CN105706924A - Fast industrialized production method for ardisia violacea - Google Patents
Fast industrialized production method for ardisia violacea Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to a fast industrialized production method for ardisia violacea. The fast industrialized production method is characterized by including the following steps that 1, stem segments, with auxiliary buds, of good single plants of ardisia violacea are selected, the dormancy auxiliary buds are induced to fast sprout; 2, the auxiliary buds are induced to differentiate adventitious buds; 3, after multiplication culture is conducted for 40 days, the auxiliary buds differentiate to generate a great number of adventitious buds, a seeding strengthening and rooting culture medium is inoculated with the single adventitious buds, after being cultured for 20 days, the adventitious buds gradually differentiate into strong regenerative seedlings, a small amount of callus tissue is generated on the base, and the upper portion of the callus tissue differentiates a few adventitious roots; the seeding strengthening and rooting culture medium is prepared from MS, N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) with the concentration of 0.10-0.15 mg/L, indolebutyric acid (IBA) with the concentration of 0.5-0.8 mg/L, activated carbon with the concentration of 0.5 g/L, saccharose with the concentration of 2% and agar with the concentration of 0.6%, and the pH value is 5.8-6.0; 4, acclimatization and transplant are conducted on the tissue culture seedlings taking roots. The production efficiency is improved, the survival rate is increased, production cost is reduced, and good characters of wild resources can be extended in the obtained regeneration seedlings.
Description
One, technical field
The invention belongs to plant tissue culture quick breeding technology field, be specifically related to violet leaf Herba Ardisiae Japonicae quicker factory
Change production method
Two, research background
Violet leaf Herba Ardisiae Japonicae (Ardisia Violacea), has another name called and wraps up in violet Herba Ardisiae Japonicae, belongs to Myrsinacea Ardisa, is
A kind of special product is in Zhejiang and the rare or endangered species in Taiwan.Violet leaf Herba Ardisiae Japonicae peel of stem taupe, by micro-pubescence, leaf
Sheet slightly in rosette-stape, the blunt circle of blade base or micro-heart, above micro-strip red, under wear lavender, edge in
Irregular wavy shallow knuckle-tooth, umbel list is born in the top of axil or stem, corolla white, the month at florescence 6-7,
Plant attitude is quiet and tastefully laid out, and Fructus Pyracanthae is gorgeous prolonged, round and smooth sparkling and crystal-clear, delicate and pretty, elegant, and Fructus Pyracanthae joins purple leaf, makes us appreciating the heart
Pleasing, and there is stronger shade tolerance and drought tolerance, can as garden by, sylvan life ground quilt and see Ye Guanguo
Little potted plant series products, has high market development and is worth.Owing to violet leaf Herba Ardisiae Japonicae is wild resource, and resource
Amount is extremely limited, conventional mode of reproduction, it is impossible to carry out batch production on the basis of maximizing protection wild resource
Producing, the foundation of group culturation rapid propagating technology system then can come in the short time with less wild resource as material
Carry out large-scale factorial praluction, it is possible to realize protection of resources and the seedling breeding of these endangered plants, and promote
The process of this species marketization.
Three, summary of the invention
The problem existed for prior art, the present invention provides the quicker factory metaplasia of a kind of violet leaf Herba Ardisiae Japonicae to produce
Method, improves production efficiency and survival rate, reduces production cost, and the regrowth obtained can be stablized
Continuity wild resource merit.
To this end, the present invention takes following technical scheme:
1) by individual plant excellent for the violet leaf Herba Ardisiae Japonicae character without pest and disease damage in time starting restoration ecosystem March, put
Maintenance in greenhouse, the week about carbendazim solution of 0.3%-0.5% of foliage spray, until May
About Fen.After sprouting is extracted out, the tender stem of clip wherein semi-lignified, blade is cut along petiole base,
Tender stem is cut into segment, first disinfects 1h with the bromo geramine+liquid detergent mixed solution stirring of 0.5%, falls
Fall disinfectant solution, under flowing water, rinse 1h, standby.In superclean bench, processed standby segment nothing
Bacterium water rinses, outwells sterilized water, with the alcoholic solution sterilization 60-90s of 70%-75%, outwells ethanol
After, then the mercuric chloride solution sterilization 5-10min with 0.1%, outwell disinfectant solution, finally repeatedly rush with sterilized water
Wash 4-5 time, standby.Aseptic water washing and during disinfecting, will shake incessantly, with
Ensure that sterilized water can contact with outer implant completely with disinfectant solution, improve disinfection efficiency；At above sterilization
The stem section managed is cut into the segment (having the axillalry bud of 1 dormancy on every section) of about 2cm, uses sterile razor blade
First partially cut-away stem section bottom otch brownization, is upwards inoculated into startup by the bud of axillary bud stem section rapidly
In culture medium.Condition of culture is: temperature 25 ± 2 DEG C, and light application time is 10h/d, and intensity of illumination is
1500-2000Lx, humidity is 60%-65%.Primary culture base is: MS+0.15-0.30mg/L chlorine pyrrole benzene
Urea (CPPU)+0.10-0.20mg/L naphthalene acetic acid (NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；
2) after Primary culture 20d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, it is indefinite to be inoculated into
In Bud polarization (propagation) culture medium.Adventitious buds differentiation culture medium is: MS+0.50-0.80mg/L chlorine pyrrole benzene
Urea (CPPU)+0.10-0.20mg/L naphthalene acetic acid (NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；
3), after enrichment culture 40d, axillalry bud differentiation produces a large amount of adventitious buds, and adventitious bud is presented in bud clump.
The adventitious bud clump that will obtain, dials gently and dissipates into single adventitious bud, and single adventitious bud is inoculated into strengthening seedling and rooting
In culture medium.Strengthening seedling and rooting condition of culture is: temperature 25 ± 2 DEG C, and light application time is 8h/d, and illumination is strong
Degree is 1000Lx, and humidity is 60%-65%.Described strengthening seedling and rooting culture medium is: MS+0.10-0.15mg/L
Forchlorfenuron (CPPU)+0.5-0.8mg/L indolebutyric acid (IBA)+0.5g/L activated carbon+2% sucrose+0.6%
Agar, pH is 5.8-6.0；
4), after strengthening seedling and rooting cultivates 20d, adventitious bud is gradually divided into the regeneration seedling of stalwartness, and base portion produces simultaneously
A small amount of callus, callus upper part dissolves a small amount of adventitious root.After cultivating 40-50d, regenerate little
The average plant height of Seedling is 4.68cm, and the root system bar number of average individual plant seedling is 4.28.The tissue culture bottle that will take root
Seedling is transferred on the seedbed in seedling exercising tunnel, natural environment lower refining seedling 10d.3-4d before transplanting, trains group
The bottle cap of bottle loosens, but does not fully open, 1-2d before transplanting, and is fully opened by the bottle cap of tissue culture bottle,
And pour into a small amount of sterilized water in tissue culture bottle, before tissue cultured seedling of taking root is transplanted, first light with tweezers Seedling of taking root
Lightly from culture medium inner clip out, the culture medium clear water carried by root cleans up completely, will clean
Good Seedling of taking root is that one clump of kind enters seedling exercising substrate (pine squama, transplants first about 1 week, with 0.8% with 3-4 strain
Disinfecting solution of potassium permanganate processes) in.Finally, transplanting taken root Miao Heji with 0.3% carbendazim solution
Matter surface uniformly sprays, and soaks completely with pine squama and is advisable, then makes the little of closing with plastic sheeting
Shed, carries out heat and moisture preserving about 40d.What seedling length to be taken root made new advances must shape root recover robust growth
After, the two ends of Small plastic shed are opened, slowly leak informaton, after about 1 week, Small plastic shed is fully opened so that it is
Grow under field conditions (factors).
The present invention is compared with traditional raising technology and conventional tissue culture technology, it is possible to during tissue-culturing rapid propagation
In strong seedling culture and bottle, two stages of root culture unite two into one, raw in making regrowth carry out bottle while increasing is strong
Root, decreases the number of times of tissue cultured seedling subculture, is greatly saved production cost while shortening the tissue cultured seedling production cycle.
Wherein adventitious bud proliferation coefficient is up to 12.8, and in bottle, rooting rate is up to 96.7%, and acclimatization and transplants survival rate can reach
More than 95%, whole fast numerous cycle time to only 4.5 months, substantially increase production efficiency, and obtain
Regrowth can be stable continuity wild resource merit.
Four, detailed description of the invention
Case study on implementation 1
On May 12nd, 2014, choose growth in greenhouse vigorous, without pest and disease damage, the violet leaf that trait expression is excellent
The wild individual plant of Herba Ardisiae Japonicae, clip newly sends out edible tender branch, cut along petiole base by blade with shears, tender stem
It is cut into segment, repeatedly rinses 3-4 time with clear water, first stir with the bromo geramine+liquid detergent mixed solution of 0.5%
Disinfect 30min, outwell disinfectant solution, under flowing water, rinse 1h, standby.In superclean bench, processed
Standby segment aseptic water washing once, outwells sterilized water, sterilizes 30s with the alcoholic solution of 70%-75%,
After outwelling ethanol, then the mercuric chloride solution sterilization 5min with 0.1%, outwell disinfectant solution, repeatedly with sterilized water finally
Rinse 4-5 time, standby.Aseptic water washing and during disinfecting, will shake, incessantly to protect
Card sterilized water can contact with outer implant completely with disinfectant solution, improves disinfection efficiency；
The stem section disinfected above is cut into the segment (having the axillalry bud of 1 dormancy on every section) of about 2cm,
First partially cut-away stem section bottom otch brownization by sterile razor blade, upwards inoculates the bud of axillary bud stem section rapidly
On Primary culture base.Condition of culture is: temperature 25 ± 2 DEG C, and light application time is 10h/d, and intensity of illumination is
1500-2000Lx, humidity is 60%-65%.Primary culture base is: MS+0.20mg/L forchlorfenuron
(CPPU)+0.10mg/L naphthalene acetic acid (NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；Primary culture 28d
After, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into adventitious buds differentiation (propagation) training
Support on base.Adventitious buds differentiation culture medium is: MS+0.50mg/L forchlorfenuron (CPPU)+0.10mg/L naphthalene acetic acid
(NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；After enrichment culture 45d, axillalry bud differentiation produces big
Amount adventitious bud, adventitious bud is presented in bud clump.The adventitious bud clump that will obtain, dial gently dissipate into single indefinite
Bud, is inoculated into single adventitious bud in strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is: temperature 25 ± 2 DEG C,
Light application time is 8h/d, and intensity of illumination is 1000Lx, and humidity is 60%-65%.Described strengthening seedling and rooting is cultivated
Base is: MS+0.10mg/L forchlorfenuron (CPPU)+0.5mg/L indolebutyric acid (IBA)+0.5g/L activated carbon
+ 2% sucrose+0.6% agar, pH is 5.8-6.0；After strengthening seedling and rooting cultivates 45d, the tissue-culture container seedling that will take root
Transfer on the seedbed in seedling exercising tunnel, according to foregoing invention content step 4) relevant technical operation refines
Transplantation of seedlings.
It is 8% that the outer implant that the implementation case obtains disinfects pollution rate, and adventitious bud proliferation coefficient reaches 11.9,
In bottle, rooting rate reaches 95.8%, and whole fast numerous cycle (starting to acclimatization and transplants to terminate from enrichment culture) is 140d.
Case study on implementation 2
On May 28th, 2014, choose growth in greenhouse vigorous, without pest and disease damage, the violet leaf that trait expression is excellent
The wild individual plant of Herba Ardisiae Japonicae, clip newly sends out edible tender branch, cut along petiole base by blade with shears, tender stem
It is cut into segment, repeatedly rinses 3-4 time with clear water, first stir with the bromo geramine+liquid detergent mixed solution of 0.5%
Disinfect 1h, outwell disinfectant solution, under flowing water, rinse 1h, standby.In superclean bench, processed standby
Segment with aseptic water washing once, outwell sterilized water, sterilize 60s with the alcoholic solution of 70%-75%, fall
After falling ethanol, then the mercuric chloride solution sterilization 8min with 0.1%, outwell disinfectant solution, finally repeatedly rush with sterilized water
Wash 4-5 time, standby.Aseptic water washing and during disinfecting, will shake, incessantly to ensure
Sterilized water can contact with outer implant completely with disinfectant solution, improves disinfection efficiency；
The stem section disinfected above is cut into the segment (having the axillalry bud of 1 dormancy on every section) of about 2cm,
First partially cut-away stem section bottom otch brownization by sterile razor blade, upwards inoculates the bud of axillary bud stem section rapidly
On Primary culture base.Condition of culture is: temperature 25 ± 2 DEG C, and light application time is 10h/d, and intensity of illumination is
1500-2000Lx, humidity is 60%-65%.Primary culture base is: MS+0.25mg/L forchlorfenuron
(CPPU)+0.15mg/L naphthalene acetic acid (NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；Primary culture 24d
After, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into adventitious buds differentiation (propagation) training
Support on base.Adventitious buds differentiation culture medium is: MS+0.70mg/L forchlorfenuron (CPPU)+0.15mg/L naphthalene acetic acid
(NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；After enrichment culture 51d, axillalry bud differentiation produces big
Amount adventitious bud, adventitious bud is presented in bud clump.The adventitious bud clump that will obtain, dial gently dissipate into single indefinite
Bud, is inoculated into single adventitious bud in strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is: temperature 25 ± 2 DEG C,
Light application time is 8h/d, and intensity of illumination is 1000Lx, and humidity is 60%-65%.Described strengthening seedling and rooting is cultivated
Base is: MS+0.10mg/L forchlorfenuron (CPPU)+0.60mg/L indolebutyric acid (IBA)+0.50g/L activity
Charcoal+2% sucrose+0.6% agar, pH is 5.8-6.0；After strengthening seedling and rooting cultivates 49d, the tissue culture bottle that will take root
Seedling is transferred on the seedbed in seedling exercising tunnel, according to foregoing invention content step 4) relevant technical operation carries out
Acclimatization and transplants.
It is 8.6% that the outer implant that the implementation case obtains disinfects pollution rate, and adventitious bud proliferation coefficient reaches 14.1,
In bottle, rooting rate reaches 98.8%, and whole fast numerous cycle (starting to acclimatization and transplants to terminate from enrichment culture) is 132d.
Case study on implementation 3
On June 15th, 2014, choose growth in greenhouse vigorous, without pest and disease damage, the violet leaf that trait expression is excellent
The wild individual plant of Herba Ardisiae Japonicae, clip newly sends out edible tender branch, cut along petiole base by blade with shears, tender stem
It is cut into segment, repeatedly rinses 3-4 time with clear water, first stir with the bromo geramine+liquid detergent mixed solution of 0.5%
Disinfect 1h, outwell disinfectant solution, under flowing water, rinse 1h, standby.In superclean bench, processed standby
Segment with aseptic water washing once, outwell sterilized water, sterilize 90s with the alcoholic solution of 70%-75%, fall
After falling ethanol, then the mercuric chloride solution sterilization 10min with 0.1%, outwell disinfectant solution, repeatedly with sterilized water finally
Rinse 4-5 time, standby.Aseptic water washing and during disinfecting, will shake, incessantly to protect
Card sterilized water can contact with outer implant completely with disinfectant solution, improves disinfection efficiency；
The stem section disinfected above is cut into the segment (having the axillalry bud of 1 dormancy on every section) of about 2cm,
First partially cut-away stem section bottom otch brownization by sterile razor blade, upwards inoculates the bud of axillary bud stem section rapidly
On Primary culture base.Condition of culture is: temperature 25 ± 2 DEG C, and light application time is 10h/d, and intensity of illumination is
1500-2000Lx, humidity is 60%-65%.Primary culture base is: MS+0.30mg/L forchlorfenuron
(CPPU)+0.20mg/L naphthalene acetic acid (NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；Primary culture 20d
After, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into adventitious buds differentiation (propagation) training
Support on base.Adventitious buds differentiation culture medium is: MS+0.80mg/L forchlorfenuron (CPPU)+0.20mg/L naphthalene acetic acid
(NAA)+3% sucrose+0.7% agar, pH is 5.8-6.0；After enrichment culture 55d, axillalry bud differentiation produces big
Amount adventitious bud, adventitious bud is presented in bud clump.The adventitious bud clump that will obtain, dial gently dissipate into single indefinite
Bud, is inoculated into single adventitious bud in strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is: temperature 25 ± 2 DEG C,
Light application time is 8h/d, and intensity of illumination is 1000Lx, and humidity is 60%-65%.Described strengthening seedling and rooting is cultivated
Base is: MS+0.15mg/L forchlorfenuron (CPPU)+0.80mg/L indolebutyric acid (IBA)+0.50g/L activity
Charcoal+2% sucrose+0.6% agar, pH is 5.8-6.0；After strengthening seedling and rooting cultivates 52d, the tissue culture bottle that will take root
Seedling is transferred on the seedbed in seedling exercising tunnel, according to foregoing invention content step 4) relevant technical operation carries out
Acclimatization and transplants.
It is 9.3% that the outer implant that the implementation case obtains disinfects pollution rate, and adventitious bud proliferation coefficient reaches 13.6,
In bottle, rooting rate reaches 99.2%, and whole fast numerous cycle (starting to acclimatization and transplants to terminate from enrichment culture) is 137d.
Claims (8)
1. violet leaf Herba Ardisiae Japonicae quicker factory production method, it is characterised in that comprise the following steps:
1) choosing violet leaf Herba Ardisiae Japonicae fine individual plant stem segment with axillary bud is outer implant, first induced dormancy axillalry bud fast-germination；
2) induction axillalry bud differentiation adventitious bud；
3) after enrichment culture 40d, axillalry bud differentiation produce a large amount of adventitious buds, adventitious bud presented in bud clump,
By step 2) the adventitious bud clump that obtains, dial and dissipate into single adventitious bud, single adventitious bud is inoculated into strong sprout
On root media, after cultivating 20d, adventitious bud is gradually divided into the regeneration seedling of stalwartness, base simultaneously
Portion produces a small amount of callus, and callus upper part dissolves a small amount of adventitious root, described strengthening seedling and rooting training
Foster base is: MS+0.10-0.15mg/L forchlorfenuron CPPU+0.5-0.8mg/L indolebutyric acid IBA+0.5g/L
Activated carbon+2% sucrose+0.6% agar, pH is 5.8-6.0；
4) tissue cultured seedling taken root is carried out acclimatization and transplants.
2. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 1, it is characterised in that step 1) in:
Choose the individual plant excellent without the violet leaf Herba Ardisiae Japonicae character of pest and disease damage, after sprouting is extracted out, clip wherein semi-lignified
Tender stem, blade is cut along petiole base, tender stem is cut into segment, carries out disinfection, by disinfected
Stem section is cut into the segment of 2cm, and every section has the axillalry bud of 1 dormancy, by sterile razor blade first by stem section bottom
Otch brownization partially cut-away, is upwards inoculated into the bud of axillary bud stem section on Primary culture base rapidly, Primary culture
Base is: MS+0.15-0.30mg/L forchlorfenuron CPPU+0.10-0.20mg/L naphthalene acetic acid NAA+3% sucrose
+ 0.7% agar, pH is 5.8-6.0.
3. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 1, it is characterised in that step 1) in
Condition of culture is: temperature 25 ± 2 DEG C, and light application time is 10h/d, and intensity of illumination is 1500-2000Lx, wet
Degree is 60%-65%.
4. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 2, it is characterised in that step 1) in
Sterilization be tender stem to be cut into after segment first with at bromo geramine+liquid detergent mixed solution stirring sterilization of 0.5%
Reason 1h, outwells disinfectant solution, rinses 1h under flowing water, standby, in superclean bench, processes standby little
Section with aseptic water washing once, outwells sterilized water, with the alcoholic solution sterilization 60-90s of 70%-75%, outwells
After ethanol, then the mercuric chloride solution sterilization 5-10min with 0.1%, outwell disinfectant solution, finally repeatedly rush with sterilized water
Wash 4-5 time.
5. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 1, it is characterised in that step 2)
In: after Primary culture 20d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, it is indefinite to be inoculated into
In Bud polarization culture medium, described adventitious buds differentiation culture medium is: MS+0.50-0.80mg/L forchlorfenuron
CPPU+0.10-0.20mg/L naphthalene acetic acid NAA+3% sucrose+0.7% agar, pH is 5.8-6.0.
6. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 1, it is characterised in that described step 3)
Middle strengthening seedling and rooting condition of culture is: temperature 25 ± 2 DEG C, and light application time is 8h/d, and intensity of illumination is 1000Lx,
Humidity is 60%-65%.
7. violet leaf Herba Ardisiae Japonicae quicker factory production method as claimed in claim 1, it is characterised in that step 4) in
3-4d before transplanting, loosens the bottle cap of tissue culture bottle, but does not fully open, and 1-2d before transplanting, by tissue culture bottle
Bottle cap fully open, and pour into a small amount of sterilized water in tissue culture bottle, before tissue cultured seedling of taking root is transplanted, first use tweezer
Son will take root Seedling lightly from culture medium inner clip out, the culture medium clear water carried by root cleans dry completely
Only, being that one clump of kind enters in seedling exercising substrate by cleaned Seedling of taking root with 3-4 strain, described seedling exercising substrate is pine squama,
Finally, with 0.3% carbendazim solution transplanting taken root Seedling and stromal surface uniformly sprays, complete with pine squama
Entirely soak and be advisable, then make the Small plastic shed of closing with plastic sheeting, carry out heat and moisture preserving about 40d.
8. the strengthening seedling and rooting culture medium of a violet leaf Herba Ardisiae Japonicae quicker factory production method, it is characterised in that by following
One-tenth is grouped into: MS+0.10-0.15mg/L forchlorfenuron CPPU+0.5-0.8mg/L indolebutyric acid IBA+0.5g/L
Activated carbon+2% sucrose+0.6% agar, pH is 5.8-6.0.
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| CN107223571B (en) * | 2017-08-04 | 2019-04-19 | 黄小燕 | The quick breeding method for tissue culture in the lobus cardiacus Japanese ardisia |
| CN109275568A (en) * | 2018-11-25 | 2019-01-29 | 钟天路 | A kind of tissue cultivation rapid breeding method of ardisia japonica |
| CN109997691A (en) * | 2019-03-19 | 2019-07-12 | 浙江省嘉兴市农业科学研究院（所） | Tissue culture and rapid propagation method of Ardisia japonica |
| CN109997691B (en) * | 2019-03-19 | 2021-01-05 | 浙江省嘉兴市农业科学研究院（所） | Tissue culture and rapid propagation method of Ardisia japonica |
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