CN104663451A - Tissue culture and rapid propagation method for lycoris albiflora - Google Patents
Tissue culture and rapid propagation method for lycoris albiflora Download PDFInfo
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Abstract
The invention discloses a tissue culture and rapid propagation method for lycoris albiflora. At present, lycoris plants in China mainly perform bulb division propagation and seed self-sowing propagation in the natural state, the propagation coefficient is low, the propagation speed is low, most bulbs can only divide 1-2 child bulbs every year, the number of bulbs is small and limited, and the requirement of commercial production of the lycoris albiflora is far from being met. In order to meet the requirement of commercial production of the lycoris albiflora, in-vitro regenerated plants of the lycoris albiflora are obtained with lycoris albiflora with basal disc bulbs used as explants through processes of adventitious bud induction, subculture, rooting, acclimatization, transplantation and the like, and a tissue culture and rapid propagation technology system for the lycoris albiflora is established to promote the commercial production of the lycoris albiflora.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of vitro of Lycoris albiflora tissue culture and rapid propagation method.
Background technology
Lycoris (
lycoris Herb) Amaryllidaceae herbaceos perennial.Lycoris plants strong adaptability, easily plants, drought-enduring, moisture-proof, barren-resistant, extensive management, and the florescence meets again most the lacking that fall of autumn in summer high temperature full-blown flowers to spend the time, therefore can be widely used in afforestation.And short-tube lycoris when flower " do not bloom during leaf, lose leaf " first spends posterior lobe, or the opening again of the withered rear flower of leaf, this is very rare and unique in phanerogams, because the most kind of lycoris plants originates in China, therefore has the title of " Chinese tulip ".In addition, Lycoris bulb contains lycorine, the multiple alkaloids such as LYCORENINE, galanthamine, Dihydrogalantnamine, ungernine, sekisanine, galantamine, Pseudolycorine, class lycorine, short-tube lycoris tincture, short-tube lycoris alcohol, there is removing toxic substances, diuresis, emetic, effect such as to eliminate the phlegm, cure mainly carbuncle sore and dislike the diseases such as core, abscess of throat, oedema, also have good effect at antitumor, anticancer aspect simultaneously.In addition, because short-tube lycoris plant alkaloid toxicity is comparatively large, can be used for the production of biopesticide, for maggot of going out, to kill mouse etc. and to prevent and treat other crop pests.
Lycoris plants commonly use bulb separation, sowing, bulb substrate cutting cuttage and etc. method breeding, natural shape
Based on a point ball, seed natural seeding under state, the normal sibling species group forming plexi distribution.But the reproduction coefficient of natural bulb separation and seed natural seeding is very low, as point ball be by around large bulb raw clove peel and plant separately, but clove poor growth, can not gather every year, generally to excavate in the fall every 3 ~ 4 years, row bulb separation again,, its reproduction speed is quite slow, and most kind is often only mitogenetic 1 ~ 2 bulbec of energy, plant ball quantity few and limited, this can not meet the needs that short-tube lycoris is commercially produced far away.Plant Tissue Breeding has that reproduction speed is fast, reproduction coefficient is large, benefit high, but the tissue culture technique of lycoris plants is still immature at present, also has a lot of problem to have to be solved.And the present invention with vitro of Lycoris albiflora band basal disc bulb for explant, through adventitious bud inducing, squamous subculture, take root, the process such as acclimatization and transplants obtains the in vitro plant again of vitro of Lycoris albiflora, set up vitro of Lycoris albiflora tissue culture rapid propagation technique system, to promote commercially producing of vitro of Lycoris albiflora.
Summary of the invention
The object of the present invention is to provide out a kind of vitro of Lycoris albiflora tissue culture and rapid propagation method, the present invention with vitro of Lycoris albiflora band basal disc bulb for explant, through adventitious bud inducing, squamous subculture, take root, the process such as acclimatization and transplants obtains the in vitro plant again of vitro of Lycoris albiflora, set up vitro of Lycoris albiflora tissue culture rapid propagation technique system, thus achieve object of the present invention.
A kind of vitro of Lycoris albiflora tissue culture and rapid propagation method of the present invention, comprises the following steps:
(1) explant sterilization: the bulb that robust growth, size are basically identical, the outermost layer of brown is divested totally, 1 ~ 3h is rinsed under flowing water, truncated, with 75% ~ 80% alcohol solution dipping 10 ~ 30s in superclean bench, aseptic water washing 3 ~ 5 times, then with 0.1 ~ 0.5% mercuric chloride solution sterilization 5 ~ 10min containing 0.01 ~ 0.05% Tween-20, blots the moisture on surface with aseptic filter paper after aseptic water washing 3 ~ 5 times for subsequent use.
(2) adventitious bud inducing: the bulb after step of learning from else's experience (1) process is first cut into four pieces with cross method, be cut into the scale that size is about 1.2cm × 1.0cm bilayer or three layers of band basal disc again, be inoculated into inducing culture in the mode that basal disc is downward and carry out adventitious bud induction culture.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 40 days.
(3) squamous subculture: step (2) Fiber differentiation is obtained indefinite bud and be inoculated into proliferated culture medium and carry out adventitious bud proliferation cultivation.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up adventitious bud proliferation situation afterwards in 40 days.
(4) culture of rootage: be seeded in root media after the Multiple Buds that step (3) induces is cut into simple bud and carry out root induction.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days.
(5) acclimatization and transplants: by root growth completely plantlet in vitro carry out hardening and transplanting, first the wide-mouth bottle that plantlet in vitro is housed unscrewed but do not open 1 ~ 3 day, a small amount of pure water is injected again in bottle, hardening was taken out after 3 ~ 5 days again, the medium on plant is cleaned with flowing water, the carbendazim solution putting into 0.1% ~ 0.2% soaks 3 ~ 5min, be transplanted to through high-temperature sterilization by sandy soil: in the matrix be mixed into of garden mould=1:1, cover preservative film and keep certain humidity, take off film after 1 week, transplant and add up survival rate after 20 days.
Inducing culture described in above-mentioned steps (2) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 3.0mg/L 6-BA+20 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+0.5 ~ 2.0mg/L 2,4-D+1.0 ~ 3.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (4) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 3.0mg/L KT+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: the lycoris plants have the laudatory title of " Chinese tulip ", is an emerging plant integrating medicinal, view and admire.But it is main for breeding mainly with the bulb separation breeding under nature and seed natural seeding at China's lycoris plants at present, its reproduction coefficient is low, and reproduction speed is slow, and most kind is often only mitogenetic 1 ~ 2 bulbec of energy, plant ball quantity few and limited, this can not meet the demand that short-tube lycoris is commercially produced far away.In order to meet the needs that short-tube lycoris is commercially produced, the present invention with vitro of Lycoris albiflora band basal disc bulb for explant, through adventitious bud inducing, squamous subculture, take root, the process such as acclimatization and transplants obtains the in vitro plant again of vitro of Lycoris albiflora, set up vitro of Lycoris albiflora tissue culture rapid propagation technique system, to promote commercially producing of vitro of Lycoris albiflora.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1
(1) explant sterilization: the bulb that robust growth, size are basically identical, the outermost layer of brown is divested totally, 1h is rinsed under flowing water, truncated, with 75% alcohol solution dipping 10s in superclean bench, aseptic water washing 3 times, then with the 0.1% mercuric chloride solution sterilization 5min containing 0.01% Tween-20, blots the moisture on surface with aseptic filter paper after aseptic water washing 3 times for subsequent use.
(2) adventitious bud inducing: the bulb after step of learning from else's experience (1) process is first cut into four pieces with cross method, be cut into the scale that size is about 1.2cm × 1.0cm bilayer or three layers of band basal disc again, be inoculated into inducing culture in the mode that basal disc is downward and carry out adventitious bud induction culture.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 23 DEG C, and relative air humidity is that to cultivate adventitious bud induction frequency after 40 days under the condition of 75% be 97.6%.Described inducing culture is: MS+0.1mg/L NAA+1.5mg/L 6-BA+25g/L sucrose+4.5g/L agar, pH is 5.5.
(3) squamous subculture: step (2) Fiber differentiation is obtained indefinite bud and be inoculated into proliferated culture medium and carry out adventitious bud proliferation cultivation.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 23 DEG C, and relative air humidity is that under the condition of 75%, cultivation adds up adventitious bud proliferation coefficient in 40 days is afterwards 10.2.Described proliferated culture medium is: MS+0.5mg/L 2,4-D+1.5mg/L 6-BA+25g/L sucrose+4.5g/L agar, pH is 5.5.
(4) culture of rootage: be seeded in root media after the Multiple Buds that step (3) induces is cut into simple bud and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 23 DEG C, and relative air humidity is that under the condition of 75%, cultivation adds up coefficient of taking root in 30 days is afterwards 8.2.Described root media is: MS+0.1mg/L NAA+2.0mg/L KT+20g/L sucrose+3.8g/L agar, pH is 5.5.
(5) acclimatization and transplants: by root growth completely plantlet in vitro carry out hardening and transplanting, first the wide-mouth bottle that plantlet in vitro is housed unscrewed but do not open 1 day, a small amount of pure water is injected again in bottle, hardening was taken out after 3 days again, the medium on plant is cleaned with flowing water, the carbendazim solution putting into 0.1%% soaks 3min, be transplanted to through high-temperature sterilization by sandy soil: in the matrix be mixed into of garden mould=1:1, cover preservative film and keep certain humidity, take off film after 1 week, transplanting survival rate after 20 days is more than 95%.
Embodiment 2
(1) explant sterilization: the bulb that robust growth, size are basically identical, the outermost layer of brown is divested totally, 2h is rinsed under flowing water, truncated, with 78% alcohol solution dipping 12s in superclean bench, aseptic water washing 5 times, then with the 0.1% mercuric chloride solution sterilization 7min containing 0.01% Tween-20, blots the moisture on surface with aseptic filter paper after aseptic water washing 5 times for subsequent use.
(2) adventitious bud inducing: the bulb after step of learning from else's experience (1) process is first cut into four pieces with cross method, be cut into the scale that size is about 1.2cm × 1.0cm bilayer or three layers of band basal disc again, be inoculated into inducing culture in the mode that basal disc is downward and carry out adventitious bud induction culture.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 1500lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate adventitious bud induction frequency after 40 days under the condition of 75% be 99.5%.Described inducing culture is: MS+0.2mg/L NAA+3.0mg/L 6-BA+30g/L sucrose+4.5g/L agar, pH is 5.5.
(3) squamous subculture: step (2) Fiber differentiation is obtained indefinite bud and be inoculated into proliferated culture medium and carry out adventitious bud proliferation cultivation.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 1500lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that under the condition of 78%, cultivation adds up adventitious bud proliferation coefficient in 40 days is afterwards 9.6.Described proliferated culture medium is: MS+0.8mg/L 2,4-D+2.0mg/L 6-BA+25g/L sucrose+5.5g/L agar, pH is 5.5.
(4) culture of rootage: be seeded in root media after the Multiple Buds that step (3) induces is cut into simple bud and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 23 DEG C, and relative air humidity is that under the condition of 75%, cultivation adds up coefficient of taking root in 30 days is afterwards 7.4.Described root media is: MS+0.5mg/L NAA+2.5mg/L KT+20g/L sucrose+4.0g/L agar, pH is 5.5.
(5) acclimatization and transplants: by root growth completely plantlet in vitro carry out hardening and transplanting, first the wide-mouth bottle that plantlet in vitro is housed unscrewed but do not open 1 day, a small amount of pure water is injected again in bottle, hardening was taken out after 3 days again, the medium on plant is cleaned with flowing water, the carbendazim solution putting into 0.1%% soaks 3min, be transplanted to through high-temperature sterilization by sandy soil: in the matrix be mixed into of garden mould=1:1, cover preservative film and keep certain humidity, take off film after 1 week, transplanting survival rate after 20 days is more than 95%.
Claims (4)
1. a vitro of Lycoris albiflora tissue culture and rapid propagation method, is characterized in that comprising the following steps:
(1) explant sterilization: the bulb that robust growth, size are basically identical; the outermost layer of brown is divested totally; 1 ~ 3h is rinsed under flowing water; truncated; with 75% ~ 80% alcohol solution dipping 10 ~ 30s in superclean bench; aseptic water washing 3 ~ 5 times, then with 0.1 ~ 0.5% mercuric chloride solution sterilization 5 ~ 10min containing 0.01 ~ 0.05% Tween-20, blots the moisture on surface with aseptic filter paper after aseptic water washing 3 ~ 5 times for subsequent use;
(2) adventitious bud inducing: the bulb after step of learning from else's experience (1) process is first cut into four pieces with cross method; be cut into the scale that size is about 1.2cm × 1.0cm bilayer or three layers of band basal disc again; be inoculated into inducing culture in the mode that basal disc is downward and carry out adventitious bud induction culture; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 40 days;
(3) squamous subculture: step (2) Fiber differentiation is obtained indefinite bud and be inoculated into proliferated culture medium and carry out adventitious bud proliferation cultivation; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up adventitious bud proliferation situation afterwards in 40 days;
(4) culture of rootage: be seeded in root media after the Multiple Buds that step (3) induces is cut into simple bud and carry out root induction; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is 23 ~ 25 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days;
(5) acclimatization and transplants: by root growth completely plantlet in vitro carry out hardening and transplanting, first the wide-mouth bottle that plantlet in vitro is housed unscrewed but do not open 1 ~ 3 day, a small amount of pure water is injected again in bottle, hardening was taken out after 3 ~ 5 days again, the medium on plant is cleaned with flowing water, the carbendazim solution putting into 0.1% ~ 0.2% soaks 3 ~ 5min, be transplanted to through high-temperature sterilization by sandy soil: in the matrix be mixed into of garden mould=1:1, cover preservative film and keep certain humidity, take off film after 1 week, transplant and add up survival rate after 20 days.
2. a kind of vitro of Lycoris albiflora tissue culture and rapid propagation method according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 3.0mg/L 6-BA+20 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. a kind of vitro of Lycoris albiflora tissue culture and rapid propagation method according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+0.5 ~ 2.0mg/L 2,4-D+1.0 ~ 3.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. a kind of vitro of Lycoris albiflora tissue culture and rapid propagation method according to claim 1, it is characterized in that the root media described in step (4) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 3.0mg/L KT+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (7)
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| CN105830920A (en) * | 2016-04-01 | 2016-08-10 | 浙江大学 | In-vitro probulb division growth method for realizing rapid proliferation of Lycoris herbs |
| CN109220807A (en) * | 2018-11-09 | 2019-01-18 | 中国农业科学院蔬菜花卉研究所 | The stock breeding method that Allium fistulosum stem tips are cultivated |
| CN110583488A (en) * | 2019-10-24 | 2019-12-20 | 杭州植物园（杭州市园林科学研究院） | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink |
| CN111109081A (en) * | 2020-01-03 | 2020-05-08 | 上海市农业科学院 | Lycoris radiata rootless tissue culture method and lycoris radiata cultivation method |
| CN112243729A (en) * | 2020-10-19 | 2021-01-22 | 上海市农业科学院 | Method for regulating and controlling flowering phase of lycoris plants |
| CN112273210A (en) * | 2020-11-13 | 2021-01-29 | 上海上房园艺有限公司 | Seedling hardening method for brocade-changing tissue culture seedlings |
| CN112602595A (en) * | 2020-12-29 | 2021-04-06 | 江苏丰收大地种业发展有限公司 | Tissue culture method for increasing number of differentiated adventitious buds of garlic growing points |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830920A (en) * | 2016-04-01 | 2016-08-10 | 浙江大学 | In-vitro probulb division growth method for realizing rapid proliferation of Lycoris herbs |
| CN109220807A (en) * | 2018-11-09 | 2019-01-18 | 中国农业科学院蔬菜花卉研究所 | The stock breeding method that Allium fistulosum stem tips are cultivated |
| CN110583488A (en) * | 2019-10-24 | 2019-12-20 | 杭州植物园（杭州市园林科学研究院） | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink |
| CN111109081A (en) * | 2020-01-03 | 2020-05-08 | 上海市农业科学院 | Lycoris radiata rootless tissue culture method and lycoris radiata cultivation method |
| CN112243729A (en) * | 2020-10-19 | 2021-01-22 | 上海市农业科学院 | Method for regulating and controlling flowering phase of lycoris plants |
| CN112243729B (en) * | 2020-10-19 | 2022-04-22 | 上海市农业科学院 | Method for regulating and controlling flowering phase of lycoris plants |
| CN112273210A (en) * | 2020-11-13 | 2021-01-29 | 上海上房园艺有限公司 | Seedling hardening method for brocade-changing tissue culture seedlings |
| CN112602595A (en) * | 2020-12-29 | 2021-04-06 | 江苏丰收大地种业发展有限公司 | Tissue culture method for increasing number of differentiated adventitious buds of garlic growing points |
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Application publication date: 20150603 |