CN106688885A - Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation - Google Patents
Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation Download PDFInfo
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- CN106688885A CN106688885A CN201611032440.0A CN201611032440A CN106688885A CN 106688885 A CN106688885 A CN 106688885A CN 201611032440 A CN201611032440 A CN 201611032440A CN 106688885 A CN106688885 A CN 106688885A
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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Abstract
The invention discloses a method for quickly obtaining masson pine vegetative propagation seedlings for afforestation. The method comprises the following steps of (1) germinating the seeds treated by ultrasonic waves on a culture medium, wherein the culture medium is prepared from cotton, konjaku flour, saccharose and the like; (2) using a top shoot as an explant, and culturing to form a cluster shoot on an MS (Murashige and Skoog) culture medium containing 6-BA, NAA and potato; (3) amplifying the cluster shoot in a culture medium containing fluorescent powder, so as to obtain a subculture shoot; (4) cutting the subculture shoot, and promoting to root by a fluorescent radiation method, so as to obtain the masson pine vegetative propagation seedling. The method has the advantages that the seed germinating time is shortened, the robust sterile seedling can be obtained after 8 to 18d, the germinating rate is 30 to 45%, the cluster shoot is subcultured for 5 generations, and the average amplification coefficient is 7.2; after the subculture shoot is rooted and cultured for 20 to 24d, the rooting rate is 95% or above, and a large amount of robust roots can be cultured; the good economic benefit, social benefit and ecological benefit can be obtained.
Description
Technical field
The present invention relates to a kind of sprouting of seed and the method field of nursery, it is more particularly related to a kind of quick
The method for obtaining masson pine vegetative propagation afforestation nursery.
Background technology
Masson pine is one of most important green barren hill reproducting tree species of south China, and in China, distribution is extremely wide.
Its economic worth is high, and purposes is wide, is the important use material in industrial and agricultural production, not only can be used to produce three plates, papermaking and chemical fibre work
Industry is manufactured, and may further be used to the raw materials of industry such as production rosin, turpentine oil.Timber is extremely water-fast wet, has saying for " pine in thousand in water ", especially
Suitable for Underwater Engineering.Masson pine studied since the eighties in last century, and by the development of decades, seed selection is substantial amounts of excellent
Clone, and by setting up masson pine Primary seed orchard, produce a large amount of masson pine breedings.But the completion building well time wants 2~4
Year, Forest field management wants 2 years, high cost.The plantation and method for passing through grafting is directly gone up a hill, can reduce to a certain degree
Cost, but survival rate is low, up to more than 50%, very big difficulty is brought to building well.
The vegetative propagation difficulty of masson pine is larger, and wherein tissue cultures are more difficult.But because the tissue cultures of plant have
Have the advantages that breeding coefficient is big, seedling growth is neat, capture its tissue culture technique significant.Being adopted traditional sterile culture more
With agar powder (bar) culture medium, it is necessary to condense three steps by agar powder (bar) heating for dissolving, sterilization and cooling, operation is more
It is cumbersome；In addition, due to there are the various problems such as percentage of seedgermination and pollution in sterile culture, the planting percent of aseptic seedling 20% with
Under, more than 80% culture medium is difficult to recycling as discarded object and is caused to waste.
Masson pine is that tissue cultures are taken root more difficult seeds.Taken root rooting rate with the masson pine Multiple Buds of Initial culture
It is ideal, but after squamous subculture, its rooting rate is decreased obviously.The multitudinous grade (2010) of Wu little Qin etc. (2009) and season hole will pass through
Simple bud is directly taken root after the Initial culture of clump bud induction and elongation, and its rooting rate is respectively 85% and 92.5%；And Xue Ying
(2006) take root taking simple bud after squamous subculture, its rooting rate is only 26.7%.Realize masson pine tissue-cultured seedling factory
Metaplasia is produced, it is necessary to is broken through subculture bud and is taken root difficult bottleneck factor.
It is current major issue urgently to be resolved hurrily that how quick, low cost, high-survival rate ground obtain masson pine afforestation nursery.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of easy to operate, with low cost and survival rate quick obtaining horse hair high
The method of loose vegetative propagation afforestation nursery, for its green barren hill afforestation provides good technical foundation.
In order to realize these purposes of the invention and further advantage, there is provided a kind of quick obtaining masson pine is without numerous property
The method for growing afforestation nursery, comprises the following steps：
1) seed is sprouted
A, making cotton culture medium
Cotton is cleaned with clear water, is positioned over after wringing out in solution a and is soaked 30min, wherein the solution a by 1L water plus
Enter 7g konjaku flours and 25g sucrose is mixed, then soaked cotton is put into blake bottle, and blake bottle is placed in refrigerator
Middle frost 30min；Non-woven fabrics is cleaned again, and is soaked in water, after 10min by non-textile mulch in blake bottle cotton it is upper
Surface, tightens bottle cap, is put into sterilizing in autoclave, and sterilising conditions are 115 DEG C, and 30min is standby, wherein, set on the non-woven fabrics
The circular hole that aperture uniformly spaced apart from each other is 1cm is equipped with, two neighboring circular hole spacing is 0.4cm；
B, disinfect
Masson pine seed is placed in the copper-bath that mass fraction is 0.4% first, ultrasonication；Then successively
It is 75% alcohol drip washing 1min with mass fraction, mass fraction is 0.05%HgCl2Solution drip washing 10min；Finally with quality point
Number soaks 20min for 3%NaClO solution, and is rinsed well with clear water, standby；
During the masson pine seed that will be disinfected in culturing room is inoculated into cotton culture medium, the circular hole of each non-woven fabrics
1 seed of inoculation, carries out seed sprouting, obtains masson pine sterile bud Z；
2) terminal bud of the masson pine sterile bud Z that length is 4~6cm is cut, used as explant, being placed in culture medium b is carried out
Culture, obtains masson pine Multiple Buds；Wherein described culture medium b by every 1LMS culture mediums add 3mg 6-BA, 0.2mg NAA,
50g potatos and 20g sucrose are mixed；
3) terminal bud or tender stem of elongation to masson pine Multiple Buds 4~6cm long are cut, plant is carried out in culture medium c
After elongation culture, elongation bud is obtained；Cut the elongation bud of 4~6cm of length terminal bud or tender stem be inoculated into culture medium d in increased
Grow, obtain subculture bud；
Wherein described culture medium c by added in every 1LMS culture mediums 20mg fluorescent material, 0.5mg NAA, 3000mg sucrose and
7000mg agar is mixed；The culture medium d is by adding 20mg fluorescent material, 1mg 2,4-D, 1mg 6- in every 1LMS culture mediums
BA, 1mg KT, 0.3mg NAA, 3g sucrose and 7g agar are mixed；
4) the subculture bud of robust growth, 3~4cm high is cut, 25~35min is soaked with fluid nutrient medium e, liquid is used after taking-up
Body culture medium f soaks 15~25min, transplants in breeding device, fluorescence treatment with irradiation, obtains masson pine apogamic seedling；
Wherein described fluid nutrient medium e is mixed by adding 75mgIBA and 30mgNAA in every 1LDCR culture mediums；Liquid
Culture medium f is mixed by adding 0.4mgABT6 and 5g talcum powder in every 1LGD culture mediums；
Wherein described fluorescence irradiation is specially：1. fluorescent material is fitted into transparent plastic bag, fills whole polybag, and seal
Mouthful；Wherein polybag is 5~10cm long, the rectangle structure of 2~4cm wide；2. polybag is pasted on the outside of breeding device, plastics
Bag lower edge is flushed with matrix of taking root top surface edge in breeding device；
5) nursery is transplanted：Well-grown masson pine apogamic seedling is transplanted to nursery.
Preferably, the method for the quick obtaining masson pine vegetative propagation afforestation nursery, solution a described in step (1)
Collocation method：Sucrose is first uniformly dissolved with water first, adds konjaku flour, treat that konjaku powder water swelling is complete.
Preferably, the method for the quick obtaining masson pine vegetative propagation afforestation nursery, cotton described in step (1) is put down
The bottom of bottle of blake bottle is layered on, thickness is 1.8~3.0cm.
Preferably, the method for the quick obtaining masson pine vegetative propagation afforestation nursery, ultrasonic wave described in step (1)
The method for the treatment of is：It is first 40KHz ultrasonication 20min with frequency, stands 10min, reuse frequency is at 80KHz ultrasonic waves
Reason 20min, stands 10min, is finally 40KHz ultrasonications 20min with frequency.
Preferably, the method for the quick obtaining masson pine vegetative propagation afforestation nursery, culture medium described in step (3)
Fluorescent material in c and culture medium d is after other raw materials are well mixed, to be eventually adding fluorescent material.
Preferably, the method for the quick obtaining masson pine vegetative propagation afforestation nursery, breeding device described in step (4)
In be equipped with solid medium, the solid medium include river sand, vermiculite, liver moss and laterite with 5:2:1:2 ratio mixing is equal
It is loaded in breeding device after even；Drench molten into Ca (H2PO4) 2 that 100g mass fractions are 2% in the culture medium of the breeding device
Liquid and the urea liquid that 100g mass fractions are 1%.
The present invention at least includes following beneficial effect：
First, for the problem that current Aseptic seedling culture is existed using agar as culture medium, the present invention is good using having
Good water suction and the cotton of water retention property, auxiliary in strong absorptive, the konjaku flour of inflatable 80~100 times of volume after water suction is sharp
Its profile is fixed with refrigerator ice freeze techniques and non-woven fabrics, the masson pine seed after sterilization is trained in being put into cotton culture medium
The aseptic seedling that can obtain robust growth is supported, aseptic seedling planting percent is 30~45%, while experimental procedure can be simplified, improve culture
Efficiency, and cotton and the repeatable utilization of non-woven fabrics, it is possible to decrease cost and environmental protection.
Second, seed is put into 0.4% copper-bath, make copper-bath internal vibration using ultrasonic wave, promote
Enter the bactericidal action of its copper-bath, be both the dormancy that can release seed, promote the sprouting of seed, 8~18d can obtain life
Healthy and strong aseptic seedling long.
3rd, using potato idiotrophic matter dominated and 6-BA and NAA synergy promote evoked callus and
The formation of Multiple Buds.
4th, using the absorbable storage luminous energy of fluorescent material, and the slow performance for discharging dim light under dark condition, coordinate 2,
The synergy of 4-D, 6-BA, KT and NAA, makes the elongation of subculture bud more balanced, the generation of Multiple Buds squamous subculture 5, average proliferation
Coefficient 7.2.
5th, using IBA, NAA, ABT6 and talcum powder, the expression and elongation of subculture bud adventitious root are promoted, root system is more thick
It is strong, using the absorbable storage luminous energy of fluorescent material, and the slow performance for discharging dim light under dark condition, promote masson pine asexual numerous
Grow the growth of nursery, rooting rate is up to more than 95%.
Further advantage of the invention, target and feature embody part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.
It should be noted that experimental technique described in following embodiments, unless otherwise specified, is conventional method, it is described
Reagent and material, unless otherwise specified, commercially obtain.
Embodiment 1：
1) seed is sprouted
A, making cotton culture medium
Cotton is cleaned with clear water, is positioned over after wringing out in solution a and is soaked 30min, be then layered on soaked cotton
The bottom of bottle of blake bottle, thickness is 1.8cm, and blake bottle is placed in 30min is freezed in refrigerator；Non-woven fabrics is cleaned again, and is soaked
By the upper surface of non-textile mulch cotton in blake bottle after Yu Shuizhong, 10min, bottle cap is tightened, be put into sterilizing in autoclave, gone out
Bacterium condition is 115 DEG C, and 30min is standby；
Wherein, the circular hole that aperture uniformly spaced apart from each other is 1cm, two neighboring circular hole spacing are provided with the non-woven fabrics
It is 0.4cm；
The collocation method of wherein described solution a：Solution a is first by adding 7g konjaku flours and 25g sucrose to mix in 1L water
First sucrose is first uniformly dissolved with water, konjaku flour is added, treats that konjaku powder water swelling is complete；
B, disinfect
Masson pine seed is placed in the copper-bath that mass fraction is 0.4% first, ultrasonication；Then successively
It is 75% alcohol drip washing 1min with mass fraction, mass fraction is 0.05%HgCl2Solution drip washing 10min；Finally with quality point
Number soaks 20min for 3%NaClO solution, and is rinsed well with clear water, standby；
The method of wherein described ultrasonication is：It is first 40KHz ultrasonication 20min with frequency, stands 10min,
Reuse frequency is 80KHz ultrasonication 20min, stands 10min, is finally 40KHz ultrasonications 20min with frequency；
During the masson pine seed that will be disinfected in culturing room is inoculated into cotton culture medium, the circular hole of each non-woven fabrics
1 seed of inoculation, carries out seed sprouting, after culture 18d, obtains masson pine sterile bud Z, and Germination And Seedling rate is 40%；
2) terminal bud of the masson pine sterile bud Z that length is 4cm is cut, as explant, is placed in culture medium b and is trained
Support, after culture 25d, obtain masson pine Multiple Buds；Wherein described culture medium b by added in every 1LMS culture mediums 3mg 6-BA,
0.2mg NAA, 50g potato and 20g sucrose are mixed；
3) terminal bud or tender stem of elongation to masson pine Multiple Buds 5cm long are cut, is planted and is extended in culture medium c
After culture, bud must be extended after culture 24d；Cut the elongation bud of length 5cm terminal bud or tender stem be inoculated into culture medium d in carry out
Propagation, obtains subculture bud after culture 24d, squamous subculture bred for 5 generations, and average coefficient of proliferation is 7.0；
Wherein described culture medium c by added in every 1LMS culture mediums 20mg fluorescent material, 0.5mg NAA, 3000mg sucrose and
7000mg agar is mixed；The culture medium d is by adding 20mg fluorescent material, 1mg 2,4-D, 1mg 6- in every 1LMS culture mediums
BA, 1mg KT, 0.3mg NAA, 3g sucrose and 7g agar are mixed；Fluorescent material is after other raw materials are well mixed, finally
Add；
4) the subculture bud of robust growth, 3cm high is cut, 25min is soaked with fluid nutrient medium e, Liquid Culture is used after taking-up
Base f soaks 15min, transplants in breeding device, fluorescence treatment with irradiation, after culture 24d, obtains masson pine apogamic seedling, takes root
Rate is 96%；
Wherein described fluid nutrient medium e is mixed by adding 75mgIBA and 30mgNAA in every 1LDCR culture mediums；Liquid
Culture medium f is mixed by adding 0.4mgABT6 and 5g talcum powder in every 1LGD culture mediums；
Solid medium is housed, the solid medium includes river sand, vermiculite, liver moss and laterite with 5 wherein in breeding device:
2:1:It is loaded in breeding device after 2 ratio is well mixed；It is 2% to be drenched in the culture medium of breeding device again into 100g mass fractions
Ca (H2PO4)2Solution and the urea liquid that 100g mass fractions are 1%.
Wherein described fluorescence irradiation is specially：1. fluorescent material is fitted into transparent plastic bag, fills whole polybag, and seal
Mouthful；Wherein polybag is 5cm long, the rectangle structure of 2cm wide；2. polybag is pasted on the outside of breeding device, polybag is following
Edge is flushed with matrix of taking root top surface edge in breeding device；
5) nursery is transplanted：Well-grown masson pine apogamic seedling is transplanted to nursery.
Embodiment 2：
1) seed is sprouted
A, making cotton culture medium
Cotton is cleaned with clear water, is positioned over after wringing out in solution a and is soaked 30min, be then layered on soaked cotton
The bottom of bottle of blake bottle, thickness is 3cm, and blake bottle is placed in 30min is freezed in refrigerator；Non-woven fabrics is cleaned again, and is soaked in
In water, by the upper surface of non-textile mulch cotton in blake bottle after 10min, bottle cap is tightened, be put into sterilizing in autoclave, sterilizing
Condition is 115 DEG C, and 30min is standby；
Wherein, the circular hole that aperture uniformly spaced apart from each other is 1cm, two neighboring circular hole spacing are provided with the non-woven fabrics
It is 0.4cm；
The collocation method of wherein described solution a：Solution a is first by adding 7g konjaku flours and 25g sucrose to mix in 1L water
First sucrose is first uniformly dissolved with water, konjaku flour is added, treats that konjaku powder water swelling is complete；
B, disinfect
Masson pine seed is placed in the copper-bath that mass fraction is 0.4% first, ultrasonication；Then successively
It is 75% alcohol drip washing 1min with mass fraction, mass fraction is 0.05%HgCl2Solution drip washing 10min；Finally with quality point
Number soaks 20min for 3%NaClO solution, and is rinsed well with clear water, standby；
The method of wherein described ultrasonication is：It is first 40KHz ultrasonication 20min with frequency, stands 10min,
Reuse frequency is 80KHz ultrasonication 20min, stands 10min, is finally 40KHz ultrasonications 20min with frequency；
During the masson pine seed that will be disinfected in culturing room is inoculated into cotton culture medium, the circular hole of each non-woven fabrics
1 seed of inoculation, carries out seed sprouting, after culture 8d, obtains masson pine sterile bud Z, and Germination And Seedling rate is 45%；
2) terminal bud of the masson pine sterile bud Z that length is 5cm is cut, as explant, is placed in culture medium b and is trained
Support, after culture 23d, obtain masson pine Multiple Buds；Wherein described culture medium b by added in every 1LMS culture mediums 3mg 6-BA,
0.2mg NAA, 50g potato and 20g sucrose are mixed；
3) terminal bud or tender stem of elongation to masson pine Multiple Buds 4cm long are cut, is planted and is extended in culture medium c
After culture, bud must be extended after culture 23d；Cut the elongation bud of length 4cm terminal bud or tender stem be inoculated into culture medium d in carry out
Propagation, obtains subculture bud after culture 22d, squamous subculture bred for 5 generations, and average coefficient of proliferation is 7.5；
Wherein described culture medium c by added in every 1LMS culture mediums 20mg fluorescent material, 0.5mg NAA, 3000mg sucrose and
7000mg agar is mixed；The culture medium d is by adding 20mg fluorescent material, 1mg 2,4-D, 1mg 6- in every 1LMS culture mediums
BA, 1mg KT, 0.3mg NAA, 3g sucrose and 7g agar are mixed；Fluorescent material is after other raw materials are well mixed, finally
Add；
4) the subculture bud of robust growth, 4cm high is cut, 35min is soaked with fluid nutrient medium e, Liquid Culture is used after taking-up
Base f soaks 20min, transplants in breeding device, fluorescence treatment with irradiation, after culture 22d, obtains masson pine apogamic seedling, takes root
Rate is 98%；
Wherein described fluid nutrient medium e is mixed by adding 75mgIBA and 30mgNAA in every 1LDCR culture mediums；Liquid
Culture medium f is mixed by adding 0.4mgABT6 and 5g talcum powder in every 1LGD culture mediums；
Solid medium is housed, the solid medium includes river sand, vermiculite, liver moss and laterite with 5 wherein in breeding device:
2:1:It is loaded in breeding device after 2 ratio is well mixed；It is 2% to be drenched in the culture medium of breeding device again into 100g mass fractions
Ca (H2PO4)2Solution and the urea liquid that 100g mass fractions are 1%.
Wherein described fluorescence irradiation is specially：1. fluorescent material is fitted into transparent plastic bag, fills whole polybag, and seal
Mouthful；Wherein polybag is 6cm long, the rectangle structure of 3cm wide；2. polybag is pasted on the outside of breeding device, polybag is following
Edge is flushed with matrix of taking root top surface edge in breeding device；
5) nursery is transplanted：Well-grown masson pine apogamic seedling is transplanted to nursery.
Embodiment 3：
1) seed is sprouted
A, making cotton culture medium
Cotton is cleaned with clear water, is positioned over after wringing out in solution a and is soaked 30min, be then layered on soaked cotton
The bottom of bottle of blake bottle, thickness is 3cm, and blake bottle is placed in 30min is freezed in refrigerator；Non-woven fabrics is cleaned again, and is soaked in
In water, by the upper surface of non-textile mulch cotton in blake bottle after 10min, bottle cap is tightened, be put into sterilizing in autoclave, sterilizing
Condition is 115 DEG C, and 30min is standby；
Wherein, the circular hole that aperture uniformly spaced apart from each other is 1cm, two neighboring circular hole spacing are provided with the non-woven fabrics
It is 0.4cm；
The collocation method of wherein described solution a：Solution a is first by adding 7g konjaku flours and 25g sucrose to mix in 1L water
First sucrose is first uniformly dissolved with water, konjaku flour is added, treats that konjaku powder water swelling is complete；
B, disinfect
Masson pine seed is placed in the copper-bath that mass fraction is 0.4% first, ultrasonication；Then successively
It is 75% alcohol drip washing 1min with mass fraction, mass fraction is 0.05%HgCl2Solution drip washing 10min；Finally with quality point
Number soaks 20min for 3%NaClO solution, and is rinsed well with clear water, standby；
The method of wherein described ultrasonication is：It is first 40KHz ultrasonication 20min with frequency, stands 10min,
Reuse frequency is 80KHz ultrasonication 20min, stands 10min, is finally 40KHz ultrasonications 20min with frequency；
During the masson pine seed that will be disinfected in culturing room is inoculated into cotton culture medium, the circular hole of each non-woven fabrics
1 seed of inoculation, carries out seed sprouting, and after culture 15d, it is 30% to obtain masson pine sterile bud Z Germination And Seedlings rate；
2) terminal bud of the masson pine sterile bud Z that length is 6cm is cut, as explant, is placed in culture medium b and is trained
Support, after culture 20d, obtain masson pine Multiple Buds；Wherein described culture medium b by added in every 1LMS culture mediums 3mg 6-BA,
0.2mg NAA, 50g potato and 20g sucrose are mixed；
3) terminal bud or tender stem of elongation to masson pine Multiple Buds 6cm long are cut, is planted and is extended in culture medium c
After culture, bud must be extended after culture 22d；Cut the elongation bud of length 6cm terminal bud or tender stem be inoculated into culture medium d in carry out
Propagation, subculture bud is obtained after culture 20d, until obtaining subculture bud 5, squamous subculture bred for 5 generations, and average coefficient of proliferation is 7.2；
Wherein described culture medium c by added in every 1LMS culture mediums 20mg fluorescent material, 0.5mg NAA, 3000mg sucrose and
7000mg agar is mixed；The culture medium d is by adding 20mg fluorescent material, 1mg 2,4-D, 1mg 6- in every 1LMS culture mediums
BA, 1mg KT, 0.3mg NAA, 3g sucrose and 7g agar are mixed；Fluorescent material is after other raw materials are well mixed, finally
Add；
4) the subculture bud of robust growth, 3.5cm high is cut, 25min is soaked with fluid nutrient medium e, trained with liquid after taking-up
Base f immersion 25min are supported, is transplanted in breeding device, fluorescence treatment with irradiation, after culture 28d, obtain masson pine apogamic seedling, it is raw
Root rate is 95%；
Wherein described fluid nutrient medium e is mixed by adding 75mgIBA and 30mgNAA in every 1LDCR culture mediums；Liquid
Culture medium f is mixed by adding 0.4mgABT6 and 5g talcum powder in every 1LGD culture mediums；
Solid medium is housed, the solid medium includes river sand, vermiculite, liver moss and laterite with 5 wherein in breeding device:
2:1:It is loaded in breeding device after 2 ratio is well mixed；It is 2% to be drenched in the culture medium of breeding device again into 100g mass fractions
Ca (H2PO4)2Solution and the urea liquid that 100g mass fractions are 1%.
Wherein described fluorescence irradiation is specially：1. fluorescent material is fitted into transparent plastic bag, fills whole polybag, and seal
Mouthful；Wherein polybag is 10cm long, the rectangle structure of 4cm wide；2. polybag is pasted on the outside of breeding device, polybag is following
Edge is flushed with matrix of taking root top surface edge in breeding device；
5) nursery is transplanted：Well-grown masson pine apogamic seedling is transplanted to nursery.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and implementation method
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 universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details.
Claims (6)
1. a kind of quick obtaining masson pine vegetative propagation is afforested the method for nursery, it is characterised in that comprised the following steps：
1) seed is sprouted
A, making cotton culture medium
Cotton is cleaned with clear water, is positioned over after wringing out in solution a and is soaked 30min, wherein the solution a in 1L water by adding 7g
Konjaku flour and 25g sucrose are mixed, and then soaked cotton is put into blake bottle, and blake bottle is placed in into ice in refrigerator
Freeze 30min；Non-woven fabrics is cleaned again, and is soaked in water, by the upper table of non-textile mulch cotton in blake bottle after 10min
Face, tightens bottle cap, is put into sterilizing in autoclave, and sterilising conditions are 115 DEG C, and 30min is standby, wherein, set on the non-woven fabrics
There is the circular hole that aperture uniformly spaced apart from each other is 1cm, two neighboring circular hole spacing is 0.4cm；
B, disinfect
Masson pine seed is placed in the copper-bath that mass fraction is 0.4% first, ultrasonication；Then matter is used successively
Amount fraction is 75% alcohol drip washing 1min, and mass fraction is 0.05%HgCl2Solution drip washing 10min；It is with mass fraction finally
3%NaClO solution soaks 20min, and is rinsed well with clear water, standby；
During the masson pine seed that will be disinfected in culturing room is inoculated into cotton culture medium, the circular hole inoculation of each non-woven fabrics
1 seed, carries out seed sprouting, obtains masson pine sterile bud Z；
2) terminal bud of the masson pine sterile bud Z that length is 4~6cm is cut, as explant, is placed in culture medium b and is cultivated,
Obtain masson pine Multiple Buds；Wherein described culture medium b is by adding 3mg 6-BA, 0.2mg NAA, 50g horses in every 1LMS culture mediums
Bell potato and 20g sucrose are mixed；
3) terminal bud or tender stem of elongation to masson pine Multiple Buds 4~6cm long are cut, is planted and is extended in culture medium c
After culture, elongation bud is obtained；Cut the elongation bud of 4~6cm of length terminal bud or tender stem be inoculated into culture medium d in bred,
Obtain subculture bud；
Wherein described culture medium c by added in every 1LMS culture mediums 20mg fluorescent material, 0.5mg NAA, 3000mg sucrose and
7000mg agar is mixed；The culture medium d is by adding 20mg fluorescent material, 1mg 2,4-D, 1mg 6- in every 1LMS culture mediums
BA, 1mg KT, 0.3mg NAA, 3g sucrose and 7g agar are mixed；
4) the subculture bud of robust growth, 3~4cm high is cut, 25~35min is soaked with fluid nutrient medium e, trained with liquid after taking-up
Support base f and soak 15~25min, transplant in breeding device, fluorescence treatment with irradiation, obtain masson pine apogamic seedling；
Wherein described fluid nutrient medium e is mixed by adding 75mgIBA and 30mgNAA in every 1LDCR culture mediums；Liquid Culture
Base f is mixed by adding 0.4mgABT6 and 5g talcum powder in every 1LGD culture mediums；
Wherein described fluorescence irradiation is specially：1. fluorescent material is fitted into transparent plastic bag, fills whole polybag, and seal；
Wherein polybag is 5~10cm long, the rectangle structure of 2~4cm wide；2. polybag is pasted on the outside of breeding device, polybag
Lower edge is flushed with matrix of taking root top surface edge in breeding device；
5) nursery is transplanted：Well-grown masson pine apogamic seedling is transplanted to nursery.
2. quick obtaining masson pine vegetative propagation as claimed in claim 1 is afforested the method for nursery, it is characterised in that also wrapped
Include：Step 1) described in solution a collocation method：Sucrose is first uniformly dissolved with water first, adds konjaku flour, treat konjaku flour
Water swelling is complete.
3. quick obtaining masson pine vegetative propagation as claimed in claim 1 is afforested the method for nursery, it is characterised in that also wrapped
Include：Step 1) described in cotton be laid in the bottom of bottle of blake bottle, thickness is 1.8~3.0cm.
4. quick obtaining masson pine vegetative propagation as claimed in claim 1 is afforested the method for nursery, it is characterised in that also wrapped
Include：Step 1) described in the method for ultrasonication be：It is first 40KHz ultrasonication 20min with frequency, stands 10min, then
It is 80KHz ultrasonication 20min with frequency, stands 10min, is finally 40KHz ultrasonications 20min with frequency.
5. quick obtaining masson pine vegetative propagation as claimed in claim 1 is afforested the method for nursery, it is characterised in that also wrapped
Include：Step 3) described in fluorescent material in culture medium c and culture medium d be after other raw materials are well mixed, to be eventually adding fluorescence
Powder.
6. quick obtaining masson pine vegetative propagation as claimed in claim 1 is afforested the method for nursery, it is characterised in that also wrapped
Include：Step 4) described in solid medium is housed in breeding device, the solid medium includes river sand, vermiculite, liver moss and laterite
With 5:2:1:It is loaded in breeding device after 2 ratio is well mixed；Drenched in the culture medium of the breeding device into 100g mass point
Number is 2% Ca (H2PO4)2Solution and the urea liquid that 100g mass fractions are 1%.
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