8 leaves, the seedling of growing thickly of 2～4 lateral buds.The seedling of these propagation can be used for the breeding of next round again.Can breed a large amount of tissue cultivating seedling quickly and efficiently with the method, every 25d is an one-period, and each segment can be induced and be formed 9～11 bud seedlings, and the strain plant can be produced 150～180 sterile test tube bud seedlings under every leaf.
Cut the tissue cultivating seedling more than high about 2cm, be inoculated on the root media of MS+IBA0.2+30g/L sucrose, strengthening seedling and rooting under 25 ℃, 2000Lx light begins behind the 7d to take root, and rooting rate reaches 100%.After cultivating 15d, select robust growth, the regeneration plant of well developed root system opens wide bottle cap hardening 7d, transplants in basin soil (vermiculite: humus soil: rural area soil=2: 1; 1) in, overlay film is preserved moisture and is cultivated 4～5d on it, removes film, natural daylight growth down, and transplanting survival rate is 100%.
Can breed a large amount of Common Leafflower Herb tissue cultured test-tube seedlings quickly and efficiently with said method, every 25d is an one-period, the reproduction coefficient of each sections is 9～15, the reproduction coefficient of every Common Leafflower Herb can reach 130～200, and this high efficiency quick breeding system is the raw material basis that the further development and use of Common Leafflower Herb provide continually, the four seasons can be used.
Claims (5)
1, Common Leafflower Herb tissue culture and rapid propagation method is an explant with the stipes and the minor matters of Common Leafflower Herb, through the bud seedling induce, seedling and bud proliferation, tissue cultivating seedling are taken root, hardening and transplanting, concrete steps are as follows:
(1) the bud seedling is induced: the segment of Common Leafflower Herb stipes and minor matters is inoculated in the upward aseptic bud seedling of differentiation of bud seedling inducing culture MSP1;
(2) seedling and bud proliferation: aseptic bud seedling stipes and minor matters segment are incubated on the seedling and bud proliferation medium MSP2, sprout the seedling of growing thickly, and the seedling of growing thickly can be used for the breeding of next round;
(3) tissue cultivating seedling is taken root: individual plant group training bud seedling is inoculated on the root media MSP3 takes root;
Nutrient media components and content proportioning are as follows in the above-mentioned steps:
Bud seedling inducing culture MSP1:
MS medium mineral salt
The organic matter of B5 medium
Vc 50～150mg/L
CH 200～400mg/L
6-BA 0.5～3.0mg/L
IBA 0.2～0.8mg/L
AgNO
3 2～10mg/L
Sucrose 30～50g/L
Agar 6.5g/L
PH value 5.8～6.2
Seedling and bud proliferation medium MSP2:
MS medium mineral salt
The B5 medium organic matter
Vc 100mg/L
CH 300mg/L
6-BA 0.5～2.0mG/L
IBA 0.2～1.0?mg/L
Sucrose 30G/L
Agar 6.5g/L
PH value 5.8～6.2
Root media MSP3:
MS or 1/2MS medium
IBA 0.2～0.5mg/L
Sucrose 20～30g/L
Agar 6.5g/L
PH value 5.8～6.2.
2. Common Leafflower Herb tissue culture and rapid propagation method according to claim 1 is characterized in that: it is that 70% ethanol soaks 30s, 0.1%HgCl with Common Leafflower Herb stipes and the flushing of minor matters flowing water that the bud seedling is induced
2Surface sterilization 7～10min, aseptic water washing cut into the long segment of 2～3cm for several times then in the sterile water that contains 50～150mg/L Vc, be inoculated on the bud seedling inducing culture MSP1, differentiate aseptic bud seedling behind the 10d successively.
3. Common Leafflower Herb tissue culture and rapid propagation method according to claim 1, it is characterized in that: seedling and bud proliferation is that the aseptic bud seedling stipes and the minor matters of will grow on the bud seedling inducing culture are cut into the segment of being with 1-2 sheet leaf, is inoculated in seedling and bud proliferation medium MSP2 and goes up cultivation 20～30d.
4. Common Leafflower Herb tissue culture and rapid propagation method according to claim 1, it is characterized in that: in hardening and the transplant step, select the whole plant of robust growth, open wide bottle cap hardening 5d, transplant in containing vermiculite: humus soil: in the basin alms bowl of rural area soil=2: 1: 1, overlay film is preserved moisture and is cultivated 5～7d, the natural lighting growth.
5. according to one of any described Common Leafflower Herb tissue culture and rapid propagation method of claim 1-3, it is characterized in that: the bud seedling is induced, 25 ℃ of the temperature of propagation and tissue cultivating seedling culture of rootage, illumination every day 12h, intensity of illumination 2000Lux.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101103700B (en) * | 2007-08-06 | 2010-08-04 | 浙江大学 | Tissue culture quick reproduction method of radix tetrastigmae |
| CN103392463A (en) * | 2013-06-25 | 2013-11-20 | 重庆市药物种植研究所 | Manual high-yield planting method of phyllanthus urinaria linn |
| CN107517612A (en) * | 2017-07-20 | 2017-12-29 | 句容市后白镇美佳多果品专业合作社 | A kind of soil management method in pear tree field |
| CN107960328A (en) * | 2017-12-21 | 2018-04-27 | 中国林业科学研究院资源昆虫研究所 | A kind of method of emblic tissue cultures |
| CN117859652A (en) * | 2024-03-12 | 2024-04-12 | 云南植虫药生物科技有限公司 | Fructus phyllanthi tissue culture rapid propagation seedling method and application |
-
2006
- 2006-03-13 CN CNB200610041910XA patent/CN100435626C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101103700B (en) * | 2007-08-06 | 2010-08-04 | 浙江大学 | Tissue culture quick reproduction method of radix tetrastigmae |
| CN103392463A (en) * | 2013-06-25 | 2013-11-20 | 重庆市药物种植研究所 | Manual high-yield planting method of phyllanthus urinaria linn |
| CN107517612A (en) * | 2017-07-20 | 2017-12-29 | 句容市后白镇美佳多果品专业合作社 | A kind of soil management method in pear tree field |
| CN107960328A (en) * | 2017-12-21 | 2018-04-27 | 中国林业科学研究院资源昆虫研究所 | A kind of method of emblic tissue cultures |
| CN107960328B (en) * | 2017-12-21 | 2020-06-26 | 中国林业科学研究院资源昆虫研究所 | Phyllanthus emblica tissue culture method |
| CN117859652A (en) * | 2024-03-12 | 2024-04-12 | 云南植虫药生物科技有限公司 | Fructus phyllanthi tissue culture rapid propagation seedling method and application |
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| Publication number | Publication date |
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| CN100435626C (en) | 2008-11-26 |
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