CN103718966B - A kind of magnolia liliiflora tissue culture method - Google Patents
A kind of magnolia liliiflora tissue culture method Download PDFInfo
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- CN103718966B CN103718966B CN201310730707.3A CN201310730707A CN103718966B CN 103718966 B CN103718966 B CN 103718966B CN 201310730707 A CN201310730707 A CN 201310730707A CN 103718966 B CN103718966 B CN 103718966B
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- 241000218394 Magnolia liliiflora Species 0.000 title claims abstract description 40
- 238000012136 culture method Methods 0.000 title abstract description 13
- 230000001954 sterilising effect Effects 0.000 claims abstract description 27
- 240000005819 Magnolia denudata Species 0.000 claims abstract description 25
- 235000003415 Michelia alba Nutrition 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 18
- 239000002609 medium Substances 0.000 claims description 36
- 239000012877 elongation medium Substances 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000005802 Mancozeb Substances 0.000 claims description 5
- 230000004069 differentiation Effects 0.000 claims description 5
- 235000013399 edible fruits Nutrition 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 5
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 229920000136 polysorbate Polymers 0.000 claims description 5
- 238000005057 refrigeration Methods 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 230000002421 anti-septic effect Effects 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000007654 immersion Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000008399 tap water Substances 0.000 claims 1
- 235000020679 tap water Nutrition 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 9
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 238000011109 contamination Methods 0.000 abstract description 4
- 230000004899 motility Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 8
- 239000000022 bacteriostatic agent Substances 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 239000003814 drug Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a kind of magnolia liliiflora tissue culture method, this magnolia liliiflora tissue culture method is carried out under open condition, the method comprising the antibacterial process of sterilizing to purple yu-lan (Magnolia liliflora) tissue cultures place, the selection of explant, the sterilization of explant, the Initial culture of explant, the step such as Multiplying culture and culture of rootage.In the present invention, magnolia liliiflora tissue culture method is carried out under open condition, can carry out in an open condition, save cost; Explant sterile contamination be ensure that to the sterilizing of purple yu-lan (Magnolia liliflora), improve it and be trained motility rate; Soak explant with 2%PVP and add 2%PVP in the medium, alleviating brownization of explant, improve its survival rate.This magnolia liliiflora tissue culture method is that the time is short simple to operate, with low cost, and survival rate is high, rooting rate is high, and the bottle outlet that can realize in a short time is transplanted, and can be kept the merit of original kind, produces lay the foundation for stock breeding and industrialization.
Description
Technical field
The present invention relates to field of plant tissue culture, relate generally to be the method for tissue culture of purple yu-lan (Magnolia liliflora).
Background technology
Purple yu-lan (Magnolia liliflora), has another name called lily magnolia, the flower bud of lily magnolia, is Chinese endemic plant, is distributed in Chinese yunnan, Fujian, Hubei, the ground such as Sichuan, be grown on the area of height above sea level 300 to 1600 meters, be generally grown on edge, hillside.Purple yu-lan (Magnolia liliflora) flower is gorgeous pleasant; fragrance is simple and elegant; isolated planting or group planting are all very attractive in appearance; excellent flower garden, street planting plant; again flowers and the Chinese medicine of Chinese tradition; but purple yu-lan (Magnolia liliflora) is not easily transplanted and maintenance, being logged " World Conservation Union " plant Red List, is the flowers and trees be of great rarity.
Now along with the development of technology, a lot of area, all in the method for carrying out purple yu-lan (Magnolia liliflora) cultivation, is want to cultivate plantation purple yu-lan (Magnolia liliflora) in this area.Traditional purple yu-lan (Magnolia liliflora) generally adopts the training methods such as sowing, cuttage, grafting, press strip and division propagation.The length consuming time of these training methods, and breed and not easily survive.More existing purple yu-lan (Magnolia liliflora) tissue cultures modes are carried out under the aseptic condition closed, and required cost is comparatively large, but also there is easy brownization of explant in tissue cultures, the problem that survival rate is not high, can not be used for the amount reproduction of purple yu-lan (Magnolia liliflora).
Summary of the invention
The present invention is directed to existing technical problem, propose a kind of magnolia liliiflora tissue culture method, the method is carried out under open condition, and a closed aseptic environment of need not purchasing, has saved cost; To purple yu-lan (Magnolia liliflora) sterilization treatment, ensure that explant sterile contamination, improving it, to be trained motility rate high; Soak explant with 2%PVP and add 2%PVP in the medium, alleviating brownization of explant, improve the survival rate of explant.This magnolia liliiflora tissue culture method is that the time is short simple to operate, with low cost, and survival rate is high, rooting rate is high, and the bottle outlet that can realize in a short time is transplanted.Method for tissue culture of the present invention can keep the merit of original kind, produces lay the foundation for stock breeding and industrialization.
Magnolia liliiflora tissue culture method of the present invention realizes above-mentioned purpose by following operation:
1, use bacteriostatic agent in sterilization fruit retention, mancozeb and hypochlorous acid, one or more do the antibacterial process of sterilizing to the place of carrying out purple yu-lan (Magnolia liliflora) tissue cultures;
2, the selection of explant: selecting diameter to be 1 ~ 2cm with the purple yu-lan (Magnolia liliflora) branch of axillalry bud is explant, cuts off the blade of branch, and is cut into the stem section of the band axillalry bud of 1 ~ 2cm;
3, the sterilization of explant: first with running water, explant is rinsed 6 ~ 10min, then explant being immersed in concentration is in the mercuric chloride solution of 0.1%, and add 2 ~ 3ml tween solution, explant is immersed in 7 ~ 13min in this mixed solution, finally with sterile water, explant is rinsed 4 ~ 6 times;
4, initial Fiber differentiation: the 2%PVP of the explant after sterilization is soaked 2 ~ 5min, then this stem section is upwards inserted in WPM minimal medium by end, at 22 ~ 26 DEG C, carry out initial incubation under light intensity 2000 ~ 3000Lx condition; Observe after 3 ~ 5 days, untainted axis section is proceeded in new medium, continue cultivation and grow to sprouting for 10 ~ 15 days;
5, cultivation is extended: the explant stem section growing sprouting turned and insert in elongation medium, namely be in minimal medium WPM, add 6-BA 0.2 ~ 0.6mg/L, NAA 0.3 ~ 0.6mg/L, GA3 0.2 ~ 0.4mg/L and NAA 0.8 ~ 1.0g/L, the elongation carrying out sprouting in elongation medium is cultivated, at 22 ~ 26 DEG C, cultivate 15 ~ 20 days under light intensity 2000 ~ 3000Lx condition, be elongated to 0.2 ~ 0.6cm to sprouting;
6, Multiplying culture: the sprouting access grown is contained in the WPM medium of growth regulatory substance BA 0.8 ~ 1.2mg/L and NAA 0.08 ~ 0.12mg/L, at 22 ~ 26 DEG C, cultivates 23 ~ 26 days under light intensity 2000 ~ 3000Lx condition;
7, culture of rootage: the plantlet in vitro obtained by Multiplying culture was 4 ~ 7 DEG C of refrigerations 10 ~ 14 days, and be transferred in root media, this root media is: add 0.8 ~ 1.0mg/LNAA in WPM minimal medium, concentrates in culture of rootage and continues cultivation 15 ~ 20 days; Again this plantlet in vitro is transferred to afterwards in the WPM medium containing 2%PVP and continues cultivation 10 ~ 15 days, after taking root, carry out hardening; Seedling of taking root moves to be received outdoor and cultivates.
Usefulness of the present invention: compared with prior art, the present invention is directed to existing technical problem, proposes a kind of magnolia liliiflora tissue culture method, and the method is carried out under open condition, and a closed aseptic environment of need not purchasing, has saved cost; Explant sterile contamination be ensure that to the sterilizing of purple yu-lan (Magnolia liliflora), improve it and be trained motility rate; Soak explant with 2%PVP and add 2%PVP in the medium, alleviating brownization of explant, improve the survival rate of explant.This magnolia liliiflora tissue culture method is that the time is short simple to operate, with low cost, and survival rate is high, rooting rate is high, and the bottle outlet that can realize in a short time is transplanted.Method for tissue culture of the present invention can keep the merit of original kind, produces lay the foundation for stock breeding and industrialization.
Embodiment
The present invention is described further below.
Embodiment 1
Use bacteriostatic agent in sterilization fruit retention, mancozeb and hypochlorous acid, one or more do the antibacterial process of sterilizing to the place of carrying out purple yu-lan (Magnolia liliflora) tissue cultures;
The selection of explant: select diameter to be the robust growth of 1.5cm with axillalry bud, the purple yu-lan (Magnolia liliflora) branch that anosis worm is poisoned is explant, cuts off the blade of branch, and is cut into the stem section of the band axillalry bud of 1.5cm;
The sterilization of explant: first with running water, explant is rinsed 6min, then explant being immersed in concentration is in the mercuric chloride solution of 0.1%, and add 2ml tween solution, explant is immersed in 7min in this mixed solution, finally with sterile water, explant is rinsed 4 times;
Initial Fiber differentiation: the explant 2%PVP after sterilization is soaked 2min, then upwards inserts in WPM minimal medium by this stem section by end, at 22 DEG C, carries out initial incubation under light intensity 2000Lx condition; Observe after 3 days, untainted axis section is proceeded in new medium, continue cultivation and grow to sprouting for 10 days;
Extend and cultivate: the explant stem section growing sprouting is turned and inserts in elongation medium, namely be in minimal medium WPM, add 6-BA 0.2mg/L, NAA 0.3mg/L, GA3 0.2mg/L and IAA 0.8g/L, the elongation carrying out sprouting in elongation medium is cultivated, at 22 DEG C, cultivate 15 days under light intensity 2000Lx condition, be elongated to 0.2cm to sprouting;
Multiplying culture: the sprouting access grown is contained in the WPM medium of growth regulatory substance BA 0.8mg/L and NAA 0.08mg/L, at 22 DEG C, cultivates 23 days under light intensity 2000Lx condition;
Culture of rootage: the plantlet in vitro obtained by Multiplying culture was 4 DEG C of refrigerations 10 days, and be transferred in root media, this root media is: add 0.8mg/LNAA in WPM minimal medium, concentrates in culture of rootage and continues cultivation 15 days; Again this plantlet in vitro is transferred to afterwards in the WPM minimal medium medium containing 2%PVP and continues cultivation 10 days, after taking root, carry out hardening; Seedling of taking root moves to be received outdoor and cultivates.
Embodiment 2
Use bacteriostatic agent in sterilization fruit retention, mancozeb and hypochlorous acid, one or more do the antibacterial process of sterilizing to the place of carrying out purple yu-lan (Magnolia liliflora) tissue cultures;
The selection of explant: select diameter to be the robust growth of 1.5cm with axillalry bud, the purple yu-lan (Magnolia liliflora) branch that anosis worm is poisoned is explant, cuts off the blade of branch, and is cut into the stem section of the band axillalry bud of 1.5cm;
The sterilization of explant: first with running water, explant is rinsed 8min, then explant being immersed in concentration is in the mercuric chloride solution of 0.1%, and add 2.5ml tween solution, explant is immersed in 10min in this mixed solution, finally with sterile water, explant is rinsed 5 times;
Initial Fiber differentiation: the explant 2%PVP after sterilization is soaked 4min, then presses end upwards in WPM minimal medium, at 24 DEG C, carries out initial incubation under light intensity 2500Lx condition by this stem section; Observe after 4 days, untainted axis section is proceeded in new medium, continue cultivation and grow to sprouting for 12 days;
Extend and cultivate: the explant stem section growing sprouting is turned and inserts in elongation medium, namely be in minimal medium WPM, add 6-BA 0.4mg/L, NAA 0.4mg/L, GA3 0.3mg/L and IAA 0.9g/L, the elongation carrying out sprouting in elongation medium is cultivated, at 24 DEG C, cultivate 18 days under light intensity 2500Lx condition, be elongated to 0.4cm to sprouting;
Multiplying culture: the sprouting access grown is contained in the WPM medium of growth regulatory substance BA 1.0mg/L and NAA 0.1mg/L, at 24 DEG C, cultivates 24 days under light intensity 2500Lx condition;
Culture of rootage: the plantlet in vitro obtained by Multiplying culture was 4 DEG C of refrigerations 12 days, and be transferred in root media, this root media is: add 0.9mg/LNAA in WPM minimal medium, concentrates in culture of rootage and continues cultivation 18 days; Again this plantlet in vitro is transferred to afterwards in the WPM minimal medium medium containing 2%PVP and continues cultivation 13 days, after taking root, carry out hardening; Seedling of taking root moves to be received outdoor and cultivates.
Embodiment 3
Use bacteriostatic agent in sterilization fruit retention, mancozeb and hypochlorous acid, one or more do the antibacterial process of sterilizing to the place of carrying out purple yu-lan (Magnolia liliflora) tissue cultures;
The selection of explant: select diameter to be the robust growth of 1.5cm with axillalry bud, the purple yu-lan (Magnolia liliflora) branch that anosis worm is poisoned is explant, cuts off the blade of branch, and is cut into the stem section of the band axillalry bud of 1.5cm;
The sterilization of explant: first with running water, explant is rinsed 10min, then explant being immersed in concentration is in the mercuric chloride solution of 0.1%, and add 3ml tween solution, explant is immersed in 13min in this mixed solution, finally with sterile water, explant is rinsed 6 times;
Initial Fiber differentiation: the explant 2%PVP after sterilization is soaked 5min, then upwards inserts in WPM minimal medium by this stem section by end, at 26 DEG C, carries out initial incubation under light intensity 3000Lx condition; Observe after 5 days, untainted axis section is proceeded in new medium, continue cultivation and grow to sprouting for 15 days;
Extend and cultivate: the explant stem section growing sprouting is turned and inserts in elongation medium, namely be add 6-BA 0.6mg/L, NAA 0.6mg/L, GA3 0.4mg/L and IAA in minimal medium WPM l.0g/L, the elongation carrying out sprouting in elongation medium is cultivated, at 26 DEG C, cultivate 20 days under light intensity 3000Lx condition, be elongated to 0.6cm to sprouting;
Multiplying culture: the sprouting access grown is contained in the WPM medium of growth regulatory substance BA 1.2mg/L and NAA 0.12mg/L, at 26 DEG C, cultivates 26 days under light intensity 3000Lx condition;
Culture of rootage: the plantlet in vitro obtained by Multiplying culture was 7 DEG C of refrigerations 14 days, and be transferred in root media, this root media is: add 1.0mg/LNAA in WPM minimal medium, concentrates in culture of rootage and continues cultivation 20 days; Again this plantlet in vitro is transferred to afterwards in the WPM minimal medium medium containing 2%PVP and continues cultivation 15 days, after taking root, carry out hardening; Seedling of taking root moves to be received outdoor and cultivates.
Compared with prior art, the present invention is directed to existing technical problem, propose a kind of magnolia liliiflora tissue culture method, the method is carried out under open condition, and a closed aseptic environment of need not purchasing, has saved cost; Explant sterile contamination be ensure that to the sterilizing of purple yu-lan (Magnolia liliflora), improve it and be trained motility rate; Soak explant with 2%PVP and add 2%PVP in the medium, alleviating brownization of explant, improve the survival rate of explant.This magnolia liliiflora tissue culture method is that the time is short simple to operate, with low cost, and survival rate is high, rooting rate is high, and the bottle outlet that can realize in a short time is transplanted.Method for tissue culture of the present invention can keep the merit of original kind, produces lay the foundation for stock breeding and industrialization.
Claims (1)
1. a method for tissue culture for purple yu-lan (Magnolia liliflora), is characterized in that, this method for tissue culture carries out under open condition, and its method comprises the following steps:
(1) to the sterilization in place carrying out purple yu-lan (Magnolia liliflora) tissue cultures, antiseptic sterilization agent is one or more in sterilization fruit retention, mancozeb and hypochlorous acid;
(2) selection of explant: selecting diameter to be 1 ~ 2cm with the purple yu-lan (Magnolia liliflora) branch of axillalry bud is explant;
(3) sterilization of explant: first use tap water 8 ~ 12min, is then mercuric chloride and the tween mixture immersion 7 ~ 13min of 0.1% by concentration, finally uses aseptic water washing 4 ~ 6 times;
(4) initial Fiber differentiation: the 2%PVP of the explant after sterilizing is soaked 2 ~ 5min, then stem section is upwards inserted in minimal medium WPM by end, at 22 ~ 26 DEG C, carry out initial incubation under light intensity 2000 ~ 3000Lx condition; Observe after 3 ~ 5 days, untainted axis section is proceeded in new medium, continue cultivation and grow to sprouting for 10 ~ 15 days;
(5) cultivation is extended: the explant stem section growing sprouting is turned the elongation cultivation of inserting and carrying out sprouting in elongation medium, sprouting elongation medium is: add 6-BA 0.2 ~ 0.6mg/L in minimal medium WPM, NAA 0.3 ~ 0.6mg/L, GA3 0.2 ~ 0.4mg/L and IAA0.8 ~ 1.0g/L, at 22 ~ 26 DEG C, cultivate 15 ~ 20 days under light intensity 2000 ~ 3000Lx condition, be elongated to 0.2 ~ 0.6cm to sprouting;
(6) Multiplying culture: the sprouting access grown is contained in the WPM minimal medium of growth regulatory substance BA1.0mg/L and NAA0.1mg/L, at 22 ~ 26 DEG C, cultivates 23 ~ 26 days under light intensity 2000 ~ 3000Lx condition;
(7) culture of rootage: the plantlet in vitro obtained by Multiplying culture, 4 ~ 7 DEG C of refrigerations 10 ~ 14 days, is transferred in the WPM medium adding NAA1.0mg/L and continues cultivation 15 ~ 20 days; Plantlet in vitro is transferred in the WPM medium adding 2%PVP and continues cultivation 10 ~ 15 days, after taking root, carry out hardening; Seedling of taking root moves to be received outdoor and cultivates.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105660410A (en) * | 2016-02-26 | 2016-06-15 | 邓珂 | Michelia alba tissue culture method |
| CN105684912A (en) * | 2016-02-26 | 2016-06-22 | 邓珂 | Michelia alba tissues culture medium |
| CN106718918A (en) * | 2016-12-29 | 2017-05-31 | 王文龙 | A kind of purple yu-lan (Magnolia liliflora) tissue cultures technique |
| CN107278892A (en) * | 2017-06-26 | 2017-10-24 | 浦江县美泽生物科技有限公司 | The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) |
| CN111448988B (en) * | 2020-05-18 | 2021-08-13 | 江苏农林职业技术学院 | Culture method and culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii |
| CN112470931A (en) * | 2020-12-04 | 2021-03-12 | 深圳市仙湖植物园管理处（深圳市园林研究中心） | Breeding method for axillary bud tissue culture of Jingning magnolia |
| CN114793892B (en) * | 2022-02-24 | 2023-08-01 | 南京林业大学 | Primary culture method for reducing pollution and browning of yellow heart night-integrated explants |
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Address after: 536000 Jilin Beihai Road, Taiwan Road, Northeast China Electronics Beihai Industrial Park incubator building, room, 3rd floor, Room 302 Patentee after: Chen Fenghua Address before: Qingxiu District, Nanning City, Nanning the Guangxi Zhuang Autonomous Region 530023 Patentee before: Chen Fenghua |
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| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191217 Address after: 215100 4th floor, building 5, No. 892, Wusong Road, Guoxiang street, Wuzhong Economic Development Zone, Suzhou City, Jiangsu Province Patentee after: Harvey Optoelectronic Technology (Suzhou) Co., Ltd Address before: 536000 Jilin Beihai Road, Taiwan Road, Northeast China Electronics Beihai Industrial Park incubator building, room, 3rd floor, Room 302 Patentee before: Chen Fenghua |