CN104472354B - Method for promoting intermediate propagation of craibiodendron yunnanense - Google Patents
Method for promoting intermediate propagation of craibiodendron yunnanense Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention provides a method for promoting intermediate propagation of craibiodendron yunnanense. The method comprises the following steps: 1) performing induction culture; 2) performing subculture; 3) performing rooting culture and 4) hardening and transplanting seedlings. The tissue culture method provided by the invention is simple and convenient, simple in step and convenient to operate, obtained craibiodendron yunnanense seedlings are strong in plant, high in growing ability and applicable to large-scale production, meanwhile the culture time is effectively shortened, and the production cost is lowered.
Description
Technical field
The present invention relates to medicinal plants field of planting, more particularly to a kind of promotion Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method.
Background technology
Yunnan Folium Craibiodendri Yunnanensiss (Craibiodendron yunnanense W.W.Smith), Photiniaserrulata section plant Yunnan Folium Craibiodendri Yunnanensiss,
Evergreen dungarunga, high 3--8 rice, sprig is without hair.Leaf alternate, heavy leather matter are oval, long 6-10 (- 13) centimetre, wide 3.5-4.5
(-- 6) centimetre, tip blunt circle or notch, base portion is blunt or subcircular, and full edge, slightly warp, two sides are dredged by black glandule without hair, the back side
Point, middle arteries are fallen under the surface, are overleaf swelled, lateral vein 14-18 pair, parallel, on surface substantially, are overleaf swelled；Petiole tubbiness,
It is about 5 millimeters.Panicle basidixed, inflorescence axial length 15 (-- 20) centimetre, by Lycoperdon polymorphum Vitt microtriche；Floral white, has fragrance；Calyx lobes
Base portion symphysis, sliver width egg shape, has pubescence；Corolla is bell, long 3-4 millimeters, have hair, 5 it is shallow split, sliver is upright；10 pieces of stamen, it is long
It is several equal to corolla, filigree tool thin hair, middle bent；Ovary is by hair.Capsule oblate spheroid, diameter of up to 12 millimeters.The month at florescence 7-10, fruit
April in October phase to next year.Yunnan Yunnan Folium Craibiodendri Yunnanensiss are puckery, pungent, temperature.There are severe toxicity, relieving superficies and warmign meridians, activating collaterals to relieve pain.Damage for traumatic injury
Wound, rheumatic numbness, myalgia, arthralgia, neurodermatitiss, affection of exogenous wind-cold.
Yunnan Folium Craibiodendri Yunnanensiss medicinal part is ripe leaf, learns according to field investigation, and Yunnan Folium Craibiodendri Yunnanensiss are shrub to dungarunga,
Tailo is typically distributed about, because mature seed quantitative proportion is low, structure is more special, natural propagation is difficult, the update cycle is longer,
In addition as demographic and economic pressure increases, the place of production is generally opened up wasteland and cultivates slope, and establishment of economic forest and stand conversion etc., Yunnan gold
The primary habitat of leaf is greatly destroyed, and wild stockss recover fragmentation and marginalisation is very serious, are hardly visible in the wild
Piece is distributed, and is hardly formed big biome, and the situation not yet causes local government's enough attention, therefore carries out biology to which
Properties study and introducing and planting and breeding research are necessary.
The present invention utilizes tissue culture technique, not by extraneous effect of natural conditions, using stipes expanding propagation, produces substantial amounts of kind
Seedling, expands seeling industry on a large scale, meets the demand of plantation.Solve the problems, such as resource scarcity.
The content of the invention
In order to solve Folium Craibiodendri Yunnanensiss growth is slow in Yunnan present in background technology, subculture can repeatedly make a variation, vitrification, germplasm decline
The problems such as moving back, the invention provides a kind of promote Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method, its technical scheme is as follows：
1) sprout-induction：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, concentration are placed in for 75-
80% alcohol solution dipping 30-40 seconds, sterilize 15-20 minutes in being subsequently placed in the mercuric chloride solution that concentration is 1-1.2%, it is aseptic
Water clean 5-6 time, cut marginal portion using scalpel, be inoculated in inducing culture, wherein inducing culture be according to
Under material rate be prepared from N68 for minimal medium additional isopentenyl gland purine (2-ip) 0.1-2.0mg/L, naphthalene acetic acid
(NAA) 0.1-1.0mg/L, gibberellins (GA) 0.5-3.0mg/L, sugared 25-30g/L, Fructus Musae 20-30%, Sucus Cocoiss 20-30%,
Mashed potatoes 20-30%, tea polyphenols 15-20%, agar 3-6g/L, pH value are 5.8, and cultivation temperature is 29-32 DEG C, and intensity of illumination is
2100-2500lux, light application time are 14-16 hours/day, obtain Yunnan Folium Craibiodendri Yunnanensiss budlet after cultivating 16-20 days；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains put in subculture medium, wherein subculture
Culture medium is prepared from according to following material rate, the additional isopentenyl gland purine (2- of 1/2MS minimal mediums
Ip) 3.0-5.0mg/L, 2,4 dichlorphenoxyacetic acid 1.0mg/L, naphthalene acetic acid (NAA) 0.2-1.0mg/L, sugared 30g/L, agar 3-
6g/L, Fructus Musae 20-30%, Sucus Cocoiss 20-30%, mashed potatoes 20-30%, tea polyphenols 15-20%, activated carbon 0.2%, pH value is
5.8, cultivation temperature is 29-32 DEG C, and incubation time is 14-16 hours/day, and intensity of illumination is 2100-2500lux, and 62-70 days left
The right side can obtain the Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation Seedling of 2cm or so height；
3) root culture of Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation：By step 2) the middle substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation Seedling for obtaining
Root media is transplanted to, wherein root media is prepared from according to following material rate, and 1/2MS is minimal medium
2-4%, auxin IBA 0.1-2.0%, sugared 2-4%, Fructus Musae 20-30%, Sucus Cocoiss 20-30%, mashed potatoes 20-30%, tea
Polyphenol 15-20%, activated carbon 0.2-0.4%, PH5.8 cultivation temperature are 28 DEG C, intensity of illumination 2100-2500lux, light application time
After cultivating 62-70 days under conditions of 14-16 hours/day, tissue cultured seedling is moved on to and cultivate under natural light, be obtained after 25-28 days
Yunnan Folium Craibiodendri Yunnanensiss with 3-4 bar roots are taken root tissue cultured seedling；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the cloud after 1-2 month
Southern Folium Craibiodendri Yunnanensiss seedling growth is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation,
Intensity of illumination is 3200-3500lux, and, in 50-60%, temperature control is at 20-25 DEG C for humid control.
Preferably, described promotion Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method, it is characterised in that step 1) in, wherein
Culture medium is：N68For minimal medium additional isopentenyl gland purine (2-ip) 0.1-2.0mg/L, naphthalene acetic acid (NAA) 0.1-
1.0mg/L, gibberellins (GA) 0.5-3.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 20-25g, Sucus Cocoiss 20-25g, Rhizoma Solani tuber osi
Mud 20-25g, tea polyphenols 18-20g.
Preferably, described promotion Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method, it is characterised in that step 2) in, wherein
Culture medium is：1/2MS minimal mediums additional isopentenyl gland purine (2-ip) 3.0-5.0mg/L, naphthalene acetic acid (NAA) 0.2-
1.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 25-30%, Sucus Cocoiss 25-30%, mashed potatoes 25-30%, tea polyphenols 15-
20%, activated carbon 0.2%.
Preferably, described promotion Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method, it is characterised in that step 3) in, wherein
Root media is：1/2MS is minimal medium, additional activity charcoal 0.4%, IBA 0.8, sugar 2%, Fructus Musae 25-30%, Cortex cocois radiciss
Juice 25-30%, mashed potatoes 25-30%, tea polyphenols 15-20%, activated carbon 0.2%.
Beneficial effects of the present invention：
1st, this method can send out the tissue cultured seedling for breeding a large amount of high-quality soon, it is to avoid because constantly repeating subculture causes tissue cultured seedling
Variation, the later stage, tissue cultured seedling robust growth, vitrification variation were reduced after nature optical culture.Simultaneously, there is provided have land for growing field crops in bottle
The similar environment of environment, improves tissue cultured seedling survival rate during the seedling exercising in later stage.The Seedling for producing high-quality meets production need
Will
2nd, growth cycle is short, and the incubation time of a subculture reduced subculture number 40-60 days time, must save significantly
About cultivation time of seedling, reach the effect for promoting seedling early growth.
3rd, reproduction speed is fast, and the strict control of breeding condition, such as illumination, temperature and cultivation time is effective must to promote seedling
Fast-growth, the seedling that is allocated to of excellent culture medium provide the required carbon source of growth, nitrogen source, inorganic salt, auxin, promote
While seedling quickly grows, germination percentage, rooting rate and seedling exercising survival rate are greatly improved.
4th, culture medium is preferred, adds different Organic substances and tea polyphenols again from variable concentrations hormone with minimal medium
Constitute in existing used culture medium, wherein the tea polyphenols for adding act on somatic cells, can progressive failure its cell wall it is complete
Whole property so that alkali phosphatase oozes out, strengthens membrane passage then, causes the seepage of metal ion, protein to make
In cell generation, gets muddled, and gradually destroys cellularity, and so as to play antibacterial effect, Sucus Cocoiss, mashed potatoes, the addition of Fructus Musae are drawn
Meet thing with natural, promote calluss, promote seedling to grow up strong and sturdy.On the basis of repeatedly attempting, according to certain principle
What is screened has induction very well to Yunnan Folium Craibiodendri Yunnanensiss, can be in the case of the good characteristic for keeping former plant, and do not make a variation situation
Under, appreciation rate is high, grows fast excellent culture medium.
Specific embodiment
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, in stream underwater cleaning
Totally, soaked 30 seconds using 75% ethanol, concentration is that 1% mercuric chloride is sterilized 15 minutes, sterile water wash 5 times, using scalpel
Marginal portion is cut, is inoculated in inducing culture, cultivation temperature is 29 DEG C, and intensity of illumination is 2100lux, and light application time is 14
Hour/day, after cultivating 16 days, visible Yunnan Folium Craibiodendri Yunnanensiss budlet sends, and wherein culture medium is：N68 is the additional isoamyl of minimal medium
Thiazolinyl adenine (2-ip) 0.3mg/L, naphthalene acetic acid (NAA) 0.02mg/L, gibberellins (GA) 2.0mg/L, sugared 30g/L, agar
4.0g/L, Fructus Musae 25g, mashed potatoes 25g, Sucus Cocoiss 25g, tea polyphenols 15g, pH value are 5.8；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains be placed in 1/2MS for minimal medium it is attached
Plus isopentenyl gland purine (2-ip) 0.5mg/L, naphthalene acetic acid (NAA) 2.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 25g,
Mashed potatoes 25g, Sucus Cocoiss 25g, tea polyphenols 15g, pH value be 5.8 culture medium in cultivate, cultivation temperature be 29 DEG C, incubation time
For 14 hours/day, intensity of illumination is 2100lux, can obtain within 44 days or so the neat Yunnan Folium Craibiodendri Yunnanensiss tissue culture of 2cm or so height
Differentiation Seedling；
3) Yunnan Folium Craibiodendri Yunnanensiss differentiation seedling rooting culture, by step 2) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings that obtains arrives
Root media, cultivation temperature be 29 DEG C, intensity of illumination 2500lux, light application time be 14 hours/day under conditions of cultivate 40
After it, tissue cultured seedling is moved on to and cultivate under natural light, the Yunnan Folium Craibiodendri Yunnanensiss with 4 roots can be obtained after 50 days and is taken root tissue cultured seedling, its
Middle root media is：1/2MS is minimal medium 4%, and Fructus Musae 20%, Rhizoma Solani tuber osi 20%, Sucus Cocoiss 20%, tea polyphenols 5% are raw
Long element IBA0.1%, sugar 2%, activated carbon 0.2%, pH value are 5.8；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan after 2 months
The growth of Folium Craibiodendri Yunnanensiss seedling is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, light
It is 3200lux according to intensity, 60%, temperature control is at 20 DEG C for humid control.
Embodiment 2
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, in stream underwater cleaning
Totally, soaked 40 seconds using 80% ethanol, concentration is that 1.2% mercuric chloride is sterilized 15 minutes, sterile water wash 6 times, using operation
Knife cuts marginal portion, is inoculated in inducing culture, and cultivation temperature is 30 DEG C, and intensity of illumination is 2200lux, and light application time is
16 hours/day, after cultivating 20 days, visible Yunnan Folium Craibiodendri Yunnanensiss budlet sends, and wherein culture medium adds different for minimal medium for N68
Pentenyl adenine (2-ip) 0.35mg/L, naphthalene acetic acid (NAA) 0.03mg/L, gibberellins (GA) 1.5mg/L, sugared 28g/L, Rhizoma Solani tuber osi
Mud 25g, Sucus Cocoiss 25g, tea polyphenols 15g, agar 3.8g/L, pH value are 5.8；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains be placed in 1/2MS for minimal medium it is attached
Plus isopentenyl gland purine (2-ip) 4.0mg/L, naphthalene acetic acid (NAA) 2.0mg/L, sugared 30g/L, agar 4.0g/L, mashed potatoes
25g, Sucus Cocoiss 25g, tea polyphenols 15g, pH value be 5.8 culture medium in cultivate, cultivation temperature is 30 DEG C, and incubation time is 16 little
When/day, intensity of illumination is 2200lux, can obtain within 70 days or so the neat Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation of 2cm or so height
Seedling；
3) Yunnan Folium Craibiodendri Yunnanensiss differentiation seedling rooting culture, by step 2) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings that obtains arrives
Root media, cultivation temperature be 30 DEG C, intensity of illumination 2500lux, light application time be 16 hours/day under conditions of cultivate 35
After it, tissue cultured seedling is moved on to and cultivate under natural light, the Yunnan Folium Craibiodendri Yunnanensiss with 4 roots can be obtained after 65 days and is taken root tissue cultured seedling, its
Middle root media is：1/2MS be minimal medium 4%, Rhizoma Solani tuber osi 20%g, Sucus Cocoiss 20%, tea polyphenols 5%g, auxin IBA
2.0%, sugar 2%, activated carbon 0.2%, pH value are 5.8；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan after 2 months
The growth of Folium Craibiodendri Yunnanensiss seedling is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, light
It is 3200lux according to intensity, 60%, temperature control is at 20 DEG C for humid control.
Embodiment 3
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, in stream underwater cleaning
Totally, soaked 35 seconds using 75% ethanol, concentration is that 1.2% mercuric chloride is sterilized 15 minutes, sterile water wash 6 times, using operation
Knife cuts marginal portion, is inoculated in inducing culture, and cultivation temperature is 32 DEG C, and intensity of illumination is 2300lux, and light application time is
16 hours/day, after cultivating 20 days, visible Yunnan Folium Craibiodendri Yunnanensiss budlet sends, and wherein culture medium is：N68 adds different for minimal medium
Pentenyl adenine (2-ip) 0.012：, naphthalene acetic acid (NAA) 0.015, gibberellins (GA) 1.0mg/L, sugared 28g/L, agar 3.8g/
L, Fructus Musae 20%, Sucus Cocoiss 20%, tea polyphenols 5%, pH value are 5.8；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains be placed in 1/2MS for minimal medium it is attached
Plus isopentenyl gland purine (2-ip) 4.0mg/L, naphthalene acetic acid (NAA) 2.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 20%,
Sucus Cocoiss 20%, tea polyphenols 5%, pH value be 5.8 culture medium in cultivate, cultivation temperature be 30 DEG C, incubation time be 16 hours/
My god, intensity of illumination is 2200lux, can obtain within 55 days or so the neat Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation Seedling of 2cm or so height；
3) Yunnan Folium Craibiodendri Yunnanensiss differentiation seedling rooting culture, by step 2) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings that obtains arrives
Root media, cultivation temperature be 30 DEG C, intensity of illumination 2500lux, light application time be 16 hours/day under conditions of cultivate 35
After it, tissue cultured seedling is moved on to and cultivate under natural light, the Yunnan Folium Craibiodendri Yunnanensiss with 4 roots can be obtained after 60 days and is taken root tissue cultured seedling, its
Middle root media is：1/2MS be minimal medium 4%, Fructus Musae 20%, Sucus Cocoiss 20%, tea polyphenols 5%, auxin IBA
2.0%, sugar 2%, activated carbon 0.2%, pH value is 5.8；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan after 2 months
The growth of Folium Craibiodendri Yunnanensiss seedling is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, light
It is 3200lux according to intensity, 60%, temperature control is at 20 DEG C for humid control.
Embodiment 4
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, in stream underwater cleaning
Totally, soaked 35 seconds using 75% ethanol, concentration is that 1.2% mercuric chloride is sterilized 15 minutes, sterile water wash 6 times, using operation
Knife cuts marginal portion, is inoculated in inducing culture, and cultivation temperature is 32 DEG C, and intensity of illumination is 2300lux, and light application time is
16 hours/day, after cultivating 20 days, visible Yunnan Folium Craibiodendri Yunnanensiss budlet sends, and wherein culture medium is：N68 adds different for minimal medium
Pentenyl adenine (2-ip)：Naphthalene acetic acid (NAA)：Gibberellins (GA) 1.5mg/L, sugared 28g/L, agar 3.Sg/L, Fructus Musae 25g,
Mashed potatoes 25g, tea polyphenols 15g, pH value are 5.8；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains be placed in 1/2MS for minimal medium it is attached
Plus isopentenyl gland purine (2-ip) 4.0mg/L, naphthalene acetic acid (NAA) 2.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 25g,
Mashed potatoes 25g, tea polyphenols 15g, pH value be 5.8 culture medium in cultivate, cultivation temperature be 30 DEG C, incubation time be 16 hours/
My god, intensity of illumination is 2200lux, can obtain within 60 days or so the neat Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation Seedling of 2cm or so height；
3) Yunnan Folium Craibiodendri Yunnanensiss differentiation seedling rooting culture, by step 2) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings that obtains arrives
Root media, cultivation temperature be 30 DEG C, intensity of illumination 2500lux, light application time be 16 hours/day under conditions of cultivate 35
After it, tissue cultured seedling is moved on to and cultivate under natural light, the Yunnan Folium Craibiodendri Yunnanensiss with 4 roots can be obtained after 45 days and is taken root tissue cultured seedling, its
Middle root media is：1/2MS be minimal medium 4%, Fructus Musae 20%, Rhizoma Solani tuber osi 20%, tea polyphenols 5%, auxin IBA
2.0%, sugar 2%, activated carbon 0.2%, pH value are 5.8；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan after 2 months
The growth of Folium Craibiodendri Yunnanensiss seedling is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, light
It is 3200lux according to intensity, 60%, temperature control is at 20 DEG C for humid control.
Embodiment 5
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, in stream underwater cleaning
Totally, soaked 35 seconds using 75% ethanol, concentration is that 1.2% mercuric chloride is sterilized 15 minutes, sterile water wash 6 times, using operation
Knife cuts marginal portion, is inoculated in inducing culture, and cultivation temperature is 32 DEG C, and intensity of illumination is 2300lux, and light application time is
16 hours/day, after cultivating 30 days, visible Yunnan Folium Craibiodendri Yunnanensiss budlet sends, and wherein culture medium is：N68 adds different for minimal medium
Pentenyl adenine (2-ip) 0.35, and naphthalene acetic acid (NAA, 0.3：Gibberellins (GA) 3mg/L, Fructus Musae 25g, mashed potatoes 25g, Cortex cocois radiciss
Juice 25g, sugared 28g/L, agar 3.8g/L, pH value are 5.8；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains be placed in 1/2MS for minimal medium it is attached
Plus isopentenyl gland purine (2-ip) 4.0mg/L, naphthalene acetic acid (NAA) 2.0mg/L, sugared 30g/L, agar 4.0g/L, Fructus Musae 25g,
Mashed potatoes 25g, Sucus Cocoiss 25g, pH value be 5.8 culture medium in cultivate, cultivation temperature be 30 DEG C, incubation time be 16 hours/
My god, intensity of illumination is 2200lux, can obtain within 70 days or so the neat Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation Seedling of 2cm or so height；
3) Yunnan Folium Craibiodendri Yunnanensiss differentiation seedling rooting culture, by step 2) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings that obtains arrives
Root media, cultivation temperature are 30 DEG C, intensity of illumination 2500lux, and light application time is culture 65 days under conditions of 16 hours/day
Afterwards, tissue cultured seedling is moved on to and cultivate under natural light, the Yunnan Folium Craibiodendri Yunnanensiss with 4 roots can be obtained after 75 days and is taken root tissue cultured seedling, wherein
Root media is：1/2MS be minimal medium 4%, auxin IBA 2.0%, sugar 2%, activated carbon 0.2%, Fructus Musae 20%,
Rhizoma Solani tuber osi 20%, Sucus Cocoiss 20%, pH value are 5.8；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry in the air
Solid carbon dioxide vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan after 2 months
The growth of Folium Craibiodendri Yunnanensiss seedling is stable, grows new root and bud can be transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, light
It is 3200lux according to intensity, 60%, temperature control is at 20 DEG C for humid control.
The tissue culture method test data contrast of embodiment 1- embodiment 5
| Sequence number | Seedling-growing time/day | Germination percentage | Subculture planting percent | Rooting rate | Seedling exercising survival rate |
| Embodiment 1 | 210 | 96% | 94% | 97% | 98% |
| Embodiment 2 | 250 | 85% | 80% | 80% | 83% |
| Embodiment 3 | 230 | 89% | 82% | 84% | 86% |
| Embodiment 4 | 220 | 90% | 85% | 86% | 90% |
| Embodiment 5 | 300 | 70% | 72% | 71% | 70% |
Although embodiment of the present invention is disclosed as above, which is not restricted to listed by description and embodiment
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Other modification is realized, therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the embodiment with description.
Claims (1)
1. it is a kind of to promote Yunnan Folium Craibiodendri Yunnanensiss rapid propagation method, it is characterised in that to comprise the following steps：
1) inducing culture：Terminal bud or axillary bud are taken in the gold leaf blastostyle of Yunnan to clean up under flowing water, concentration are placed in for 75-80%
The alcohol solution dipping 30-40 seconds, sterilize 15-20 minutes in being subsequently placed in the mercuric chloride solution that concentration is 1-1.2%, sterilized water is clear
Washing 5-6 time, marginal portion being cut using scalpel, be inoculated in inducing culture, wherein inducing culture is according to following
Material rate is prepared from, N68 be minimal medium additional isopentenyl gland purine (2-ip) 0.1-2.0mg/L, naphthalene acetic acid
(NAA) 0.1-1.0mg/L, gibberellins (GA) 0.5-3.0mg/L, sugared 25-30g/L, Fructus Musae 20-30%, Sucus Cocoiss 20-30%,
Mashed potatoes 20-30%, tea polyphenols 15-20%, agar 3-6g/L, pH value are 5.8, and cultivation temperature is 29-32 DEG C, and intensity of illumination is
2100-2500lux, light application time are 14-16 hours/day, obtain Yunnan Folium Craibiodendri Yunnanensiss budlet after cultivating 16-20 days；
2) successive transfer culture：By step 1) the substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss budlet that obtains put in subculture medium, wherein successive transfer culture
Base is prepared from according to following material rate, 1/2MS minimal mediums additional isopentenyl gland purine (2-ip) 3.0-
5.0mg/L, 2,4 dichlorphenoxyacetic acid 1.0mg/L, naphthalene acetic acid (NAA) 0.2-1.0mg/L, sugared 30g/L, agar 3-6g/L, Fructus Musae
20-30%, Sucus Cocoiss 20-30%, mashed potatoes 20-30%, tea polyphenols 15-20%, activated carbon 0.2%, pH value are 5.8, culture
Temperature is 29-32 DEG C, and incubation time is 14-16 hours/day, and intensity of illumination is 2100-2500lux, obtains 2cm within 62-70 days high
The Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation Seedling of degree；
3) root culture of Yunnan Folium Craibiodendri Yunnanensiss tissue culture differentiation：By step 2) the middle substantial amounts of Yunnan Folium Craibiodendri Yunnanensiss differentiation transplantation of seedlings for obtaining
To root media, wherein root media is prepared from according to following material rate, and 1/2MS is minimal medium, raw
Long element IBA 0.1-2.0%, sugared 2-4%, Fructus Musae 20-30%, Sucus Cocoiss 20-30%, mashed potatoes 20-30%, tea polyphenols 15-
20%, activated carbon 0.2-0.4%, pH5.8 cultivation temperature are 28 DEG C, and intensity of illumination 2100-2500lux, light application time are 14-16
After cultivating 62-70 days under conditions of hour/day, tissue cultured seedling is moved on to and cultivate under natural light, obtained after 25-28 days with 3-4 bars
The Yunnan Folium Craibiodendri Yunnanensiss of root are taken root tissue cultured seedling；
4) seedling exercising and transplanting：Wash step 3) in the Yunnan Folium Craibiodendri Yunnanensiss that obtain take root the culture medium of tissue cultured seedling root, dry water
Vapour, Folium Craibiodendri Yunnanensiss the take root whole plant of tissue cultured seedling band root in Yunnan is transplanted on the seedbed of carrying substrates, the Yunnan gold after 1-2 month
The growth of leaf seedling is stable, grows new root and bud is just transplanted in planting greenhouse, suitably will shelter from heat or light during greenhouse cultivation, and illumination is strong
Spend for 3200-3500lux, in 50-60%, temperature control is at 20-25 DEG C for humid control.
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