CN105475129A - Tissue-culture rapid propagation method for arundina graminifolia - Google Patents
Tissue-culture rapid propagation method for arundina graminifolia Download PDFInfo
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Abstract
A tissue-culture rapid propagation method for arundina graminifolia is characterized by comprising step 1, selecting and disinfecting explant materials, concretely selecting a high bud as an explant, and performing disinfection for usage; step 2, performing inducing culture to obtain adventitious bud, concretely, cutting the top of the high bud, inoculating an adventitious bud culture medium with the high bud, so as to obtain the adventitious bud through inducing; step 3, performing propagation culture to clustered buds, concretely, inoculating a clustered-bud induction medium with the adventitious bud, so as to obtain the clustered buds, and then inoculating a propagation medium with the clustered buds, so as to obtain a clustered-bud block mass; step 4, performing seedling strengthening and rooting culture, concretely, cutting the large clustered-bud block mass obtained in the step 3 into small block masses, then inoculating the seedling-strengthening rooting medium with the small clustered-bud block masses obtained in the step 3, wherein the small clustered-bud block mass grow vigorously and new roots are grown; and step 5, performing seedling hardening and domestication transplanting, when a seedling grows to 4-8 cm and has 1-3 roots, performing seedling-hardening culture, then taking the seedling out, immersing in a thiophanate methyl solution, and finally transplanting by using moss. The variation probability of arundina graminifolia during subculture proliferation is reduced.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly a kind of method of purpleback murdannia herb tissue-culturing rapid propagation.
Background technology
Purpleback murdannia herb (AluminagraminifoliaHochr) is the orchid family leaf of bamboo Cymbidium herbaceos perennial, mainly be distributed in south east asia, originate in the ground such as Fujian, Jiangxi, Zhejiang, Guangdong, Hainan, Guangxi, Sichuan, Guizhou, Hunan, Yunnan in China.Its flower beauty is elegant, and have light fragrance, the florescence is long, and ornamental value is high, can be potted plant, also can be used for courtyard greening.Purpleback murdannia herb also has very high medical value, all herbal medicine. there is effect of compensate qi and blood, clearing heat and detoxicating, wines used as antirheumatic, anti-inflammatory, diuresis.Can be used for treatment rheumatic lumbocrural pain, stomachache, urinary tract infections, venomous snake bite, food poisoning etc.Therefore, viewing and admiring of abundant development and utilization purpleback murdannia herb has wide market prospects with medical value.
Purpleback murdannia herb seed development is complete, without endosperm, lively not to sprout, and needs in the wild could sprout with fungal component symbiosis and germination rate is extremely low.Therefore, traditional purpleback murdannia herb artificial propagation means can only take division propagation, cottage propagation and high-order bud to breed.Wherein, high-order bud breeding can adopt following two kinds of methods:
on the stem branch blossomed and beared fruit or impaired old branch, grow high-order bud when growing to 10 cm, taken and insert in the seedbed of seedling medium, just can transplant after root grows;
also high-order bud can be allowed to grow together with maternal plant, after growing aerial root, take transplanting again.Traditional artificial fecundation method is bred 1-2 time and is needed the maternal plant of a large amount of cultivation 2-3 for 1 year, therefore its reproductive-cost is high, and efficiency is low, cannot meet need of production.Current, have and utilize plant tissue culture technique to breed the report of purpleback murdannia herb seedling.Old woodss etc. (2006) utilize purpleback murdannia herb seed to carry out cultured in vitro, successfully obtain purpleback murdannia herb protocorm and test-tube plantlet.Zhang Wenzhu etc. (2011) for explant carries out aseptic seeding with purpleback murdannia herb seed, also successfully induce protocorm and obtain regeneration plant, and establishing with seed is the tissue-culturing rapid propagation system of explant.But the test-tube plantlet that aseptically sowing seeds obtains is seedling, and its proterties is easily separated, and can not obtain the seedling that genetic material is consistent.And with protocorm occurring mode tissue-culturing rapid propagation seedling, through dedifferentiation and atomization again, easily there is genetic variation in seedling, usual aberration rate is about 8%.
Summary of the invention
The object of the invention is for above the deficiencies in the prior art, provide one to utilize the high-order bud of purpleback murdannia herb as explant, adopt Multiple Buds occurring mode to carry out a kind of method of purpleback murdannia herb tissue-culturing rapid propagation.
Technical scheme of the present invention is: it is characterized in that:
Step 1 explant material is selected and surface sterilization:
With high-order bud distance cultivation matrix upper surface more than 10 centimetres and length 3-5 centimetre of full high-order bud for chosen material, cut high-order bud as explant, first rinse well under running water, afterwards aseptically, with 70-75% alcohol disinfecting 30-60 second, aseptic water washing once, then with 0.1% mercuric chloride sterilization 8-12 minute, last aseptic water washing 3-5 time, aseptic filter paper blots for subsequent use;
The Fiber differentiation of step 2 indefinite bud:
Aseptically, 1-3 centimetre, the high-order bud top position excision first will disinfected, again remaining part according to base portion down mode be inoculated into containing plant growth regulator 6-BA2.0-3.0mg/L+NAA0.1mg/L combine or TDZ0.2-0.3mg/L+NAA0.1mg/L combination MS adventitious bud induction culture base on, illumination cultivation in culturing room, keep intensity of illumination 1500-2000LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the high-order bastem portion at excision top induces indefinite bud;
The Multiplying culture of step 3 Multiple Buds:
Aseptically, product step 2 obtained is inoculated into containing on the MS inducing clumping bud medium that plant growth regulator 6-BA2.0mg/L+NAA0.1mg/L combines or TDZ0.2mg/L+NAA0.1mg/L combines, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, after temperature 24-26 DEG C, 45-50 days, obtain Multiple Buds; Again above-mentioned Multiple Buds is inoculated into containing on the MS adventitious buds proliferation medium that plant growth regulator 6-BA0.5-1.0mg/L+NAA0.1mg/L combines or TDZ0.05-0.1mg/L+NAA0.1mg/L combines, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-50 days, obtain a large amount of Multiple Buds agglomerates;
Step 4 strong sprout and culture of rootage:
Aseptically, Multiple Buds large crumb more than seedling 4 strain first step 3 obtained cuts the little agglomerate of Multiple Buds of seedling 2-4 strain, be inoculated into containing on MS+ banana puree 50g/L+ activated carbon 1.0g/L strong sprout of IBA0.5mg/L or NAA0.1mg/L and root media together with the little agglomerate of Multiple Buds of the seedling 2-4 strain directly obtained with step 3 again, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 16 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the little agglomerate of Multiple Buds of seedling 2-4 strain grows sturdy and grows new root;
The hardening of step 5 plantlet in vitro and rooting culture:
In the little agglomerate of Multiple Buds of the seedling 2-4 strain of step 4 acquisition, seedling grows to plant height 4-8 centimetre, can move into seeding room and carry out hardening cultivation in 7-14 days, to improve its adaptive capacity to environment during root 1-3 bar.After hardening terminates, first from blake bottle, take out above-mentioned Multiple Buds agglomerate, clean root medium, then 15-20 minute in the thiophanate methyl liquid being immersed in dilution 800 times, finally transplant by liver moss matrix.
the invention has the advantages that:
Because this patent adopts high-order bud as explant, explant material genetic material homogeneity can be guaranteed, thus ensure that the genetic stability of purpleback murdannia herb tissue-culturing rapid propagation seedling.And high-order shoot regeneration ability is strong, little by microbiological effect in cultivation matrix, surface sterilization is effective, easily sets up aseptic strain; Because this patent adopts Multiple Buds occurring mode tissue-culturing rapid propagation purpleback murdannia herb seedling, without dedifferentiation and atomization again, thus reduce the probability morphed in the cultivation of purpleback murdannia herb shoot proliferation.
The present invention is by being explant with high-order bud, adopting Multiple Buds occurring mode, shortening the technical measures such as induction time, simplified culture process, plantlet in vitro aberration rate can be reduced, save plantlet in vitro production cost, enhance productivity and profit, therefore the method reproduction speed of purpleback murdannia herb tissue-culturing rapid propagation of the present invention is fast, seedling aberration rate is low, and production cost is low.
Embodiment
Embodiment one:
The method of purpleback murdannia herb tissue-culturing rapid propagation of the present invention comprises the following steps:
Step 1 explant material is selected and surface sterilization:
With high-order bud distance cultivation matrix upper surface more than 10 centimetres and length 3-5 centimetre of full high-order bud for chosen material, cut high-order bud as explant, first rinse well under running water, afterwards aseptically, with 70-75% alcohol disinfecting 30-60 second, aseptic water washing once, then with 0.1% mercuric chloride sterilization 8-12 minute, last aseptic water washing 3-5 time, aseptic filter paper blots for subsequent use.
The Fiber differentiation of step 2 indefinite bud:
Aseptically, 1-3 centimetre, the high-order bud top position excision first will disinfected, again remaining part according to base portion down mode be inoculated into containing plant growth regulator 6-BA3.0mg/L+NAA0.1mg/L combine MS medium on, illumination cultivation in culturing room, keep intensity of illumination 1500-2000LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the high-order bastem portion at excision top can induce indefinite bud.
The Multiplying culture of step 3 Multiple Buds:
Aseptically, product step 2 obtained is inoculated on the MS medium containing plant growth regulator 6-BA2.0mg/L+NAA0.1mg/L combination, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, after temperature 24-26 DEG C, 45-50 days, obtain Multiple Buds; Again above-mentioned Multiple Buds is inoculated on the MS medium containing plant growth regulator 6-BA1.0mg/L+NAA0.1mg/L combination, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-50 days, average proliferation coefficient can reach about 3.2.
Step 4 strong sprout and culture of rootage:
Aseptically, Multiple Buds large crumb more than seedling 4 strain first step 3 obtained cuts the little agglomerate of Multiple Buds of seedling 2-4 strain, be inoculated on the MS+ banana puree 50g/L+ activated carbon 1.0g/L medium containing IBA0.5mg/L or NAA0.1mg/L together with the little agglomerate of Multiple Buds of the seedling 2-4 strain directly obtained with step 3 again, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 16 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the Multiple Buds agglomerate of 2-4 strain grows sturdy and grows new root.
The hardening of step 5 plantlet in vitro and rooting culture:
In the Multiple Buds agglomerate of the seedling 2-4 strain of step 4 acquisition, seedling grows to plant height 4-8 centimetre, can move into seeding room and carry out hardening cultivation in 7-14 days, to improve its adaptive capacity to environment during root 1-3 bar.After hardening terminates, first from blake bottle, take out above-mentioned Multiple Buds agglomerate, clean root medium, then 15-20 minute in the thiophanate methyl liquid being immersed in dilution 800 times, finally transplant by liver moss matrix.
Embodiment two:
Step 1 explant material is selected and surface sterilization:
With high-order bud distance cultivation matrix upper surface more than 10 centimetres and length 3-5 centimetre of full high-order bud for chosen material, cut high-order bud as explant, first rinse well under running water, afterwards aseptically, with 70-75% alcohol disinfecting 30-60 second, aseptic water washing once, then with 0.1% mercuric chloride sterilization 8-12 minute, last aseptic water washing 3-5 time, aseptic filter paper blots for subsequent use.
The Fiber differentiation of step 2 indefinite bud:
Aseptically, 1-3 centimetre, the high-order bud top position excision first will disinfected, again remaining part according to base portion down mode be inoculated into containing plant growth regulator TDZ0.3mg/L+NAA0.1mg/L combine MS medium on, illumination cultivation in culturing room, keep intensity of illumination 1500-2000LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the high-order bastem portion at excision top can induce indefinite bud.
The Multiplying culture of step 3 Multiple Buds:
Aseptically, product step 2 obtained is inoculated on the MS medium containing plant growth regulator TDZ0.2mg/L+NAA0.1mg/L combination, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, after temperature 24-26 DEG C, 45-50 days, the Multiple Buds of acquisition; Again above-mentioned Multiple Buds is inoculated on the MS medium containing plant growth regulator TDZ0.1mg/L+NAA0.1mg/L, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-50 days, average proliferation coefficient can reach about 3.5.
Step 4 strong sprout and culture of rootage:
Aseptically, Multiple Buds large crumb more than seedling 4 strain first step 3 obtained cuts the little agglomerate of Multiple Buds of seedling 2-4 strain, be inoculated on the MS+ banana puree 50g/L+ activated carbon 1.0g/L medium containing IBA0.5mg/L or NAA0.1mg/L together with the little agglomerate of Multiple Buds of the seedling 2-4 strain directly obtained with step 3 again, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 16 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the Multiple Buds agglomerate of 2-4 strain grows sturdy and grows new root.
The hardening of step 5 plantlet in vitro and rooting culture:
In the little agglomerate of Multiple Buds of the seedling 2-4 strain of step 4 acquisition, seedling grows to plant height 4-8 centimetre, can move into seeding room and carry out hardening cultivation in 7-14 days, to improve its adaptive capacity to environment during root 1-3 bar.After hardening terminates, first from blake bottle, take out described plantlet in vitro, clean root medium, then 15-20 minute in the thiophanate methyl liquid being immersed in dilution 800 times, finally transplant by liver moss matrix.
Because the present invention adopts higher concentration basic element of cell division 6-BA2.0-3.0mg/L and low-consistency plant somatotropin NAA0.1mg/L combines or new and effective plant growth regulator TDZ0.2-0.3mg/L and auxin NAA0.1mg/L combines MS medium as the medium of inducing high-order bud to produce indefinite bud, so effectively promote inducing clumping bud, shorten induction time; Because the present invention adopts the MS medium of low concentration plant growth regulator 6-BA0.5-1.0mg/L and NAA0.1mg/L combination or TDZ0.05-0.1mg/L and NAA0.1mg/L combination as proliferated culture medium, so effectively can promote that adventitious buds proliferation can improve again shoot proliferation gained Multiple Buds quality; Because strong seedling culture and culture of rootage two process are merged into strong sprout and a process of taking root, so simplify incubation by the present invention; Because the present invention adopts the MS+ banana puree 50g/L+ activated carbon 1.0g/L medium of IBA0.5mg/L or NAA0.1mg/L as strong seedling culture base also by Multiple Buds large crumb more than seedling 4 strain being cut the little agglomerate of Multiple Buds of seedling 2-4 strain, reduce strong sprout and rooting process inoculum density, for in the little agglomerate of Multiple Buds, seedling growth provides adequate nutrients and growing space, so Multiple Buds strong sprout effectively enough can be urged and take root.
The present invention is by being explant with high-order bud, adopting Multiple Buds occurring mode, shortening the technical measures such as induction time, simplified culture process, can reduce within group cultivates seedling aberration rate to 3%, save plantlet in vitro production cost, enhance productivity and profit, therefore the method reproduction speed of purpleback murdannia herb tissue-culturing rapid propagation of the present invention is fast, seedling aberration rate is low, and production cost is low.
Claims (2)
1. a method for purpleback murdannia herb tissue-culturing rapid propagation, is characterized in that: it is characterized in that:
Step 1 explant material is selected and surface sterilization:
With high-order bud distance cultivation matrix upper surface more than 10 centimetres and length 3-5 centimetre of full high-order bud for chosen material, cut high-order bud as explant, first rinse well under running water, afterwards aseptically, with 70-75% alcohol disinfecting 30-60 second, aseptic water washing once, then with 0.1% mercuric chloride sterilization 8-12 minute, last aseptic water washing 3-5 time, aseptic filter paper blots for subsequent use;
The Fiber differentiation of step 2 indefinite bud:
Aseptically, 1-3 centimetre, the high-order bud top position excision first will disinfected, again remaining part according to base portion down mode be inoculated into containing plant growth regulator 6-BA2.0-3.0mg/L+NAA0.1mg/L combine or TDZ0.2-0.3mg/L+NAA0.1mg/L combination MS adventitious bud induction culture base on, illumination cultivation in culturing room, keep intensity of illumination 1500-2000LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the high-order bastem portion at excision top induces indefinite bud;
The Multiplying culture of step 3 Multiple Buds:
Aseptically, product step 2 obtained is inoculated into containing on the MS inducing clumping bud medium that plant growth regulator 6-BA2.0mg/L+NAA0.1mg/L combines or TDZ0.2mg/L+NAA0.1mg/L combines, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, after temperature 24-26 DEG C, 45-50 days, obtain Multiple Buds; Again above-mentioned Multiple Buds is inoculated into containing on the MS adventitious buds proliferation medium that plant growth regulator 6-BA0.5-1.0mg/L+NAA0.1mg/L combines or TDZ0.05-0.1mg/L+NAA0.1mg/L combines, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 12 hours every days of light application time, temperature 24-26 DEG C, after 45-50 days, obtain a large amount of Multiple Buds agglomerates;
Step 4 strong sprout and culture of rootage:
Aseptically, Multiple Buds large crumb more than seedling 4 strain first step 3 obtained cuts the little agglomerate of Multiple Buds of seedling 2-4 strain, be inoculated into containing on MS+ banana puree 50g/L+ activated carbon 1.0g/L strong sprout of IBA0.5mg/L or NAA0.1mg/L and root media together with the little agglomerate of Multiple Buds of the seedling 2-4 strain directly obtained with step 3 again, illumination cultivation in culturing room, keep intensity of illumination 2000-2500LX, 16 hours every days of light application time, temperature 24-26 DEG C, after 45-60 days, the little agglomerate of Multiple Buds of seedling 2-4 strain grows sturdy and grows new root;
The hardening of step 5 plantlet in vitro and rooting culture:
In the little agglomerate of Multiple Buds of the seedling 2-4 strain of step 4 acquisition, seedling grows to plant height 4-8 centimetre, can move into seeding room and carry out hardening cultivation in 7-14 days, to improve its adaptive capacity to environment during root 1-3 bar.
2. after hardening terminates, first from blake bottle, take out above-mentioned Multiple Buds agglomerate, clean root medium, then 15-20 minute in the thiophanate methyl liquid being immersed in dilution 800 times, finally transplant by liver moss matrix.
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| CN105830928A (en) * | 2016-05-05 | 2016-08-10 | 宁波大学 | Method for tissue culture and rapid propagation of purple-flower toadlily |
| CN107637494A (en) * | 2017-10-20 | 2018-01-30 | 三明市农业科学研究院 | A kind of method for improving oncidiumLuridum tissue-cultured seedling transplanting survival rate |
| CN107646684A (en) * | 2017-10-23 | 2018-02-02 | 广东省农业科学院环境园艺研究所 | A kind of breeding method of purpleback murdannia herb and its application |
| CN110301355A (en) * | 2019-07-31 | 2019-10-08 | 三明市农业科学研究院 | A kind of Chinese cymbidium method for tissue culture |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
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| CN107646684A (en) * | 2017-10-23 | 2018-02-02 | 广东省农业科学院环境园艺研究所 | A kind of breeding method of purpleback murdannia herb and its application |
| CN110301355A (en) * | 2019-07-31 | 2019-10-08 | 三明市农业科学研究院 | A kind of Chinese cymbidium method for tissue culture |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
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