CN107232060A - A kind of method for spending imperial Vitro Quick Reproduction in vain - Google Patents
A kind of method for spending imperial Vitro Quick Reproduction in vain Download PDFInfo
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- CN107232060A CN107232060A CN201710544482.0A CN201710544482A CN107232060A CN 107232060 A CN107232060 A CN 107232060A CN 201710544482 A CN201710544482 A CN 201710544482A CN 107232060 A CN107232060 A CN 107232060A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
-
- 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
Abstract
The invention discloses a kind of method for spending imperial Vitro Quick Reproduction in vain, comprise the steps of：(1) seed is sprouted；(2) squamous subculture；(3) strong sprout and culture of rootage；(4) hardening and transplanting.Removed in the method for the present invention for spending imperial Vitro Quick Reproduction in vain and spend dragon species shell in vain to increase gas permeability, water penetration, endosperm reduction germination inhibitor and the material for causing seed browning are removed to the influence of seed, germination rate, survival rate is improved and shortens sprout time.Optimize the hormone kind of proliferated culture medium and the proportioning of concentration to improve growth coefficient.Optimize rooting method, take root minimal medium and hormone kind, the proportioning of concentration number and shorten rootage duration to improve rooting rate, increase and take root.The method of the present invention for spending imperial Vitro Quick Reproduction in vain realizes cultured in vitro and quickly bred, and is laid the foundation to spend seedling supply of the dragon in terms of afforestation, side slope reparation, medicinal and grease are used in vain.
Description
Technical field
The present invention relates to a kind of method for in-vitro rapid propagation of plant, and in particular to a kind of to spend imperial Vitro Quick Reproduction in vain
Method.
Background technology
Spend imperial (Styrax faeri Perk.) category Styracaceae (Styracaceae) Styraax (Styrax) in vain, point
It is distributed in the provinces such as Anhui, Hubei, Jiangsu, Zhejiang, Hunan, Jiangxi, Guangdong, Guangxi and Taiwan.Spend imperial tree-like grace, late spring in vain
Open white flowers, it is pure and fresh free from vulgarity, spend small, bennet long, it is piece sagging when the flowers are in blossom piece, be hung on branch slightly like lantern one by one.In Guangdong
Area, the seeds that spring blooms are mainly the kapok of red, flame wood, and the Golden Bell Tree of yellow, the Chinese redbud of pink colour etc. is spent in vain
Dragon is to open the ornamental tree species of one's native land of white flowers, form rich in peculiar fun spring few in number, be particularly suitable for cell, park,
The greening of road etc..Spend the vanguard tree seed under dragon or the arid habitat of the strong light of south subtropicses in vain, be common in height above sea level 100-600m's
Low-relief terrain and hills shrubbery, are the seeds that good slope ecological is repaired.
In addition to viewing and admiring, spending dragon in vain also has very high economic value.Spend in vain dragon leaf can be used for stop blooding, myogenic, detumescence,
Fruit can control dizzy heating, and root can be used for controlling epigastric pain, seed oil can soap system and lubricating oil, be good medicinal and grease
With plant, with wide DEVELOPMENT PROSPECT.Vitro Plant culture quickly breeding be obtain a large amount of high quality seedlings effective way it
One, the present invention it is proposed it is vertical spend imperial high-efficiency in-vitro culture rapid propagation system in vain, lay the foundation, promote for the seedling supply of spending dragon in vain
Spend application of the dragon in terms of afforestation, side slope reparation, medicinal and grease are used in vain.
Spend dragon in vain and mainly use cutting propagation at this stage, but survival rate is low and branch demand is big, and sapling multiplication speed is not
The market demand can be met.Spend in vain and bloom the imperial 4-6 months, 8-10 month results, seed production is very big, but because kind of shell is hard and with one
Fixed hibernation feature, sprouts, germination rate is low, sprout time is long in or so Second Year 4-5 months in its natural state.Congener
(Styraax, Styrax) seed also more hibernation feature, should not be with adopting with broadcasting, such as styrax tonkinensis Craib ex Hart (Styrax tonkinensis
(Pierre) Craib ex Hartw.) seed through under normal temperature store 5 months after, germination percentage is only 20%；Hanging bead is spent
(Styrax dasyanthus Perk) is through 1200mg/L GA324h is pre-processed, is only with reference to low temperature sand storage lamination germination percentage
44%.So far, not yet there is the report for spending imperial artificial seeding's breeding and Propagation In Vitro in vain.
The content of the invention
It is an object of the invention to overcome weak point that prior art is present and provide one kind spend in vain it is imperial in vitro quick numerous
The method grown.
To achieve the above object, the technical scheme taken of the present invention is：A kind of method for spending imperial Vitro Quick Reproduction in vain, bag
Containing following steps：
(1) seed is sprouted：Dragon species will be spent in vain with alcohol-pickled 40~80s, the alcohol of the surface of the seed is washed away with sterilized water,
Outer kind of shell is divested, mercuric chloride soaks 4~6min, the mercury of the surface of the seed is washed away with sterilized water, is dried, and removes the endosperm of seed, obtain outer
Sprouting induction is carried out in implant, the sprouting inducing culture that explant is seeded to, aseptic seedling is obtained；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 1~3 section of band is seeded to inducing clumping bud
Inducing clumping bud is carried out in culture medium；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 1.0~2.0cm that selection step (2) is obtained are inoculated in strong sprout training
Support and 30~45 days are cultivated on base with strong sprout；Well-grown seedling is cut into the stem section of 1~3 section of band for direct after selection strong sprout
Culture of rootage or indirect culture of rootage；
(4) hardening and transplanting：By plant obtained by step (3), hardening in cool canopy is placed in, is transplanted after 6~8 days to matrix,
Imperial seedling must be spent in vain.
The method of the present invention for spending imperial Vitro Quick Reproduction in vain removes kind of shell to increase the saturating of seed to spending dragon species in vain
Gas, water penetration, improve germination rate.It is 105Kpa that used medium of the present invention, which is both needed in pressure, and temperature is under conditions of 121 DEG C
Carry out autoclaving 20min.The concentration of alcohol can be 75% alcohol, and mercuric chloride can be 0.1% mercuric chloride.To removing kind of a shell
Dragon species of spending in vain be removed endosperm processing, reducing germination inhibitor and causes the material of seed browning to the shadow of seed
Ring, germination rate, survival rate can be effectively improved and sprout time is shortened.Direct culture of rootage of the present invention is directly will
Bud, which is inoculated in corresponding culture medium, carries out culture of rootage, and indirect culture of rootage is the IBA that stem section base portion is first placed in high concentration
10~20min is soaked in solution, returns again to and is cultivated in corresponding culture medium.It is of the present invention to spend imperial Vitro Quick Reproduction in vain
The obtained seed germination rate of method and planting percent it is high, the survival rate of seedling is high, can realize that cultured in vitro is quickly bred, to spend in vain
Seedling supply of the dragon in terms of afforestation, side slope reparation, medicinal and grease are used lays the foundation.
It is described in step (1) as the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain
Sprout inducing culture and include MS, 0.5~4.0mg/LGA3, 20~35g/L sucrose, 3.5~7.5g/L agar, described sprouting
The pH value of inducing culture is 5.5~6.2.The proportioning of hormone kind and concentration in sprouting inducing culture of the present invention can
To improve the germination rate and planting percent of seed.
As the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, in step (2), the clump
Inducing culture of sprouting includes MS, 0.1~3.0mg/L BA, 0~0.5mg/L NAA, 0~0.5mg/L TDZ, 20~35g/L
Sucrose and 3.5~7.5g/L agar, the pH value of the inducing clumping bud culture medium is 5.5~6.2.Multiple Buds of the present invention are lured
Growth coefficient can be improved by leading the proportioning of hormone kind and concentration in culture medium.
It is described strong in step (3) as the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain
Seedling culture medium includes MS, 0.1~1.5mg/L BA, 0.01~0.5mg/L NAA, 0.05~1.5mg/LGA3, 20~35g/L sugarcanes
Sugar and 3.5~7.5g/L agar, the pH value of the strong seedling culture base is 5.5~6.2.Strong seedling culture can be such that aseptic seedling is more good for
It is strong, so as to be conducive to improving the survival rate of rooting rate and acclimatization and transplantses.
Optimize the hormone kind of proliferated culture medium and the proportioning of concentration to improve growth coefficient.Optimize rooting method, life
Foundation basal culture medium and hormone, the proportioning of concentration count and shortened rootage duration to improve rooting rate, increase to take root.Realize in vitro
The quick breeding of culture, base is established to spend seedling supply of the dragon in terms of afforestation, side slope reparation, medicinal and grease are used in vain
Plinth.
It is described straight in step (3) as the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain
The culture medium for connecing culture of rootage includes 1/4MS~MS, 0.5~5.0mg/L IBA, 20~35g/L sucrose and 3.5~7.5g/L fine jades
Fat, the pH value of the direct root media is 5.5~6.2.
As the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, in step (3), it is described between
The method for connecing culture of rootage is：First the base portion of stem section is placed in the IBA that concentration is 50~500mg/L and soaks 10~20min, then
It is transferred on indirect root media and cultivates.Compared with direct culture of rootage, the rooting rate of indirect culture of rootage is higher, number of taking root
More and rootage duration is shorter.
As the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the concentration of the IBA is
50~500mg/L.Rooting rate is as the increase of IBA concentration is in downward trend after first rising.Root can be suppressed if excessive concentration
The growth of former base, so as to reduce rooting rate, reduce take root number and suppression root growth.It is 50~500mg/L in above-mentioned IBA concentration
When, be conducive to improving rooting rate, increase take root number and shortening rootage duration.
It is used as the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the indirect culture of rootage
Base includes 1/4MS~MS, 20~35g/L sucrose and 3.5~7.5g/L agar, and the pH value of the indirect root media is 5.5
~6.2.The method of the species of hormone in indirect root media of the present invention and the proportioning and indirect culture of rootage of concentration
Mutually promote, rooting rate can be effectively improved, increase take root number and shortening rootage duration.
As the preferred embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, in step (4), the base
Matter is the mixture of peat soil, perlite and sand, and the ratio of the peat soil, perlite and sand is：Peat soil：Perlite：It is husky
=3:1:1.The survival rate of seedling highest in this matrix, the matrix is spent imperial seedling in vain for culture and is adapted to the most.
The beneficial effects of the present invention are：The invention provides a kind of method for spending imperial Vitro Quick Reproduction in vain, the present invention
Removed in the method for spending imperial Vitro Quick Reproduction in vain and spend dragon species shell in vain to increase gas permeability, water penetration, remove endosperm reduction
Germination inhibitor and the material for causing seed browning improve germination rate, survival rate and shorten sprouting to the influence of seed
Time.Optimize the hormone kind of proliferated culture medium and the proportioning of concentration to improve growth coefficient.Optimize rooting method, base of taking root
Basal culture medium and hormone kind, the proportioning of concentration count and shortened rootage duration to improve rooting rate, increase to take root.It is of the present invention
Spend the method for imperial Vitro Quick Reproduction in vain and realize cultured in vitro and quickly breed, to spend dragon in vain in afforestation, side slope reparation, medicine
Laid the foundation with the seedling supply in terms of being used with grease.
Brief description of the drawings
Fig. 1 is the growing state in some stage in embodiment 1,2,7,8, comparative example；
Wherein 1A is the sprouting situation after being inoculated with 45d days with albuminous seed described in comparative example；Figure 1B is to be planted described in embodiment 2
Son sprouts the sprouting situation after 7d；Fig. 1 C are inoculated with the growing state of seedling after 45d for inducing clumping bud described in embodiment 1；Fig. 1 D are
The growing state of root after the direct culture of rootage 45d of seedling in embodiment 2；Fig. 1 E are the indirect culture of rootage 45d of seedling in embodiment 8
The growing state of root afterwards；Fig. 1 F are the growing state after transplantation of seedlings 30d in embodiment 8.
Embodiment
English initialism of the present invention is explained as follows：GA3It is that gibberellin, BA are that 6- benzylaminopurines, NAA are naphthalenes
Acetic acid, TDZ are that Thidiazuron, IBA are indolebutyric acids.
The preparation of culture medium：
Minimal medium MS, 1/2MS constituent in the present invention is as shown in table 1：
Table 1. spends imperial cultured in vitro in vain and quickly breeds use minimal medium list
Exemplified by preparing 1L culture mediums：The accurate various compounds weighed described in table 1, plus appropriate distilled water dissolving, are used
Glass bar stirs dissolution, adjusts pH to 6.0 with NaOH or HCL, last constant volume to 1L.
Culture medium of the present invention be both needed to pressure be 105Kpa, temperature be 121 DEG C under conditions of carry out autoclaving
20min；The condition of medium culture of the present invention is：25 ± 1 DEG C of cultivation temperature, intensity of illumination 2000lx, light application time
12h/d。
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiments and the drawings pair
The present invention is described further.
Embodiment 1
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 40s, sterile water wash 1 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 5min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：MS、1.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the sprouting
The pH value of inducing culture is 6.0；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 2 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base includes MS, 0.5mg/L BA, 0.05mg/L NAA, 30g/L sucrose and 7.0g/L agar, the inducing clumping bud culture medium
PH value is 6.0；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the direct culture of rootage of stem section progress of 2 sections of band after selection strong sprout；Often
Individual 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base is included：MS、0.5mg/L BA、0.1mg/L NAA、
0.5mg/LGA3, 30g/L sucrose and 7.0g/L agar, the pH value of the strong seedling culture base is 6.0；The rooting induction culture medium
Comprising MS, 1.0mg/LIBA, 30g/L sucrose and 7.0g/L agar, the pH is 6.0；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Embodiment 2
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 60s, sterile water wash 2 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 6min, sterile water wash 7 times is placed on filter paper and dried, finally the seed with endosperm
It is inoculated into corresponding sprout and sprouting induction is carried out in inducing culture culture medium.Each 15 bottles of processing, 2 explants of every bottle of inoculation
Body；The sprouting inducing culture is included：MS、2.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the sprouting induction training
The pH value for supporting base is 6.0；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 2 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture.Each 15 bottles of processing, 2 explants of every bottle of inoculation；The inducing clumping bud culture
Base is included：MS, 1.0mg/L BA, 0.05mg/L NAA, 30g/L sucrose and 7.0g/L agar, the inducing clumping bud culture medium
PH value be 6.0；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the direct culture of rootage of stem section progress of 2 sections of band after selection strong sprout；Often
Individual 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base comprising MS, 0.5mg/L BA, 0.1mg/L NAA,
1.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the pH value of the strong seedling culture base is 6.0；The rooting induction culture
Base includes MS, 3.0mg/LIBA, 30g/L sucrose and 7.0g/L agar, and the pH is 6.0；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening.Transplanted after 6~8d in matrix, the matrix is the mixture of peat soil, perlite and sand, the peat soil, treasure
Zhu Yan and husky ratio are：Peat soil：Perlite：Husky=3:1:1.
Embodiment 3
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 40s, sterile water wash 1 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 4min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：3.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the sprouting is lured
The pH value for leading culture medium is 6.0；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 1 section of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base is included：MS, 2.0mg/L BA, 0.05mg/L NAA, 30g/L sucrose and 7.0g/L agar, the inducing clumping bud culture medium
PH value be 6.0；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 1.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the direct culture of rootage of stem section progress of 1 section of band after selection strong sprout；Often
Individual 8 bottles of processing, 5 explants of every bottle of inoculation；The composition of the strong seedling culture base is：MS、0.5mg/L BA、0.1mg/L NAA、
1.5mg/L GA3, 20g/L sucrose and 3.5g/L agar, the pH value of the strong seedling culture base is 6.0；The rooting induction culture
Base MS, 5.0mg/L IBA, 30g/L sucrose and 7.0g/L agar, the pH value of the rooting induction culture medium is 6.0；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Embodiment 4
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the present embodiment and embodiment 1 are not
The difference of inducing clumping bud medium component is only that with part, inducing clumping bud culture medium is included described in the present embodiment：MS、
0.5mg/L BA, 0.01mg/L TDZ, 0.05mg/L NAA, 30g/L sucrose and 7.0g/L agar.
Embodiment 5
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 40s, sterile water wash 1 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 5min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：MS、1.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the sprouting
The pH value of inducing culture is 6.0；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 2 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base includes MS, 0.5mg/L BA, 0.1mg/L NAA, 30g/L sucrose and 7.0g/L agar, the inducing clumping bud culture medium
PH value is 6.0；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the indirect culture of rootage of stem section progress of 2 sections of band after selection strong sprout：First
Stem section base portion is placed in the IBA solution that concentration is 100mg/L and soaks 15min, returns again to and is cultivated in corresponding culture medium；
Each 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base is included：MS、1.0mg/L BA、0.1mg/L NAA、
0.5mg/LGA3, 30g/L sucrose and 7.0g/L agar, the pH value of the strong seedling culture base is 6.0；The rooting induction culture medium
Comprising MS, 30g/L sucrose and 7.0g/L agar, the pH is 6.0；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Embodiment 6
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the present embodiment and embodiment 5 are not
It is only that with part in the difference of IBA concentration in step (3), the present embodiment step (3), IBA solution concentrations are 200mg/L.
Embodiment 7
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 40s, sterile water wash 1 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 5min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：MS、1.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, the sprouting
The pH value of inducing culture is 6.0；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 2 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base includes MS, 0.5mg/L BA, 0.1mg/L NAA, 0.1mg/L TDZ, 30g/L sucrose and 7.0g/L agar, the Multiple Buds
The pH value of inducing culture is 6.0；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the indirect culture of rootage of stem section progress of 2 sections of band after selection strong sprout：First
Stem section base portion is placed in the IBA solution that concentration is 100mg/L and soaks 15min, returns again to and is cultivated in corresponding culture medium；
Each 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base is included：MS、1.0mg/L BA、0.1mg/L NAA、
0.5mg/LGA3, 30g/L sucrose and 7.0g/L agar, the pH value of the strong seedling culture base is 6.0；The rooting induction culture medium
Comprising 1/2MS, 30g/L sucrose and 7.0g/L agar, the pH is 6.0；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Embodiment 8
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the present embodiment and embodiment 7 are not
It is only that with part in the difference of IBA concentration in step (3), the present embodiment step (3), IBA solution concentrations are 200mg/L.
Embodiment 9
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the present embodiment and embodiment 7 are not
It is only that with part in the difference of IBA concentration in step (3), the present embodiment step (3), IBA solution concentrations are 300mg/L.
Embodiment 10
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, the present embodiment and embodiment 7 are not
It is only that with part in the difference of IBA concentration in step (3), the present embodiment step (3), IBA solution concentrations are 400mg/L.
Embodiment 11
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 80s, sterile water wash 2 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 6min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：MS、0.5mg/L GA3, 20g/L sucrose and 3.5g/L agar, the sprouting
The pH value of inducing culture is 5.5；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 3 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base is：Inducing clumping bud culture medium includes MS, 0.1mg/L BA, 20g/L sucrose and 3.5g/L agar, the inducing clumping bud training
The pH value for supporting base is 5.5；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the indirect culture of rootage of stem section progress of 1 section of band after selection strong sprout：First
Stem section base portion is placed in the IBA solution that concentration is 50mg/L and soaks 10min, returns again to and is cultivated in corresponding culture medium；
Each 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base comprising MS, 0.1mg/L BA, 0.01mg/L NAA,
0.05mg/LGA3, 20g/L sucrose and 3.5g/L agar, the pH value of the strong seedling culture base is 5.5；The indirect culture of rootage
Base includes 1/4MS, 20g/L sucrose and 3.5g/L agar, and the pH value of the indirect root media is 5.5；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Embodiment 12
A kind of embodiment of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 80s, sterile water wash 2 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 6min, sterile water wash 6 times is placed on filter paper and dried, and then removes the embryo of seed
Breast, is finally seeded to corresponding sprout by seed and sprouting induction is carried out in inducing culture；Each 10 bottles of processing, every bottle of inoculation 3
Individual explant；The sprouting inducing culture is included：MS、4.0mg/L GA3, 35g/L sucrose and 7.5g/L agar, the sprouting
The pH value of inducing culture is 6.2；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 3 sections of band is inoculated into corresponding Multiple Buds
Inducing clumping bud is carried out in inducing culture；Each 10 bottles of processing, 3 explants of every bottle of inoculation；The inducing clumping bud culture
Base is：Inducing clumping bud culture medium comprising MS, 3.0mg/L BA, 0.5mg/L NAA, 0.5mg/L TDZ, 35g/L sucrose and
7.5g/L agar, the pH value of the inducing clumping bud culture medium is 6.2；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
Upper culture 45d is with strong sprout；Well-grown seedling is cut into the indirect culture of rootage of stem section progress of 1 section of band after selection strong sprout：First
Stem section base portion is placed in the IBA solution that concentration is 500mg/L and soaks 10min, returns again to and is cultivated in corresponding culture medium；
Each 8 bottles of processing, 5 explants of every bottle of inoculation；The strong seedling culture base comprising MS, 1.5mg/L BA, 0.5mg/L NAA,
1.5mg/LGA3, 35g/L sucrose and 7.5g/L agar, the pH value of the strong seedling culture base is 6.2；The indirect root media
Comprising 1/4MS, 35g/L sucrose and 7.5g/L agar, the pH value of the indirect root media is 6.2；
(4) hardening and transplanting：The plant for selecting stalwartness, the lateral root of step (3) acquisition flourishing, opens bottle cap, is placed in cool canopy
Interior hardening；Transplanted after 6~8d to matrix, matrix is the mixture of peat soil, perlite and sand, the peat soil, perlite
It is with husky ratio：Peat soil：Perlite：Husky=3:1:1.
Comparative example
A kind of comparative example of the method for the present invention for spending imperial Vitro Quick Reproduction in vain, comprises the following steps：
(1) seed is sprouted：On superclean bench, dragon species 75% alcohol-pickled 60s, sterile water wash 2 will be spent in vain
Time, outer kind shell is peeled off, 0.1% mercuric chloride immersion 6min, sterile water wash 7 times is placed on filter paper and dried, finally the seed with endosperm
It is inoculated into progress sprouting induction in corresponding culture medium.Each 15 bottles of processing, 2 explants of every bottle of inoculation；Described sprouting is lured
Culture medium is led to include：MS、2.0mg/L GA3, 30g/L sucrose and 7.0g/L agar, pH is 6.0.Because the sprouting of step (1) is induced
Planting percent be 0, it is impossible to carry out step (2), (3), (4).
Embodiment 13
Situation in embodiment 1 after inducing clumping bud 45d is shown in Fig. 1 C, and as can be seen from the figure the growth of Multiple Buds is vigorous；
The situation that seed is sprouted after 7d in embodiment 2 is shown in Figure 1B, and the directly situation of taking root is shown in Fig. 1 D, from figure：The quantity of root is fewer,
Unrooted hair is produced；The situation of root of being delivered a child in the middle of embodiment 8 is shown in Fig. 1 E, from figure：The quantity of root is more, and has root hair generation；Move
Plant the situation after 30d and see Fig. 1 F, as can be seen from the figure the upgrowth situation of seedling is good.
Seed just starts to sprout after the seed of comparative example is inoculated with one month, and cotyledon starts browning after sprouting 15d, final complete
Portion is dead, and Figure 1A is the situation after inoculation 45d.
Embodiment 14
Spend imperial Vitro Quick Reproduction situation described in observed and recorded embodiment 1~12 in vain：
1st, the upgrowth situation of the germination rate of seed, planting percent and seedling after 30~45d is inoculated with statistic procedure (1)；
2nd, the growing state of the growth coefficient after 30~45d, average height of seedling and Multiple Buds is inoculated with statistic procedure (2)；
3rd, culture of rootage is inoculated with rooting rate after 30~45d, the growth for counting and recording root of averagely taking root in statistic procedure (3)
Situation；
4th, the survival rate that seedling is counted after 30~40d is transplanted in statistic procedure (4).
Statistical result is shown in Table 2.
Imperial Vitro Quick Reproduction situation is spent in vain described in the embodiment 1~12 of table 2
As it can be seen from table 1 spend that imperial method for in-vitro rapid propagation obtains described in embodiment 1~12 in vain spend in vain Long Douyou compared with
High survival rate, from the contrast of embodiment 1~3,11,12 step (1) result：With GA3The rise of concentration, spends dragon species in vain
Sub- germination rate and planting percent are in downward trend, GA after first rising3Concentration when being 1~2mg/L, germination rate and planting percent compared with
It is high.Comparative example 1~12 and comparative example are understood：The seed sprout time for removing endosperm is short, and germination rate and planting percent are high；And band
Albuminous seed sprout time is long, and cotyledon starts browning after sprouting 15d, final all dead.Therefore, the endosperm for spending dragon in vain may
In sprout and cause the dead material of seedling browning containing suppressing seed, must go to remove in germination in vitro.Comparative example 1,
The result of step (2) is understood in 12：BA and NAA ratios ratio is relatively low and absolute concentration of BA and NAA also than it is relatively low when, can obtain
Higher growth coefficient；Addition high concentration TDZ easily produces callus.The step of comparative example 1~3 and embodiment 7~12
Suddenly the result of (3) is understood：Rise of the imperial rooting rate with IBA concentration is spent in vain, in downward trend after first rising.Comparative example 1
~6 and (3) are understood the step of embodiment 7~12：Indirect rooting method is conducive to improving that rooting rate, increase take root number and shortening is taken root
Time.In addition, when minimal medium Concentrations are relatively low, being conducive to root hair generation.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (9)
1. a kind of method for spending imperial Vitro Quick Reproduction in vain, it is characterised in that comprise the steps of：
(1) seed is sprouted：Dragon species will be spent in vain with alcohol-pickled 40~80s, the alcohol of the surface of the seed is washed away with sterilized water, is divested
Outer kind of shell, soaks 4~6min with mercuric chloride, the mercury of the surface of the seed is washed away with sterilized water, is dried, and removes the endosperm of seed, obtains explant
Sprouting induction is carried out in body, the sprouting inducing culture that explant is seeded to, aseptic seedling is obtained；
(2) squamous subculture：The stem section that the aseptic seedling that step (1) is obtained is cut into 1~3 section of band is seeded to inducing clumping bud culture
Inducing clumping bud is carried out in base；
(3) strong sprout and culture of rootage：The Multiple Buds for a height of 1.0~2.0cm that selection step (2) is obtained are inoculated in strong seedling culture base
It is upper to cultivate 30~45 days with strong sprout；Well-grown seedling is cut into the stem section of 1~3 section of band for directly taking root after selection strong sprout
Culture or indirectly culture of rootage；
(4) hardening and transplanting：By plant obtained by step (3), hardening in cool canopy is placed in, is transplanted after 6~8 days to matrix, obtains white
Spend imperial seedling.
2. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (1), described sprouting
Inducing culture includes MS, 0.5~4.0mg/LGA3, 20~35g/L sucrose, 3.5~7.5g/L agar, described sprouting induction
The pH value of culture medium is 5.5~6.2.
3. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (2), the Multiple Buds
Inducing culture includes MS, 0.1~3.0mg/L BA, 0~0.5mg/L NAA, 0~0.5mg/L TDZ, 20~35g/L sucrose
With 3.5~7.5g/L agar, the pH value of the inducing clumping bud culture medium is 5.5~6.2.
4. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (3), the strong sprout training
Support base and include MS, 0.1~1.5mg/L BA, 0.01~0.5mg/L NAA, 0.05~1.5mg/LGA3, 20~35g/L sucrose and
3.5~7.5g/L agar, the pH value of the strong seedling culture base is 5.5~6.2.
5. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (3), the direct life
The culture medium of root culture includes 1/4MS~MS, 0.5~5.0mg/L IBA, 20~35g/L sucrose and 3.5~7.5g/L agar,
The pH value of the direct root media is 5.5~6.2.
6. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (3), it is described between deliver a child
The method of root culture is：First the base portion of stem section is placed in the IBA that concentration is 50~500mg/L and soaks 10~20min, then is transferred
Cultivated on to indirect root media.
7. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 6, it is characterised in that the concentration of the IBA is 50~
500mg/L。
8. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 6, it is characterised in that the indirect root media bag
Containing 1/4MS~MS, 20~35g/L sucrose and 3.5~7.5g/L agar, the pH value of the indirect root media for 5.5~
6.2。
9. spend the method for imperial Vitro Quick Reproduction in vain as claimed in claim 1, it is characterised in that in step (4), the matrix is
The mixture of peat soil, perlite and sand, the peat soil, perlite and husky ratio are：Peat soil：Perlite：Husky=3:
1:1。
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Cited By (2)
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| CN109984038A (en) * | 2019-04-08 | 2019-07-09 | 鲁东大学 | Rapidly promote Chinese wax stem apex proliferation and the take root culture medium being completed at the same time and purposes |
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