CN105706924B - Violet leaf Japanese ardisia quicker factory production method - Google Patents
Violet leaf Japanese ardisia quicker factory production method Download PDFInfo
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- CN105706924B CN105706924B CN201610068368.0A CN201610068368A CN105706924B CN 105706924 B CN105706924 B CN 105706924B CN 201610068368 A CN201610068368 A CN 201610068368A CN 105706924 B CN105706924 B CN 105706924B
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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
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention relates to a kind of violet leaf Japanese ardisia quicker factory production method, it is characterised in that comprises the following steps：1. selection violet leaf Japanese ardisia fine individual plant stem segment with axillary bud is explant, first induced dormancy axillary bud fast-germination；2. induce axillary bud differentiation adventitious bud；3. after Multiplying culture 40d, axillary bud differentiation produces a large amount of adventitious buds, single adventitious bud is inoculated on strengthening seedling and rooting culture medium, after culture 20d, adventitious bud is gradually divided into the regeneration seedling of stalwartness, base portion produces a small amount of callus simultaneously, and callus top differentiates a small amount of adventitious root, and described strengthening seedling and rooting culture medium is：The agar of+2% sucrose of MS+0.10 0.15mg/L forchlorfenuron CPPU+0.5 0.8mg/L indolebutyric acid IBA+0.5g/L activated carbons+0.6%, pH are 5.8 6.0；4. pair tissue-cultured seedling taken root carries out acclimatization and transplantses.The present invention improves production efficiency and survival rate, reduces production cost, and the merit for continuing wild resource that the regrowth obtained can be stablized.
Description
First, technical field
The invention belongs to Plant Tissue Breeding quick breeding technology field, and in particular to violet leaf Japanese ardisia quicker factory metaplasia
Production method
2nd, research background
The violet leaf Japanese ardisia (Ardisia Violacea), also known as the violet Japanese ardisia is wrapped up in, belong to Myrsinacea Ardisa, be a kind of
Special product is in the rare or endangered species in Zhejiang and Taiwan.Violet leaf Japanese ardisia stem skin taupe, by micro- pubescence, blade is slightly in rosette-stape,
Blade base blunt circle or micro-heart, above micro-strip it is red, below band lavender, edge is in irregular wavy shallow knuckle-tooth, umbrella shape flower
Sequence list is born in the top of axil or stem, and corolla white, the month at florescence 6-7, plant posture is quiet and tastefully laid out, and haw is gorgeous prolonged, round and smooth sparkling and crystal-clear,
Delicate and pretty, elegant, haw matches somebody with somebody purple leaf, makes us pleasing, and has stronger shade tolerance and drought tolerance, with being used as garden
By, sylvan life quilt and see leaf and see the small potted plant class product of fruit, there is the high market development to be worth.Because the violet leaf Japanese ardisia is wild
Resource, and stock number is extremely limited, conventional mode of reproduction, can not carry out factory on the basis of protection wild resource is maximized
Metaplasia is produced, and the foundation of group culturation rapid propagating technology system can then be carried out big using less wild resource as material in the short time
The factorial praluction of scale, can realize protection of resources and the seedling breeding of the endangered plants, and promote the species marketization
Process.
3rd, the content of the invention
The problem of existing for prior art, the present invention provide a kind of quicker factory production method in the violet leaf Japanese ardisia,
Production efficiency and survival rate are improved, reduces production cost, and the continuity wild resource that the regrowth obtained can be stablized
Merit.
Therefore, the present invention takes following technical scheme：
1) the excellent individual plant of the violet leaf Japanese ardisia character of no disease and pests harm is put it to when March starts restoration ecosystem
Conserved in greenhouse, week about 0.3%-0.5% of foliage spray carbendazim solution, until or so May.Treat that sprouting is taken out
After going out, the tender stem of clip wherein semi-lignified, blade is cut along petiole base, tender stem is cut into segment, first with 0.5% it is new clean
You go out+and the stirring of liquid detergent mixed solution disinfects 1h, outwells thimerosal, 1h is rinsed under flowing water, and it is standby.Superclean bench
In, treated standby segment once, outwells sterilized water with aseptic water washing, and 60- is sterilized with 70%-75% alcoholic solution
90s, after outwelling alcohol, then with 0.1% mercuric chloride solution sterilize 5-10min, outwell thimerosal, finally rinsed repeatedly with sterilized water
It is 4-5 times, standby.Aseptic water washing and during disinfecting, will shake, to ensure sterilized water and thimerosal incessantly
It can be contacted completely with explant, improve disinfection efficiency；The segment that the stem section disinfected by more than is cut into 2cm or so is (every
Have the axillary bud of 1 dormancy in section), with sterile razor blade first the partially cut-away of stem section bottom otch browning, rapidly by axillary bud stem
The bud of section is inoculated into primary culture medium upwards.Condition of culture is：25 ± 2 DEG C, light application time 10h/d of temperature, intensity of illumination
For 1500-2000Lx, humidity 60%-65%.Primary culture medium is：MS+0.15-0.30mg/L forchlorfenurons (CPPU)+
The agar of 0.10-0.20mg/L methyl α-naphthyl acetates (NAA)+3% sucrose+0.7%, pH 5.8-6.0；
2) after Primary culture 20d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into adventitious bud point
Change on (propagation) culture medium.Adventitious buds differentiation culture medium is：MS+0.50-0.80mg/L forchlorfenurons (CPPU)+0.10-
The agar of 0.20mg/L methyl α-naphthyl acetates (NAA)+3% sucrose+0.7%, pH 5.8-6.0；
3) after Multiplying culture 40d, axillary bud differentiation produces a large amount of adventitious buds, and adventitious bud exists in the form of bud clump.Will
The adventitious bud clump arrived, is gently dialled and dissipates into single adventitious bud, and single adventitious bud is inoculated on strengthening seedling and rooting culture medium.Strengthening seedling and rooting
Condition of culture is：25 ± 2 DEG C, light application time 8h/d, intensity of illumination 1000Lx of temperature, humidity 60%-65%.Described
Strengthening seedling and rooting culture medium is：MS+0.10-0.15mg/L forchlorfenurons (CPPU)+0.5-0.8mg/L indolebutyric acids (IBA)+
The agar of+2% sucrose of 0.5g/L activated carbons+0.6%, pH 5.8-6.0；
4) after strengthening seedling and rooting culture 20d, adventitious bud is gradually divided into the regeneration seedling of stalwartness, while base portion produces on a small quantity
Callus, callus top differentiate a small amount of adventitious root.After cultivating 40-50d, the average plant height for regenerating seedling is
4.68cm, the root system bar number of average individual plant seedling is 4.28.The tissue-culture container seedling taken root is transferred on the seedbed in hardening tunnel,
Natural environment lower refining seedling 10d.3-4d before transplanting, the bottle cap of tissue culture bottle is loosened, but do not opened completely, 1-2d before transplanting, will
The bottle cap of tissue culture bottle is opened completely, and toward a small amount of sterilized water is poured into tissue culture bottle, first will with tweezers before tissue culture of taking root transplantation of seedlings
Rooted seedling is lightly come out from culture medium inner clip, and the culture medium of root institute band is cleaned up completely with clear water, will be cleaned
Rooted seedling enters hardening matrix (loose squama, 1 week or so before transplanting, at 0.8% disinfecting solution of potassium permanganate by one clump of kind of 3-4 strains
Reason) in.Finally, the rooted seedling and stromal surface of transplanting are uniformly sprayed with 0.3% carbendazim solution, soaked completely with loose squama
It is wet to be advisable, the Small plastic shed of closing is then made with plastic sheeting, carries out heat and moisture preserving 40d or so.Under growth root seedling grows new
Must shape root and after recovering robust growth, the both ends of Small plastic shed are opened, slowly leaks informaton, after about 1 week, Small plastic shed is opened completely,
It is set to grow under field conditions (factors).
It is of the invention with traditional raising technology and compared with conventional tissue culture technology, can be by the strong sprout during tissue-culturing rapid propagation
Two stages of culture of rootage are combined into one in culture and bottle, make regrowth carry out taking root in bottle while increasing strong, shorten tissue culture
Reduce the number of tissue-cultured seedling subculture while the seedling production cycle, greatly save production cost.Wherein adventitious bud proliferation coefficient
Up to 12.8, rooting rate is up to 96.7% in bottle, and acclimatization and transplantses survival rate can reach more than 95%, and whole fast numerous cycle time arrives
Only 4.5 months, substantially increase production efficiency, and the Optimality for continuing wild resource that the regrowth obtained can be stablized
Shape.
4th, embodiment
Case study on implementation 1
On May 12nd, 2014, chosen in greenhouse and grow vigorous, no disease and pests harm, the excellent violet leaf Japanese ardisia of trait expression is wild
Raw individual plant, clip are newly sent out edible tender branch, cut blade along petiole base with scissors, tender stem is cut into segment, anti-with clear water then
Rinse again 3-4 times, first disinfect 30min with 0.5% bromogeramine+liquid detergent mixed solution stirring, outwell thimerosal,
1h is rinsed under flowing water, it is standby.In superclean bench, treated standby segment once, outwells sterilized water with aseptic water washing, uses
70%-75% alcoholic solution sterilization 30s, after outwelling alcohol, then with 0.1% mercuric chloride solution sterilize 5min, outwell thimerosal,
Finally rinsed 4-5 times repeatedly with sterilized water, it is standby.Aseptic water washing and during disinfecting, will shake incessantly,
To ensure that sterilized water and thimerosal can contact with explant completely, disinfection efficiency is improved；
The stem section disinfected by more than is cut into 2cm or so segment (axillary bud for having 1 dormancy on every section), and use is sterile
The bud of axillary bud stem section is inoculated into primary culture medium by blade upwards rapidly first the partially cut-away of stem section bottom otch browning
On.Condition of culture is：25 ± 2 DEG C, light application time 10h/d, intensity of illumination 1500-2000Lx of temperature, humidity 60%-
65%.Primary culture medium is：MS+0.20mg/L forchlorfenurons (CPPU)+0.10mg/L methyl α-naphthyl acetates (NAA)+3% sucrose+0.7%
Agar, pH 5.8-6.0；After Primary culture 28d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into not
On normal bud differentiation (propagation) culture medium.Adventitious buds differentiation culture medium is：MS+0.50mg/L forchlorfenurons (CPPU)+0.10mg/L
The agar of methyl α-naphthyl acetate (NAA)+3% sucrose+0.7%, pH 5.8-6.0；After Multiplying culture 45d, axillary bud differentiation produces largely not
Normal bud, adventitious bud exist in the form of bud clump.The adventitious bud clump that will be obtained, gently dials and dissipates into single adventitious bud, will be single indefinite
Bud is inoculated on strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is：25 ± 2 DEG C, light application time 8h/d of temperature, illumination is strong
Spend for 1000Lx, humidity 60%-65%.Described strengthening seedling and rooting culture medium is：MS+0.10mg/L forchlorfenurons (CPPU)+
The agar of+2% sucrose of 0.5mg/L indolebutyric acids (IBA)+0.5g/L activated carbons+0.6%, pH 5.8-6.0；Strengthening seedling and rooting culture
After 45d, the tissue-culture container seedling taken root is transferred on the seedbed in hardening tunnel, according to foregoing invention content step 4) related
Technical operation carries out acclimatization and transplantses.
The explant that the implementation case obtains disinfects pollution rate as 8%, and adventitious bud proliferation coefficient reaches 11.9, in bottle
Rooting rate reaches 95.8%, and whole fast numerous cycle (terminating since Multiplying culture to acclimatization and transplantses) is 140d.
Case study on implementation 2
On May 28th, 2014, chosen in greenhouse and grow vigorous, no disease and pests harm, the excellent violet leaf Japanese ardisia of trait expression is wild
Raw individual plant, clip are newly sent out edible tender branch, cut blade along petiole base with scissors, tender stem is cut into segment, anti-with clear water then
Rinse again 3-4 times, first disinfect 1h with 0.5% bromogeramine+liquid detergent mixed solution stirring, outwell thimerosal, flowing
Underwater flushing 1h, it is standby.In superclean bench, treated standby segment once, outwells sterilized water with aseptic water washing, uses
70%-75% alcoholic solution sterilization 60s, after outwelling alcohol, then with 0.1% mercuric chloride solution sterilize 8min, outwell thimerosal,
Finally rinsed 4-5 times repeatedly with sterilized water, it is standby.Aseptic water washing and during disinfecting, will shake incessantly,
To ensure that sterilized water and thimerosal can contact with explant completely, disinfection efficiency is improved；
The stem section disinfected by more than is cut into 2cm or so segment (axillary bud for having 1 dormancy on every section), and use is sterile
The bud of axillary bud stem section is inoculated into primary culture medium by blade upwards rapidly first the partially cut-away of stem section bottom otch browning
On.Condition of culture is：25 ± 2 DEG C, light application time 10h/d, intensity of illumination 1500-2000Lx of temperature, humidity 60%-
65%.Primary culture medium is：MS+0.25mg/L forchlorfenurons (CPPU)+0.15mg/L methyl α-naphthyl acetates (NAA)+3% sucrose+0.7%
Agar, pH 5.8-6.0；After Primary culture 24d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into not
On normal bud differentiation (propagation) culture medium.Adventitious buds differentiation culture medium is：MS+0.70mg/L forchlorfenurons (CPPU)+0.15mg/L
The agar of methyl α-naphthyl acetate (NAA)+3% sucrose+0.7%, pH 5.8-6.0；After Multiplying culture 51d, axillary bud differentiation produces largely not
Normal bud, adventitious bud exist in the form of bud clump.The adventitious bud clump that will be obtained, gently dials and dissipates into single adventitious bud, will be single indefinite
Bud is inoculated on strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is：25 ± 2 DEG C, light application time 8h/d of temperature, illumination is strong
Spend for 1000Lx, humidity 60%-65%.Described strengthening seedling and rooting culture medium is：MS+0.10mg/L forchlorfenurons (CPPU)+
The agar of+2% sucrose of 0.60mg/L indolebutyric acids (IBA)+0.50g/L activated carbons+0.6%, pH 5.8-6.0；Strengthening seedling and rooting is trained
After supporting 49d, the tissue-culture container seedling taken root is transferred on the seedbed in hardening tunnel, according to foregoing invention content step 4) it is related
Technical operation carry out acclimatization and transplantses.
It is 8.6% that the explant that the implementation case obtains, which disinfects pollution rate, and adventitious bud proliferation coefficient reaches 14.1, bottle
Interior rooting rate reaches 98.8%, and whole fast numerous cycle (terminating since Multiplying culture to acclimatization and transplantses) is 132d.
Case study on implementation 3
On June 15th, 2014, chosen in greenhouse and grow vigorous, no disease and pests harm, the excellent violet leaf Japanese ardisia of trait expression is wild
Raw individual plant, clip are newly sent out edible tender branch, cut blade along petiole base with scissors, tender stem is cut into segment, anti-with clear water then
Rinse again 3-4 times, first disinfect 1h with 0.5% bromogeramine+liquid detergent mixed solution stirring, outwell thimerosal, flowing
Underwater flushing 1h, it is standby.In superclean bench, treated standby segment once, outwells sterilized water with aseptic water washing, uses
70%-75% alcoholic solution sterilization 90s, after outwelling alcohol, then with 0.1% mercuric chloride solution sterilize 10min, outwell sterilization
Liquid, finally rinsed 4-5 times repeatedly with sterilized water, it is standby.Aseptic water washing and during disinfecting, will shake incessantly
It is dynamic, to ensure that sterilized water and thimerosal can contact with explant completely, improve disinfection efficiency；
The stem section disinfected by more than is cut into 2cm or so segment (axillary bud for having 1 dormancy on every section), and use is sterile
The bud of axillary bud stem section is inoculated into primary culture medium by blade upwards rapidly first the partially cut-away of stem section bottom otch browning
On.Condition of culture is：25 ± 2 DEG C, light application time 10h/d, intensity of illumination 1500-2000Lx of temperature, humidity 60%-
65%.Primary culture medium is：MS+0.30mg/L forchlorfenurons (CPPU)+0.20mg/L methyl α-naphthyl acetates (NAA)+3% sucrose+0.7%
Agar, pH 5.8-6.0；After Primary culture 20d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off, is inoculated into not
On normal bud differentiation (propagation) culture medium.Adventitious buds differentiation culture medium is：MS+0.80mg/L forchlorfenurons (CPPU)+0.20mg/L
The agar of methyl α-naphthyl acetate (NAA)+3% sucrose+0.7%, pH 5.8-6.0；After Multiplying culture 55d, axillary bud differentiation produces largely not
Normal bud, adventitious bud exist in the form of bud clump.The adventitious bud clump that will be obtained, gently dials and dissipates into single adventitious bud, will be single indefinite
Bud is inoculated on strengthening seedling and rooting culture medium.Strengthening seedling and rooting condition of culture is：25 ± 2 DEG C, light application time 8h/d of temperature, illumination is strong
Spend for 1000Lx, humidity 60%-65%.Described strengthening seedling and rooting culture medium is：MS+0.15mg/L forchlorfenurons (CPPU)+
The agar of+2% sucrose of 0.80mg/L indolebutyric acids (IBA)+0.50g/L activated carbons+0.6%, pH 5.8-6.0；Strengthening seedling and rooting is trained
After supporting 52d, the tissue-culture container seedling taken root is transferred on the seedbed in hardening tunnel, according to foregoing invention content step 4) it is related
Technical operation carry out acclimatization and transplantses.
It is 9.3% that the explant that the implementation case obtains, which disinfects pollution rate, and adventitious bud proliferation coefficient reaches 13.6, bottle
Interior rooting rate reaches 99.2%, and whole fast numerous cycle (terminating since Multiplying culture to acclimatization and transplantses) is 137d.
Claims (7)
1. violet leaf Japanese ardisia quicker factory production method, it is characterised in that comprise the following steps：
1) it is explant to choose violet leaf Japanese ardisia fine individual plant stem segment with axillary bud, first induced dormancy axillary bud fast-germination, by axillary bud
The bud of stem section is inoculated into primary culture medium upwards, and primary culture medium is：MS+0.15-0.30mg/L forchlorfenurons CPPU+
The agar of 0.10-0.20mg/L methyl α-naphthyl acetate NAA+3% sucrose+0.7%, pH 5.8-6.0；
2) axillary bud differentiation adventitious bud is induced, after Primary culture 20d, the axillary bud sprouting of dormancy, the tender shoots newly sprouted is scaled off,
It is inoculated on adventitious buds differentiation culture medium, described adventitious buds differentiation culture medium is：MS+0.50-0.80mg/L forchlorfenurons
The agar of CPPU+0.10-0.20mg/L methyl α-naphthyl acetate NAA+3% sucrose+0.7%, pH 5.8-6.0；
3) after differentiation culture 40d, axillary bud differentiation produces a large amount of adventitious buds, and adventitious bud exists in the form of bud clump, by step 2)
Obtained adventitious bud clump, dial dissipate into single adventitious bud, single adventitious bud is inoculated on strengthening seedling and rooting culture medium, culture 20d with
Afterwards, adventitious bud is gradually divided into the regeneration seedling of stalwartness, while base portion produces a small amount of callus, and callus top differentiates
A small amount of adventitious root, described strengthening seedling and rooting culture medium are：MS+0.10-0.15mg/L forchlorfenuron CPPU+0.5-0.8mg/L Yin
The agar of+2% sucrose of diindyl butyric acid IBA+0.5g/L activated carbons+0.6%, pH 5.8-6.0；
4) acclimatization and transplantses are carried out to the tissue-cultured seedling taken root.
2. violet leaf Japanese ardisia quicker factory production method as claimed in claim 1, it is characterised in that in step 1)：Choose nothing
The excellent individual plant of the violet leaf Japanese ardisia character of pest and disease damage, after sprouting is extracted out, the tender stem of clip wherein semi-lignified, by blade edge
Petiole base is cut, and tender stem is cut into segment, is carried out disinfection, and the stem section disinfected is cut into 2cm segment, has 1 on every section
The axillary bud of individual dormancy, with sterile razor blade first by the partially cut-away of stem section bottom otch browning.
3. violet leaf Japanese ardisia quicker factory production method as claimed in claim 1, it is characterised in that culture bar in step 1)
Part is：25 ± 2 DEG C, light application time 10h/d, intensity of illumination 1500-2000Lx of temperature, humidity 60%-65%.
4. violet leaf Japanese ardisia quicker factory production method as claimed in claim 2, it is characterised in that the sterilization in step 1)
It is that tender stem is cut into after segment first to disinfect 1h with 0.5% bromogeramine+liquid detergent mixed solution stirring, outwells sterilization
Liquid, 1h is rinsed under flowing water, standby, in superclean bench, treated standby segment once, is outwelled sterile with aseptic water washing
Water, 60-90s is sterilized with 70%-75% alcoholic solution, after outwelling alcohol, then with 0.1% mercuric chloride solution sterilizes 5-10min,
Thimerosal is outwelled, is finally rinsed 4-5 times repeatedly with sterilized water.
5. violet leaf Japanese ardisia quicker factory production method as claimed in claim 1, it is characterised in that strengthened in the step 3)
Seedling rooting condition of culture is：25 ± 2 DEG C, light application time 8h/d, intensity of illumination 1000Lx of temperature, humidity 60%-65%.
6. violet leaf Japanese ardisia quicker factory production method as claimed in claim 1, it is characterised in that before being transplanted in step 4)
3-4d, the bottle cap of tissue culture bottle is loosened, but do not opened completely, 1-2d before transplanting, the bottle cap of tissue culture bottle opened completely, and it is past
A small amount of sterilized water is poured into tissue culture bottle, before tissue culture of taking root transplantation of seedlings, first with tweezers by rooted seedling lightly from culture medium inner clip
Out, the culture medium of root institute band is cleaned up completely with clear water, cleaned rooted seedling is entered into refining by one clump of kind of 3-4 strains
In seedling matrix, the hardening matrix is loose squama, and finally, the rooted seedling and stromal surface of transplanting are entered with 0.3% carbendazim solution
Row is uniformly sprayed, and being soaked completely with loose squama is advisable, and the Small plastic shed of closing is then made with plastic sheeting, carries out heat and moisture preserving 40d
Left and right.
7. a kind of strengthening seedling and rooting culture medium of violet leaf Japanese ardisia quicker factory production method, it is characterised in that by following component group
Into：The sucrose of MS+0.10-0.15mg/L forchlorfenuron CPPU+0.5-0.8mg/L indolebutyric acid IBA+0.5g/L activated carbons+2%+
0.6% agar, pH 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 |
| 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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| CN105028208B (en) * | 2015-08-25 | 2017-03-08 | 广西壮族自治区农业科学院生物技术研究所 | A kind of Rhizoma Ardisiae Gigantifoliae callus induction and tissue culture sprout quick breeding method |
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