CN108142284A - A kind of tissue culture and rapid propagation method of five leaflets maple - Google Patents
A kind of tissue culture and rapid propagation method of five leaflets maple Download PDFInfo
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- CN108142284A CN108142284A CN201611110432.3A CN201611110432A CN108142284A CN 108142284 A CN108142284 A CN 108142284A CN 201611110432 A CN201611110432 A CN 201611110432A CN 108142284 A CN108142284 A CN 108142284A
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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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- 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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present invention discloses a kind of tissue culture and rapid propagation method of five leaflets maple, includes the following steps：(1) explant is chosen and sterilizes：Five leaflet maple branches is taken to clean, sterilizing cuts stem with bud and obtains explant；(2) Initial culture：The explant that the step (1) obtains is inoculated into Initial culture base and carries out Initial culture, induces axillary buds formation；(3) squamous subculture：The axillary bud that the step (2) obtains is inoculated into proliferated culture medium and carries out squamous subculture, evoking adventive bud generation；(4) culture of rootage：Squamous subculture：The adventitious bud that the step (3) obtains is inoculated into root media and carries out culture of rootage, obtains rooted seedling.Used medium formula is simple, and tissue culture simple flow, incubation time is short, and proliferation frequency is high, reduces cost of labor, easily operated, can carry out industrialization production.
Description
Technical field
The present invention relates to plant tissue culture seedling-raising technique fields, and in particular to a kind of tissue culture and rapid propagation method of five leaflets maple.
Background technology
Five leaflet maples (Acer pentaphyllum Diels) are Aceraceae Acer deciduous trees, are distinctive states of China
Family first class of protection plant.Distribution be only limitted to Sichuan Yalongjiang River and its tributary (in wood, Kowloon, OK a karaoke club and wheat field dragon) height above sea level 2200~
The river valley area of 3000m.Person poultry activity's serious threat is on the point of to the existence of five leaflet maples according to World Conservation Union (IUCN)
Classification standard of endangering (IUCN 1994) is already belonging to pole danger species, faces the edge of extinction, instant need of first aid protection.Five leaflet maples pair
The origin and succession for studying Aceraceae plant have very important significance, while are also rare cities and towns garden landscape trees
Kind.
At present, it is mainly seed propagation seedling technology to the research of the mode of reproduction of five leaflet maples.This method repoductive time compared with
Long, material cost is higher, and amount reproduction has some limitations.
Invention content
In view of this, the application provides a kind of tissue culture and rapid propagation method of five leaflets maple, and used medium formula is simple, tissue culture
Simple flow, incubation time is short, and proliferation frequency is high, and seedling is neat, and convenient for the later stage, unified transplanting and Cultivate administration, reduce people
Work cost can guarantee the consistency of grown seedling plants in shape, easily operated, can carry out industrialization production.
A kind of tissue culture and rapid propagation method of five leaflets maple, includes the following steps：
(1) explant is chosen and sterilizes：Five leaflet maple branches is taken to clean, sterilizing cuts stem with bud and obtains explant；
(2) Initial culture：By the explant that the step (1) obtains be inoculated into the culture bottle equipped with Initial culture base into
Row Initial culture, generates axillary bud, and Initial culture base composition includes：1/2MS minimal mediums add 0.05~0.5mg/L 6-
BA, 0.02~0.2mg/L TDZ, 10~40g/L sucrose, 4~7g/L agar；
(3) squamous subculture：The axillary bud that the step (2) obtains is inoculated into proliferated culture medium and carries out squamous subculture, it is raw
Into adventitious bud, the proliferated culture medium composition includes：WPM minimal mediums, additional 0.05~0.5mg/L 6-BA, 0.2~
1.0mg/L CPPU, 10~40g/L sucrose, 4~7g/L agar；
(4) culture of rootage：It the step (3) is obtained adventitious bud is inoculated into root media to carry out culture of rootage, obtain
To rooted seedling, the root media composition includes：WPM minimal mediums add 0.2~1.0mg/L NAA, 10~40g/L
Sucrose, 4~7g/L agar.
Preferably, the explant is chosen and sterilization process is specific：Five leaflet maple edible tender branch are taken, blade is removed, uses wine
Smart oscillation treatment 30s, sterile water wash 1 time, then with mercuric chloride 6~10min of oscillation treatment, sterile water wash 3~5 times, sterile filter
Paper sucks moisture, and the stem with bud for cutting into 1~2cm obtains the explant.
Preferably, the alcohol by volume percentage is 75%.
Preferably, the mercuric chloride mass percent is 0.1%.
Preferably, the Initial culture process intensity of illumination is 800~1200Lux, light application time 14h/d., and culture is warm
Spend is 23~27 DEG C.
Preferably, the Initial culture process intensity of illumination is 1000Lux.
Preferably, 25 DEG C of the Initial culture process cultivation temperature.
Preferably, the Subculture and the process of rooting culture intensity of illumination are 1300~1800Lux, illumination
Time is 14h/d., and cultivation temperature is 23~27 DEG C.
Preferably, the Subculture and the process of rooting culture intensity of illumination are 1500Lux.
Preferably, the Subculture and the process of rooting culture cultivation temperature are 25 DEG C.
Preferably, the Initial culture time is 4~5 weeks.
Preferably, the squamous subculture time is 4~5 weeks.
Preferably, the culture of rootage time is 3~5 weeks.
Preferably, the culture of rootage time is 4 weeks.
Preferably, the Initial culture base composition includes：1/2MS minimal mediums, additional 0.05~0.2mg/L 6-BA,
0.02~0.1mg/L TDZ, 10~30g/L sucrose, 4~6g/L agar.
Preferably, the Initial culture base composition includes：1/2MS minimal mediums, additional 0.1~0.2mg/L6-BA,
0.05~0.1mg/L TDZ, 20~30g/L sucrose, 6g/L agar.
Preferably, the Initial culture base composition includes：1/2MS minimal mediums add 0.1mg/L6-BA, 0.05mg/
L TDZ, 30g/L sucrose, 6g/L agar.
Preferably, pH is 5.5~6.5 before the Initial culture base sterilizing.
Preferably, the proliferated culture medium composition includes：WPM minimal mediums, additional 0.05~0.2mg/L 6-BA,
0.2~1.0mg/L CPPU, 10~30g/L sucrose, 4~6g/L agar.
Preferably, the proliferated culture medium composition includes：WPM minimal mediums add 0.1~0.2mg/L 6-BA, 0.5
~1.0mg/L CPPU, 20~30g/L sucrose, 6g/L agar.
Preferably, the proliferated culture medium composition includes：WPM minimal mediums, supplement 0.1mg/L 6-BA, 0.5mg/L
CPPU, 30g/L sucrose, 6g/L agar.
Preferably, the root media composition includes：WPM minimal mediums, add 0.2~1.0mg/L NAA, 10~
30g/L sucrose, 4~6g/L agar.
Preferably, the root media composition includes：WPM minimal mediums, add 0.5~1.0mg/L NAA, 10~
20g/L sucrose, 6g/L agar.
Preferably, the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L, 20g/L sucrose,
6g/L agar.
Herein described WPM minimal mediums be xylophyta culture medium (WPM, woody plant medium), nothing
Machine salinity is moderate, and other nutritional ingredients are sufficient.It disclosure satisfy that the nutrition and physiological requirements of plant cell growth, especially suitable for
The tissue-culturing rapid propagation of xylophyta.
MS minimal mediums have higher inorganic salt concentration, can ensure the mineral nutrition needed for tissue growth, moreover it is possible to
Accelerate the growth of callus, be more stable ionic equilibrium solution, its nitrate content is high, the quantity and ratio of nutrient
Properly.Herein described 1/2MS minimal mediums are that MS minimal medium a great number of elements halves, other components unchangeds.With nothing
Machine salinity is relatively low, but can ensure the mineral nutrition needed for tissue growth, meets the nutrition and physiological requirements of plant cell, often
For Plant Tissue Breeding.
Herein described 6-BA is 6- benzyl aminoadenines, is a plant growth regulators, and main function is to promote
The formation of bud, can also evoked callus occur, promote cell division, promote the differentiation of undifferentiated tissue, promote in organism
The accumulation of substance, promotes lateral bud, prevents aging, is most common a kind of cell division in plant tissue and cell culture
Element.
The TDZ is a plant growth regulators, has very strong cytokine activity, can promote plant sprout
Regeneration and breeding, that breaks bud stops eye, and seed is promoted to sprout, promotes callus growth, delays plant senescence etc., can pass through
The growth and development process of plant is adjusted to the effect of other plant modifying agents and physiological activator, can be used as plant tissue
Culture.
Herein described NAA is methyl α-naphthyl acetate, is a plant growth regulators, makes when plant is using cuttage breeding
With, it can also be used to Plant Tissue Breeding can promote cell division with expanding, induced synthesis adventitious root.
Herein described CPPU is chloropyuril, is a kind of phenyl urea derivatives of high activity, is a kind of complete new and effective
Plant growth regulator, have cytokine activity.There is the conjunction for promoting cell division and expansion, orga- nogenesis and protein
Into, improve photosynthetic efficiency, enhancing resistance, anti-aging, plant bud differentiation, flower and fruit protecting can be promoted, raising is beared fruit
Rate promotes the effects that Fruit.
Compared with prior art, detailed description are as follows by the application：Technical scheme provides a kind of five leaflet maples
Tissue culture and rapid propagation method, the step of selection and sterilizing, Initial culture, squamous subculture including explant, culture of rootage, by right
The screening of Initial culture base, proliferated culture medium, root media obtains best nutrient media components and proportioning, using aforementioned plant
Object growth regulator matches the culture medium to be formed, and formula is simple, and culture medium cost is low, coordinates the condition of culture in each stage, obtains
Seedling repoductive time it is short, cultivate simple flow, improve the efficiency of five leaflet maples breeding, growth coefficient up to 3.0~5.0,
Survival rate is high, can be quickly obtained five consistent leaflet maple seedlings of inhereditary feature, using tissue culture technique, carries out explant culture,
Can not be changed by seasonal climate, natural calamity is influenced, large-scale industrialization nursery and deep processing can be carried out.
Specific embodiment
In order to which those skilled in the art is made to more fully understand technical scheme of the present invention, with reference to specific embodiment pair
The present invention is described in further detail.
Embodiment 1
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
1000Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 10g/L sucrose, 6g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, incubation intensity of illumination
For 1500Lux, light application time 14h/d, cultivation temperature is 25 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 10g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, in incubation intensity of illumination be 1500Lux, illumination
Time is 14h/d, and cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums add NAA0.5mg/
L, 10g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 14 days, 32 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 21 days, 32 days after
It is commissioned to train at the end of supporting, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 15 days start to give birth to
Root, root growth is to 3~5cm, rooting rate 87% at 21 days.
Embodiment 2
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
1000Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 6g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, incubation intensity of illumination is
1500Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the proliferated culture medium composition includes：WPM minimal mediums,
0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 6g/L agar are supplemented, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, and incubation intensity of illumination is 1500Lux, during illumination
Between for 14h/d, cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L,
20g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 14 days, 28 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 18 days, 28 days after
It is commissioned to train to support and terminate, adventitious bud grows to 3~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 14 days start to take root,
Root growth is to 3~5cm, rooting rate 87% at 21 days.
Embodiment 3
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
1000Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the proliferated culture medium composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 40g/L sucrose, 6g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
For 1500Lux, light application time 14h/d, cultivation temperature is 25 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 40g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, and incubation intensity of illumination is 1500Lux, during illumination
Between for 14h/d, cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L,
40g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 16 days, 30 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 21 days, 30 days after
It is commissioned to train to support and terminate, adventitious bud grows to 3~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 16 days start to take root,
Root growth is to 3~5cm, rooting rate 80% at 25 days.
Embodiment 4
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
1000Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 4g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, incubation intensity of illumination is
1500Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the proliferated culture medium composition includes：WPM minimal mediums,
0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 4g/L agar are supplemented, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, in incubation intensity of illumination be 1500Lux, illumination
Time is 14h/d, and cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums add NAA0.5mg/
L, 20g/L sucrose, 4g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 21 days, 29 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 21 days, 32 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 14 days start to take root,
Root growth is to 3~5cm, rooting rate 87% at 22 days.
Embodiment 5
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
1000Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 7g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
It is 1500, light application time 14h/d, cultivation temperature is 25 DEG C, and the proliferated culture medium composition includes：WPM minimal mediums are mended
0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 7g/L agar are filled, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, and incubation intensity of illumination is 1500Lux, during illumination
Between for 14h/d, cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L,
20g/L sucrose, 7g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 21 days, 30 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 22 days, 33 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 14 days start to take root,
For root growth to 3~5cm, acclimatization and transplants rooting rate is 85% at 23 days.
Embodiment 6
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, incubation intensity of illumination is
800Lux, light application time 14h/d, cultivation temperature are 25 DEG C, and the Initial culture base composition includes：1/2MS minimal mediums,
Additional 0.1mg/L6-BA, 0.05mg/L TDZ 30g/L sucrose, 6g/L agar, pH is 5.5~6.5 before Initial culture base sterilizing,
Generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
For 1300Lux, light application time 14h/d, cultivation temperature is 25 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, and incubation intensity of illumination is 1300Lux, during illumination
Between for 14h/d, cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L,
20g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 23 days, 32 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 23 days, 35 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 13 days start to take root,
Root growth is to 3~5cm, rooting rate 87% at 24 days.
Embodiment 7
Take five leaflet maple edible tender branch, the alcohol oscillation treatment 30s that removal blade percentage by volume is 75%, sterile water
Cleaning 1 time, then with mercuric chloride 6~10min of oscillation treatment, sterile water wash 3~5 times, aseptic filter paper sucks moisture, cut into 1~
The stem with bud of 2cm obtains the explant.
The obtained explant is inoculated into the culture bottle equipped with Initial culture base and carries out Initial culture, is cultivated
Process intensity of illumination is 1200Lux, and light application time 14h/d, cultivation temperature is 25 DEG C, and the Initial culture base composition includes：
1/2MS minimal mediums, add 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 6g/L agar, and Initial culture base is gone out
PH is 5.5~6.5 before bacterium, generates axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
For 1800Lux, light application time 14h/d, cultivation temperature is 25 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, in incubation intensity of illumination be 1800Lux, illumination
Time is 14h/d, and cultivation temperature is 25 DEG C, and the root media composition includes：WPM minimal mediums add NAA0.5mg/
L, 20g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 21 days, 29 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 21 days, 30 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 11 days start to take root,
Root growth is to 3~5cm, rooting rate 85% at 24 days.
Embodiment 8
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, intensity of illumination is in incubation
1000Lux, light application time 14h/d, cultivation temperature are 23 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 6g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
For 1500Lux, light application time 14h/d, cultivation temperature is 23 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, in incubation intensity of illumination be 1500Lux, illumination
Time is 14h/d, and cultivation temperature is 23 DEG C, and the root media composition includes：WPM minimal mediums add NAA0.5mg/
L, 20g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 21 days, 32 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 23 days, 33 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 16 days start to take root,
Root growth is to 3~5cm, rooting rate 85% when 26.
Implement 9
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The obtained explant is inoculated into the Initial culture base and is cultivated, intensity of illumination is in incubation
1000Lux, light application time 14h/d, cultivation temperature are 27 DEG C, and the Initial culture base composition includes：1/2MS is cultivated substantially
Base adds 0.1mg/L6-BA, 0.05mg/L TDZ, 30g/L sucrose, 6g/L agar, before Initial culture base sterilizing pH for 5.5~
6.5, generate axillary bud；
The obtained axillary bud is inoculated into proliferated culture medium and carries out squamous subculture, intensity of illumination in incubation
For 1500Lux, light application time 14h/d, cultivation temperature is 27 DEG C, and the proliferated culture medium composition includes：WPM is cultivated substantially
Base, supplement 0.1mg/L 6-BA, 0.5mg/L CPPU, 30g/L sucrose, 6g/L agar, generates adventitious bud.
Adventitious bud, which is inoculated into root media, carries out culture of rootage, and incubation intensity of illumination is 1500Lux, during illumination
Between for 14h/d, cultivation temperature is 27 DEG C, and the root media composition includes：WPM minimal mediums, additional NAA0.5mg/L,
20g/L sucrose, 6g/L agar obtain rooted seedling, carry out acclimatization and transplants.
In the present embodiment, during Initial culture, Initial culture knot when the time of first axillary buds formation is 21 days, 29 days
Beam, axillary bud growth to 2.5~3.5cm；In Subculture, when the time of first adventitious bud generation is 20 days, 30 days after
It is commissioned to train to support and terminate, adventitious bud grows to 2~5cm, and growth coefficient is 3.0~5.0；In process of rooting culture, 15 days start to take root,
Root growth is to 3~5cm, rooting rate 87% at 24 days.
Embodiment 10
The influence of plant growth regulator ingredient and concentration to five leaflet maple axillary bud growths in Initial culture base
It takes five leaflet maple edible tender branch, removes blade, it is sterile with percentage by volume is 75% alcohol oscillation treatment 30s
Water cleans 1 time, then with mercuric chloride 6~10min of oscillation treatment, and sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into 1
The stem with bud of~2cm obtains the explant.
The explant for taking growing state consistent is several, is inoculated into Initial culture base after sterilizing is dried, incubation
Middle intensity of illumination is 1000Lux, and light application time is that 14h/d cultivation temperatures are 23~27 DEG C.Wherein Initial culture base uses 1/2MS
Minimal medium adds 6-BA, NAA, sucrose, agar.In 1/2MS minimal mediums, additional sucrose, the item of agar all same
It under part, is grouped according to the ingredient of plant growth regulator and concentration, observes and records 4~5 weeks wild Oryza species China and foreign countries of inoculation
The culture situation of implant, specific grouping and cultivation results are shown in Table 1.
Plant growth regulator ingredient and concentration are to the influence result of five leaflet maple axillary bud growths in 1 Initial culture base of table
As can be seen from the above table, 6-BA and TDZ uses differentiation rate during be applied alone any than the two generally high simultaneously, average
Axillary bud length is also longer；6-BA is 0.01mg/L, and when TDZ is 0.05mg/L, differentiation rate highest is being most suitable for for Initial culture base
Plant growth regulator composition condition.
Embodiment 11
The influence that plant growth regulator ingredient and concentration are proliferated five leaflet maples in proliferated culture medium
Consistent several by the axillary bud of Initial culture of growing state are taken, is inoculated on proliferated culture medium and cultivates 4~5 weeks,
Intensity of illumination is 1500Lux in incubation, and light application time 14h/d, cultivation temperature is 23~27 DEG C.Wherein proliferated culture medium
Using WPM minimal mediums, 6-BA, CPPU, sucrose, agar are added；In WPM minimal mediums, additional sucrose, agar is homogeneous
With under conditions of, it is grouped according to the ingredient of plant growth regulator and concentration, observes and records the training of axillary bud in culture medium
The situation of supporting, specific grouping and cultivation results are shown in Table 2.
The influence result of plant growth regulator ingredient and concentration to proliferation in 2 proliferated culture medium of table
As can be seen from the above table, 6-BA, CPPU use proliferation rate during exclusive use more any than the two is high to obtain simultaneously
It is more, average plant height also higher；The two uses simultaneously, and 6-BA 0.1mg/L, when CPPU is 0.5mg/L, proliferation rate highest is
The most suitable plant growth regulator composition condition of proliferated culture medium.
Embodiment 12
Influence of the plant growth regulator concentration to taking root in root media
The single plant for the adventitious bud by Multiplying culture for taking growing state consistent is several, is inoculated on root media and cultivates
3~5 weeks, intensity of illumination was 1500Lux in incubation, and light application time 14h/d, cultivation temperature is 23~27 DEG C.It is wherein raw
Root culture medium uses WPM minimal mediums, adds NAA, sucrose, agar.In WPM minimal mediums, additional sucrose, agar is equal
Under the same conditions, it is grouped according to the concentration of plant growth regulator, observes and records the culture of adventitious bud in culture medium
Situation, and the rooted seedling that adventitious bud rooting culture is obtained carries out acclimatization and transplants.Record rooting rate, specific grouping, cultivation results
3 are shown in Table with rooting rate.
Plant growth regulator ingredient and concentration are to the influence result taken root in 3 root media of table
| Grouping | NAA(mg/L) | Inoculation number (a) | It takes root and counts (a) | It averagely takes root and counts (item/strain) | Average root long (cm) | Rooting rate (%) |
| 1 | 0.2 | 30 | 24 | 3 | 3 | 80 |
| 2 | 0.5 | 30 | 26 | 5 | 5 | 87 |
| 3 | 0.7 | 30 | 24 | 4 | 4 | 80 |
| 4 | 1 | 30 | 25 | 3 | 3 | 83 |
As can be seen from the above table, it during a concentration of 0.2~1mg/L of NAA, can effectively take root, during a concentration of 0.5mg/L of NAA,
Rooting rate reaches 87%, and growing way is preferable, and several and average root long of averagely taking root is higher, is the most suitable plant of root media
Object growth regulating agent concentration.
It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
The limitation of the present invention, protection scope of the present invention should be subject to claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
Protection scope of the present invention is also should be regarded as into retouching.
Claims (10)
1. a kind of tissue culture and rapid propagation method of five leaflets maple, which is characterized in that include the following steps：
(1) explant is chosen and sterilizes：Five leaflet maple branches is taken to clean, sterilizing cuts stem with bud and obtains explant；
(2) Initial culture：The explant that the step (1) obtains is inoculated into the culture bottle equipped with Initial culture base and is carried out just
It is commissioned to train foster, axillary buds formation, Initial culture base composition includes：1/2MS minimal mediums, additional 0.05~0.5mg/L 6-BA,
0.02~0.2mg/L TDZ, 10~40g/L sucrose, 4~7g/L agar；
(3) squamous subculture：The axillary bud that the step (2) obtains is inoculated into proliferated culture medium and carries out squamous subculture, adventitious bud
Generation, the proliferated culture medium composition include：WPM minimal mediums add 0.05~0.5mg/L 6-BA, 0.2~1.0mg/L
CPPU, 10~40g/L sucrose, 4~7g/L；
(4) culture of rootage：The adventitious bud that the step (3) obtains is inoculated into root media and carries out culture of rootage, is obtained
Rooted seedling, root media composition include：WPM minimal mediums add 0.2~1.0mg/L NAA, 10~40g/L sucrose, 4
~7g/L.
2. the tissue culture and rapid propagation method of a kind of five leaflets maple according to claim 1, which is characterized in that the explant is chosen
And sterilization process is specific：It takes five leaflet maple edible tender branch, removes blade, with alcohol oscillation treatment 30s, sterile water wash 1 time, then
With mercuric chloride 6~10min of oscillation treatment, sterile water wash 3~5 times, aseptic filter paper sucks moisture, cuts into the stem segment with bud of 1~2cm
Section obtains the explant.
A kind of 3. tissue culture and rapid propagation method of five leaflets maple according to claim 1, which is characterized in that the Initial culture mistake
Journey intensity of illumination is 800~1200Lux, and light application time 14h/d., cultivation temperature is 23~27 DEG C.
A kind of 4. tissue culture and rapid propagation method of five leaflets maple according to claim 1, which is characterized in that the squamous subculture mistake
Journey and the process of rooting culture intensity of illumination are 1300~1800Lux, and light application time 14h/d., cultivation temperature is 23~27
℃。
5. the tissue culture and rapid propagation method of a kind of five leaflets maple according to claim 1, which is characterized in that during the Initial culture
Between be 4~5 weeks.
6. the tissue culture and rapid propagation method of a kind of five leaflets maple according to claim 1, which is characterized in that during the squamous subculture
Between be 4~5 weeks.
7. the tissue culture and rapid propagation method of a kind of five leaflets maple according to claim 1, which is characterized in that during the culture of rootage
Between be 3~5 weeks.
A kind of 8. tissue culture and rapid propagation method of five leaflets maple according to claim 1, which is characterized in that the Initial culture base
Composition includes：1/2MS minimal mediums add 0.05~0.2mg/L 6-BA, 0.02~0.1mg/L TDZ, 10~30g/L sugarcanes
Sugar, 4~6g/L agar.
A kind of 9. tissue culture and rapid propagation method of five leaflets maple according to claim 1, which is characterized in that the proliferated culture medium
Composition includes：WPM minimal mediums add 0.05~0.2mg/L 6-BA, 0.2~1.0mg/L CPPU, 10~30g/L sugarcanes
Sugar, 4~6g/L agar.
A kind of 10. tissue culture and rapid propagation method of five leaflets maple according to claim 1, which is characterized in that the culture of rootage
Base composition includes：WPM minimal mediums add 0.2~1.0mg/L NAA, 10~30g/L sucrose, 4~6g/L agar.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020107888A1 (en) * | 2018-11-30 | 2020-06-04 | 四川七彩林科股份有限公司 | Ex vitro rooting method for acer pentaphyllum tissue culture |
| WO2020107875A1 (en) * | 2018-11-30 | 2020-06-04 | 四川七彩林科股份有限公司 | Acer pentaphyllum cutting propagation method |
| CN114711143A (en) * | 2022-04-21 | 2022-07-08 | 东北林业大学 | Method for asexually and rapidly propagating acer truncatum seedlings through tissue culture |
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| CN104509439A (en) * | 2014-12-19 | 2015-04-15 | 湖南师范大学 | Method suitable for tissue rapid cultivation of Acer rubrum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020107888A1 (en) * | 2018-11-30 | 2020-06-04 | 四川七彩林科股份有限公司 | Ex vitro rooting method for acer pentaphyllum tissue culture |
| WO2020107875A1 (en) * | 2018-11-30 | 2020-06-04 | 四川七彩林科股份有限公司 | Acer pentaphyllum cutting propagation method |
| CN114711143A (en) * | 2022-04-21 | 2022-07-08 | 东北林业大学 | Method for asexually and rapidly propagating acer truncatum seedlings through tissue culture |
| CN114711143B (en) * | 2022-04-21 | 2023-08-08 | 东北林业大学 | Method for asexually and rapidly propagating Acer ginnala Maxim seedlings through tissue culture |
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Address after: 636600 Tissue Culture Workshop Building of Rare and Colorful Leaf Plant Varieties in Guangwu Mountain, Sanshe, Changtan Village, Zhengzheng Town, Nanjiang County, Bazhong City, Sichuan Province Applicant after: Sichuan Qicai Forestry Co.,Ltd. Address before: 636611 six Huai Shu Village, Dongyu Town, Nanjiang County, Bazhong, Sichuan Applicant before: SICHUAN COLORFUL FORESTRY DEVELOPMENT CO.,LTD. |
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Application publication date: 20180612 |